Scientific discovery has traditionally relied on human-led iterative loops of observation, modeling, and intervention. This thesis explores the possibility of automating components of this loop using artificial intelligence (AI), particularly in systems characterized by non-equilibrium dynamics, high dimensionality, and emergent behaviors. Two foundational challenges are addressed: automating physical modeling and enabling adaptive interaction with dynamic experimental systems, and generalizing agent behavior across tasks and contexts without retraining.
\r\n\r\nTo address the first challenge, we introduce a hierarchical AI framework for controlling active biomolecular matter, exemplified by microtubule\u2013kinesin networks driven by light-activated motors. At the foundation are predictive models that learn the system\u2019s response to static light patterns, enabling inverse design by selecting inputs that yield desired structural outcomes. Building on this, dynamic models construct low-dimensional representations of the system\u2019s evolving state under time-varying stimuli, supporting forward simulation and real-time tracking. At the highest level, reinforcement learning agents\u2014trained in simulation\u2014discover and execute closed-loop control policies that achieve fine-grained manipulation objectives. These agents are deployed across ~100 parallel experimental setups, demonstrating autonomous operation with robustness, scalability, and reliable transfer.
\r\n\r\nTo address the second challenge, we investigate how generalist reinforcement learning agents can be constructed by leveraging the geometry of policy parameter space. We show that agents trained on distinct tasks self-organize into functionally segregated regions of weight space that encode both task identity and strategic variability. This insight enables the design of a hypernetwork\u2014a network that generates the weights of other networks\u2014that can interpolate smoothly between tasks and strategies via a single scalar input. Combined with a meta-controller, this architecture enables real-time modulation of agent behavior\u2014ranging from conservative to risk-seeking\u2014without retraining.
\r\n\r\nTogether, these contributions demonstrate that intelligent systems can both design and control physical experiments in real time, and adapt cognitive strategies across tasks through principled representations in policy space. This work establishes a foundation for closed-loop scientific autonomy, programmable biomaterials, and generalist AI agents, converging at the intersection of machine learning, biophysics, and automation.
" }, { "name": "Caldera, Luis Fernando", "degree": "PhD", "year": "2026", "title": "Engineering Immunological Solutions for Pandemic-Level Threats", "advisor": "Bjorkman, Pamela J.; Mayo, Stephen L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05312026-053755214", "creators": [ { "name": { "family": "Caldera", "given": "Luis Fernando" }, "id": "Caldera-Luis-Fernando", "orcid": "0009-0005-3254-6563", "display_name": "Caldera, Luis Fernando" } ], "advisors": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "advisor", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "co-advisor", "display_name": "Mayo, Stephen L." } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "chair", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Bois", "given": "Justin S." }, "id": "Bois-J-S", "orcid": "0000-0001-7137-8746", "role": "member", "display_name": "Bois, Justin S." } ], "option_major": [ "bioeng" ], "doi": "10.7907/wgps-rp81", "abstract": "Pandemics remain among the most serious threats to global public health, especially when viruses can efficiently spread and persist in human populations. Two major examples are the AIDS pandemic caused by HIV-1, which emerged in the early 1980s, and the COVID-19 pandemic caused by SARS-CoV-2, which began in late 2019. Despite their differences in timescale and transmission, HIV-1 and SARS-CoV-2 exhibit high antigenic diversity that drives an ongoing arms race between viral escape and the development of vaccines and therapeutics. This thesis presents three molecular engineering strategies to confront that problem. For SARS-CoV-2, we utilized computational tools to generate mosaic nanoparticle vaccines displaying engineered SARS-CoV-2 or selected sarbecovirus receptor-binding domains (RBDs) sharing conserved antigenic features to drive immunity toward eliciting cross-reactive responses. In na\u00efve and pre-vaccinated mice, our lead candidate, mosaic-7COM, elicited broader and more potent cross-reactive antibody responses compared to our prior mosaic-8b candidate. For HIV-1, we engineered a stabilized CD4-Ig reagent specific to HIV-1 envelope (Env) glycoprotein with greatly reduced off-target recognition of MHC class II. The final CD4 design had markedly increased thermostability (>20\u00b0C) and a nearly 50-fold improvement in mammalian expression compared to the wild-type construct, highlighting its potential as a therapeutic biologic. Additionally, we developed a yeast-display screening platform to isolate nanobody (VHH) domains against the caldera, a conserved, glycan-free epitope exposed on Env upon engagement with CD4. This campaign yielded five VHH-Fc candidates that bind receptor-bound Env and established a foundation for future biologic development targeting a previously inaccessible site on the virus." }, { "name": "Carilli, Maria Theresa Natalina", "degree": "PhD", "year": "2026", "title": "Genetic Interrogation of Expression Regulation", "advisor": "Pachter, Lior S.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06012026-033232974", "creators": [ { "name": { "family": "Carilli", "given": "Maria Theresa Natalina" }, "id": "Carilli-Maria-Theresa-Natalina", "orcid": "0000-0002-8977-7224", "display_name": "Carilli, Maria Theresa Natalina" } ], "advisors": [ { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "advisor", "display_name": "Pachter, Lior S." } ], "committee": [ { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "chair", "display_name": "Phillips, Robert B." }, { "name": { "family": "Yue", "given": "Yisong" }, "id": "Yue-Yisong", "orcid": "0000-0001-9127-1989", "role": "member", "display_name": "Yue, Yisong" }, { "name": { "family": "Engelhardt", "given": "Barbara E." }, "id": "Engelhardt-Barbara-E", "orcid": "0000-0002-6139-7334", "role": "member", "display_name": "Engelhardt, Barbara E." }, { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "member", "display_name": "Pachter, Lior S." } ], "option_major": [ "biochem" ], "doi": "10.7907/qnzh-rf18", "abstract": "Understanding the effects of variation in the genome on organisms' phenotypes is a central goal of biology. For the past several decades, this has been done by performing large-scale association tests to identify links between genetic variants across the genome and particular diseases or traits. As most variants are in non-coding regions of the genome and act only in specific contexts, there arose the intermediate step of associating variants with gene expression patterns in particular tissues using bulk RNA-sequencing data and, with the recent widespread adoption of single-cell RNA-sequencing, particular cell types. However, associative tests are generally restricted to variants within some sequence distance of the gene, as testing tens of millions of distal variants against tens of thousands of genes in hundreds of contexts is computationally and statistically burdensome. Moreover, associating these proximal variants to changes in average gene expression using scRNA-seq data is far from pinpointing the cellular process they may affect. To move beyond analysis of averages, recent work has advocated for a mechanistic approach to analyzing scRNA-seq by modeling the underlying biological processes of transcription, splicing, and degradation.
\r\n\r\nIn this thesis, we first show how biophysical models are useful for identifying changes in biophysical processes not accessible at the level of mean expression. We next develop accelerated inference procedures for these models to make feasible their application at the scale required for genetic association tests. We then propose a framework for testing for the presence or absence of proximal or distal gene regulation using homozygous crosses. Finally, we couple the genetic testing framework and biophysical models to identify regulatory strategies of biophysical processes across cell types in eight tissues of eight genetically diverse mouse strains.
" }, { "name": "Chadly, Duncan Matthew", "degree": "PhD", "year": "2026", "title": "High-Resolution Phylogenetic Lineage Recording with CRISPR Base Editors", "advisor": "Elowitz, Michael B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:12042025-191849199", "creators": [ { "name": { "family": "Chadly", "given": "Duncan Matthew" }, "id": "Chadly-Duncan-Matthew", "orcid": "0000-0002-8417-1522", "display_name": "Chadly, Duncan Matthew" } ], "advisors": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "advisor", "display_name": "Elowitz, Michael B." } ], "committee": [ { "name": { "family": "Cai", "given": "Long" }, "id": "Cai-Long", "orcid": "0000-0002-7154-5361", "role": "chair", "display_name": "Cai, Long" }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "member", "display_name": "Guttman, Mitchell" }, { "name": { "family": "Lois", "given": "Carlos" }, "id": "Lois-Carlos", "orcid": "0000-0002-7305-2317", "role": "member", "display_name": "Lois, Carlos" }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" } ], "option_major": [ "bioeng" ], "doi": "10.7907/0afd-8p19", "abstract": "Dividing and differentiating cells form exquisitely organized structures across every facet of multicellular life. If we could measure the complete history of cells as they divide, change transcriptional state, and move spatially, we could address critical questions about stem cell differentiation, development, and the onset of disease. However, determining cellular ontologies is challenging except in rare cases where continual optical access is possible. Base editing technology enables the generation of stochastic, heritable mutations into genomic DNA while cells grow and divide. Comparing mutation patterns between cells allows inference of their lineage relationships in a manner analogous to evolutionary phylogenetic reconstruction. Here, we present two phylogenetic recording systems that enable high resolution lineage reconstruction over long time scales. In the first system, termed baseMEMOIR, we introduce a multiplexed, genomically dispersed set of editable targets that can be read out by imaging in situ. This system preserves spatial organization of cells and is compatible with downstream transcriptional measurements. In the second system, which we term the hypercascade, we take advantage of the predictability of A-to-G base editing to create a system in which edits not only alter bases but also generate new editable target sites in synthetic sequences. This behavior linearizes the rate at which mutations accumulate, improving lineage reconstruction. These methods enable analysis of temporal dynamics in diverse biological contexts." }, { "name": "Chan, Yeuk Chin Ailene", "degree": "PhD", "year": "2026", "title": "Seeing Beyond Sight: Multisensory Inference under Degraded Visual Input", "advisor": "Shimojo, Shinsuke", "url": "https://resolver.caltech.edu/CaltechTHESIS:05182026-165950389", "creators": [ { "name": { "family": "Chan", "given": "Yeuk Chin Ailene" }, "id": "Chan-Yeuk-Chin-Ailene", "orcid": "0000-0001-9522-0417", "display_name": "Chan, Yeuk Chin Ailene" } ], "advisors": [ { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "orcid": "0000-0002-1290-5232", "role": "advisor", "display_name": "Shimojo, Shinsuke" } ], "committee": [ { "name": { "family": "O'Doherty", "given": "John P." }, "id": "O'Doherty-J-P", "orcid": "0000-0003-0016-3531", "role": "chair", "display_name": "O'Doherty, John P." }, { "name": { "family": "Rutishauser", "given": "Ueli" }, "id": "Rutishauser-U", "orcid": "0000-0002-9207-7069", "role": "member", "display_name": "Rutishauser, Ueli" }, { "name": { "family": "Shams", "given": "Ladan" }, "id": "Shams-L", "orcid": "0000-0001-9303-9921", "role": "member", "display_name": "Shams, Ladan" }, { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "orcid": "0000-0002-1290-5232", "role": "member", "display_name": "Shimojo, Shinsuke" } ], "option_major": [ "cns" ], "doi": "10.7907/fws6-8361", "abstract": "Navigating a complex environment requires the human nervous system to continuously integrate noisy and often conflicting sensory signals. To do so, the brain must solve the causal inference problem: determining whether signals arise from a common source and should be integrated, or from independent sources and should remain segregated. While Bayesian Causal Inference (BCI) provides a principled framework for this computation, it remains unclear how these mechanisms operate under conditions of severely degraded or absent visual input.
\r\n\r\nThis dissertation investigates multisensory inference across a spectrum of visual uncertainty using complementary behavioral and computational approaches in three regimes: clinical low vision, physiological absence at the blind spot, and controlled degradation in immersive virtual reality with visuotactile interactions. In the low vision study, we find that multisensory illusions are not enhanced despite chronic impairment. Instead, BCI accurately captures behavior at high-visibility locations, where inference parameters remain comparable to sighted controls. At low-visibility locations, model fits become substantially weaker and behavioral responses more variable, limiting reliable interpretation of fitted parameters. Importantly, this likely reflects increased behavioral noise and the exploratory nature of the experimental design, which was optimized for broad spatial sampling rather than exhaustive inclusion of conditions required for robust model fitting at each location.
\r\n\r\nIn the blind spot, where visual input is structurally absent, illusory percepts persist and remain highly structured. BCI provides an excellent account of behavior, revealing stable binding tendencies alongside increased visual uncertainty and broader priors. This demonstrates that multisensory filling-in arises from probabilistic inference under uncertainty rather than strengthened multisensory binding.
\r\n\r\nIn a virtual reality visuotactile task, visual reliability is systematically manipulated in a naturalistic 3D environment. Here, BCI continues to explain behavior best under high reliability, but its predictive power decreases as visual uncertainty increases. We see a similar pattern of increased visual uncertainty and broader priors in response to experimentally reduced visual reliability. Notably, BCI model fit scales with task performance, with higher accuracy observed in individuals whose behavior more closely approximates a Bayes-optimal observer, indicating more effective multisensory integration.
\r\n\r\nAcross all three studies, the results reveal a consistent pattern: the brain\u2019s causal inference architecture remains stable across vastly different visual conditions. Rather than increasing binding tendency, the system adapts by recalibrating sensory uncertainty and prior expectations. However, as uncertainty increases, behavior deviates from Bayes-optimal predictions, reflecting both computational and representational limitations.
\r\n\r\nTogether, this work refines our understanding of sensory compensation by showing that multisensory interactions are shaped primarily by local sensory reliability rather than broad changes in binding tendency. By identifying when and why BCI succeeds or falls short, this dissertation provides a more nuanced account of perception under degraded conditions and offers useful considerations for the design of sensory substitution systems.
" }, { "name": "Chen, Linlin", "degree": "PhD", "year": "2026", "title": "A Platform for High Throughput Discovery of Sequence Defined Affinity Reagents", "advisor": "Guttman, Mitchell", "url": "https://resolver.caltech.edu/CaltechTHESIS:06022026-035443468", "creators": [ { "name": { "family": "Chen", "given": "Linlin" }, "id": "Chen-Linlin", "orcid": "0000-0003-2401-2420", "display_name": "Chen, Linlin" } ], "advisors": [ { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "advisor", "display_name": "Guttman, Mitchell" } ], "committee": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "chair", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "member", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "member", "display_name": "Guttman, Mitchell" } ], "option_major": [ "bioeng" ], "doi": "10.7907/xrr0-ck24", "abstract": "Protein-level measurement of biological systems remains constrained by the availability of specific, validated affinity reagents. While next-generation sequencing has enabled transcriptome-wide measurement at single-cell and spatial resolution, analogous protein measurement is limited by the scarcity of high-quality binders against most of the proteome. Conventional antibody generation is a one-target-at-a-time process in which specificity must be assessed after selection, reproducibility depends on biological source material that is finite and sequence-undefined, and effort scales linearly with target number.
\r\n\r\nThis thesis describes a platform for high-throughput nanobody discovery that addresses these limitations through two innovations within a single in vitro workflow. The first is pooled ribosome display selection: a synthetic nanobody library is panned against many proteins simultaneously in a single reaction, eliminating the need for sequential per-target campaigns. The second is split-pool DNA barcoding for target deconvolution: protein identity is assigned to each enriched nanobody by combinatorial barcode intersection without physical separation, enabling target number to scale logarithmically with experimental complexity. Critically, because enrichment is quantified across all targets simultaneously, specificity emerges as a comparative property of the data \u2014 a nanobody is called as a specific binder not by passing a threshold in isolation, but because it enriches preferentially against one target relative to the others in the pool. Selected nanobodies are sequence-defined and therefore renewable and reproducible by construction.
\r\n\r\nThe platform was applied to a panel of human cell-surface extracellular domain proteins. Controlled experiments established that pooled selection correctly enriches and deconvolves specific binders and that a combined enrichment-specificity scoring framework distinguishes genuine hits from promiscuous sequences. De novo binder discovery was demonstrated across panels of 3, 24, and 120 proteins, with called hits validated by multiplexed quantitative binding assay, flow cytometry on cells expressing the native membrane-anchored protein, and immunoprecipitation from cell lysate. CDR sequence convergence analysis provided independent evidence of affinity-driven selection, with multiple distinct convergent clusters per target indicating sampling of different epitopes. Split-pool barcoding produced concordant results with physical split deconvolution and enabled scale-up to 120 proteins.
\r\n\r\nThe platform demonstrates that throughput and specificity in binder discovery need not trade off and provides a practical path toward systematic reagent generation against large protein panels. These reagents are directly applicable to the interaction mapping assays developed in this laboratory, to sequencing-based protein measurement platforms currently bottlenecked by binder availability, and to the systematic characterization of the cell-surface proteome more broadly.
" }, { "name": "Cheung, Yuen Man Kathy", "degree": "PhD", "year": "2026", "title": "Neural Coding of Fear: From Genes to Brain-Wide Dynamics", "advisor": "Anderson, David J.; Shapiro, Mikhail G.", "url": "https://resolver.caltech.edu/CaltechTHESIS:12182025-233110599", "creators": [ { "name": { "family": "Cheung", "given": "Yuen Man Kathy" }, "id": "Cheung-Yuen-Man-Kathy", "orcid": "0009-0007-1204-2178", "display_name": "Cheung, Yuen Man Kathy" } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "co-advisor", "display_name": "Anderson, David J." }, { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "co-advisor", "display_name": "Shapiro, Mikhail G." } ], "committee": [ { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "chair", "display_name": "Adolphs, Ralph" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Oka", "given": "Yuki" }, "id": "Oka-Yuki", "orcid": "0000-0003-2686-0677", "role": "member", "display_name": "Oka, Yuki" }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "orcid": "0000-0002-7371-4675", "role": "member", "display_name": "Prober, David A." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "member", "display_name": "Shapiro, Mikhail G." } ], "option_major": [ "neurobio" ], "doi": "10.7907/vcr9-gp26", "abstract": "Fear is essential for survival. Predator threats, aggressive conspecifics, and environmental dangers elicit innate defensive responses that do not require learning. Although these responses are hardwired, animals display remarkable flexibility in selecting the most adaptive behaviors. The neural circuits that enable such dynamic representations of internal fear states remain poorly understood. The dorsomedial and central subdivisions of the ventromedial hypothalamus (VMHdm) integrate multisensory threat-related inputs and are both necessary and sufficient to drive defensive responses. In this thesis, I investigate how VMHdm neurons encode internal states associated with predator fear, across multiple biological scales\u2014from transcriptomic profile, single-cell neural dynamics to brain-wide activity patterns.
\r\n\r\nFirst, I characterized the transcriptomic profile of VMH neurons activated by predator exposure using activity-dependent single-cell RNA sequencing (act-seq). I then combined optogenetic perturbation of VMHdm with act-seq to identify state-dependent transcriptomic correlates in a downstream output, the periaqueductal gray (PAG). To investigate the real-time dynamics of these neurons during naturalistic predator encounters, I developed a novel behavioral paradigm and performed microendoscopic single-cell calcium imaging. Contrary to the long-held view of VMHdm as a uniformly threat-activated population, my analyses revealed functionally distinct clusters that encode not only threat, but also safety, arousal or novelty or neophobia, predator imminence, and anxiety. Moreover, I found that individual variation in behavioral defensiveness was correlated with VMHdm neural dynamics.
\r\n\r\nFinally, to assess how local hypothalamic activation influences global brain states, I combined VMHdm optogenetic stimulation with functional ultrasound imaging (fUSI), which permits high-resolution recording of brain-wide hemodynamics. This approach revealed the spatiotemporal propagation of neural activity from the hypothalamus to distributed brain regions. These findings demonstrate how a genetically defined hypothalamic subpopulation can engage a dynamic, brain-wide ensemble to orchestrate defensive responses.
\r\n \r\nTogether, these studies provide a multi-modal, multi-scale analysis of the innate fear state and its flexible representation via the hypothalamus, embedded within a dynamic global network of interacting regions. These findings offer insight into how internal states are encoded and broadcast, with potential implications for understanding the neural basis of human psychiatric disorders and the distributed computation of affective states.
" }, { "name": "Condiotte, Zevin Joseph", "degree": "PhD", "year": "2026", "title": "Cooperative Microbe-Host Carbon Metabolism Drives Drosophila Regenerative Response", "advisor": "Goentoro, Lea A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03262026-171915471", "creators": [ { "name": { "family": "Condiotte", "given": "Zevin Joseph" }, "id": "Condiotte-Zevin-Joseph", "orcid": "0000-0002-2028-5993", "display_name": "Condiotte, Zevin Joseph" } ], "advisors": [ { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "orcid": "0000-0002-3904-0195", "role": "advisor", "display_name": "Goentoro, Lea A." } ], "committee": [ { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "orcid": "0000-0002-7033-0844", "role": "chair", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "orcid": "0000-0002-3904-0195", "role": "member", "display_name": "Goentoro, Lea A." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." }, { "name": { "family": "Orphan", "given": "Victoria J." }, "id": "Orphan-V-J", "orcid": "0000-0002-5374-6178", "role": "member", "display_name": "Orphan, Victoria J." } ], "option_major": [ "biology" ], "doi": "10.7907/2h1e-ew42", "abstract": "The gut microbiome is a collaborative intermediary between host diet and metabolism. Previous work from our lab in multiple species including in Drosophila, demonstrates that modulating nutrients can promote regeneration processes. Motivated by the roles of nutrients, in my thesis, I examined the role of the microbiome. I found that systemic administration of a single strain of Lactobacillus brevis promotes activation of regeneration processes in injured limbs. The regeneration-promoting effect of Lactobacillus can be recapitulated by lactate, TCA metabolites, and genetically overexpressing lactate dehydrogenase. Finally, in collaboration with Gloria Bates, we found that directly supplying lactate can promote partial limb regrowth. These experiments support growing evidence that bacteria can promote host regeneration processes, and propose a role for lactate in fuelling this host-microbe interaction." }, { "name": "DeLaitsch, Andrew T.", "degree": "PhD", "year": "2026", "title": "Antibody Responses to HIV-1 Immunogens and Viruses", "advisor": "Bjorkman, Pamela J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01152026-213540802", "creators": [ { "name": { "family": "DeLaitsch", "given": "Andrew T." }, "id": "DeLaitsch-Andrew-T", "orcid": "0000-0002-3458-3449", "display_name": "DeLaitsch, Andrew T." } ], "advisors": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "advisor", "display_name": "Bjorkman, Pamela J." } ], "committee": [ { "name": { "family": "Voorhees", "given": "Rebecca M." }, "id": "Voorhees-R-M", "orcid": "0000-0003-1640-2293", "role": "chair", "display_name": "Voorhees, Rebecca M." }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." }, { "name": { "family": "Clemons", "given": "William M." }, "id": "Clemons-W-M", "orcid": "0000-0002-0021-889X", "role": "member", "display_name": "Clemons, William M." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." } ], "option_major": [ "biochem" ], "doi": "10.7907/jz5g-9h97", "abstract": "The Envelope (Env) trimer mediates fusion of the HIV-1 viral membrane with that of the target cell. As such, it is the sole target for neutralizing antibodies against the virus. Broadly neutralizing antibodies (bnAbs) that are capable of blocking entry of diverse viral strains hold therapeutic potential for people living with HIV-1 and may be utilized for prophylaxis, either through passive immunization or as templates for immunogen design. Chapter 1 of this thesis introduces relevant background to the HIV-1 virus, the Env trimer, antibodies, and antibody-antigen interactions, with an emphasis on bivalent antibody binding to enveloped viruses. Chapter 2 then describes the results of a preclinical sequential immunization study aimed at eliciting, and subsequently boosting, antibodies directed to the V3 glycan patch on Env. Electron microscopy-based polyclonal epitope mapping demonstrated initial V3-targeting, followed by an increase in off-target responses with boosting that was associated with the disassociation of trimeric Env immunogens. Nonetheless, monoclonal antibodies that competed with the V3-targeting bnAb 10-1074 and exhibited weak, heterologous neutralizing activity were isolated after the third boost. Cryo-electron microscopy structures of two of these monoclonal antibodies, reported in Chapter 3, revealed targeting of altered Env conformations. The results presented in Chapters 2 and 3 provide insight into Env structural dynamics and inform strategies for improved immunogen design.
\r\n\r\nChapters 4 and 5 of this thesis present new best-in-class human bnAbs targeting the CD4-binding site and V3 glycan patch on Env, respectively. These antibodies were identified from top elite neutralizers out of an international cohort of over 2,300 people living with HIV-1. Multiple, distinct VRC01-class, CD4-binding site bnAbs were identified from one of these individuals. These bnAbs differed in the presence and length of framework region insertions and CDRH3 lengths. One bnAb, 04_A06, exhibited an 11-amino acid long insertion, and structural analyses demonstrated this resulted in a protruding CDRH1 loop that recognized highly conserved residues on the adjacent gp120, contributing to the near pan-neutralizing breadth and resilience to escape mutations by 04_A06. Chapter 5 then presents 007, a highly broad and potent V3 bnAb that binds Env independently of the gp120 N332 glycan. 007 exhibited weak monovalent binding affinity, and pseudovirus neutralization assays demonstrated antibody bivalency contributes to potency. Structures of 007 IgG in complex with engineered, soluble Env ectodomains revealed a dimer of Env trimers, crosslinked by three IgG molecules. The results presented in Chapters 4 and 5 advance our mechanistic understanding of Env recognition and neutralization by bnAbs and inform the development of antibody therapy and vaccines for HIV-1.
" }, { "name": "Du, Rongrong", "degree": "PhD", "year": "2026", "title": "Build Synthetic Circuits at Different Scales", "advisor": "Elowitz, Michael B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01152026-202412598", "creators": [ { "name": { "family": "Du", "given": "Rongrong" }, "id": "Du-Rongrong", "orcid": "0009-0003-4942-3020", "display_name": "Du, Rongrong" } ], "advisors": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "advisor", "display_name": "Elowitz, Michael B." } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Cai", "given": "Long" }, "id": "Cai-Long", "orcid": "0000-0002-7154-5361", "role": "member", "display_name": "Cai, Long" }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." } ], "option_major": [ "bioeng" ], "doi": "10.7907/1ksn-sb30", "abstract": "Multicellular organisms rely on the coordinated actions of diverse organs to sustain life. Each organ comprises cells that communicate with each other to execute physiological functions, and each cell encodes gene regulatory networks that shape its gene expression programs. The intrinsic complexity of biological systems, including features such as redundancy that endow them with robustness, also makes them difficult to study using reductionist approaches alone.
\r\n\r\nTo elucidate quantitative design principles underlying multicellular organization, I adopted a bottom-up approach and built synthetic circuits at multiple scales. In the first project, I engineered a single-gene incoherent feedforward circuit that leverages multispecific microRNA targeting to achieve dosage-invariant and tunable protein expression across wide ranges of gene copy numbers. In the second project, I constructed a multicellular reaction\u2013diffusion circuit that integrates juxtacrine and paracrine signaling to generate self-organized, periodic Turing patterns.
\r\n \r\nTogether, these studies introduce new tools for engineering regulatory behaviors, reveal general principles that govern biological organization across scales, and pave the way for potential translational applications.
" }, { "name": "Gerber, Bryan Michael", "degree": "PhD", "year": "2026", "title": "Synthetic Control of the Biological Central Dogma", "advisor": "Wang, Kaihang", "url": "https://resolver.caltech.edu/CaltechTHESIS:04282026-200138296", "creators": [ { "name": { "family": "Gerber", "given": "Bryan Michael" }, "id": "Gerber-Bryan-Michael", "orcid": "0000-0002-3979-1095", "display_name": "Gerber, Bryan Michael" } ], "advisors": [ { "name": { "family": "Wang", "given": "Kaihang" }, "id": "Wang-Kaihang", "orcid": "0000-0001-7657-8755", "role": "advisor", "display_name": "Wang, Kaihang" } ], "committee": [ { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "chair", "display_name": "Murray, Richard M." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." }, { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "member", "display_name": "Shapiro, Mikhail G." }, { "name": { "family": "Wang", "given": "Kaihang" }, "id": "Wang-Kaihang", "orcid": "0000-0001-7657-8755", "role": "member", "display_name": "Wang, Kaihang" } ], "option_major": [ "biology" ], "doi": "10.7907/1t51-1e91", "abstract": "The Central Dogma of biology dictates the flow of information within all living organisms. By focusing on the construction of synthetic DNA, the transcription of target RNA, and the translation of select proteins, we can improve the synthetic control of engineered organisms. Prioritizing the mis-connection rate present when attaching two DNA molecules, I propose two conceptual improvements to DNA assembly technologies. The high efficiency of the first technique, called Sidewinder, is demonstrated through the construction of a GFP mutation library from DNA oligos, whose diversity is confirmed by high-fidelity sequencing and functional phenotypic analysis. The second DNA assembly technique, High Temperature (HighT) assembly, is demonstrated by high efficiency plasmid ligation, direct integration into bacterial genomes by two independent recombinase HighT assembly-to-integration systems, and the construction of multiple eukaryotic genes including MAPT, VEGFA, BRCA1 and the Sonic Hedgehog embryonic development gene 10-kB genomic DNA segment from the functionally extinct Northern White Rhino. Applications of synthetic DNA are then explored through the import of orthogonal transcription and translation molecular machinery into cells, where they directly regulate protein production. On the transcription level, variations in inducible split-T7 polymerase systems are used to create an orthogonal signaling pathway for low leak and tunable transcriptional control of target genes. Unnatural amino acid incorporation is used to translationally regulate genomically modified essential genes, where exposure to this molecule is demonstrated to enable translation of select essential genes. Through the lens of the Central Dogma, in this thesis I will explore various frameworks to build and fine tune the cells we may aspire to create." }, { "name": "Horak, Richard Davis", "degree": "PhD", "year": "2026", "title": "Metabolic Rewiring Promotes Bacterial Survival Under Oxidative and Reductive Stress", "advisor": "Newman, Dianne K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05292026-023647406", "creators": [ { "name": { "family": "Horak", "given": "Richard Davis" }, "id": "Horak-Richard-Davis", "orcid": "0000-0003-0630-5481", "display_name": "Horak, Richard Davis" } ], "advisors": [ { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "advisor", "display_name": "Newman, Dianne K." } ], "committee": [ { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "orcid": "0000-0002-7033-0844", "role": "chair", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Ruby", "given": "Edward G." }, "id": "Ruby-Edward", "orcid": "0000-0002-4112-4830", "role": "member", "display_name": "Ruby, Edward G." }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "orcid": "0000-0003-2713-1513", "role": "member", "display_name": "Mazmanian, Sarkis K." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." } ], "option_major": [ "microbio" ], "doi": "10.7907/240t-cx19", "abstract": "Across the tree of life, all cells must follow unifying metabolic rules. Namely, organisms must balance electron flow to couple energy conservation with energy expenditure. Historically, studies in bacterial metabolism focused on exponential growth where cells are awash in nutrients and electron acceptors, exhibiting high bioenergetic levels. Therefore, from the perspective of both human biology and these fast-growing microbes, loss of redox balance is purely detrimental, leading to suppressed energetic states, growth arrest, and even death. Yet bacteria are commonly found under such conditions across diverse environments from industrial bioreactors to chronic infections to agricultural fields. This thesis was motivated by the remaining mystery behind how and why bacteria exist in such low energy survival states. Specifically, I focus on metabolic shifts during non-growth survival in the opportunistic pathogen Pseudomonas aeruginosa due to redox imbalance, as well as the potential benefits to such transitions. In the first section, I focus on oxidative stress, exploring bacterial survival during oxic nutrient starvation. I find that phenazines and toxoflavin \u2013 endogenous redox-active metabolites produced by P. aeruginosa and Burkholderia species respectively \u2013 lower the bioenergetic state of P. aeruginosa. Such bioenergetic self-poisoning would be traditionally deemed detrimental. Yet I find this phenomenon provides cells with increased tolerance to a variety of clinical antibiotics, suggesting cells might have agency over lowering their energetic state and that there is a benefit to doing so. In the following chapters, I turn my attention to reductive stress, examining the metabolic strategies P. aeruginosa uses to support anaerobic survival in the absence of terminal electron-acceptors. I discover that P. aeruginosa uses a phosphoketolase-mediated alternative glucose catabolic pathway under reductive stress, reminiscent of fermentative growth metabolisms in many obligate anaerobes. Moreover, this phosphoketolase plays a key role in mediating ribonucleotide homeostasis during survival-triggered macromolecule turnover. I find that many bacteria unable to grow in the absence of respiration contain phosphoketolases and show that at least two of these species, Dyella japonica and Paraburkholderia graminis, similarly rely on these enzymes for anaerobic survival. Finally, I speculate a generalizable role for phosphoketolases in supporting ribonucleotide turnover across bacterial taxa. These studies expose the large gaps remaining in our understanding of growth arrest metabolisms, even in well-studied model organisms. I hope this thesis motivates further exploration of these enigmatic yet important bacterial lifestyles." }, { "name": "Huang, Jianyi", "degree": "PhD", "year": "2026", "title": "Construction of Unconstructable DNA Constructs in Synthetic Chassis", "advisor": "Wang, Kaihang", "url": "https://resolver.caltech.edu/CaltechTHESIS:05272026-230304616", "creators": [ { "name": { "family": "Huang", "given": "Jianyi" }, "id": "Huang-Jianyi", "orcid": "0009-0006-1492-7693", "display_name": "Huang, Jianyi" } ], "advisors": [ { "name": { "family": "Wang", "given": "Kaihang" }, "id": "Wang-Kaihang", "orcid": "0000-0001-7657-8755", "role": "advisor", "display_name": "Wang, Kaihang" } ], "committee": [ { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "orcid": "0000-0002-7033-0844", "role": "chair", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Demirer", "given": "Gozde S." }, "id": "Demirer-G\u00f6zde-S", "orcid": "0000-0002-3007-1489", "role": "member", "display_name": "Demirer, Gozde S." }, { "name": { "family": "Karthikeyan", "given": "Smruthi" }, "id": "Karthikeyan-Smruthi", "orcid": "0000-0001-6226-4536", "role": "member", "display_name": "Karthikeyan, Smruthi" }, { "name": { "family": "Wang", "given": "Kaihang" }, "id": "Wang-Kaihang", "orcid": "0000-0001-7657-8755", "role": "member", "display_name": "Wang, Kaihang" } ], "option_major": [ "bioeng" ], "doi": "10.7907/dd90-sp96", "abstract": "Synthetic biology increasingly depends on the ability to construct, maintain, and verify DNA molecules that encode complex biological functions. Many of the most valuable genetic programs, however, are difficult to propagate in conventional cloning hosts because the same cells used to amplify the DNA are also exposed to the functions encoded by that DNA. This coupling is especially problematic for toxic genes, antibacterial proteins, lysis genes, nucleases, and complete bacteriophage genomes, where the desired activity can damage or kill the host and select for mutants that have lost the intended function.
\r\n\r\nThis thesis develops a synthetic-chassis strategy for separating DNA amplification from functional gene expression. Using a bacterial host with a refactored genetic code, natural-rule coding sequences containing selected codons can be maintained as DNA while expression of their protein products are silenced. Replicon backbones and selection markers are encoded in mutually compatible rule so that they remain active in the synthetic chassis, allowing toxic cargoes to be propagated, sequence-verified, and transferred into an execution context where the standard decoding rule restores function. This framework is applied to the construction of otherwise difficult DNA, including toxic genes and bacteriophage genomes, and is extended toward the assembly of increasingly complex phage systems.
\r\n\r\nThe thesis also presents SynPl-Seq, a rapid colony-to-consensus workflow for whole-plasmid sequence verification. By combining backbone-specific whole-plasmid PCR, multiplexed barcoding and Nanopore sequencing, SynPl-Seq enables high-throughput validation of candidate clones within a single working day and supports the iterative construction workflows required for large or unstable genetic systems.
\r\n\r\nTogether, these studies advance a central concept: the genetic code can be used not only to expand or contain biological function, but also to route when and where a genetic program is expressed. Refactored-code chassis provide a protected environment for constructing DNA whose activity must be preserved but temporarily silenced, offering a general platform for phage engineering, toxic-cargo cloning, and future biological systems that require context-dependent expression control.
" }, { "name": "Jackson, Cameron Richard", "degree": "PhD", "year": "2026", "title": "Accessing the Developing CNS: Advancing AAV-Mediated Prenatal Gene Editing in the Brain", "advisor": "Gradinaru, Viviana", "url": "https://resolver.caltech.edu/CaltechTHESIS:05302026-011912053", "creators": [ { "name": { "family": "Jackson", "given": "Cameron Richard" }, "id": "Jackson-Cameron-Richard", "orcid": "0009-0008-7212-616X", "display_name": "Jackson, Cameron Richard" } ], "advisors": [ { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "advisor", "display_name": "Gradinaru, Viviana" } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Lois", "given": "Carlos" }, "id": "Lois-Carlos", "orcid": "0000-0002-7305-2317", "role": "member", "display_name": "Lois, Carlos" }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "member", "display_name": "Gradinaru, Viviana" } ], "option_major": [ "neurobio" ], "doi": "10.7907/881q-x239", "abstract": "Targeted gene delivery to defined neural populations is a central challenge in neuroscience and a major barrier to gene therapy for neurodevelopmental and congenital disorders. In the developing central nervous system, this is further complicated by dynamic changes in accessibility, vector tropism, and cellular composition across developmental stages. This thesis develops new approaches for systemic prenatal CNS access, capsid engineering, and in utero genome editing in the embryonic brain.
\r\n\r\nWe introduce UseqFISH, a spatial transcriptomic platform that enables high-sensitivity, multiplexed detection of endogenous transcripts and barcoded viral genomes in intact tissue at single-cell resolution. This allows quantitative in situ mapping of AAV tropism and scalable evaluation of viral libraries in embryonic contexts. Using new and existing techniques, we characterize systemic AAV biodistribution across development and show that adult brain-biased serotypes do not maintain specificity in late-mid gestation embryos. We engineer modified capsids, including \u201cnull\u201d scaffolds with reduced native tropism, which can be reprogrammed via targeting motifs. High-throughput barcoded in vivo selection identifies sequence features associated with improved CNS enrichment during embryogenesis.
\r\n\r\nTo expand access to midgestational delivery, we develop a surgical method enabling systemic AAV administration at embryonic day 12.5 via uterine microdissection of the vitelline circulation, achieving widespread and viable transduction through gestational development. Finally, we established an in utero genome editing platform using AAV-mediated CRISPR and homology-directed repair. We demonstrate efficient gene disruption, epitope tagging, and precise genome modification, including endogenous tagging and introduction of disease-associated alleles across cortical cell types, enabling modeling of neurodevelopmental phenotypes.
\r\n\r\nTogether, this work provides a framework for engineering AAV-based gene delivery and genome editing in the developing CNS, advancing tools for studying brain development, modeling disease, and enabling early therapeutic strategies.
" }, { "name": "Kapasiawala, Manisha Kaushik", "degree": "PhD", "year": "2026", "title": "Design Considerations for Synthetic Cells", "advisor": "Murray, Richard M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08252025-232825764", "creators": [ { "name": { "family": "Kapasiawala", "given": "Manisha Kaushik" }, "id": "Kapasiawala-Manisha-Kaushik", "orcid": "0000-0002-0302-2921", "display_name": "Kapasiawala, Manisha Kaushik" } ], "advisors": [ { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "advisor", "display_name": "Murray, Richard M." } ], "committee": [ { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "chair", "display_name": "Thomson, Matthew" }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Voorhees", "given": "Rebecca M." }, "id": "Voorhees-R-M", "orcid": "0000-0003-1640-2293", "role": "member", "display_name": "Voorhees, Rebecca M." }, { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "orcid": "0000-0002-5899-7523", "role": "member", "display_name": "Winfree, Erik" } ], "option_major": [ "bioeng" ], "doi": "10.7907/zfhy-bk03", "abstract": "Efforts to understand life as we know it and life as it can be have culminated in the field of synthetic cell research, which aims to build life from the bottom up using individual biological components. Recent progress in the field has enabled the reconstitution of many functions of living cells in synthetic cells, from cell-cell communication to membrane protein expression and function. However, future progress in the field is limited by many challenges, including irreproducibility, lack of predictability, difficulties in integrating existing synthetic cell modules (or subsystems), and the need for autonomous functionalities.
\r\n\r\nIn this work, I describe my efforts towards addressing these challenges. In Chapter 2, I describe sources of variability in transcription-translation (TX-TL) systems, the biological machinery used to implement biomolecular programs in synthetic cells. In Chapter 3, I describe a novel methodology for readily building more predictive models of TX-TL performance. In Chapter 4, I present a design for a proof-of-concept for integrating an energy regeneration subsystem and a motility subsystem to achieve autonomous programmable motility and highlight some early successes towards achieving that goal. Throughout this work, I highlight many design principles for building synthetic cells reproducibly, more predictably, and with novel functionalities.
" }, { "name": "Kratz, Matthieu Francois", "degree": "PhD", "year": "2026", "title": "Naturally-Inspired Circuits for Microbial Composition Control and Biosensing", "advisor": "Murray, Richard M.; Elowitz, Michael B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:11242025-191228061", "creators": [ { "name": { "family": "Kratz", "given": "Matthieu Francois" }, "id": "Kratz-Matthieu-Francois", "display_name": "Kratz, Matthieu Francois" } ], "advisors": [ { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "co-advisor", "display_name": "Murray, Richard M." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "co-advisor", "display_name": "Elowitz, Michael B." } ], "committee": [ { "name": { "family": "Demirer", "given": "G\u00f6zde S." }, "id": "Demirer-G\u00f6zde-S", "orcid": "0000-0002-3007-1489", "role": "chair", "display_name": "Demirer, G\u00f6zde S." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Bois", "given": "Justin S." }, "id": "Bois-J-S", "orcid": "0000-0001-7137-8746", "role": "member", "display_name": "Bois, Justin S." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." } ], "option_major": [ "bioeng" ], "doi": "10.7907/d42b-jh46", "abstract": "When considering the design of gene circuits, there are many possible sources of inspiration. Many early synthetic gene circuits used nature as an inspiration, seeking to recreate biological behaviors with non-native components. As the field grew, alternative approaches sourcing designs from adjacent engineering fields and computational approaches emerged and grew in prominence. Despite this shift, there remains a great deal of naturally-inspired circuits that provide useful functions for biotechnology. Indeed nature has often been uniquely capable of exploiting typically undesirable phenomena, e.g. noise to create biologically useful function. This thesis presents two projects directly inspired by natural systems. Each project aims to replicate a behavior or circuit topology found in nature, leveraging its unique dynamics to address key challenges in biotechnology. Chapters 2 and 3 will cover the development of a circuit emulating the microbial behavior of phase variation, whereby individual cells reversibly and stochastically transition between distinct phenotypes. We recreate this behavior using serine recombinases and demonstrate how it can enable stable, bulk control of phenotype composition\u2014a task of great relevance to biotechnology. Chapter 4 lays the groundwork for applying the biologically-relevant feed-forward loop topology to the problem of spurious biosensor activation. We realize this topology in a modular manner using small transcription activating RNAs (STARs) and provide a preliminary characterization of its dynamical properties. Finally, we discuss alternative implementations that may provide more directly applicable properties than the current STAR implementation" }, { "name": "Larios-Colorado, David Antonio", "degree": "PhD", "year": "2026", "title": "Reverse Engineering the Programming Logic of Cytoskeletal Dynamics", "advisor": "Thomson, Matthew", "url": "https://resolver.caltech.edu/CaltechTHESIS:05312026-103739938", "creators": [ { "name": { "family": "Larios-Colorado", "given": "David Antonio" }, "id": "Larios-Colorado-David-Antonio", "orcid": "0009-0002-9277-5438", "display_name": "Larios-Colorado, David Antonio" } ], "advisors": [ { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "advisor", "display_name": "Thomson, Matthew" } ], "committee": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "chair", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." }, { "name": { "family": "Parker", "given": "Joseph" }, "id": "Parker-J", "orcid": "0000-0001-9598-2454", "role": "member", "display_name": "Parker, Joseph" }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" } ], "option_major": [ "biology" ], "doi": "10.7907/yp1w-1f12", "abstract": "Eukaryotic cells generate mechanical force through cytoskeletal filaments actively reorganized by families of molecular motors. Despite extensive characterization of filament-motor self-organization, how variations in motor sequence and structure translate into filament organization dynamics remains poorly understood. Here we develop ActiveDROPS (Active Dynamic Reprogrammable self-Organizing Protein System), a cell-free approach to reconstitute microtubule dynamics driven by genetically encoded kinesin variants in bacterial lysate droplets. Across twelve diverse kinesin-1 homologs, microtubule dynamics collapse onto three classes: \"Slow-Sustained\" flows that activate near 8-10 h and persist to ~30 h, \"Fast-Burst\" contractions initiating within minutes and dissipating within ~1 h, and \"Multiphase\" progression through nematic, rotational, and contractile flows over ~30 h. Microtubule gliding assays and molecular dynamics simulations on AlphaFold-predicted structures show that Fast-Burst motors couple high ATP hydrolysis rate with strong microtubule binding, while Slow-Sustained motors couple low turnover with weak binding. By recombining two motors across these classes, we generated a \"Fast-Sustained\" chimera with rapid microtubule flows lasting ~15 h, revealing the protein domain configurations that specify the velocity (Slow/Fast) and duration (Sustained/Burst) of macroscopic dynamics. These results uncover a constrained modular logic by which kinesin sequence specifies microtubule self-organization, providing a framework for dissecting the mechanical repertoire available to cellular systems." }, { "name": "Leslie, Kent L.", "degree": "PhD", "year": "2026", "title": "Autophagy Proteins Direct STING Trafficking and Innate Immune Signaling Independently of Canonical Autophagy", "advisor": "Chou, Tsui-Fen", "url": "https://resolver.caltech.edu/CaltechTHESIS:05132026-165833672", "creators": [ { "name": { "family": "Leslie", "given": "Kent L." }, "id": "Leslie-Kent-L", "orcid": "0009-0000-8680-6459", "display_name": "Leslie, Kent L." } ], "advisors": [ { "name": { "family": "Chou", "given": "Tsui-Fen" }, "id": "Chou-Tsui-Fen", "orcid": "0000-0003-2410-2186", "role": "advisor", "display_name": "Chou, Tsui-Fen" } ], "committee": [ { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "chair", "display_name": "Guttman, Mitchell" }, { "name": { "family": "Chong", "given": "Shasha" }, "id": "Chong-Shasha", "orcid": "0000-0002-5372-311X", "role": "member", "display_name": "Chong, Shasha" }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "member", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "orcid": "0000-0002-7371-4675", "role": "member", "display_name": "Prober, David A." }, { "name": { "family": "Chou", "given": "Tsui-Fen" }, "id": "Chou-Tsui-Fen", "orcid": "0000-0003-2410-2186", "role": "member", "display_name": "Chou, Tsui-Fen" } ], "option_major": [ "biology" ], "doi": "10.7907/27n6-za50", "abstract": "Autophagy is a conserved lysosome-mediated degradation pathway that maintains cellular homeostasis by recycling cytoplasmic components and responding to metabolic stress. Initiation of autophagy is coordinated by the ULK1 complex, comprising ULK1, FIP200, ATG13, and ATG101, which is regarded as the earliest regulatory node controlling autophagosome formation. Emerging evidence, however, suggests that components of this complex also function in signaling processes beyond canonical autophagy. This thesis identifies and describes a previously unrecognized role of the ATG9A\u2013ATG13\u2013ATG101 module in mediating a Golgi-to-lysosome trafficking pathway essential for the degradation and signal termination of STING, a central adaptor in the cGAS\u2013STING innate immune pathway. We demonstrate that loss of ATG13, ATG101, or the Golgi-resident membrane protein ATG9A impairs STING turnover, resulting in constitutive and cell-autonomous interferon activation. Mechanistically, STING exiting the Golgi recruits ATG9A-positive vesicles that depend on the ATG13\u2013ATG101 subcomplex to enable entry into the endolysosomal pathway. Notably, this function is separable from canonical autophagy initiation, revealing a noncanonical trafficking role for autophagy-associated proteins in immune signal regulation. Complementing these findings, we identify Heat Shock Factor Binding Protein 1 (HSBP1) as a direct interactor of FIP200 that associates with coiled-coil scaffolds and vesicle-tethering factors, including ATG16L1 and EEA1, suggesting a role in organizing multimeric membrane trafficking assemblies. HSBP1 is intrinsically short-lived and undergoes rapid proteasome-dependent degradation during metabolic stress, indicating a regulatory mechanism independent of lysosomal autophagy. Together, this thesis describes previously unrecognized functions of autophagy initiation machinery in coordinating vesicular trafficking and innate immune regulation, establishing ATG9A\u2013ATG13\u2013ATG101 as a Golgi-to-lysosome trafficking module for STING." }, { "name": "Li, Hongyi Richard", "degree": "PhD", "year": "2026", "title": "Acoustically Targeted Gene Delivery for Non-Invasive Neuroengineering", "advisor": "Shapiro, Mikhail G.", "url": "https://resolver.caltech.edu/CaltechTHESIS:09212025-192830865", "creators": [ { "name": { "family": "Li", "given": "Hongyi Richard" }, "id": "Li-Hongyi-Richard", "orcid": "0000-0001-6970-0230", "display_name": "Li, Hongyi Richard" } ], "advisors": [ { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "000-0002-0291-4215", "role": "advisor", "display_name": "Shapiro, Mikhail G." } ], "committee": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "chair", "display_name": "Lester, Henry A." }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "member", "display_name": "Andersen, Richard A." }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." }, { "name": { "family": "Tsao", "given": "Doris Y." }, "id": "Tsao-D-Y", "orcid": "0000-0003-1083-1919", "role": "member", "display_name": "Tsao, Doris Y." }, { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "member", "display_name": "Shapiro, Mikhail G." } ], "option_major": [ "biology" ], "doi": "10.7907/qbrx-4132", "abstract": "Noninvasive, spatially targeted gene delivery to the brain holds tremendous promise for addressing some of the most pressing neurological and psychiatric conditions of our time, including Parkinson\u2019s disease, treatment-resistant epilepsy, obsessive-compulsive disorder, and addictions. While adeno-associated viruses (AAVs) are the leading vectors for gene therapy in the state of the art, their clinical translation is hindered by the need for invasive injections to achieve site-specific delivery in the brain. Over the past two decades, focused ultrasound blood-brain barrier opening (FUS-BBBO) has emerged as a compelling alternative \u2014 enabling targeted entry of biomolecules, nanoparticles, and even small viral vectors like AAVs from the bloodstream into the brain without surgical intervention. Yet, natural AAV serotypes have shown only modest success with this method, often displaying low transduction efficiency and undesirable off-target expression in peripheral organs.
\r\n\r\nTo overcome these limitations, we have developed a new framework for acoustically targeted gene delivery \u2014 a noninvasive, spatially and cell-type-specific approach for delivering genetic material to the brain. In this thesis, I will describe how we harnessed high-throughput in vivo directed evolution to engineer AAV variants optimized for neuronal transduction specifically at the site of ultrasound targeting. In rodent models, these newly evolved vectors demonstrate significantly improved performance \u2014 achieving efficient, localized gene delivery to neurons while minimizing peripheral expression. Building on these successes, we advanced the platform toward clinical relevance by extending our evolutionary screening to non-human primates (NHPs). This allowed us to identify AAV variants with enhanced translational potential and establish a strong foundation for future studies in human clinical trials.
\r\n\r\nIn the final part of this thesis, I will showcase how these engineered AAVs can be further empowered by combining them with acoustic reporter genes \u2014 specifically, gas vesicle (GV) proteins \u2014 enabling non-invasive imaging of molecular activity deep within the brain. Using this powerful platform, we have also developed a novel therapeutic strategy for treating opioid addiction, in which biomolecular ultrasound coalesces with chemogenetic neuromodulation. Taken together, I hope to convince you that the technique of ultrasound-based acoustically targeted gene delivery, paired with engineered delivery vectors, unlocks a new frontier in non-invasive neurotherapeutics and brings us one step closer to precise, personalized neuroengineering in interfacing the human brain.
" }, { "name": "Li, Kejun", "degree": "PhD", "year": "2026", "title": "User-Aligned and Robust Bipedal Locomotion", "advisor": "Ames, Aaron D.; Yue, Yisong", "url": "https://resolver.caltech.edu/CaltechTHESIS:10232025-045548517", "creators": [ { "name": { "family": "Li", "given": "Kejun" }, "id": "Li-Kejun", "orcid": "0000-0002-0823-9839", "display_name": "Li, Kejun" } ], "advisors": [ { "name": { "family": "Ames", "given": "Aaron D." }, "id": "Ames-A-D", "orcid": "0000-0003-0848-3177", "role": "advisor", "display_name": "Ames, Aaron D." }, { "name": { "family": "Yue", "given": "Yisong" }, "id": "Yue-Yisong", "orcid": "0000-0001-9127-1989", "role": "co-advisor", "display_name": "Yue, Yisong" } ], "committee": [ { "name": { "family": "Burdick", "given": "Joel Wakeman" }, "id": "Burdick-J-W", "orcid": "0000-0002-3091-540X", "role": "chair", "display_name": "Burdick, Joel Wakeman" }, { "name": { "family": "Tucker", "given": "Maegan L." }, "id": "Tucker-Maegan-Lindsay", "orcid": "0000-0001-7363-6809", "role": "member", "display_name": "Tucker, Maegan L." }, { "name": { "family": "Ames", "given": "Aaron D." }, "id": "Ames-A-D", "orcid": "0000-0003-0848-3177", "role": "member", "display_name": "Ames, Aaron D." }, { "name": { "family": "Yue", "given": "Yisong" }, "id": "Yue-Yisong", "orcid": "0000-0001-9127-1989", "role": "member", "display_name": "Yue, Yisong" } ], "option_major": [ "cns" ], "doi": "10.7907/nfhg-dq67", "abstract": "Bipedal robots are uniquely positioned to operate in environments designed for humans. From humanoids traversing unstructured terrain to robotic exoskeletons assisting individuals with paralysis, these systems highlight the promise of legged locomotion. Yet enabling walking that is both robust and aligned with user needs remains a fundamental challenge, owing to hybrid dynamics, hardware limitations, and the variability across individuals in assistive devices.
\r\n\r\nThe first part of this dissertation addresses user-aligned locomotion. A gait that is theoretically stable may still be rejected in practice if it feels unnatural, uncomfortable, or strenuous. To bridge this gap, we integrate musculoskeletal modeling with trajectory optimization to generate anthropomorphic, dynamically feasible walking gaits, and extend preference-based learning with an active learning formulation that efficiently elicits user feedback within a region of interest while maintaining comfort. Together, these methods enable systematic design of gaits that not only achieve stable walking but also capture the nuanced trade-offs users make between comfort, effort, and naturalness of movement.
\r\n\r\nThe second part of this dissertation focuses on robust locomotion in the face of model mismatch, external disturbances, and environmental variability. We develop robustness strategies spanning multiple layers of the control hierarchy: offline trajectory design informed by robustness metrics grounded in hybrid forward invariance, online adaptation through a data-driven predictive framework, and feedback policies learned in massively parallel simulation using reinforcement learning guided by control Lyapunov functions. While independent, these approaches together provide complementary strategies for handling uncertainty, spanning from offline design to real-time adaptation.
\r\n\r\nAlthough motivated by the challenges of exoskeleton locomotion, the methods are validated on other bipedal platforms such as humanoids and lower-limb prostheses, highlighting their broad applicability to diverse bipedal platforms. Overall, this dissertation shows that principled integration of model-based and data-driven approaches enables locomotion strategies that are robust, adaptive, and aligned with human needs, advancing the deployment of bipedal robots and assistive devices.
" }, { "name": "Lipschitz, Mikel", "degree": "PhD", "year": "2026", "title": "SLIM: Stochastic Lineage-Based Iterative Minimization", "advisor": "Wang, Kaihang", "url": "https://resolver.caltech.edu/CaltechTHESIS:08212025-162748066", "creators": [ { "name": { "family": "Lipschitz", "given": "Mikel" }, "id": "Lipschitz-Mikel", "orcid": "0000-0002-5764-1648", "display_name": "Lipschitz, Mikel" } ], "advisors": [ { "name": { "family": "Wang", "given": "Kaihang" }, "id": "Wang-Kaihang", "orcid": "0000-0001-7657-8755", "role": "advisor", "display_name": "Wang, Kaihang" } ], "committee": [ { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "chair", "display_name": "Phillips, Robert B." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "member", "display_name": "Shapiro, Mikhail G." }, { "name": { "family": "Wang", "given": "Kaihang" }, "id": "Wang-Kaihang", "orcid": "0000-0001-7657-8755", "role": "member", "display_name": "Wang, Kaihang" } ], "option_major": [ "bioeng" ], "doi": "10.7907/q80m-3t75", "abstract": "The bacterial genomes we encounter today have been shaped by billions of years of genome altering events which involve rewriting, addition, and removal of genomic elements. The resulting product is a complex network of interactions composed of elements which are defined by contextual necessity. The elucidation of a minimal set of elements, comprised of just those essential for sustaining life, has long been sought after. This set, or minimal genome, has been proposed to be a representation for the foundation of life itself. Genome minimization, the pursuit of this foundation, is a process by which genomic segments deemed unnecessary, or non-essential, in an environmental context dependent manner are identified and removed, leaving only DNA that provides the cell with the resources and processes it needs to stay alive and reproduce. Numerous genome minimization efforts have been undertaken previously. However, each of these studies has resulted in the generation of a single genome-reduced strain derived from a single wild-type bacteria in a single environment. While these methods have shown a great deal of promise in their ability to identify foundational genomic pieces in this extremely narrow context, they lack the throughput and generalizability to identify foundational pieces of all bacterial life.
\r\n \r\nBuilding upon prior genome minimization efforts, we developed SLIM (Stochastic Lineage-based Iterative Minimization), a modular genome reduction system designed for unbiased DNA removal, high-throughput parallelization and cross-species compatibility. In this study we utilize SLIM to generate a library of ten genome-reduced E. coli strains. We then rigorously interrogate the library to identify patterns in deleted segments. We assess the effects that these deletions have on remaining genomic components and explore how these effects can result in substantial fitness changes in different environments. Finally, we demonstrate the modularity of SLIM by generating two additional libraries of genome-reduced strains from two phylogenetically distinct parent bacteria, S. flexneri and P. putida. This work highlights the power and promise of generating diverse libraries of genome-reduced strains; substantially expanding the number of minimized genomes that can be achieved, while simultaneously reducing the time to generation.
" }, { "name": "Liu, Shichen", "degree": "PhD", "year": "2026", "title": "Reconstituting Cellular Intelligence in Nonliving Biomolecular Matter", "advisor": "Thomson, Matthew", "url": "https://resolver.caltech.edu/CaltechTHESIS:05302026-225700411", "creators": [ { "name": { "family": "Liu", "given": "Shichen" }, "id": "Liu-Shichen", "display_name": "Liu, Shichen" } ], "advisors": [ { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "advisor", "display_name": "Thomson, Matthew" } ], "committee": [ { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "chair", "display_name": "Phillips, Robert B." }, { "name": { "family": "Dabiri", "given": "John O." }, "id": "Dabiri-J-O", "orcid": "0000-0002-6722-9008", "role": "member", "display_name": "Dabiri, John O." }, { "name": { "family": "Brady", "given": "John F." }, "id": "Brady-J-F", "orcid": "0000-0001-5817-9128", "role": "member", "display_name": "Brady, John F." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" } ], "option_major": [ "bioeng" ], "doi": "10.7907/x80h-cs27", "abstract": "Living cells convert environmental and internal information into physical and biochemical action. This dissertation focuses on two layers of cell-like control that can be rebuilt in nonliving biomolecular systems: active physical machinery, where cytoskeletal architecture turns molecular force generation into mechanics and transport, and molecular sense-and-response, where compartment architecture turns environmental cues into biochemical output. The common problem is control. Active cytoskeletal materials remodel while generating force, so force transmission must remain predictable in a scaffold that also acts as a machine. Synthetic compartments face a related boundary problem: a compartment must preserve internal state while admitting inputs, sustaining production, and releasing functional output.
\r\n\r\nStudy 1 asks how nanoscale motor forces become network-scale mechanical work. We use PRC1-mediated microtubule crosslinking to tune architecture in kinesin-driven active materials and show that connectivity alone does not guarantee mechanical output. Low-connectivity networks dissipate motor activity locally. Connected but mechanically floppy networks reorganize collectively but fail to sustain long-range force propagation or perform substantial work. Mechanically persistent networks support coherent contraction and extractable work. Velocity correlations reveal a transition from local motion to collective organization, while work measurements and a semiflexible-network model separate connectivity from rigidity. The result is a force-to-work rule for active matter: motors generate force, but crosslinkers determine whether force dissipates locally or becomes coherent, extractable mechanical work.
\r\n\r\nStudy 2 asks how spatial patterning can make active matter predictable enough for task execution despite nonlinear dynamics. Motor--microtubule active fluids generate autonomous motion, but useful engineering requires flow fields specifying where material moves, which objects separate, and how fluids mix. We use light to pattern contracting motor--filament networks and use predictive modeling to compose hydrodynamic outputs into micrometre-scale flow fields. Programmed flows move and separate cell clusters, impose extensional deformation on polymers and giant vesicles, and mix fluids at low Reynolds number. The result is a force-to-flow rule: optical geometry converts internally generated active forces into predictable transport functions.
\r\n\r\nStudy 3 builds an acellular sense-and-response system. A giant unilamellar vesicle, or GUV, supplies a cell-sized lipid boundary for preserving internal biochemical state. Cell-free transcription--translation, or TX-TL, supplies a nonliving biochemical engine for reading DNA and synthesizing proteins outside a cell. We engineer input-output separation across the compartment boundary: small-molecule access admits lactate and nutrients, lactate sensing switches internal productive state, TX-TL synthesizes a bispecific T-cell engager and a genetically encoded membrane gate, and controlled release exports macromolecular output. Lactate-conditioned GUVs recruit T cells to kill CD19-positive target cells. Study 3 brings selective input, productive internal state, controlled output, and target-cell killing into one nonliving compartment.
\r\n\r\nAcross the three studies, we rebuild cellular functions outside living cells. Molecular architecture controls force-to-work conversion, optical patterning controls force-to-flow conversion, and compartment architecture controls cue-to-output conversion. In each case, nonliving biomolecular matter uses molecular, spatial, or compartmental structure to link input with output.
" }, { "name": "Lu, Jialiang", "degree": "PhD", "year": "2026", "title": "Neural Code for Dynamic Visual Experience in the Primate Brain", "advisor": "Tsao, Doris Y.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05232026-010040610", "creators": [ { "name": { "family": "Lu", "given": "Jialiang" }, "id": "Lu-Jialiang", "orcid": "0000-0001-5403-3228", "display_name": "Lu, Jialiang" } ], "advisors": [ { "name": { "family": "Tsao", "given": "Doris Y." }, "id": "Tsao-D-Y", "orcid": "0000-0003-1083-1919", "role": "advisor", "display_name": "Tsao, Doris Y." } ], "committee": [ { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "orcid": "0000-0003-2136-6506", "role": "chair", "display_name": "Meister, Markus" }, { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "member", "display_name": "Adolphs, Ralph" }, { "name": { "family": "Tsao", "given": "Doris Y." }, "id": "Tsao-D-Y", "orcid": "0000-0003-1083-1919", "role": "member", "display_name": "Tsao, Doris Y." }, { "name": { "family": "Rutishauser", "given": "Ueli" }, "id": "Rutishauser-U", "orcid": "0000-0002-9207-7069", "role": "member", "display_name": "Rutishauser, Ueli" } ], "option_major": [ "cns" ], "doi": "10.7907/3ewf-6795", "abstract": "Neuroscience increasingly needs experimental tools that can study vision in naturalistic, continuous, and interactive scenes. This thesis presents a methodological and scientific framework for studying naturalistic vision and social scene representation in the primate brain. I developed MINOS, a Unity-based platform that integrates complex stimulus generation, behavioral control, hardware communication, synchronization, and analysis-ready data recording in one modular environment. I also developed a compact piezo-actuated Neuropixels insertion system for acute large-scale electrophysiology in awake macaques, designed to support precise, reproducible, and flexible multi-probe recordings in limited chamber space. Using these tools, I recorded neural populations from inferotemporal cortex and ventrolateral prefrontal cortex while a monkey freely viewed a continuous social scene in which one or two characters entered, waited, interacted, and left. This design kept precise event labels while reducing the strong visual transients of discrete image presentation. The results showed a clear functional difference between the two regions. Inferotemporal cortex mainly represented the currently fixated face and updated rapidly after fixation changes. Ventrolateral prefrontal cortex integrated information across time and scene state, represented current and recently relevant identities, encoded entering and leaving events, and maintained identity information in different low-dimensional formats depending on whether a person was fixated or not. It also carried a stable and largely identity-independent code for action category and action role across sessions. Together, these findings suggest that prefrontal cortex helps organize dynamic social scenes by combining context-dependent identity representations with a more general relational code. This thesis therefore contributes new experimental tools for primate systems neuroscience and opens a route for studying how the brain represents structured events during continuous visual experience." }, { "name": "Markarian, Nicholas", "degree": "PhD", "year": "2026", "title": "Mixtures of Latent Variable Models for Interpreting Gene Expression Covariation from Pathways to Transcriptomes", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02262026-191401810", "creators": [ { "name": { "family": "Markarian", "given": "Nicholas" }, "id": "Markarian-Nicholas", "orcid": "0000-0003-1347-2392", "display_name": "Markarian, Nicholas" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "chair", "display_name": "Pachter, Lior S." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Bois", "given": "Justin S." }, "id": "Bois-J-S", "orcid": "0000-0001-7137-8746", "role": "member", "display_name": "Bois, Justin S." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" } ], "option_major": [ "biology" ], "doi": "10.7907/dcbd-wy35", "abstract": "This thesis centers on interpretable subspace learning and latent variable models for characterizing covariation modulated by categorical variables in the context of biology. First, it introduces a probabilistic model with ties to Principal Component Analysis and k-means clustering, k-spaces, which has implications across different biological analyses through its interpretations as a subspace learning technique, a latent variable model, and a dimension reduction technique. Second, it establishes the problem of simultaneously characterizing gene covariation and expression level in known pathways in human tissue samples and applies k-spaces to GTEx data to lay the foundations for this line of research. Finally, it outlines a path forward to being able to use such data as references for clinical samples from patients." }, { "name": "Martinez, Zachary A.", "degree": "PhD", "year": "2026", "title": "Tokens, Topologies, Taxa: Towards Declarative Biology and Bioengineering", "advisor": "Thomson, Matthew W.; Murray, Richard M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06022026-081334128", "creators": [ { "name": { "family": "Martinez", "given": "Zachary A." }, "id": "Martinez-Zachary-A", "orcid": "0000-0002-7830-3162", "display_name": "Martinez, Zachary A." } ], "advisors": [ { "name": { "family": "Thomson", "given": "Matthew W." }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "co-advisor", "display_name": "Thomson, Matthew W." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "co-advisor", "display_name": "Murray, Richard M." } ], "committee": [ { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "orcid": "0000-0003-2713-1513", "role": "chair", "display_name": "Mazmanian, Sarkis K." }, { "name": { "family": "Wang", "given": "Kaihang" }, "id": "Wang-Kaihang", "orcid": "0000-0001-7657-8755", "role": "member", "display_name": "Wang, Kaihang" }, { "name": { "family": "Bois", "given": "Justin" }, "id": "Bois-Justin", "orcid": "0000-0001-7137-8746", "role": "member", "display_name": "Bois, Justin" }, { "name": { "family": "Thomson", "given": "Matthew W." }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew W." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." } ], "option_major": [ "bioeng" ], "doi": "10.7907/b5ye-jy33", "abstract": "Contemporary deep-learning models for the life-sciences have outpaced the tooling that lets experimentalists compose them. Three contributions are presented in response, a software platform, exemplary tasks built on it, and a predicted structural proteome of a defined gut microbiome. The underlying argument is that for experimentalists who use rather than build deep-learning methods, difficulties with composition and usability now outpace availability.
\r\n\r\nTRILL, a platform for AI-based protein engineering and analysis, is open-source, runs locally, and wraps models/methods behind a uniform vocabulary of thirteen top-level commands. Furthermore, TRILL is scalable, ranging from parallel fine-tuning of large models on a supercomputer to democratized, parameter off-loading in compute-limited scenarios. Models can be swapped with a one-argument change rather than a pipeline rewrite, and fast predictions can be paired with physics-based validation where overconfidence costs most.
\r\n\r\nProtein language models were fine-tuned using a homology-aware strategy, decreasing data leakage when evaluating generated proteins. Classifiers for cellulase, antimicrobial, and toxin activity were trained and applied to a scan of over two hundred million proteins from the NCBI non-redundant catalogue. An end-to-end pipeline carried seventeen predicted toxins of unknown function through structure prediction, binder design, and molecular dynamics on nearly nine hundred designed complexes.
\r\n\r\nThe third contribution targets hCom2, a defined synthetic gut consortium. We present a structural resource, where roughly four hundred thousand structures of its proteome were predicted using TRILL, segmented into eight hundred thousand domains, and assigned CATH designations. A case study demonstrating the utility of this structural database identifies nineteen carriers of the Helicobacter pylori virulence-factor TIPalpha fold across fourteen strains where sequence-only annotation fails.
" }, { "name": "Moiseyenko, Anastasiya O.", "degree": "PhD", "year": "2026", "title": "Gut Microbiota as Modulators and Therapeutic Targets in Parkinson\u2019s Disease", "advisor": "Mazmanian, Sarkis K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:11052025-233903791", "creators": [ { "name": { "family": "Moiseyenko", "given": "Anastasiya O." }, "id": "Moiseyenko-Anastasiya-O", "orcid": "0000-0001-5379-7808", "display_name": "Moiseyenko, Anastasiya O." } ], "advisors": [ { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "orcid": "0000-0003-2713-1513", "role": "advisor", "display_name": "Mazmanian, Sarkis K." } ], "committee": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "chair", "display_name": "Lester, Henry A." }, { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "orcid": "0000-0002-7033-0844", "role": "member", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "orcid": "0000-0003-2713-1513", "role": "member", "display_name": "Mazmanian, Sarkis K." } ], "option_major": [ "biology" ], "doi": "10.7907/3yxw-z226", "abstract": "The gastrointestinal (GI) tract is a unique junction of the nervous system, immune system, and the gut microbiome. The gut microbiome, a complex community of bacteria, fungi, and viruses, is able to regulate host development, behavior, immunity, and disease. In Parkinson\u2019s disease (PD), a neurodegenerative disorder characterized by motor dysfunction, \u03b1-synuclein (\u03b1Syn) pathology, and common GI symptoms, the gut bacterial composition is significantly altered, with depletions in beneficial, anti-inflammatory taxa compared to healthy controls. This thesis explores whether specific gut bacteria may be disease-protective. We first assembled a consortium of taxa that are reduced in individuals with PD across multiple cohorts and geographies. We find that both therapeutic and prophylactic oral administration of this consortium to Thy-1-\u03b1Syn overexpressing (Thy1-ASO) mice, a preclinical model of PD, improves select motor and GI deficits and reduces \u03b1Syn pathology in the brain. We next identified three taxa that independently drive motor function improvements, with Faecalibacterium prausnitzii producing the most pronounced effects. Further characterization of treatment with F. prausnitzii revealed improvements in GI symptoms, reduced \u03b1Syn aggregates in the brain, remodeling of the gut microbiome, and induction of anti-inflammatory and tissue-regenerative pathways in the colon. Collectively, these findings provide a foundation for developing specific bacterial species as novel therapeutics for PD and highlight the broader potential of the gut microbiome to transform the way we understand and treat human health and disease." }, { "name": "Olson, Blade A.", "degree": "PhD", "year": "2026", "title": "Synthetic Antigen-Presenting Vesicles for Selective Immunomodulation", "advisor": "Mayo, Stephen L.; Murray, Richard M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08212025-191338101", "creators": [ { "name": { "family": "Olson", "given": "Blade A." }, "id": "Olson-Blade-A", "orcid": "0000-0002-1526-1399", "display_name": "Olson, Blade A." } ], "advisors": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "co-advisor", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "co-advisor", "display_name": "Murray, Richard M." } ], "committee": [ { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "chair", "display_name": "Thomson, Matthew" }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "member", "display_name": "Gradinaru, Viviana" } ], "option_major": [ "bioleng" ], "doi": "10.7907/fxzx-yn04", "abstract": "The rapid advancement of generative artificial intelligence has enabled unprecedented progress for the field of computational protein design. A forthcoming challenge for generative protein design algorithms is the immunocompatibility of these de novo designed molecules with organism physiology, namely humans. A separate, but related, aspirational goal for synthetic biology is to perform cellular reprogramming in vivo so that cell-based therapies and biologics are generated endogenously by patients rather than being externally manufactured or expanded before delivery, as is the case with biologics, T cell therapies, and stem cell therapies; again, a major hurdle for the in vivo production of these therapies and in vivo cellular reprogramming is immunogenicity.
\r\n\r\nTo address these challenges, we first demonstrate a cell-like, cell-free approach for in vivo cellular reprogramming with the induced release of pMHCI and pMHCII-loaded synthetic antigen-presenting vesicles that are secreted from non-immune cells by DNA and mRNA transfection to facilitate the selective expansion or silencing of immune responses. Next, we show initial results for the use of human tonsil organoids as a quantitative assay for adenoviral vector immunogenicity, enabling future directed evolution approaches for immunogenicity reduction as well as generation of an immunogenicity dataset to tailor modern computational protein design algorithms for human immunocompatibility. Together, these projects represent complementary methods to control protein immunogenicity, either through rationally engineered or directedly evolved modifications identified by physiologically-relevant in vitro models, or with an administered mRNA therapeutic that selectively modifies the immune response to a protein that cannot be computationally redesigned.
" }, { "name": "Pang Wan Rion, Marion", "degree": "PhD", "year": "2026", "title": "Deep Profiling of the Single-Cell Proteome", "advisor": "Chou, Tsui-Fen; Roukes, Michael Lee", "url": "https://resolver.caltech.edu/CaltechTHESIS:02172026-235945229", "creators": [ { "name": { "family": "Pang Wan Rion", "given": "Marion" }, "id": "Pang-Wan-Rion-Marion", "orcid": "0000-0002-0158-2976", "display_name": "Pang Wan Rion, Marion" } ], "advisors": [ { "name": { "family": "Chou", "given": "Tsui-Fen" }, "id": "Chou-Tsui-Fen", "orcid": "0000-0003-2410-2186", "role": "co-advisor", "display_name": "Chou, Tsui-Fen" }, { "name": { "family": "Roukes", "given": "Michael Lee" }, "id": "Roukes-M-L", "orcid": "0000-0002-2916-6026", "role": "co-advisor", "display_name": "Roukes, Michael Lee" } ], "committee": [ { "name": { "family": "Cai", "given": "Long" }, "id": "Cai-Long", "orcid": "0000-0002-7154-5361", "role": "chair", "display_name": "Cai, Long" }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-Matthew", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Chou", "given": "Tsui-Fen" }, "id": "Chou-Tsui-Fen", "orcid": "0000-0003-2410-2186", "role": "member", "display_name": "Chou, Tsui-Fen" }, { "name": { "family": "Roukes", "given": "Michael Lee" }, "id": "Roukes-M-L", "orcid": "0000-0002-2916-6026", "role": "member", "display_name": "Roukes, Michael Lee" } ], "option_major": [ "bioeng" ], "doi": "10.7907/27fk-fd20", "abstract": "The proteome encodes the functional state of a cell with a resolution that transcripts cannot fully capture, yet single-cell and spatial proteomics remain constrained by limited sensitivity, throughput, and analytical scalability. This thesis presents a comprehensive framework for deep profiling of the single-cell proteome, spanning methodological development, biological application, and technological innovation.
\r\n\r\nIn Part I, a robust pipeline for single-cell proteomics is established through systematic optimization of sample preparation, including deparaffinization, lysis and extraction, and miniaturized digestion workflows in high-throughput multi-well plate formats. These advances enable efficient protein recovery from both fluorescence-activated cell sorting (FACS) isolated single cells and laser capture microdissection (LCM) tissue specimens. Complementary complexity reduction strategies and scpViz, an open-source platform for visualization and analysis of sparse single-cell proteomic datasets, further improve sensitivity and data interpretability. Together, these approaches enable quantification of approximately 2,500 proteins from single cells, with recent benchmarks on next-generation instrumentation exceeding 4,000 proteins per cell.
\r\n\r\nIn Part II, the developed framework is applied to interrogate biological systems at cellular and tissue scales. In a colorectal cancer model, single-cell proteomics reveals proteomic heterogeneity between parental and drug-resistant lines, identifying divergent response programs undetectable at bulk resolution, and implicating loss of signalling plasticity as a hallmark of resistance. In the nervous system, spatially resolved LCM proteomics uncovers region-specific protein dysregulation at near single-cell resolution. Additional investigations into developmental systems demonstrate the capacity to capture spatiotemporal proteomic dynamics across biological contexts.
\r\n\r\nIn Part III, a nanoDESI-based platform is developed toward subcellular spatial proteomics. A first-principles model of fluid and analyte transport provides quantitative scaling laws governing extraction efficiency as a function of probe geometry and flow parameters. This theoretical framework is coupled with nanoDESI probe microfabrication and a custom instrumentation platform enabling controlled, high-resolution sampling.
\r\n\r\nCollectively, this thesis integrates experimental, computational, and theoretical approaches to enable deep, spatially resolved profiling of the single-cell proteome, providing both the experimental infrastructure and the physical framework necessary for next-generation proteomics technologies.
" }, { "name": "R\u00f6schinger, Tom", "degree": "PhD", "year": "2026", "title": "Illuminating the Regulatory Dark Matter of E. coli with Massively Parallel Reporter Assays", "advisor": "Phillips, Robert B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04302026-154155078", "creators": [ { "name": { "family": "R\u00f6schinger", "given": "Tom" }, "id": "R\u00f6schinger-Tom", "orcid": "0000-0002-4900-3216", "display_name": "R\u00f6schinger, Tom" } ], "advisors": [ { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "advisor", "display_name": "Phillips, Robert B." } ], "committee": [ { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "chair", "display_name": "Thomson, Matthew" }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Bois", "given": "Justin S." }, "id": "Bois-J-S", "orcid": "0000-0001-7137-8746", "role": "member", "display_name": "Bois, Justin S." }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." } ], "option_major": [ "biochem" ], "doi": "10.7907/3qy4-em46", "abstract": "All cells respond to changes in their environment through the regulation of their genes. Despite decades of effort in Escherichia coli, huge gaps remain in our knowledge of both the function of many genes - the so-called y-ome - and how they are regulated. For roughly 40% of genes, no function has been assigned, and for the majority we do not know which, if any, transcription factors control their expression. Here we describe a joint experimental and theoretical dissection of the regulation of 117 promoters in E. coli across 39 diverse environments, enabling us to identify the binding sites and transcription factors that mediate regulatory control at base-pair resolution.
\r\n\r\nUsing Reg-Seq - a combination of saturation mutagenesis, massively parallel reporter assays, mass spectrometry, and tools from information theory - we go from complete ignorance of a promoter's environment-dependent regulatory architecture to detailed models of its behavior. We first develop the theoretical framework for interpreting the information footprints that are the primary readout of Reg-Seq, using toy models to establish the expected scale of mutual information at binding site positions and how the noise floor scales with sequencing depth. We then describe improvements to the genome-integrated Reg-Seq protocol, including redesigned constructs and an expanded condition panel spanning carbon source shifts, antibiotic stress, anaerobiosis, osmotic shock, and stationary phase.
\r\n\r\nAs proof of principle, we chose a combination of gold standard promoters with well-characterized regulation, genes from the y-ome, toxin-antitoxin pairs, and genes hypothesized to be part of regulatory modules. At well-characterized promoters, Reg-Seq recovers known binding sites for LexA, CpxR, MarA, Rob, MprA, and CRP under the expected conditions, while also revealing previously unreported features: new transcription start sites, a novel CRP binding site at the uncharacterized gene yadI, and condition-specific activation patterns not predicted by existing annotations. Extending the method to 34 promoters with no prior regulatory information, we discovered a host of new insights into the regulatory landscape of the y-ome. A cluster of osmotically induced promoters shares a conserved binding motif for an unidentified transcription factor, and genome-wide scanning with this motif identifies a putative regulon spanning osmoprotectant transport, trehalose metabolism, and envelope modification. At the anaerobic gene ybiY, we identify YciT as a repressor - correcting the existing database annotation - and reveal an unidentified activator that does not correspond to any characterized anaerobic regulator. At the cryptic prophage gene yagB, mass spectrometry identifies both XynR and H-NS, and the overlapping architecture of the repressor and sigma^S binding sites explains the stationary phase specificity of expression.
\r\n\r\nA systematic survey of single-mutation effects across the library reveals that many promoter regions harbor latent sequences one base change away from creating a functional sigma^70 promoter. These de novo promoters are strongly enriched at loci that require a specific activator, consistent with the signal only being detectable against a silent background. In a complementary set of results, we find that several loci with multiple annotated transcription start sites resolve to fewer active sites under physiological conditions.
\r\n\r\nTogether, these results demonstrate that Reg-Seq can systematically annotate the regulatory architecture of uncharacterized genes, correct existing annotations, and generate testable hypotheses about transcription factor identity and condition-specificity, bridging the gap between single-gene studies and the largely uncharacterized regulatory landscape of E. coli.
" }, { "name": "Salmon, Gabriel L.", "degree": "PhD", "year": "2026", "title": "To see a World in a Grain of Cells: Statistical Auguries of Nonequilibrium", "advisor": "Phillips, Robert B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05222026-005425424", "creators": [ { "name": { "family": "Salmon", "given": "Gabriel L." }, "id": "Salmon-Gabriel-L", "orcid": "0000-0003-2163-8399", "display_name": "Salmon, Gabriel L." } ], "advisors": [ { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "advisor", "display_name": "Phillips, Robert B." } ], "committee": [ { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "orcid": "0000-0002-5899-7523", "role": "chair", "display_name": "Winfree, Erik" }, { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "orcid": "0000-0002-3904-0195", "role": "member", "display_name": "Goentoro, Lea A." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." } ], "option_major": [ "bioeng" ], "doi": "10.7907/ehyd-3072", "abstract": "Precisely what new mathematical and biological behaviors are unlocked as cells---and their collectives---operate out of equilibrium? In this thesis, we take simple steps towards accounting for the characteristic scales of biological energy expenditures and tracing their functional destinies.
\r\n\r\nAfter embarking on an initial Census of lively dissipation (Chapter 1) to set the stage, Part I explores cells on the bioenergetic brink of survival and death. In Chapter 2 we explore the sweep of the smallest measured cellular metabolic rates; reflect on their empirical and philosophical underpinnings; and endeavor an initial order of magnitude accounting of the biophysical processes that could most plausibly dominate the energy budgets of starving cells. Followed by this tour, Chapter 3 hones in on fresh, provocative experiments probing anaerobic maintenance metabolism of Pseudomonas, proposing simple math to predict how microbes die in balance with their energetic resources and interpreting measured reactions to salt.
\r\n\r\nPart II travels to intracellular gene regulation. Among other regulatory settings, in Chapter 4 we investigate the most common regulated architecture of gene regulation in prokaryotes and ask how its behaviors categorically change under different investments in biochemical drive. Chapter 5 narrates how graph theory gives powerful tools for humanly thinking about even large regulatory state graphs.
\r\n\r\nIn Part III, we work closely with inspiring new experimental collaborations and data on patterns of microtubules made by molecular motors. Chapter 6 reports on measuring biochemical energy profiles, and their rates of change, over time and space. Using this phenomenology, in Chapter 7 we develop theory to ask about the fundamental costs required to build or maintain biochemical gradients.
\r\n\r\nLast, in Chapter 8 (Part IV) we ask how a common mathematics might unify a large class of biological dynamics with disperse initial conditions but coherent final conditions, a setting we refer to as exploratory dynamics.
\r\n\r\nCollectively, we hope these case studies give quantitative glimpses of precisely how energy so exquisitely animates biology.
" }, { "name": "Shaker, Sammy", "degree": "PhD", "year": "2026", "title": "3D Vat Photopolymerization of Microarchitected Magnetic Metal Alloys for Chemotherapy Capture Filters", "advisor": "Greer, Julia R.", "url": "https://resolver.caltech.edu/CaltechTHESIS:09132025-054711849", "creators": [ { "name": { "family": "Shaker", "given": "Sammy" }, "id": "Shaker-Sammy", "orcid": "0000-0003-1751-4908", "display_name": "Shaker, Sammy" } ], "advisors": [ { "name": { "family": "Greer", "given": "Julia R." }, "id": "Greer-J-R", "orcid": "0000-0002-9675-1508", "role": "advisor", "display_name": "Greer, Julia R." } ], "committee": [ { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "chair", "display_name": "Shapiro, Mikhail G." }, { "name": { "family": "Kornfield", "given": "Julia A." }, "id": "Kornfield-J-A", "orcid": "0000-0001-6746-8634", "role": "member", "display_name": "Kornfield, Julia A." }, { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "member", "display_name": "Guttman, Mitchell" }, { "name": { "family": "Greer", "given": "Julia R." }, "id": "Greer-J-R", "orcid": "0000-0002-9675-1508", "role": "member", "display_name": "Greer, Julia R." } ], "option_major": [ "bioeng" ], "doi": "10.7907/gc1t-5869", "abstract": "Primary liver cancer constitutes an object of concern for communities across the globe. With the majority of new diagnoses of the most common subtype of primary liver cancer, hepatocellular carcinoma, inoperable at diagnosis, the standard of care revolves around the use of liver-directed therapies to treat tumors. The most popular therapy is termed transarterial chemoembolization, wherein the unique anatomy of the liver is exploited to deliver chemotherapy and embolic agents selectively to a large tumor, leading to tumor cell death and improved survival for patients. However, the chemotherapeutics used in this procedure have toxic effects on organs outside of the liver, and are as such dose-restricted on the basis of these side effects. In order to increase the amount of drug used and thus increase the chance of tumor cell death, chemotherapy capture devices are necessary. While some materials have been developed for this application, these devices regularly suffer from such restrictions as passive capture mechanisms necessitating the design of devices with poor hemodynamics or the use of immunogenic materials such as heteroDNA. Magnetic nanoparticles conjugated to chemotherapeutics as well as magnetic nanoparticle chemotherapy capture agents, delivered with the chemotherapeutics, present a potential way out of this conundrum, but the application of these materials requires the design of magnetic capture devices with favorable hemodynamic and magnetic properties. Architected magnetic metal devices can potentially provide the sought after solution to these difficulties, but techniques to such devices with high spatial resolution are lacking. An additive manufacturing technique that can provide high spatial resolution utilizes vat photopolymerization in tandem with thermal processing to produce well-resolved metal lattices that can provide for this ongoing need.
\r\n\r\nThis thesis applies this technique to the synthesis of magnetic lattices for particle capture. Lattices are synthesized in iron, nickel-iron, and copper-nickel-iron compositions and characterized structurally and magnetically. Simulations of particle capture in these lattices under various conditions are performed. In addition, attempts at particle capture are described and methods of characterization of particle capture are discussed. This technique is also explored with regards to the synthesis of iron-nickel and iron-cobalt lattices and the characterization of the resultant products is discussed and evaluated. Finally, a modification to this technique is used to generate metal-carbon microcomposites with unusual magnetic properties when compared with their counterparts synthesized using the unmodified procedure.
" }, { "name": "Sharma, Tarun", "degree": "PhD", "year": "2026", "title": "Quantifying Insect Behavior Across Scales Using Computer Vision", "advisor": "Parker, Joseph; Dickinson, Michael H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01132026-022337021", "creators": [ { "name": { "family": "Sharma", "given": "Tarun" }, "id": "Sharma-Tarun", "orcid": "0009-0008-8945-3896", "display_name": "Sharma, Tarun" } ], "advisors": [ { "name": { "family": "Parker", "given": "Joseph" }, "id": "Parker-J", "orcid": "0000-0001-9598-2454", "role": "advisor", "display_name": "Parker, Joseph" }, { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "orcid": "0000-0002-8587-9936", "role": "co-advisor", "display_name": "Dickinson, Michael H." } ], "committee": [ { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "orcid": "0000-0001-8837-678X", "role": "chair", "display_name": "Siapas, Athanassios G." }, { "name": { "family": "Parker", "given": "Joseph" }, "id": "Parker-J", "orcid": "0000-0001-9598-2454", "role": "member", "display_name": "Parker, Joseph" }, { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "orcid": "0000-0002-8587-9936", "role": "member", "display_name": "Dickinson, Michael H." }, { "name": { "family": "Hong", "given": "Elizabeth J." }, "id": "Hong-Elizabeth-J", "orcid": "0000-0003-3866-418X", "role": "member", "display_name": "Hong, Elizabeth J." } ], "option_major": [ "cns" ], "doi": "10.7907/yphx-fh43", "abstract": "Insects exhibit extraordinary behavioral diversity from rapid sensorimotor control required for flight to coordinated behavior across large societies. While some behaviors are best studied in controlled lab settings and require precise fine-grained measurements, others demand long-term observations in more natural settings that cannot be replicated in lab. In this thesis, I demonstrate the applications of computer vision to quantify insect behavior across scales, from the level of individuals to colonies, in both lab and field conditions.
\r\n\r\nIn the first part, I examine the role of mechanosensory cells, campaniform sensilla, on the stabilization response of the fruit fly, Drosophila melanogaster. Using tethered flies mounted on a rotating arena, I measure head and wing equilibrium responses using marker less pose estimation and edge tracking. By genetically silencing different subsets of campaniform sensilla, I show a linear relationship between the number of cells silenced and the magnitude of head and wing response, providing direct experimental evidence for their role in the equilibrium response.
\r\n \r\nIn the second part, I apply computer vision techniques to study colony scale movement dynamics at the nest entrance of the ant species, Liometopum occidentale, in the Angeles National Forest. I introduce a custom, low-cost, field deployable multisensory camera trap, called the Ethocam, and collect an extensive dataset of hourly videos from three ant nests spanning over 100 days. Using computer vision methods for detection and multi-object tracking, I quantify circadian activity patterns, \tdirectional traffic imbalances, environmental drivers of activity and walking speed, and spatiotemporal movement dynamics across distinct trails.
\r\n \r\nTogether, by integrating lab experiments, long term field data, and quantitative analysis using computer vision, this thesis presents a general framework for studying insect behavior across scales in both controlled and natural environments.
" }, { "name": "Shi, Yuelin", "degree": "PhD", "year": "2026", "title": "The Brain\u2019s Second Look: Generative Feedback and Dynamic Coding in Primate Vision", "advisor": "Tsao, Doris Y.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04012026-192212616", "creators": [ { "name": { "family": "Shi", "given": "Yuelin" }, "id": "Shi-Yuelin", "orcid": "0000-0002-6788-976X", "display_name": "Shi, Yuelin" } ], "advisors": [ { "name": { "family": "Tsao", "given": "Doris Y." }, "id": "Tsao-D-Y", "orcid": "0000-0003-1083-1919", "role": "advisor", "display_name": "Tsao, Doris Y." } ], "committee": [ { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "orcid": "0000-0003-2136-6506", "role": "chair", "display_name": "Meister, Markus" }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "member", "display_name": "Andersen, Richard A." }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "member", "display_name": "Perona, Pietro" }, { "name": { "family": "Tsao", "given": "Doris Y." }, "id": "Tsao-D-Y", "orcid": "0000-0003-1083-1919", "role": "member", "display_name": "Tsao, Doris Y." } ], "option_major": [ "neurobio" ], "doi": "10.7907/v1xv-2152", "abstract": "Vision must infer the latent causes of retinal input from signals that are incomplete, noisy, and often ambiguous. This thesis asks whether primate vision is best understood as a predominantly feedforward computation or as a generative, recurrent process in which higher-order hypotheses help shape sensory representations over time. I propose that the visual brain is best described as performing analysis by synthesis: internal scene hypotheses generate predicted sensory structure, and perception emerges through iterative interactions between these predictions and incoming evidence.
\r\n\r\nThe first part of the thesis develops this proposal at the conceptual and neurobiological levels. Drawing on phenomena such as imagery, dreaming, active vision, bistable perception, perceptual completion, and the effects of prior knowledge, I argue that a purely feedforward account cannot fully explain the coordinated, context-sensitive, and inference-like nature of visual experience. I then review computational families of generative models and neuroscientific evidence for feedback, iterative dynamics, and laminar circuitry, proposing that these features are well suited to implement hypothesis-driven visual inference in the brain.
\r\n\r\nThe second part tests this framework in the macaque face-patch system using simultaneous Neuropixels recordings from face patches ML and AM. To ask whether degraded-face recognition engages feedback-supported inference, I measured the time at which degraded-face responses become aligned with intact identity representations. Under passive viewing of a wide range of degradations, both response timing and intact-to-degraded generalization followed the canonical posterior-to-anterior ordering, with ML preceding AM. Thus, degradation alone did not reveal an anterior-leading signature of top-down inference within inferotemporal cortex. In contrast, learning to associate ambiguous Mooney faces with their intact counterparts selectively reshaped ML population activity, increasing both representational separability and cross-condition generalization for upright Mooney faces. These findings suggest that top-down recruitment in the ventral stream is constrained and may depend not simply on degraded input, but on the availability of learned priors that can disambiguate it.
\r\n\r\nThe final part shows that high-level visual coding itself is dynamically reformatted over time. Large-scale recordings from ML and AM during viewing of faces and non-face objects revealed that face-selective neurons do not use a single, fixed encoding axis. Instead, responses to faces initially align with a domain-general object code, consistent with rapid face detection, but then undergo a rapid, concerted switch within 20 ms. This switch includes reversal of tuning in low dimensions of object space, emergence of new tuning in higher-dimensional face space, increased response sparsity, and improved reconstruction and discrimination of individual faces. The effect is stimulus-gated, appearing for faces but not for non-face objects, and resolves a long-standing debate by showing that inferotemporal face coding is both domain general and domain specific, but at different moments in time.
\r\n\r\nTogether, these studies support a view of primate vision as a dynamic and knowledge-sensitive process. Rather than relying on a static feedforward code alone, the visual system appears to use recurrent computations that allow sensory representations to be revised, sharpened, and reformatted as incoming evidence interacts with internal models and prior knowledge.
" }, { "name": "Subramanian, Arjuna Michael", "degree": "PhD", "year": "2026", "title": "Rewriting the Sequence and Structure Rules of Deep Protein Space", "advisor": "Thomson, Matthew", "url": "https://resolver.caltech.edu/CaltechTHESIS:09162025-184128136", "creators": [ { "name": { "family": "Subramanian", "given": "Arjuna Michael" }, "id": "Subramanian-Arjuna-Michael", "orcid": "0009-0004-2790-0209", "display_name": "Subramanian, Arjuna Michael" } ], "advisors": [ { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "advisor", "display_name": "Thomson, Matthew" } ], "committee": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "chair", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "orcid": "0000-0002-5899-7523", "role": "member", "display_name": "Winfree, Erik" } ], "option_major": [ "biochem" ], "doi": "10.7907/p4st-m614", "abstract": "With a 20-letter alphabet, conceivable protein sequence-space is enormous; sparks of structure and function are vanishingly rare. Despite massive advances in AI-guided protein design, we remain largely ignorant of the sequences and structures that populate the depths of protein space more than a handful of mutations away from what nature has tried. In this work, we leverage the potential of one specific class of AI protein model \u2014 the protein language model, or PLM \u2014 to internalize the essential features of the protein sequence-structure map while retaining the capacity to explore its extremes. Guided by a \"novelty first, fitness next\" mentality, we harness this balance towards systematic discovery of new-to-nature sequences and structures throughout deep protein space.
\r\n\r\nIn the first section, we dissect the ability of PLMs to explore natural and novel regimes of sequence and structure during free generation. We find that while these models readily emit novel sequences encoding artificial proteins that appear biophysically feasible in silico, they fail to completely or representatively capture the known distribution of natural protein structures. We expose a fundamental tradeoff between the ability of a PLM to generate with sequence novelty or structural coverage but not both simultaneously; prioritizing sampling of far-from-natural sequences triggers a collapse to a handful of simple structural motifs and disordered regions.
\r\n\r\nTurning this sequence novelty vs. structural breadth tradeoff to our advantage, the second section is devoted to the development of \"foldtuning\" \u2014 a structure-preserving, sequence-remodeling engine for navigating the far corners of sequence-space with PLM-based probes. We successfully scale and deploy foldtuning for >700 targets, pushing artificial sequences past the point of detectable homology to any real protein documented in nature, discovering novel sequence-level semantics and grammar for mimicking known protein folds, and accessing potential reservoirs of downstream structural and functional innovation. Experimental validation of select targets reveals that foldtuning produces realizable and functional binders in contexts including a toxin/antitoxin system and peptide hormone signaling.
\r\n\r\nShifting to focus on structural novelty, the final section introduces two PLM-driven methods for the discovery of new-to-nature structures. We show that with appropriate steering functions, PLMs readily yield well-structured domains (featuring diverse secondary and supersecondary elements) outside the several thousand such families cataloged from among known proteins. Overall, this work makes substantial inroads towards the challenge of locating viable far-from-natural regions of protein density across the global sequence-structure map, and revises our notions of the physical constraints on sequence and structure in valid proteins. Moreover, it sets the stage for future assembly of synthetic biological systems composed fully of new-to-nature parts and ultimately for modeling efforts that close the design loop from sequence all the way to complex phenotype.
" }, { "name": "Talukder, Sabera", "degree": "PhD", "year": "2026", "title": "Beyond Text: The Rudiments of Next Generation Foundation Models", "advisor": "Yue, Yisong; Gkioxari, Georgia", "url": "https://resolver.caltech.edu/CaltechTHESIS:12152025-232943789", "creators": [ { "name": { "family": "Talukder", "given": "Sabera" }, "id": "Talukder-Sabera", "display_name": "Talukder, Sabera" } ], "advisors": [ { "name": { "family": "Yue", "given": "Yisong" }, "id": "Yue-Yisong", "orcid": "0000-0001-9127-1989", "role": "advisor", "display_name": "Yue, Yisong" }, { "name": { "family": "Gkioxari", "given": "Georgia" }, "id": "Gkioxari-Georgia", "role": "co-advisor", "display_name": "Gkioxari, Georgia" } ], "committee": [ { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "chair", "display_name": "Perona, Pietro" }, { "name": { "family": "Yue", "given": "Yisong" }, "id": "Yue-Yisong", "orcid": "0000-0001-9127-1989", "role": "member", "display_name": "Yue, Yisong" }, { "name": { "family": "Gkioxari", "given": "Georgia" }, "id": "Gkioxari-Georgia", "role": "member", "display_name": "Gkioxari, Georgia" }, { "name": { "family": "Wierman", "given": "Adam C." }, "id": "Wierman-A-C", "orcid": "0000-0002-5923-0199", "role": "member", "display_name": "Wierman, Adam C." } ], "option_major": [ "neurobio" ], "doi": "10.7907/s12m-5692", "abstract": "This thesis builds non-text and multimodal foundation models that overcome the difficulties of non-text data. These challenges are namely image\u2019s and time series\u2019 (e.g. audio\u2019s and video\u2019s): data heterogeneity, data continuity, and large memory requirements. In order to overcome these attributes we must build models that are information dense, generalizable, and multimodal. By the end of this thesis we will empirically demonstrate that the recipe for performant non-text and multimodal foundation models is: create discrete information dense representations, train models with large scale data in the most generalizable manner possible, and fuse data modalities early in the modeling stack." }, { "name": "Wang, Tongtong", "degree": "PhD", "year": "2026", "title": "The Neural Basis of Brain-Body Communication", "advisor": "Oka, Yuki", "url": "https://resolver.caltech.edu/CaltechTHESIS:03162026-211941584", "creators": [ { "name": { "family": "Wang", "given": "Tongtong" }, "id": "Wang-Tongtong", "orcid": "0000-0002-0408-2571", "display_name": "Wang, Tongtong" } ], "advisors": [ { "name": { "family": "Oka", "given": "Yuki" }, "id": "Oka-Yuki", "orcid": "0000-0003-2686-0677", "role": "advisor", "display_name": "Oka, Yuki" } ], "committee": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "chair", "display_name": "Lester, Henry A." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "orcid": "0000-0003-2713-1513", "role": "member", "display_name": "Mazmanian, Sarkis K." }, { "name": { "family": "Oka", "given": "Yuki" }, "id": "Oka-Yuki", "orcid": "0000-0003-2686-0677", "role": "member", "display_name": "Oka, Yuki" } ], "option_major": [ "neurobio" ], "doi": "10.7907/a8mn-0e75", "abstract": "Understanding how the nervous system orchestrates physiology across the body has long been a central question in neuroscience. While neural mechanisms underlying behavior have been extensively characterized, the cellular and circuit principles that mediate brain-body communication remain underexplored. In this thesis, I investigate how internal physiological signals are detected and translated into coordinated regulation of organ functions through specialized sensory and autonomic pathways.
\r\n\r\nUsing molecular, behavioral, and genetic perturbation approaches, I first examine how changes in body fluid balance are detected by central sensory neurons. I identify distinct neuronal populations within forebrain circumventricular regions that detect hyperosmotic and hypovolemic challenges and drive modality-specific fluid consumption behaviors. Then I show how water signals in the gut are encoded by a dedicated vagal afferent population, providing feed-forward inputs that contribute to thirst satiation. These studies demonstrate that internal states are monitored through specialized channels spanning central and peripheral circuits.
\r\n \r\nNext, I investigate the circuit logic of sympathetic regulation in the abdomen, identifying molecularly defined neuronal populations that project selectively to visceral organs and differentially regulate gastrointestinal transit and digestive processes. These results demonstrate that sympathetic outputs are organized into discrete pathways that enable precise and independent control of physiology. I then synthesize current knowledge of autonomic organization, highlighting its molecular diversity and modular architecture as key features enabling selective regulation of organ function.
\r\n \r\nTogether, these findings reveal that brain-body communication is mediated by structured sensory pathways and modular autonomic circuits to achieve precise yet flexible control of physiology. This work provides a framework for understanding how neural systems coordinate internal stability and offers insight into how disruptions of these processes may contribute to diseases.
" }, { "name": "Wexler, Helen", "degree": "PhD", "year": "2026", "title": "Algae as a Platform for Sustainable Biocomposites: Process\u2013Structure\u2013Property Relations", "advisor": "Daraio, Chiara", "url": "https://resolver.caltech.edu/CaltechTHESIS:10312025-170502666", "creators": [ { "name": { "family": "Wexler", "given": "Helen" }, "id": "Wexler-Helen", "orcid": "0000-0003-4030-9603", "display_name": "Wexler, Helen" } ], "advisors": [ { "name": { "family": "Daraio", "given": "Chiara" }, "id": "Daraio-C", "orcid": "0000-0001-5296-4440", "role": "advisor", "display_name": "Daraio, Chiara" } ], "committee": [ { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "chair", "display_name": "Thomson, Matthew" }, { "name": { "family": "Daraio", "given": "Chiara" }, "id": "Daraio-C", "orcid": "0000-0001-5296-4440", "role": "member", "display_name": "Daraio, Chiara" }, { "name": { "family": "Burdick", "given": "Joel Wakeman" }, "id": "Burdick-J-W", "orcid": "0000-0002-3091-540X", "role": "member", "display_name": "Burdick, Joel Wakeman" }, { "name": { "family": "McAniff", "given": "Peter" }, "id": "McAniff-Peter-J", "role": "member", "display_name": "McAniff, Peter" }, { "name": { "family": "Saigal", "given": "Anil" }, "id": "Saigal-Anil", "orcid": "0000-0001-7911-8674", "role": "member", "display_name": "Saigal, Anil" } ], "option_major": [ "bioeng" ], "doi": "10.7907/ba11-m769", "abstract": "This thesis investigates whole-cell algae as a possible binder matrix for fully bio-based composites and evaluates agricultural residues as reinforcing fillers. The study quantifies how feedstock morphology and preprocessing govern microstructure and mechanical response under compression molding that uses only water, heat, and pressure. Candidate algal feedstocks include food grade algae, single strain wastewater algae, and mixed wastewater communities." }, { "name": "White, Jonathan Alexander", "degree": "PhD", "year": "2026", "title": "Untangling Overlapping Barcodes in Image-Based Spatial\r\nGenomics", "advisor": "Cai, Long", "url": "https://resolver.caltech.edu/CaltechTHESIS:05132026-192926992", "creators": [ { "name": { "family": "White", "given": "Jonathan Alexander" }, "id": "White-Jonathan-Alexander", "orcid": "0000-0002-7009-3332", "display_name": "White, Jonathan Alexander" } ], "advisors": [ { "name": { "family": "Cai", "given": "Long" }, "id": "Cai-Long", "orcid": "0000-0002-7154-5361", "role": "advisor", "display_name": "Cai, Long" } ], "committee": [ { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "chair", "display_name": "Thomson, Matthew" }, { "name": { "family": "Van Valen", "given": "David A." }, "id": "Van-Valen-D", "orcid": "0000-0001-7534-7621", "role": "member", "display_name": "Van Valen, David A." }, { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "member", "display_name": "Guttman, Mitchell" }, { "name": { "family": "Cai", "given": "Long" }, "id": "Cai-Long", "orcid": "0000-0002-7154-5361", "role": "member", "display_name": "Cai, Long" } ], "option_major": [ "bioeng" ], "doi": "10.7907/r2z1-jj33", "abstract": "Difficulty in resolving spatially overlapping barcodes is a major bottleneck for imaging-based spatial genomics methods. Here, we present an approach for untangling overlapping barcodes by using strong encoding and global optimization to reduce spurious solutions resulting from recombinations of barcodes. We demonstrate experimentally that cellular regions with average local densities of 127 barcodes per \u00b5m\u00b2 can be decoded with an estimated FDR of less than 4%, enabling a new type of super-resolution microscopy by coding." }, { "name": "Xu, Yue", "degree": "PhD", "year": "2026", "title": "New Approaches to Characterize Psychological Traits and States", "advisor": "Adolphs, Ralph", "url": "https://resolver.caltech.edu/CaltechTHESIS:05212026-183448162", "creators": [ { "name": { "family": "Xu", "given": "Yue" }, "id": "Xu-Yue", "orcid": "0000-0003-2366-8807", "display_name": "Xu, Yue" } ], "advisors": [ { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "advisor", "display_name": "Adolphs, Ralph" } ], "committee": [ { "name": { "family": "O'Doherty", "given": "John P." }, "id": "O'Doherty-J-P", "orcid": "0000-0003-0016-3531", "role": "chair", "display_name": "O'Doherty, John P." }, { "name": { "family": "Eberhardt", "given": "Frederick D." }, "id": "Eberhardt-Frederick", "orcid": "0009-0008-0557-5926", "role": "member", "display_name": "Eberhardt, Frederick D." }, { "name": { "family": "Rutishauser", "given": "Ueli" }, "id": "Rutishauser-U", "orcid": "0000-0002-9207-7069", "role": "member", "display_name": "Rutishauser, Ueli" }, { "name": { "family": "Hong", "given": "Elizabeth J." }, "id": "Hong-Elizabeth-J", "orcid": "0000-0003-3866-418X", "role": "member", "display_name": "Hong, Elizabeth J." }, { "name": { "family": "Katz", "given": "Jonathan N." }, "id": "Katz-J-N", "orcid": "0000-0002-5287-3503", "role": "member", "display_name": "Katz, Jonathan N." }, { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "member", "display_name": "Adolphs, Ralph" } ], "option_major": [ "cns" ], "doi": "10.7907/cvwt-1962", "abstract": "This thesis addresses two central questions about psychological traits (stable individual differences such as personality) and states (transient fluctuations such as momentary emotions). First, how can we quantitatively distinguish traits from states in empirical data? Second, how are traits structured when we infer them about others, and is that structure shared across perceivers from different cultural backgrounds? To address the first question (Chapter 2), I applied statistical models to longitudinal self-report data collected during the COVID-19 pandemic, demonstrating that psychological measures are mixtures of trait-like and state-like components rather than pure categories, and decomposing their variability into temporally stable between-individual differences and within-individual fluctuations. To address the second question (Chapters 3\u20134), I developed a label-free experimental paradigm to recover the latent structure of trait impressions from faces without relying on experimenter-imposed trait words, revealing a low-dimensional representational geometry that is largely shared across perceiver racial groups. In the discussion (Chapter 5), I situate these findings within a broader measurement framework, comparing self- versus other-inference and examining how traits and states can be studied in self-report, task-based, neurobiological, and experimental-manipulation approaches across humans, nonhuman animals, and large language models. I outline future directions in person\u2013situation interactions, cross-cultural generalizability, and multimodal measurements. Together, these findings provide a quantitative framework for modeling psychological traits and states in both self-evaluation and social perception, advancing a unified view of psychological structure grounded in statistical organization rather than semantic categories." }, { "name": "Yu, Changhua", "degree": "PhD", "year": "2026", "title": "Understanding Kinase-Substrate Interaction with Deep Learning and High-Throughput Scanning", "advisor": "Van Valen, David A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05152026-160124985", "creators": [ { "name": { "family": "Yu", "given": "Changhua" }, "id": "Yu-Changhua", "orcid": "0000-0003-4799-4535", "display_name": "Yu, Changhua" } ], "advisors": [ { "name": { "family": "Van Valen", "given": "David A." }, "id": "Van-Valen-D", "orcid": "0000-0001-7534-7621", "role": "advisor", "display_name": "Van Valen, David A." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Yue", "given": "Yisong" }, "id": "Yue-Yisong", "orcid": "0000-0001-9127-1989", "role": "member", "display_name": "Yue, Yisong" }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Van Valen", "given": "David A." }, "id": "Van-Valen-D", "orcid": "0000-0001-7534-7621", "role": "member", "display_name": "Van Valen, David A." } ], "option_major": [ "bioeng" ], "doi": "10.7907/2m7a-nx91", "abstract": "Catalyzed by more than 500 human kinases, protein phosphorylation contributes to almost every aspect of cellular signaling. Despite advances in the past decades, a profound gap persists between the scale of the kinase signaling network and our ability to characterize it: over 95% of human phosphosites lack an assigned kinase, approximately one-third of the human kinome remains functionally understudied, and mechanisms by which kinase domains select substrates remain under-explored. Consequently, the druggable landscape for targeting phosphorylation rewiring events in disease contexts remains limited.
\r\n\r\nThis thesis seeks to address these challenges through developments spanning machine learning, high-throughput interactome screening, and deep mutational scanning. We develop KINBERT, a transformer-based protein language model that jointly encodes paired kinase domain and substrate peptide sequences and demonstrate its utility through disease variant interpretation and identification of host kinase targets during viral infection. We develop PhosphoPCA, a barcoded yeast-based assay that links kinase\u2013substrate phosphorylation to cell growth for high-throughput pooled profiling of kinase specificity and apply it to identify novel substrates for understudied kinases and to engineer validated live-cell kinase biosensors. We leveraged saturated mutagenesis to enable deep mutational scanning of kinase domains with measurable components of variant fitness from protein stability, phosphorylation activity, and substrate selectivity.
\r\n\r\nTogether, the thesis builds an integrated framework for systematically decoding the kinase\u2013substrate interaction space, providing a diverse set of novel technologies for illuminating the dark kinome, interpreting pathogenic phosphoSNVs, uncovering the mutational effect of kinase variants, and enabling generalizable engineering of kinase biosensors.
" }, { "name": "Zhang, Raymond J.", "degree": "PhD", "year": "2026", "title": "Construction of Bacterial Genome Chimeras", "advisor": "Wang, Kaihang", "url": "https://resolver.caltech.edu/CaltechTHESIS:06012026-205812005", "creators": [ { "name": { "family": "Zhang", "given": "Raymond J." }, "id": "Zhang-Raymond-J", "orcid": "0009-0003-1404-3239", "display_name": "Zhang, Raymond J." } ], "advisors": [ { "name": { "family": "Wang", "given": "Kaihang" }, "id": "Wang-Kaihang", "orcid": "0000-0001-7657-8755", "role": "advisor", "display_name": "Wang, Kaihang" } ], "committee": [ { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "chair", "display_name": "Shapiro, Mikhail G." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Wang", "given": "Kaihang" }, "id": "Wang-Kaihang", "orcid": "0000-0001-7657-8755", "role": "member", "display_name": "Wang, Kaihang" } ], "option_major": [ "biology" ], "doi": "10.7907/074j-1k29", "abstract": "Bacterial genomes are natural mosaics subject to whole genome engineering by horizontal gene transfer in the wild. Existing approaches to whole-genome engineering have concentrated either on the de novo synthesis of single-source genomes from synthetic DNA, or on small-scale editing at the level of nucleotides to kilobases. The intermediate domain, combining large, megabase-scale segments of DNA from different genomes within the same chromosome for the deliberate construction of chimeric bacterial genomes, has been underexplored. This thesis covers this research gap by presenting methods for constructing megabase-scale bacterial genome chimeras.
\r\n\r\nChapter 2 presents methods for addition chimera construction using the Additive Conjugative-CAST Engineering (ACE) technology to expand recipient genomes with up to 2-Mb of a donor sequence in a single step. Transfers were demonstrated over large phylogenetic distance, namely 1-Mb cross-order and 100-kb cross-class transfers from Escherichia coli to Vibrio natriegens and to Agrobacterium tumefaciens, respectively. 522-kb cross-order transfers from Pseudomonas protegens to E. coli provide a window to rebooting foreign genome segments in a different genetic background.
\r\n\r\nChapter 3 extends these methods to substitution chimeras using the technique Replacements by ACE (ReplACE) to rewrite native recipient genome segments with corresponding donor genome segments. We show the recoding of a 1-Mb segment of the E. coli MDS42 genome with E. coli Syn61, and the two-step hybridization of the MDS42 genome with Shigella flexneri to create a Shigoli chimera strain with 50% of its genomic material from both parent strains. We investigate the expression changes from this chimerization event before proceeding to rewrite the genome of MDS42 systematically with that of gut commensal E. coli EcAZ1 to explore genetic determinants of colonization.
\r\n\r\nChapter 4 articulates the natural barriers of horizontal gene transfer removed with the invention of these two techniques and summarizes the benefits and drawbacks of each method and the biological questions they can address as well as the engineering possibilities they provide. Experimental limitations are considered and future experiments are proposed.
" }, { "name": "Zhang, Yameng", "degree": "PhD", "year": "2026", "title": "Neural Circuits Underlying Salt-Taste Valence", "advisor": "Oka, Yuki", "url": "https://resolver.caltech.edu/CaltechTHESIS:12102025-235050354", "creators": [ { "name": { "family": "Zhang", "given": "Yameng" }, "id": "Zhang-Yameng", "orcid": "0009-0005-7038-7049", "display_name": "Zhang, Yameng" } ], "advisors": [ { "name": { "family": "Oka", "given": "Yuki" }, "id": "Oka-Yuki", "orcid": "0000-0003-2686-0677", "role": "advisor", "display_name": "Oka, Yuki" } ], "committee": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "chair", "display_name": "Anderson, David J." }, { "name": { "family": "Lois", "given": "Carlos" }, "id": "Lois-Carlos", "orcid": "0000-0002-7305-2317", "role": "member", "display_name": "Lois, Carlos" }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Oka", "given": "Yuki" }, "id": "Oka-Yuki", "orcid": "0000-0003-2686-0677", "role": "member", "display_name": "Oka, Yuki" } ], "option_major": [ "neurobio" ], "doi": "10.7907/kp37-4v28", "abstract": "Salt consumption is unique among the five basic tastes. The perception of salty taste revealed a concentration-dependent and internal-state-dependent valence pattern. Low concentrations of salt trigger sodium-specific taste receptors while high concentrations recruit bitter and sour pathways, which have been investigated by previous research and proven with daily experience. I focus on the dynamic nature of salt perception, in the perspective of physiological states.
\r\n\r\nIn calorie restricted state, food cues become more appetitive, but in sodium depletion state, the hedonic value of salt fundamentally reversed. High concentrations of salt induce innate aversion under sated states, whereas such aversive stimuli transform into appetitive ones under sodium depletion. Neural mechanisms underlying this state-dependent salt valence switch are poorly understood. Using transcriptomics state-to-cell-type mapping and neural manipulations, we show that positive and negative valences of salt are controlled by anatomically distinct neural circuits in the mammalian brain. The hindbrain interoceptive circuit regulates sodium-specific appetitive drive, whereas behavioral tolerance of aversive salts is encoded by a dedicated class of neurons in the forebrain lamina terminalis (LT) expressing prostaglandin E2 (PGE2) receptor, Ptger3. We show that these LT neurons regulate salt tolerance by selectively modulating aversive taste sensitivity, partly through a PGE2-Ptger3 axis. These results reveal the bimodal regulation of appetitive and tolerance signals toward salt, which together dictate the amount of sodium consumption under different internal states.
\r\n\r\nMaintaining the fluid balance requires complex crosstalk within the neural circuits and endocrine systems through the brain-body axis. Despite the prevalence of fluid balance dysregulation and salt overconsumption in modern life, its health relevance remains underappreciated. I have been attracted to the global salt overconsumption crisis from the start of my Ph. D. study. The current approaches to regulate salt intake rely heavily on imperfect salt substitutes. The PGE2-Ptger3 brain-body axis posed a new top-down approach to reduce salt intake. Interestingly, PGE2 is a critical biomarker in pro-inflammation state. It has been investigated that high salt intake induces chronic inflammation, desensitization of salty tastes, and intensified craving for consumption. I regard my research of the tolerance circuit as an entry point and hope future research into inflammation and its role in salt intake can be translated into concrete salt reduction solutions.
" }, { "name": "Zou, Olivia Aoli", "degree": "PhD", "year": "2026", "title": "Engineering DNA liquids Towards Macroscopic Separation of Biomolecules", "advisor": "Rothemund, Paul W. K.; Qian, Lulu", "url": "https://resolver.caltech.edu/CaltechTHESIS:02092026-215015195", "creators": [ { "name": { "family": "Zou", "given": "Olivia Aoli" }, "id": "Zou-Olivia-Aoli", "orcid": "0009-0007-1149-130X", "display_name": "Zou, Olivia Aoli" } ], "advisors": [ { "name": { "family": "Rothemund", "given": "Paul W. K." }, "id": "Rothemund-P-W-K", "orcid": "0000-0002-1653-3202", "role": "advisor", "display_name": "Rothemund, Paul W. K." }, { "name": { "family": "Qian", "given": "Lulu" }, "id": "Qian-Lulu", "orcid": "0000-0003-4115-2409", "role": "co-advisor", "display_name": "Qian, Lulu" } ], "committee": [ { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "orcid": "0000-0002-5899-7523", "role": "chair", "display_name": "Winfree, Erik" }, { "name": { "family": "Qian", "given": "Lulu" }, "id": "Qian-Lulu", "orcid": "0000-0003-4115-2409", "role": "member", "display_name": "Qian, Lulu" }, { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "member", "display_name": "Pierce, Niles A." }, { "name": { "family": "Fygenson", "given": "Deborah K." }, "id": "Fygenson-Deborah-K", "orcid": "0000-0002-5681-3938", "role": "member", "display_name": "Fygenson, Deborah K." }, { "name": { "family": "Rothemund", "given": "Paul W. K." }, "id": "Rothemund-P-W-K", "orcid": "0000-0002-1653-3202", "role": "member", "display_name": "Rothemund, Paul W. K." } ], "option_major": [ "bioeng" ], "doi": "10.7907/3639-5v91", "abstract": "Liquid-liquid phase separation (LLPS) is a thermodynamic process by which a mixture of solutions de-mixes into separate, coexisting phases. In cells, macromolecules such as proteins and nucleic acids can undergo LLPS to form membraneless structures known as biomolecular condensates. These condensates are usually liquid-like droplets highly concentrated in the species they are composed of. Condensates are crucial to many cellular processes and functions, such as protein assembly, gene regulation, subcellular organization, storage, and stress response. On a larger scale, condensates are also implicated in various neurodegenerative diseases, such as Huntington's and Alzheimer's disease.
\r\n\r\nDNA condensates are easy to engineer due to the programmable nature of the molecule. DNA nanostars are multi-armed junctions with double-stranded arms and typically single-stranded, palindromic overhangs or 'sticky ends' that allow for transient interactions between the molecules. They can phase-separate to form liquid-like droplets at the microscopic scale. Centrifugation of a high concentration solution of nanostars coalesces condensates into a macroscopic liquid that is visible to the naked eye.
\r\n\r\nIn biology, one of the key functions of condensates is to act as compartments and localize various molecules, such as enzymes and nucleic acids. Our goal is to engineer macroscopic DNA liquids to similarly act as compartments that can separate multiple biomolecular targets. We propose that these macroscopic DNA liquids are a potentially novel method for multiplexed separation that is fast, simple to use, and biocompatible with various high-value targets, such as protein therapeutics.
\r\n\r\nWe designed multiple immiscible DNA liquids of different densities to act as macroscopic compartments. We present a set of design principles for engineering nanostars to create liquid layers in a microcentrifuge tube. We show via UV absorbance measurements how various nanostar features, such as arm length, sticky end strength, and valency, affect liquid phase density, which determines the order of layering between liquids. We further show that the interface quality between pairwise liquids is determined by a combination of density differences between liquids and the orthogonality of nanostar sticky ends, and we devise a metric to calculate this orthogonality. Using these design principles, we are currently able to create up to five orthogonal liquid layers in a tube.
\r\n\r\nWith these DNA liquid layers, we envision separation to be a two step process. The first step is to localize specific target biomolecules into these layers. We demonstrate localization of oligos in a multilayer DNA liquid system by modifying nanostars with tag regions complementary to the target strand. We also demonstrate localization of fluorescent streptavidin to a single DNA liquid layer by modifying nanostars with a streptavidin-binding aptamer. The second step is to release targets from the liquid layers so that they can be collected for downstream use. After localization of the aforementioned targets, we added strands complementary to either the tag region or the aptamer. This causes targets to be displaced from their binding moiety and released into the supernatant.
" }, { "name": "Bhattacharya, Paulomi", "degree": "PhD", "year": "2025", "title": "RNA-Mediated Toxicity In Neurodegeneration: The Mechanistic Role Of The C9ORF72 Repeat Expansion In ALS Molecular Pathogenesis", "advisor": "Guttman, Mitchell", "url": "https://resolver.caltech.edu/CaltechTHESIS:03182025-023751137", "creators": [ { "name": { "family": "Bhattacharya", "given": "Paulomi" }, "id": "Bhattacharya-Paulomi", "display_name": "Bhattacharya, Paulomi" } ], "advisors": [ { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "advisor", "display_name": "Guttman, Mitchell" } ], "committee": [ { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "chair", "display_name": "Shapiro, Mikhail G." }, { "name": { "family": "Ichida", "given": "Justin K." }, "id": "Ichida-Justin-K", "orcid": "0000-0002-8827-8087", "role": "member", "display_name": "Ichida, Justin K." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" } ], "option_major": [ "bioleng" ], "doi": "10.7907/2ywx-7a47", "abstract": "The G4C2 hexanucleotide repeat expansion in the first intron of the C9ORF72 gene is the most common genetic mutation linked to ALS, accounting for ~40 percent of familial and 10 percent of sporadic cases. Yet, its functional contribution to molecular pathogenesis remains unknown. The prevailing model is that this expansion leads to transcription of a novel RNA (C9-repeat RNA) that leads to disease either through its RNA product or translation of dipeptide repeat proteins it encodes (\u201cgain-of-function\u201d). However, recent attempts to degrade the C9-repeat RNA in several major clinical trials have failed to show any improvement in C9-ALS patients, raising questions about what role, if any, the C9-repeat RNA plays in ALS pathogenesis. Here, we demonstrate that the C9-repeat RNA is not detectable in C9-ALS patient-derived iPSNs or postmortem brain tissue. We show that transcription of the C9ORF72 gene initiates downstream of the G4C2 repeat sequence with the repeat expansion residing at a promoter-proximal region and displaying chromatin signatures of an enhancer. Because this region is GC-rich and has been reported to be preferentially methylated in C9-ALS patients, we explored whether this repeat expansion might lead to reduced C9ORF72 gene expression. We show that the C9-repeat is associated with reduced allele-specific expression of the C9ORF72 gene, consistent with the GC-rich features of the repeat expansion and previous reports of preferential DNA methylation in C9-ALS patients. Taken together, our findings challenge the prevailing gain-of-function models in C9-ALS and instead suggest that the repeat expansion region may function as a regulatory element that silences C9ORF72 expression from the mutant allele." }, { "name": "Chakravorty, Arun", "degree": "PhD", "year": "2025", "title": "Bridging Space and Time: Resolving the Temporal Dynamics of the Seminiferous Epithelial Cycle Using Spatial Transcriptomics", "advisor": "Cai, Long", "url": "https://resolver.caltech.edu/CaltechTHESIS:05112025-035044867", "creators": [ { "name": { "family": "Chakravorty", "given": "Arun" }, "id": "Chakravorty-Arun", "orcid": "0000-0003-2890-0855", "display_name": "Chakravorty, Arun" } ], "advisors": [ { "name": { "family": "Cai", "given": "Long" }, "id": "Cai-Long", "orcid": "0000-0002-7154-5361", "role": "advisor", "display_name": "Cai, Long" } ], "committee": [ { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "chair", "display_name": "Guttman, Mitchell" }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Cai", "given": "Long" }, "id": "Cai-Long", "orcid": "0000-0002-7154-5361", "role": "member", "display_name": "Cai, Long" } ], "option_major": [ "sysbiol" ], "doi": "10.7907/2rcd-0v79", "abstract": "Biology is inherently spatial, with tissue architecture and cell\u2013cell interactions shaping dynamic developmental and homeostatic processes. In this thesis, we harness high-resolution spatial transcriptomics via RNA seqFISH+ to show how spatial information can be used to resolve temporal information in complex tissues, using adult mouse spermatogenesis as a model. By profiling 2,638 genes in over 216,000 cells, we find that each seminiferous tubule cross-section represents a distinct timepoint of the seminiferous epithelial cycle, and collectively all tubules form a circular topology in gene expression space that precisely aligns with the known 12-stage progression. Intriguingly, Sertoli cells exhibit a robust cyclic transcriptional program synchronized with germ cell differentiation, raising the question of whether this cycle is driven solely by germ cells or whether Sertoli cells display an intrinsic cyclic expression profile. To address this, we ablate differentiating germ cells using a DNA alkylating agent, busulfan. In this model, despite the lack of differentiating germ cells, Sertoli cells maintain much of their cyclic expression suggesting an autonomous cycle that partially dephases without germ cell input. Integrative analyses suggest that the underlying mechanism of this oscillation may involve an innate retinoic acid metabolic cycle and/or an interconnected transcription factor network. Finally, we discuss how these findings broaden our understanding of tissue processes and propose that spatial transcriptomics can be adopted to reconstruct temporal dynamics for many tissues from static snapshots." }, { "name": "Coughlin, Gerard Michael", "degree": "PhD", "year": "2025", "title": "Spatial Biology Tools to Accelerate and Refine Adeno-Associated Virus Engineering and Application", "advisor": "Gradinaru, Viviana", "url": "https://resolver.caltech.edu/CaltechTHESIS:04172025-154334670", "creators": [ { "name": { "family": "Coughlin", "given": "Gerard Michael" }, "id": "Coughlin-Gerard-Michael", "orcid": "0000-0003-0644-4721", "display_name": "Coughlin, Gerard Michael" } ], "advisors": [ { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "advisor", "display_name": "Gradinaru, Viviana" } ], "committee": [ { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "orcid": "0000-0002-7371-4675", "role": "chair", "display_name": "Prober, David A." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Lois", "given": "Carlos" }, "id": "Lois-Carlos", "orcid": "0000-0002-7305-2317", "role": "member", "display_name": "Lois, Carlos" }, { "name": { "family": "Oka", "given": "Yuki" }, "id": "Oka-Yuki", "orcid": "0000-0003-2686-0677", "role": "member", "display_name": "Oka, Yuki" }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "member", "display_name": "Gradinaru, Viviana" } ], "option_major": [ "neurobio" ], "doi": "10.7907/7gfy-hn24", "abstract": "The transfer of exogenous genetic material into living cells is a fundamental technique for basic research and, increasingly, for the treatment of human disease. Adeno-associated viruses (AAVs) are small, unenveloped viruses that can carry a limited DNA cargo of 4.4 kb (plus 0.3 kb inverted terminal repeats). These vectors are workhorses for in vivo gene transfer into mammalian systems, both for fundamental research and for therapeutic purposes. Natural serotypes of AAVs generally show broad tropism for easy to access tissues. Engineering of AAVs, through modification to the capsid surface and/or to the DNA genome, can enable access to otherwise privileged organs (e.g., brain) and can refine tropism to specific cell types (e.g., Purkinje cells of the cerebellum). Such engineering efforts can generate hundreds to thousands of interesting variants, but there is a dearth of high-throughput methods to characterize these variants. Furthermore, despite widespread usage, including in human patients, many questions on fundamental AAV biology remain unanswered.
\r\n\r\nIn this thesis, I attempt to address some of these outstanding bottlenecks and open questions. In Chapter 2, I address the lack of high-throughput methods for broadly characterizing engineered AAV vectors in vivo, by developing and applying high-throughput spatial transcriptomics for AAV transcripts. In Chapter 3, I focus on understanding the biology of AAV genome processing, illuminated by novel spatial genomics methods. Using these novel methods, I then profile and mechanistically dissect transcriptional crosstalk between codelivered AAV vectors (Chapter 4). Finally, in Chapter 5, I address the limited packaging capacity of AAV vectors by leveraging AAV transcriptional crosstalk to enable minimally invasive, all-AAV cell type-specific gene editing in wildtype animals, with enough efficiency to recapitulate known phenotypes.
\r\n\r\nThe work presented in this thesis will help to accelerate and refine AAV engineering and application. Furthermore, this thesis highlights potential confounds for AAV genome engineering, but also opens new avenues for AAV-powered functional genetics in mammalian systems.
" }, { "name": "Courellis, Hristos Spiridonos", "degree": "PhD", "year": "2025", "title": "A Study on the Content, Format, and Implementation of Neural Representations That Underlie Flexible Human Cognition", "advisor": "Adolphs, Ralph; Rutishauser, Ueli", "url": "https://resolver.caltech.edu/CaltechTHESIS:06122024-185146643", "creators": [ { "name": { "family": "Courellis", "given": "Hristos Spiridonos" }, "id": "Courellis-Hristos-Spiridonos", "orcid": "0000-0001-5963-679X", "display_name": "Courellis, Hristos Spiridonos" } ], "advisors": [ { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "advisor", "display_name": "Adolphs, Ralph" }, { "name": { "family": "Rutishauser", "given": "Ueli" }, "id": "Rutishauser-U", "orcid": "0000-0002-9207-7069", "role": "co-advisor", "display_name": "Rutishauser, Ueli" } ], "committee": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "chair", "display_name": "Andersen, Richard A." }, { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "orcid": "0000-0003-2136-6506", "role": "member", "display_name": "Meister, Markus" }, { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "member", "display_name": "Adolphs, Ralph" }, { "name": { "family": "Rutishauser", "given": "Ueli" }, "id": "Rutishauser-U", "orcid": "0000-0002-9207-7069", "role": "member", "display_name": "Rutishauser, Ueli" } ], "option_major": [ "bioleng" ], "doi": "10.7907/edk1-kb22", "abstract": "Humans are the most capable cognitive generalists to walk the earth. They have a remarkable capacity for flexibility reallocating cognitive resources to rapidly acquire and execute an effectively infinite number of tasks. By utilizing the opportunity to record single-neuron activity in the frontal and temporal lobes of awake, behaving neurosurgical patients, we aim to elucidate the principles by which task representations are organized at the neural-circuit level to give rise to flexible cognition and behavior.
\r\n\r\nOur research program consists of four inter-related projects, each of which seeks to clarify the content, format, and single-neuron implementation of the representations that underlie different aspects of cognition and behavior that are uniquely human. In the first project, we demonstrate that the emergence of disentangled task representations in the hippocampus correlate with the ability of an individual to discover and perform inference on the state of latent context variables in their environment. In the second project, we describe differences in the temporal stability of instructed task representations in the hippocampus and medial frontal cortex, and show that they rely on persistent activity of single-neurons that lasts for 1-2 orders of magnitude longer than is typically studied in working-memory tasks. In the third project, we study the neural mechanisms of task-switching costs, and show that the state of medial frontal cortical context-representing neurons immediately following instructions is predictive of switching cost. In the fourth project, we evaluate the extent to which frontal cortical task representations inherit the compositional structure of natural language, and attempt to predict the neural representation of novel tasks as patients perform zero-shot generalization in a large task space.
\r\n\r\nTogether, these projects constitute a first step in understanding the neural computations that underlie cognitive processing used by humans to solve complex, multi-task environments.
" }, { "name": "Davidson, Samuel Ryan", "degree": "PhD", "year": "2025", "title": "Localized Catalytic DNA Circuits for Integrated Information Processing in Molecular Machines", "advisor": "Qian, Lulu", "url": "https://resolver.caltech.edu/CaltechTHESIS:04092025-234838672", "creators": [ { "name": { "family": "Davidson", "given": "Samuel Ryan" }, "id": "Davidson-Samuel-Ryan", "orcid": "0000-0002-8081-3591", "display_name": "Davidson, Samuel Ryan" } ], "advisors": [ { "name": { "family": "Qian", "given": "Lulu" }, "id": "Qian-Lulu", "orcid": "0000-0003-4115-2409", "role": "advisor", "display_name": "Qian, Lulu" } ], "committee": [ { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "chair", "display_name": "Pierce, Niles A." }, { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "orcid": "0000-0002-5899-7523", "role": "member", "display_name": "Winfree, Erik" }, { "name": { "family": "Wang", "given": "Kaihang" }, "id": "Wang-Kaihang", "orcid": "0000-0001-7657-8755", "role": "member", "display_name": "Wang, Kaihang" }, { "name": { "family": "Qian", "given": "Lulu" }, "id": "Qian-Lulu", "orcid": "0000-0003-4115-2409", "role": "member", "display_name": "Qian, Lulu" } ], "option_major": [ "bioeng" ], "doi": "10.7907/831v-vq95", "abstract": "This thesis supports the long-term goal of engineering molecular devices with computational complexity akin to cells. Like cells, artificial molecular devices can benefit from integrating multiple computational modalities.
\r\n \r\nTo that end, this thesis advances molecular computing systems in three modalities: dynamic molecular assembly, well-mixed circuits, and spatially-organized cascades. Specifically, it introduces methods to enhance control over DNA structural assembly, well-mixed DNA circuits, and DNA circuits localized to a DNA origami surface.
\r\n \r\nAs DNA structural assembly grows increasingly complex, so too grows the potential for off-target structures. This issue can be addressed through developmental self-assembly, where components join a growing structure in a programmed sequence under controlled kinetics. The scope of developmental self-assembly is here expanded by a method enabling specific pathway selection among multiple encoded options.
\r\n \r\nWell-mixed DNA circuits require catalytic motifs for signal restoration and amplification. A catalytic motif is presented where two input strands cooperate to control catalysis. This motif could enhance AND gates and thresholding, and could enable adaptive memories and learning behaviors in DNA-based neural networks.
\r\n\r\nLocalized DNA circuits lack cascadable catalytic mechanisms for signal restoration and amplification. Two designs for a localized catalytic mechanism are presented. Each omits any intermediate diffusible species to support nanodevices compatible with uncontrolled environments, as in biomedical contexts. This constraint leads to design lessons; principally, we respond to leak in the first design through geometric constraints in the second design.
" }, { "name": "Fang, Meichen", "degree": "PhD", "year": "2025", "title": "A Biophysical Approach to Normalization and Trajectory Inference in Single-Cell RNA Sequencing Data Analysis", "advisor": "Pachter, Lior S.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06032025-002120461", "creators": [ { "name": { "family": "Fang", "given": "Meichen" }, "id": "Fang-Meichen", "orcid": "0000-0002-8217-0710", "display_name": "Fang, Meichen" } ], "advisors": [ { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "advisor", "display_name": "Pachter, Lior S." } ], "committee": [ { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "chair", "display_name": "Thomson, Matthew" }, { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "member", "display_name": "Pachter, Lior S." }, { "name": { "family": "Bois", "given": "Justin S." }, "id": "Bois-J-S", "orcid": "0000-0001-7137-8746", "role": "member", "display_name": "Bois, Justin S." }, { "name": { "family": "Chong", "given": "Shasha" }, "id": "Chong-Shasha", "orcid": "0000-0002-5372-311X", "role": "member", "display_name": "Chong, Shasha" } ], "option_major": [ "bioeng" ], "doi": "10.7907/asek-t904", "abstract": "Single-cell genomics assays, particularly single-cell RNA sequencing that enables genome-wide profiling of gene expression, have been driven forward by a combination of technological and computational advances. While producing extraordinary large amounts of data for biological discovery, methods for mining results currently rely heavily on heuristics and lack of modeling has resulted in limited mechanistic biological insight. This thesis presents two models for normalization and trajectory inference in single-cell RNA sequencing analysis to demonstrate how biophysical modeling, when combined with principled statistical inference, can yield interpretable insights grounded in rigorous theoretical frameworks.
\r\n\r\nWe begin by explaining the two cultures in single-cell RNA sequencing analysis. Next, we present the chemical master equation, which forms the theoretical foundation for biophysically informed stochastic models of gene expression, and explore an existing gap in developing uniform approximations over time under the large-volume limit. Returning to single-cell RNA sequencing data analysis, we introduce two mechanistic models for normalization and trajectory inference, which are essential components of single-cell RNA sequencing analysis.
" }, { "name": "Flores-Bautista, Emanuel", "degree": "PhD", "year": "2025", "title": "The Topology of Cellular Ontogeny", "advisor": "Thomson, Matthew", "url": "https://resolver.caltech.edu/CaltechTHESIS:06022025-234901151", "creators": [ { "name": { "family": "Flores-Bautista", "given": "Emanuel" }, "id": "Flores-Bautista-Emanuel", "orcid": "0000-0002-2810-1757", "display_name": "Flores-Bautista, Emanuel" } ], "advisors": [ { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "advisor", "display_name": "Thomson, Matthew" } ], "committee": [ { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "orcid": "0000-0002-7371-4675", "role": "chair", "display_name": "Prober, David A." }, { "name": { "family": "Marcolli", "given": "Matilde" }, "id": "Marcolli-M", "orcid": "0000-0002-2045-2907", "role": "member", "display_name": "Marcolli, Matilde" }, { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "member", "display_name": "Pachter, Lior S." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" } ], "option_major": [ "biochem" ], "doi": "10.7907/t8hc-yq15", "abstract": "A fundamental goal of modern biology is to build global, predictive models of gene regulation that encompass diverse physiological contexts. Single-cell transcriptomics has enabled the creation of developmental cell atlases--detailed catalogs of gene expression patterns and differentiation trajectories at an organismal scale. The widespread availability of cell atlases across metazoan model organisms presents an opportunity to construct global theories of cell-state control. In this thesis, we introduce a framework that uses persistent homology to decompose cell atlases into topological structures that provide signatures of gene regulation at the scale of an organism. Using this framework, we found that the topological structure of a broad set of developmental atlases contains only a discrete set of topological structures\u2014such as clusters, trees, and loops\u2014-revealing the recurrent use of global gene regulatory strategies. Our analysis revealed that the tree topology, while predominant, is not universal. Indeed, we identified non-trivial topologies containing loops in the development of human immune cells, seam-hypodermal cells in \\textit{C. elegans}, and the cnidocytes of multiple cnidarians. Analysis of cell-state manifolds with non-trivial topology demonstrated an important role of convergent structures in increasing cellular diversity along paths to a common cell fate, and of cyclic structures in self-renewal of progenitor-like states. Together, this work provides a global perspective on principles of cell-state regulation, and suggests that loops are important organizing structures for controlling cell differentiation." }, { "name": "Gillespie, Sarah Knox", "degree": "PhD", "year": "2025", "title": "Structure-Guided SCHEMA Recombination of VRC01-Class Antibodies for Reduced Polyreactivity", "advisor": "Mayo, Stephen L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06022025-062448836", "creators": [ { "name": { "family": "Gillespie", "given": "Sarah Knox" }, "id": "Gillespie-Sarah-Knox", "display_name": "Gillespie, Sarah Knox" } ], "advisors": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "advisor", "display_name": "Mayo, Stephen L." } ], "committee": [ { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "orcid": "0000-0002-3904-0195", "role": "member", "display_name": "Goentoro, Lea A." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "chair", "display_name": "Bjorkman, Pamela J." } ], "option_major": [ "bioch" ], "doi": "10.7907/93qk-7n49", "abstract": "The therapeutic administration of monoclonal antibodies (mAbs) has revolutionized treatment options for many diseases over the last decade. Recent findings from clinical trials have demonstrated that broadly neutralizing antibodies (bNAbs) could have a potential role in the future treatment and prevention of HIV-1. There is a group of broad and potent bNAbs that target the CD4-binding site (CD4bs) on the envelope glycoprotein gp120. These VRC01-class antibodies are notable for both their breadth and potency. The Bjorkman lab has designed a remarkably broad and potent bNAb, 45-46m2, that unfortunately cannot currently be used clinically due to its increased polyreactivity and short in vivo half-life. In this study we designed a SCHEMA-guided recombination library composed of sequence fragments of the VRC01-class bNAbs 45-46m2 and 3BNC117, aiming to create a bNAb that is both potent and broad but not polyreactive. We endeavored to maintain the strong binding of 45-46m2 while gaining the low polyreactivity of 3BNC117. Our analysis of this family shuffled library of chimeric antibodies revealed the sequence elements that led to strong binding to gp120 for the chimeras in our library. We also identify three key framework regions that can be modified to significantly reduce polyreactivity. Furthermore, we report three novel chimeras from the family shuffled library that bind as strongly to gp120 as 45-46m2 but are significantly reduced in polyreactivity." }, { "name": "Guareschi, Matteo Michele", "degree": "PhD", "year": "2025", "title": "Enriching Architectures for Biosensing and Motor-Filament Systems Through the Programmability of DNA", "advisor": "Rothemund, Paul W. K.; Pierce, Niles A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05052025-211734908", "creators": [ { "name": { "family": "Guareschi", "given": "Matteo Michele" }, "id": "Guareschi-Matteo-Michele", "orcid": "0000-0002-5197-3158", "display_name": "Guareschi, Matteo Michele" } ], "advisors": [ { "name": { "family": "Rothemund", "given": "Paul W. K." }, "id": "Rothemund-P-W-K", "orcid": "0000-0002-1653-3202", "role": "advisor", "display_name": "Rothemund, Paul W. K." }, { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "co-advisor", "display_name": "Pierce, Niles A." } ], "committee": [ { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "member", "display_name": "Pierce, Niles A." }, { "name": { "family": "Qian", "given": "Lulu" }, "id": "Qian-Lulu", "orcid": "0000-0003-4115-2409", "role": "chair", "display_name": "Qian, Lulu" }, { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "orcid": "0000-0002-5899-7523", "role": "member", "display_name": "Winfree, Erik" }, { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "member", "display_name": "Shapiro, Mikhail G." }, { "name": { "family": "Rothemund", "given": "Paul W. K." }, "id": "Rothemund-P-W-K", "orcid": "0000-0002-1653-3202", "role": "member", "display_name": "Rothemund, Paul W. K." } ], "option_major": [ "bioeng" ], "doi": "10.7907/fmhp-r892", "abstract": "Since its inception, the field of DNA nanotechnology has focused on studying the fundamental behaviors and capabilities of engineered nucleic acids. A deep understanding of this toolkit has enabled advancements in several fields, for research tools and in translational applications. Together with its programmability and nanometric resolution, the great promise of DNA nanotechnology lies in the incorporation of structure and function in a single molecule. In this work, we show how these advantages can be leveraged to expand the capabilities of two different systems: a sensor for biomarkers and a motor-filament architecture. During our exploration, we also discover and work to overcome some of the less obvious limitations of the technology, shining light on more foundational questions.
\r\n\r\nWe demonstrate an electrochemical biosensor based on a DNA origami that can detect and quantify nucleic acids and proteins in a package easily adaptable to different analytes by simply replacing the binder molecules. Upon target binding, the structure undergoes a large conformational change, bringing a multitude of redox reporters to the electrode surface where an electric current can be measured. The high number of reporter molecules on a single detector results in improved signal gain per binding event, allowing for the detection of low analyte concentrations, while the conformational change yields an unprecedented gain between the off and on state. We demonstrate how the system can be readily adapted to different analyte molecules and reused over several cycles to analyze multiple samples. We then run simulations of the detector molecule to understand structural deformations intrinsic to this design, in order to optimize the number and placement of the redox reporters. We discover and investigate a phenomenon that causes significant curling of the DNA origami, possibly limiting the contribution of many of the reporter molecules. We explore experimental directions to mitigate the issue by changing the configuration of the redox molecules and by designing stiffer sensors.
\r\n\r\nWe then set out to integrate DNA origami-based nanostructures with an engineered dynein protein that can bind to and kick double-stranded DNA instead of tubulin. Motor-filament architectures have been studied as the main mechanism for cellular transport and as a system that can exhibit mesoscopic active matter behaviors in biology, but the relative difficulty of engineering microtubules has hindered the exploration of their properties. The high-resolution programmability of DNA nanostructures makes them prime candidates to overcome this obstacle and this study has been enabled by the recent development of new protein motors where the tubulin binding domain is replaced by a DNA binding domain. We first look at DNA nanotubes, structures that resemble microtubules, but that retain a level of programmability that is typical of DNA nanotechnology. By exploiting the DNA strand displacement technique, we incorporate machinery that enables new behaviors, with a focus on different ways to turn gliding on and off by stopping the DNA nanotubes.
\r\n\r\nWe then turn our focus to more complex gliders designed with DNA origami. We explore the space of DNA origami polymers in order to assemble superstructures that can be detected under light microscopy, encountering again issues of deformations due to the addition of overhangs. We then assess the gliding capabilities of DNA origami, designing ways to incorporate motor binding sequences on them, but we find that DNA origami sticks nonspecifically to the engineered dynein motors. After testing several different hypotheses, we gather evidence that this interaction might be caused by the large sequence variability of the scaffold strand in DNA origami, coupled with the recognition of spurious binding sequences by the motor proteins.
" }, { "name": "Hazu, Masami", "degree": "PhD", "year": "2025", "title": "Mechanistic Studies of Membrane Protein Biogenesis at the ER and Mitochondria", "advisor": "Voorhees, Rebecca M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02082025-232945882", "creators": [ { "name": { "family": "Hazu", "given": "Masami" }, "id": "Hazu-Masami", "orcid": "0000-0002-6527-9597", "display_name": "Hazu, Masami" } ], "advisors": [ { "name": { "family": "Voorhees", "given": "Rebecca M." }, "id": "Voorhees-R-M", "orcid": "0000-0003-1640-2293", "role": "advisor", "display_name": "Voorhees, Rebecca M." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Shan", "given": "Shu-ou" }, "id": "Shan-Shu-ou", "orcid": "0000-0002-6526-1733", "role": "member", "display_name": "Shan, Shu-ou" }, { "name": { "family": "Voorhees", "given": "Rebecca M." }, "id": "Voorhees-R-M", "orcid": "0000-0003-1640-2293", "role": "member", "display_name": "Voorhees, Rebecca M." } ], "option_major": [ "biology" ], "doi": "10.7907/snc5-jf69", "abstract": "Eukaryotic cells are organized into membrane-enclosed compartments with elaborate networks of integral membrane proteins. From synthesis, localization, and insertion into designated cellular membranes, to proper folding and assembly of the membrane proteins, the successful biogenesis of membrane proteins is crucial for defining the organellar compartments and for overall proteostasis. Recent advances in the field of membrane protein biogenesis in both the endoplasmic reticulum (ER) and the mitochondria have identified novel machineries involved in the membrane insertion step. These are the ER membrane protein complex (EMC) at the ER and the mitochondrial carrier homolog 2 (MTCH2) at the outer mitochondrial membrane (OMM). In this thesis, we employ a combination of biochemical, cell biological, structural, and genetic techniques to explore in mechanistic detail the insertase function of the EMC and MTCH2 at the molecular level. In the first part of the thesis, our work on the EMC maps out the pathway of a tail-anchored (TA) protein through the insertase and revealed a selectivity filter that provides the biochemical basis for how the EMC protects compartment integrity. The selectivity filter of the EMC limits TA protein mislocalization and prevents topological errors of multi-pass membrane proteins. In the second part, ongoing work on MTCH2 reveals the absence of a prominent selectivity mechanism and provides insight into a regulatory mechanism of MTCH2, which seems to be conserved across metazoan MTCH2 homologs." }, { "name": "Honson, Drew Daniel", "degree": "PhD", "year": "2025", "title": "Development and Application of Proteomic and Genomic Methods in RNA Biology", "advisor": "Guttman, Mitchell", "url": "https://resolver.caltech.edu/CaltechTHESIS:12092024-181948039", "creators": [ { "name": { "family": "Honson", "given": "Drew Daniel" }, "id": "Honson-Drew-Daniel", "orcid": "0000-0002-4654-8974", "display_name": "Honson, Drew Daniel" } ], "advisors": [ { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "advisor", "display_name": "Guttman, Mitchell" } ], "committee": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "chair", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Fejes Toth", "given": "Katalin" }, "id": "Fejes-Toth-K", "orcid": "0000-0001-6558-2636", "role": "member", "display_name": "Fejes Toth, Katalin" }, { "name": { "family": "Zernicka-Goetz", "given": "Magdalena" }, "id": "Zernicka-Goetz-M", "orcid": "0000-0002-7004-2471", "role": "member", "display_name": "Zernicka-Goetz, Magdalena" }, { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "member", "display_name": "Guttman, Mitchell" } ], "option_major": [ "biology" ], "doi": "10.7907/08dq-1q63", "abstract": "This thesis contains three interrelated projects. Chapter 1 describes the development of a novel RNA-proteomics method: RNA-antisense purification followed by mass spectrometry (RAP-MS 2.0). It contains results of a RAP-MS 2.0 study profiling the protein partners of eight RNAs (7SL, 7SK, RMRP, U1, U2, U6, U7, and Xist) as well as a detailed, step-by-step protocol for the new method. Chapter 2 describes a quality control method for Split and Pool Identification of RBP targets (SPIDR). It identifies an underappreciated failure point in SPIDR experiments (the equal loading of antibody-IDs onto beads), and describes a method for monitoring and resolving this issue. Chapter 3 describes the application of SPIDR to ribosome-associated proteins in human cells. The study both validates existing structures and identifies novel interactions between nucleolar proteins and immature ribosomal RNA, and between protein trafficking factors and the large ribosomal subunit." }, { "name": "Isella, Emma Xueqian", "degree": "Senior Thesis", "year": "2025", "title": "Investigating the Biological Mechanism of N\u2082O Emissions from Arid Southern Californian Drylands", "advisor": "Newman, Dianne K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06122025-192128192", "creators": [ { "name": { "family": "Isella", "given": "Emma Xueqian" }, "id": "Isella-Emma-Xueqian", "orcid": "0009-0000-2709-8333", "display_name": "Isella, Emma Xueqian" } ], "advisors": [ { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "advisor", "display_name": "Newman, Dianne K." } ], "committee": [ { "name": { "family": "None", "given": "None" }, "display_name": "None, None" } ], "option_major": [ "biology", "geobiol" ], "doi": "10.7907/9a4y-mm41", "abstract": "Nitrous oxide (N\u2082O) is a powerful greenhouse gas, each molecule capable of warming the atmosphere 273 times more effectively than CO\u2082. Arid soils that have been rewetted by rainfall events can produce some of the highest instantaneous N\u2082O emission rates recorded globally. Recent work has shown that the majority of these emissions are biologically produced. While these emissions have classically been attributed to bacterial and fungal denitrification catalyzed by catabolic nitric oxide (NO) reductases (e.g. NOR), measured N\u2082O isotopic fingerprinting (site preference, SP) more closely matches flavohemoglobin enzymes involved in nitric oxide detoxification (e.g. Fhp). Analysis of the microbial community of the site demonstrates that fhp is significantly more phylogenetically abundant than nor. We hypothesize that NO detoxification pathways are responsible for the initial pulse of N\u2082O production after rainfall, with denitrification only becoming dominant after a few hours. N\u2082O production is only triggered once some critical saturation with the water is reached, suggesting that the soil community has to receive enough water to become anaerobic. Using coupled measurements of oxygen and N\u2082O concentration in soils, we show that N\u2082O production begins only once the added water depletes the soil of oxygen. Initial measurements of N\u2082O production from Pseudomonas synxantha, a bacterium isolated from soil, demonstrate clear differences in the timing and quantity of gas production following rewetting via the detoxification and denitrification pathways. We thus suggest that previously overlooked detoxification pathways may play key roles in observed biogeochemical events, as appears to be the case with soil N\u2082O emissions." }, { "name": "Kadlec, Kelly Marie", "degree": "PhD", "year": "2025", "title": "Distinct Patterns of Overlapping Neural Representation Of Sensorimotor Variables in Primary and Associative Motor Areas: Insights from Chronic Intracortical Recordings in the Human Brain", "advisor": "Andersen, Richard A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:07292024-181336718", "creators": [ { "name": { "family": "Kadlec", "given": "Kelly Marie" }, "id": "Kadlec-Kelly-Marie", "orcid": "0000-0002-8765-7253", "display_name": "Kadlec, Kelly Marie" } ], "advisors": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "00000-00002-7947-0472", "role": "advisor", "display_name": "Andersen, Richard A." } ], "committee": [ { "name": { "family": "Camerer", "given": "Colin F." }, "id": "Camerer-C-F", "orcid": "0000-0003-4049-1871", "role": "chair", "display_name": "Camerer, Colin F." }, { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "orcid": "0000-0002-1290-5232", "role": "member", "display_name": "Shimojo, Shinsuke" }, { "name": { "family": "Rutishauser", "given": "Ueli" }, "id": "Rutishauser-U", "orcid": "0000-0002-9207-7069", "role": "member", "display_name": "Rutishauser, Ueli" }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "member", "display_name": "Andersen, Richard A." } ], "option_major": [ "neurobio" ], "doi": "10.7907/e3x8-t812", "abstract": "Although many of the movements we make are produced without much conscious thought, motor control requires the coordination of multiple brain areas and several complex processes to occur as seamlessly as it does, two of which are primary motor cortex (MC) and posterior parietal cortex (PPC). Traditional views of the organization of these areas have mapped separate parts of the body, or effectors, onto separate areas of cortex. However, recent findings that show extensively overlapping representations of different effectors within small populations of neurons in both motor and posterior parietal cortices have reignited a debate over the organization of each area. The studies in this thesis aim to reconcile these conflicting records through a unique opportunity to directly compare between single neuron recordings in both areas in human participants chronically implanted with intracortical electrode arrays. The functional organization of these areas was investigated during movement of different parts of the body in different contexts. In the first study, I found that the entire body is represented within small patches of both MC and PPC, but with a clear emphasis on a single part of the body in MC. In PPC, although single neurons showed specialization for particular effectors, there were an equal number of neurons specialized for every effector resulting in an equal strength in representation of the population across effectors. In the second study, I investigated how spatial information was represented across different effectors. In particular, it has previously been reported that some areas within PPC represent location of an object in space relative to the position of one's eyes, or in an eye-centered coordinate frame, while other areas represent location in space as relative to the position of one's body, for example a hand-centered coordinate frame. We find that the population in PPC flexibly changes the coordinate frame it encodes the location of a visual target in from hand centered during a reach paradigm to eye-centered during a delayed saccade paradigm. In contrast to the multiple coordinate frames coded by the population in PPC, in MC the population predominantly encoded spatial location in hand-centered coordinates during reaches. The flexibility seen in the population results in PPC motivate the study of Chapter 4, where I explore these changing coordinate frames in more detail at the single neuron level. I found that the distinct coordinate frames are encoded by almost entirely separate sets of neurons, with very few neurons engaged in both task. Overall, these results show clearly distinct organization of motor variables within MC and PPC, and offer important insights into the possible functions of each region both within and beyond motor control. In addition, they highlight a need to continue exploring how neurons within a defined region respond beyond their traditionally associated functional roles." }, { "name": "Kolhe, Rohan Rajendra", "degree": "Senior Thesis", "year": "2025", "title": "Batik: a Vision Language Model for End-to-End Social Behavior Discovery, Interpretation and Annotation", "advisor": "Anderson, David J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06062025-053817901", "creators": [ { "name": { "family": "Kolhe", "given": "Rohan Rajendra" }, "id": "Kolhe-Rohan-Rajendra", "display_name": "Kolhe, Rohan Rajendra" } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "advisor", "display_name": "Anderson, David J." } ], "committee": [ { "name": { "family": "None", "given": "None" }, "display_name": "None, None" } ], "option_major": [ "cns" ], "doi": "10.7907/cmvx-1n97", "abstract": "Quantitative analysis of animal behavior is a burgeoning field. By converting behavior into measurable features, the field replaces anecdotal observations with precise, data-driven insights into how animals interact with their environment and with one another. Through certain analyses, reproducible structure and diversity within behaviors are revealed, illuminating complex behavioral patterns. Most current state-of-the-art methods focus on annotation and segmentation of behavior using pose-estimation; these methods attach nodes to body parts of mice which then compute a features space. This feature space is then used for discovery of behavior classes or training supervised behavior classifiers. However, this excludes the time-consuming task of interpreting resulting behavioral syllables and has multiple failure modes, such as an inability to attend to frames where there are other objects of interest or frames where the nodes are all on top of one another. The majority of these methods use a convolutional neural network structure. In recent years, a new set of feed-forward neural networks called transformers have been proven to surpass CNNs on most vision-related tasks. Batik addresses the long time-commitments of interpreting these syllables by using multimodal transformers to extract unsupervised features directly from raw video, and perform end-to-end analysis, bypassing pose estimation. Alongside state-of-the-art supervised annotation, Batik leverages fine-tuned large language models to automate discovery and provide expert human-level interpretation of behavior syllables, offering researchers a transformative UI-based tool for behavioral analysis through vision-language models. Through these methods, we show a 96% accuracy for syllables like attack and mount, a large jump from previous methods (85%). We also accurately identify differences in behavior in different metabolic states, as well as an interpretation with sub behaviors for the broad investigative behavior. We further apply our method to other species datasets, correctly classifying distinct fly aggressive behaviors with no additional fine-tuning of the underlying model, showing our model\u2019s generalizability." }, { "name": "Kondapaneni, Neehar", "degree": "PhD", "year": "2025", "title": "Aligning and Comparing Vision Representations to Improve Understanding and Performance", "advisor": "Perona, Pietro", "url": "https://resolver.caltech.edu/CaltechTHESIS:06032025-000834048", "creators": [ { "name": { "family": "Kondapaneni", "given": "Neehar" }, "id": "Kondapaneni-Neehar", "orcid": "0000-0002-2782-3977", "display_name": "Kondapaneni, Neehar" } ], "advisors": [ { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "advisor", "display_name": "Perona, Pietro" } ], "committee": [ { "name": { "family": "Yue", "given": "Yisong" }, "id": "Yue-Yisong", "orcid": "0000-0001-9127-1989", "role": "chair", "display_name": "Yue, Yisong" }, { "name": { "family": "Gkioxari", "given": "Georgia" }, "id": "Gkioxari-Georgia", "role": "member", "display_name": "Gkioxari, Georgia" }, { "name": { "family": "Mac Aodha", "given": "Oisin" }, "id": "Mac-Aodha-Oidin", "orcid": "0000-0002-5787-5073", "role": "member", "display_name": "Mac Aodha, Oisin" }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "display_name": "Perona, Pietro" } ], "option_major": [ "cns" ], "doi": "10.7907/0crh-zb71", "abstract": "Recent advances in large artificial intelligence (AI) models have enabled these models to perform a wide range of real-world tasks with skill levels comparable to or surpassing those of humans. In this thesis, we develop methods to compare, analyze, and align data representations from these powerful models. In Part 1, we develop methods for estimating human knowledge during a learning task and for comparing various data representations. These methods are steps towards a system designed to help us learn from AI.
\r\n\r\nIn Part 2, we show how aligning models can be useful in two separate domains. First, we discover and fix a misalignment in the inputs to a powerful foundation model and show how it improves performance. Second, we show that biologically inspired object manipulation tasks can be used as a training signal for learning human-aligned representations of number. Our results demonstrate the potential for alignment and comparison methods to improve the overall performance of AI models, improve our understanding of biological intelligence, and help us discover new patterns in the natural world.
" }, { "name": "Lanfranchi, Francesco (Frank)", "degree": "PhD", "year": "2025", "title": "A Multispecies Perspective on the Evolution of Form Vision", "advisor": "Tsao, Doris Y.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01282025-190331921", "creators": [ { "name": { "family": "Lanfranchi", "given": "Francesco (Frank)" }, "id": "Lanfranchi-Francesco", "orcid": "0000-0001-8176-320X", "display_name": "Lanfranchi, Francesco (Frank)" } ], "advisors": [ { "name": { "family": "Tsao", "given": "Doris Y." }, "id": "Tsao-D-Y", "orcid": "0000-0003-1083-1919", "role": "advisor", "display_name": "Tsao, Doris Y." } ], "committee": [ { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "chair", "display_name": "Perona, Pietro" }, { "name": { "family": "Tsao", "given": "Doris Y." }, "id": "Tsao-D-Y", "orcid": "0000-0003-1083-1919", "role": "member", "display_name": "Tsao, Doris Y." }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "member", "display_name": "Andersen, Richard A." }, { "name": { "family": "Lois", "given": "Carlos" }, "id": "Lois-Carlos", "orcid": "0000-0002-7305-2317", "role": "member", "display_name": "Lois, Carlos" }, { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "member", "display_name": "Shapiro, Mikhail G." }, { "name": { "family": "Rutishauser", "given": "Ueli" }, "id": "Rutishauser-U", "orcid": "0000-0002-9207-7069", "role": "member", "display_name": "Rutishauser, Ueli" } ], "option_major": [ "cns" ], "doi": "10.7907/154t-x703", "abstract": "In the mammalian visual system, photons captured by the retina are transformed into meaningful internal percepts of surroundings through a hierarchy of interconnected visual areas. Understanding the representation of visual information at each node of the hierarchy has been a central quest of visual systems neuroscience over the past 50 years. The primate visual system, with its over two dozen distinct areas broadly organized into a dorsal stream for visuo-motor transformations and a ventral stream for object recognition, has served as the gold standard for studying the organization of the visual system. Recent advances in artificial neural networks modeled on the primate visual system for object recognition have prompted the question, is hierarchical representation necessary, and if so, can we observe it across all highly visual mammalian species? Hierarchical organization appears to be a key architectural principle of both artificial and biological networks, enabling stepwise construction of a structured and compact representation from raw sensory input. Here we present a series of efforts to determine the cortical organization and connectivity of the tree shrew visual system and directly compare to that of the primate. This cross-species study sheds light on the evolution and mechanisms of vision in a close relative of primates. Using high-density Neuropixels recordings, we demonstrate that the tree shrew ventral visual pathway exhibits primate-like hierarchical processing, with progressively larger receptive fields, increasing response latencies, and enhanced selectivity for complex stimuli along the visual pathway. Area V2 in the tree shrew performs key functions similar to those of the primate inferotemporal (IT) cortex. Specifically, V2 contains strongly face-selective cells, supports a complete representation of high-level object space, and achieves the most accurate object identity decoding and reconstruction among all tree shrew visual areas. Yet we also found significant differences from the canonical template for hierarchical organization observed in the primate, including maintenance of relatively small, focal receptive fields throughout the hierarchy, and better decoding of latent variables in late deep neural network (DNN) layers by area V2 compared to other areas.
\r\n\r\nThe hierarchical organization of the visual system describes the arrangement of areas but does not reveal how information flows between them. Understanding the type of processing carried out at each node raised the next question of whether information that is transmitted across nodes is differentiated between feedforward and feedback connections. To explore this, we combined electrical microstimulation and extracellular recordings to identify the directionality of projections which is applicable in various species. We used this technique to first study the connections between the first two nodes of the tree shrew cortical hierarchy, V1 and V2. We found that V2 feedback neurons carry a full visual representation on par to other V2 cells. These feedback neurons were distinct with regards to their spatial features, including distinct locations and sizes of their receptive fields. We also found that both feedforward and feedback V2 neurons were modulated by perceptual conflict arising when distinct textures were presented to each eye, suggesting they could refine V1 processing to perceptual inconsistencies.
\r\n\r\nThese studies provide insights into how the tree shrew visual system generates object representations through a hierarchy of interconnected nodes, employing strategies adapted to its cortical constraints. In addition, by combining electrical microstimulation with electrophysiology we set the foundation for cross-species studies to determine the role of feedforward and feedback processing along the visual hierarchy. Together, this work reveals conserved principles of visual processing across species while showcasing unique adaptations in the tree shrew, offering insights into the evolutionary origins and functional organization of the primate visual system.
" }, { "name": "Larsson, Elin Maria", "degree": "PhD", "year": "2025", "title": "Domestication of Environmental Bacteria for Biosensing Applications", "advisor": "Murray, Richard M.; Newman, Dianne K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06012025-210705210", "creators": [ { "name": { "family": "Larsson", "given": "Elin Maria" }, "id": "Larsson-Elin-Maria", "orcid": "0000-0003-1341-5937", "display_name": "Larsson, Elin Maria" } ], "advisors": [ { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "co-advisor", "display_name": "Murray, Richard M." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "co-advisor", "display_name": "Newman, Dianne K." } ], "committee": [ { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "orcid": "0000-0002-7033-0844", "role": "chair", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Cao", "given": "Mengyi" }, "id": "Cao-Mengyi", "orcid": "0000-0002-3117-3401", "role": "member", "display_name": "Cao, Mengyi" }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." } ], "option_major": [ "bioeng" ], "doi": "10.7907/m077-7633", "abstract": "The field of synthetic biology has made impressive progress in the past 25 years, but is still lacking when it comes to our capability to predictably engineer organisms outside of a small group of lab model organisms. In this thesis, I present the efforts to domesticate two soil bacteria important in agriculture for biosensing. The first, Pseudomonas synxantha, a wheat-colonizing bacterium that helps fight off fungal disease, was engineered into a bioreporter for phosphorus limitation. We also made cell-free extract from this organism, to enable rapid characterization of genetic elements. For the second, Xenorhabdus griffiniae, we asked the question of whether this bacterium can sense the presence of its entomopathogenic nematode host Steinernema hermaphroditum. We learned that X. griffiniae is able to sense its host and we were able to build an early variant of a nematode reporter by first characterizing genetic elements in X. griffiniae." }, { "name": "Li, Can", "degree": "PhD", "year": "2025", "title": "Gene Regulatory Analysis of the Developing Enteric Nervous System of Zebrafish (Danio rerio)", "advisor": "Bronner, Marianne E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02172025-204256436", "creators": [ { "name": { "family": "Li", "given": "Can" }, "id": "Li-Can", "orcid": "0000-0002-5352-6212", "display_name": "Li, Can" } ], "advisors": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "advisor", "display_name": "Bronner, Marianne E." } ], "committee": [ { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "orcid": "0000-0001-6597-2036", "role": "chair", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "member", "display_name": "Pachter, Lior S." }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "orcid": "0000-0002-7371-4675", "role": "member", "display_name": "Prober, David A." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "member", "display_name": "Bronner, Marianne E." } ], "option_major": [ "biology" ], "doi": "10.7907/mc6z-3k03", "abstract": "Neural crest cells give rise to the neurons of the enteric nervous system (ENS) that innervate the gastrointestinal tract to regulate gut motility. The immense size and distinct subregions of the gut present a challenge to understanding the spatial organization and sequential differentiation of different neuronal subtypes. To tackle this, we profile enteric neurons and progenitors at single cell resolution during zebrafish embryonic and larval development to provide a near complete picture of transcriptional changes that accompany emergence of ENS neurons throughout the gastrointestinal tract. Multiplex spatial RNA transcript analysis was then used to reveal the temporal order and distinct localization patterns of neuronal subtypes along the length of the gut. Next, we show that functional perturbation of select transcription factors Ebf1a, Gata3 and Satb2 alters the cell fate choice, respectively, of inhibitory, excitatory and serotonergic neuronal subtypes in the developing ENS. To decipher the molecular mechanism underlying the development of ENS, we further performed single cell ATAC-seq to profile the epigenetic landscape of the developing ENS. Together with CUT&RUN results, we found the master regulator Phox2bb harbors extensive binding sites throughout the genome and plays versatile roles in neuronal differentiation, including regulating progenitor gene Sox10, activating transcription factors Phox2a and Insm1b for early neural development and regulating genes Etv1 and Hmx3a for neuronal differentiation. Integrated with single cell RNA-seq analysis, we further reconstruct a putative gene regulatory circuit involving in the specification and maturation of ENS neurons." }, { "name": "Li, Francesca-Zhoufan", "degree": "PhD", "year": "2025", "title": "Evaluation of the Generalizability of Machine Learning-Assisted Protein Engineering Methods", "advisor": "Arnold, Frances Hamilton; Yue, Yisong", "url": "https://resolver.caltech.edu/CaltechTHESIS:04292025-041809434", "creators": [ { "name": { "family": "Li", "given": "Francesca-Zhoufan" }, "id": "Li-Francesca-Zhoufan", "orcid": "0000-0002-5710-9512", "display_name": "Li, Francesca-Zhoufan" } ], "advisors": [ { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "advisor", "display_name": "Arnold, Frances Hamilton" }, { "name": { "family": "Yue", "given": "Yisong" }, "id": "Yue-Yisong", "orcid": "0000-0001-9127-1989", "role": "advisor", "display_name": "Yue, Yisong" } ], "committee": [ { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "chair", "display_name": "Murray, Richard M." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "co-chair", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Yang", "given": "Kevin K." }, "id": "Yang-Kevin-K", "orcid": "0000-0001-9045-6826", "role": "member", "display_name": "Yang, Kevin K." }, { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "member", "display_name": "Arnold, Frances Hamilton" }, { "name": { "family": "Yue", "given": "Yisong" }, "id": "Yue-Yisong", "orcid": "0000-0001-9127-1989", "role": "member", "display_name": "Yue, Yisong" } ], "option_major": [ "bioeng" ], "doi": "10.7907/yzb2-cb66", "abstract": "Engineered proteins can carry out a vast array of functions and have become indispensable across numerous industrial applications. To accelerate wet-lab protein engineering efforts, machine learning-based methods have advanced rapidly. However, a gap remains between state-of-the-art machine learning methods and their practical adoption. A key factor contributing to this disconnect is the lack of application-relevant benchmarking and generalizable insights across protein engineering tasks. This thesis evaluates machine learning-assisted protein engineering approaches to identify generalizable strategies. The central problem considered is learning the mapping from protein sequence to function\u2014known as the fitness landscape\u2014to enable the prediction of unseen variant fitness. Chapter 1 introduces the background and context for machine learning-assisted protein engineering and highlights the practical constraint of limited experimental budgets. Chapter 2 investigates transfer learning, which leverages models pretrained on large protein sequence databases to generate informative representations for modeling task specific sequence-function relationships. Evaluation across ten diverse tasks shows that while transfer learning is effective in structure prediction, it underperforms in variant fitness prediction\u2014a key objective in protein engineering. Chapter 3 evaluates alternative strategies with a focus on combinatorial fitness landscapes, a common setting in protein engineering. Across 16 diverse landscapes, focused training improves the performance of various machine learning approaches by strategically selecting training variants using zero-shot predictors, which estimate variant fitness from auxiliary information without relying on experimental data. Building on these insights, Chapter 4 addresses the specific challenge of engineering enzymes\u2014proteins that convert substrates into products\u2014for novel chemistries. While six general zero-shot predictors without substrate information can predict enzyme activity on non-native substrates, they fail on more out-of-distribution, new-to-nature chemistries. Incorporating substrate information into zero-shot predictors leads to more generalizable performance across all tested chemistries, spanning 22 substrates. Overall, this thesis identifies generalizable strategies for machine learning-assisted protein engineering by systematically evaluating and improving how sequence-to-function relationships are modeled across diverse tasks." }, { "name": "Louren\u00e7o, Alexandre Luiz", "degree": "PhD", "year": "2025", "title": "Building Closed-Loop Frameworks for AI-Guided Protein Design", "advisor": "Thomson, Matthew", "url": "https://resolver.caltech.edu/CaltechTHESIS:05312025-073148050", "creators": [ { "name": { "family": "Louren\u00e7o", "given": "Alexandre Luiz" }, "id": "Louren\u00e7o-Alexandre-Luiz", "orcid": "0009-0005-0758-2968", "display_name": "Louren\u00e7o, Alexandre Luiz" } ], "advisors": [ { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "advisor", "display_name": "Thomson, Matthew" } ], "committee": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "chair", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" } ], "option_major": [ "biochem" ], "doi": "10.7907/8can-jz97", "abstract": "The design of proteins with tailored properties remains a central challenge in protein engineering, with profound implications for therapeutics, sustainable manufacturing, and environmental remediation. Recent advances in artificial intelligence have dramatically improved our ability to design novel proteins, yet the precision required for many applications remains elusive. This thesis details the development and implementation of closed-loop frameworks that integrate AI-guided protein design with quantitative experimental data to iteratively improve design outcomes.
\r\n\r\nFirst, I present Protein CREATE (Computational Redesign via an Experiment-Augmented Training Engine), a high-throughput platform that combines phage display with molecular counting techniques to generate quantitative binding data at scale. This platform enables rapid evaluation of thousands (and is in the process of being scaled to millions) of designed protein variants against multiple targets simultaneously.
\r\n\r\nIn subsequent chapters, I explore two separate strands of protein design as they reach for each other to close the loop. One thread focuses on collecting data on binders I engineered to the interleukin 7 receptor alpha (IL7RA) and Insulin receptor while the other investigates the value data, even when limited, adds to improve the design process of enzymes to solve a pressing environmental remediation problem: cleaning up per and polyfluoroalkyl substances (PFAS).
\r\n\r\nWhile all of the targets discussed so far have benefited from developments in artificial intelligence, I explore one target where the benefits are limited, the human sweet taste receptor. Here, I leverage alternative computational methods coupled to experimental testing to chart a course for design.
\r\n\r\nFinally, I discuss the technologies we are integrating within the Protein CREATE framework to enable rapid in vitro and in vivo testing.
\r\n\r\nThroughout my PhD, I have been bringing the two threads of computational design and experimental characterization closer together for not only theoretically interesting, but also practically relevant, engineering cases. The methodologies developed here represent a significant advancement in our ability to design proteins with precisely tailored properties for diverse applications.
" }, { "name": "Loving Ngo, Rebekah Kiana", "degree": "PhD", "year": "2025", "title": "Methods for Long Read RNA-Seq Transcriptomics", "advisor": "Pachter, Lior S.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06012025-204136107", "creators": [ { "name": { "family": "Loving Ngo", "given": "Rebekah Kiana" }, "id": "Loving-Ngo-Rebekah-Kiana", "orcid": "0000-0001-8725-0376", "display_name": "Loving Ngo, Rebekah Kiana" } ], "advisors": [ { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "advisor", "display_name": "Pachter, Lior S." } ], "committee": [ { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "chair", "display_name": "Thomson, Matthew" }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "member", "display_name": "Perona, Pietro" }, { "name": { "family": "Mortazavi", "given": "Ali" }, "id": "Mortazavi-Ali", "orcid": "0000-0002-4259-6362", "role": "member", "display_name": "Mortazavi, Ali" }, { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "member", "display_name": "Pachter, Lior S." } ], "option_major": [ "biology" ], "doi": "10.7907/3nz8-3c83", "abstract": "While short read RNA-seq dominated the field for decades, long read RNA-seq is particularly useful for isoform-level expression analysis, genome annotation, detecting novelly splicing transcripts, identifying exact breakpoints in gene fusions, and discovering chimeric RNAs. Long read RNA-seq has rapidly scaled to the point of producing terabytes of data from a single set of experiments. Technological advances in RNA and DNA sequencing library preparation, chemistry used in the Oxford nanopores, and basecalling algorithms have reduced long read sequencing error rates to sub-1% error. Further, the cost of long read sequencing has dropped to about one hundred US dollars per human genome. These two factors have lead to the mass production of high-throughput, long read, and single-cell RNA-seq data. While recent tools for long read RNA-seq have been developed, they have not kept pace in scalability and accuracy with long read RNA-seq in the fashion that short read RNA-seq tools have met computational scalability and accuracy challenges. To address this, in this thesis, we leverage long k-mers and pseudoalignment for mapping and quantifying long reads in the novel algorithm implemented within lr-kallisto, which yields both efficiency and higher accuracy for long read mapping and quantification than previous tools. We demonstrate that long read RNA-seq has reached sufficient depth and accuracy to yield accurate quantification of isoform-level expression for differential expression analysis. Furthermore, we explore the feasibilty of also utilizing long k-mers and pseudoalignment in both transcript discovery in dn-kallisto and gene fusion and immune receptor sequence discovery with fugi with measured success. Thus, our tools will enable a more complete, accurate, and scalable analysis of single-cell and bulk RNA-seq than has hitherto been possible in both quantifications and differential expression analysis as well as investigation of gene fusions, chimeric RNAs, and immune receptor sequences without bias." }, { "name": "Lu, Andrew C.", "degree": "PhD", "year": "2025", "title": "Engineered Protein Circuits for Cancer Therapy", "advisor": "Elowitz, Michael B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06092025-211524859", "creators": [ { "name": { "family": "Lu", "given": "Andrew C." }, "id": "Lu-Andrew-C", "orcid": "0000-0002-7594-6445", "display_name": "Lu, Andrew C." } ], "advisors": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "advisor", "display_name": "Elowitz, Michael B." } ], "committee": [ { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "chair", "display_name": "Shapiro, Mikhail G." }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "member", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Voorhees", "given": "Rebecca M." }, "id": "Voorhees-R-M", "orcid": "0000-0003-1640-2293", "role": "member", "display_name": "Voorhees, Rebecca M." }, { "name": { "family": "Dawson", "given": "David" }, "id": "Dawson, David", "orcid": "0000-0002-0215-5861", "role": "member", "display_name": "Dawson, David" }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." } ], "option_major": [ "sysbiol" ], "doi": "10.7907/kxvr-r291", "abstract": "Engineered protein circuits seek to treat cancer by directly rewiring oncogenic signaling to cell death. However, it has remained unclear what circuit designs could operate effectively, and what advantages protein circuits could provide compared to existing small molecule inhibitors. Here, we introduce Ras-targeting circuits that accurately discriminate and kill Ras-mutant cells, circumventing drug resistance mechanisms and suppressing cancer in vivo. These circuits combine three modules: a protease-based sensor that responds to a broad spectrum of clinically relevant Ras mutations, an optional protease amplifier, and protease-triggered cell death effectors of the apoptosis and pyroptosis cell death pathways. When delivered as mRNA in lipid nanoparticles (LNPs), the circuits were effective against Ras-mutant human cancer cell lines with minimal off-target killing of wild-type Ras cells. In immunocompetent mice bearing aggressive, multifocal Ras-driven liver tumors, systemically delivered mRNA-LNP circuits strongly reduced tumor burden. Further, therapeutic circuits provided more complete killing of Ras-mutant cancer cells than the Ras inhibitors Sotorasib and RMC-7977 and did not require oncogene addiction. They also exhibited increased potency against Sotorasib-resistant cells. These results establish a programmable mechanism for treating cancer and other human diseases." }, { "name": "McGill, Mason Benjamin", "degree": "PhD", "year": "2025", "title": "Visual Systems and the Forces That Shape Them", "advisor": "Perona, Pietro", "url": "https://resolver.caltech.edu/CaltechTHESIS:06092025-113042101", "creators": [ { "name": { "family": "McGill", "given": "Mason Benjamin" }, "id": "McGill-Mason-Benjamin", "orcid": "0000-0002-2782-3977", "display_name": "McGill, Mason Benjamin" } ], "advisors": [ { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "advisor", "display_name": "Perona, Pietro" } ], "committee": [ { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "chair", "display_name": "Thomson, Matthew" }, { "name": { "family": "Parker", "given": "Joseph" }, "id": "Parker-J", "orcid": "0000-0001-9598-2454", "role": "member", "display_name": "Parker, Joseph" }, { "name": { "family": "Rutishauser", "given": "Ueli" }, "id": "Rutishauser-U", "orcid": "0000-0002-9207-7069", "role": "member", "display_name": "Rutishauser, Ueli" }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "member", "display_name": "Perona, Pietro" } ], "option_major": [ "cns" ], "doi": "10.7907/y27w-m760", "abstract": "Vision neuroscience provides a unique opportunity to draw a correspondance between the physical world and its neural representation. But despite the amazing advances in neural recording technology that have occurred over the past two decades, we can't yet simultaneously record from more than a tiny fraction of the neurons in most of the visual systems currently being studied, which limits our ability to develop a holistic cause-and-effect understanding of how they operate. So it may make sense, as a complement to directly studying a visual system found in nature, to also study synthetic visual systems that in some way resemble it but are easier to inspect. This document describes four lines of work aimed at improving our ability to learn about biological visual systems using models optimized in ways that are analogous to the selective pressures that biological visual systems face, like the pressures to relay accurate information about the world, minimize energy consumption, and withstand perturbation. The first two of these lines of work---discussed in chapters 2 and 3---focus on expanding the space of selective forces that can be factored into optimization-guided models, and the other two---discussed in chapters 4 and 5---focus on modeling particular visual systems (in the macaque and the fruit fly, respectively). Taken together, optimization-guided modeling is shown to be a promising approach to advancing our understanding of visual processing across the animal kingdom, allowing us to leverage hypotheses about the high-level properties of visual systems to amplify the value of sparse neural data." }, { "name": "Nair, Aditya", "degree": "PhD", "year": "2025", "title": "The Neural Computation of Internal Affective States", "advisor": "Anderson, David J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10162024-031702349", "creators": [ { "name": { "family": "Nair", "given": "Aditya" }, "id": "Nair-Aditya", "orcid": "0000-0001-5242-5527", "display_name": "Nair, Aditya" } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "advisor", "display_name": "Anderson, David J." } ], "committee": [ { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "chair", "display_name": "Perona, Pietro" }, { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "member", "display_name": "Adolphs, Ralph" }, { "name": { "family": "Rutishauser", "given": "Ueli" }, "id": "Rutishauser-U", "orcid": "0000-0002-9207-7069", "role": "member", "display_name": "Rutishauser, Ueli" }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." } ], "option_major": [ "cns" ], "doi": "10.7907/wryk-rb16", "abstract": "The study of neural computation has long concentrated on our cognitive abilities, with extensive research dissecting the mechanisms of memory, decision-making, and navigation. In contrast, the realm of social innate behavior and emotion has often been treated as a simpler problem, overlooking the immense complexity and biological significance it entails. This thesis aims to bring neural computation into the domain of emotional or affective states, employing data-driven modeling methods that approximate neural activity as dynamical systems. The application of these methods has uncovered brain representations that encode key qualities of persistence and escalation associated with aggressive states, formalized as line attractors. These emergent features of neural circuits arise from the complex interplay of connectivity and network dynamics, challenging long-held notions of subcortical computation. This discovery led us to rigorously test various key properties of line attractor dynamics. Through closed-loop modeling and holographic neural activation, we demonstrate that the line attractor is intrinsic to the mammalian hypothalamus, providing some of the first causal evidence of this property for any continuous attractor. These experiments also suggest that functional connectivity within the hypothalamus underpins the stability of this attractor. Furthermore, using a new cell-type-specific gene-editing system, we show that the implementation of this line attractor depends on neuropeptides, indicating a non-canonical mechanism that contributes to the robustness of this innate attractor. Finally, we reveal that line attractors encode emotional states beyond aggression, including states of sexual receptivity in the female hypothalamus. Longitudinal recordings of neural data across the estrus cycle show that the line attractor disappears during non-estrus states, suggesting long-timescale modulation of attractor dynamics by hormones. Together, these studies present a new paradigm for understanding subcortical computation underlying internal states and suggest a canonical motif that the brain reuses to encode diverse internal affective states." }, { "name": "Narayanan, Aditi Kalpagam", "degree": "PhD", "year": "2025", "title": "Diversity, Activity, and Adaptations of Phage Communities in Anoxic Hydrocarbon-Rich Marine Sediments", "advisor": "Orphan, Victoria J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05272025-172840679", "creators": [ { "name": { "family": "Narayanan", "given": "Aditi Kalpagam" }, "id": "Narayanan-Aditi-Kalpagam", "orcid": "0000-0003-0627-1859", "display_name": "Narayanan, Aditi Kalpagam" } ], "advisors": [ { "name": { "family": "Orphan", "given": "Victoria J." }, "id": "Orphan-V-J", "orcid": "0000-0002-5374-6178", "role": "advisor", "display_name": "Orphan, Victoria J." } ], "committee": [ { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "chair", "display_name": "Newman, Dianne K." }, { "name": { "family": "Parker", "given": "Joseph" }, "id": "Parker-J", "orcid": "0000-0001-9598-2454", "role": "member", "display_name": "Parker, Joseph" }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." }, { "name": { "family": "Orphan", "given": "Victoria J." }, "id": "Orphan-V-J", "orcid": "0000-0002-5374-6178", "role": "member", "display_name": "Orphan, Victoria J." } ], "option_major": [ "microbio" ], "doi": "10.7907/v5x8-7447", "abstract": "The viruses of the global ocean, especially those infecting prokaryotic taxa, are known to play an important role in maintaining the genetic and taxonomic diversity of their host communities and in the cycling of atmospheric carbon and key nutrients like nitrogen and iron. However, the vast majority of these conclusions are drawn from the surface ocean and upper water column, while the sediments, which constitute one of the largest biomes on earth, are understudied in comparison. Of special interest are areas on the ocean floor where methane and other hydrocarbons are produced and released by geological activity and oxidized by a consortium of archaea and bacteria. Using direct genomic sequencing of the viruses from a variety of simplified sediment-free enrichments of hydrocarbon oxidizers, I compare viral communities sampled from different locations and incubated under a range of temperatures to understand the role these parameters might play in shaping distribution and community structure. I then present the most comprehensive picture thus far of viral diversity and distribution from a methane cold seep and discuss whether the viral assemblages are influenced by the steep geochemical gradients that characterize seep sediments. From these datasets, I propose that viral communities in methane-oxidizing sediments are tailored specifically to the physical constraints of the sediment matrix rather than to the dominant members of the cellular community or to other physicochemical parameters such as temperature, sampling location, or depth below the seafloor. I then outline the development of two methods, stable-isotope probing coupled to nanoSIMS and biorthogonal non-canonical amino acid tagging, to work in heterogenous sediment virus samples rather than the liquid pure cultures on which they had previously relied. Implementation of these methods, which allow us to temporally constrain viral production and virus-influenced nutrient flow, resulted in the hypothesis that viral production likely responds to shifts in the major metabolic processes within an ecosystem and may influence cellular community composition." }, { "name": "Robinson, Noah Evan", "degree": "PhD", "year": "2025", "title": "Construction of Long, Complex, and Diverse DNA Sequences", "advisor": "Wang, Kaihang", "url": "https://resolver.caltech.edu/CaltechTHESIS:05282025-171526879", "creators": [ { "name": { "family": "Robinson", "given": "Noah Evan" }, "id": "Robinson-Noah-Evan", "orcid": "0009-0000-2481-9596", "display_name": "Robinson, Noah Evan" } ], "advisors": [ { "name": { "family": "Wang", "given": "Kaihang" }, "id": "Wang-Kaihang", "orcid": "0000-0001-7657-8755", "role": "advisor", "display_name": "Wang, Kaihang" } ], "committee": [ { "name": { "family": "Parker", "given": "Joseph" }, "id": "Parker-J", "orcid": "0000-0001-9598-2454", "role": "chair", "display_name": "Parker, Joseph" }, { "name": { "family": "Wang", "given": "Kaihang" }, "id": "Wang-Kaihang", "orcid": "0000-0001-7657-8755", "role": "member", "display_name": "Wang, Kaihang" }, { "name": { "family": "Qian", "given": "Lulu" }, "id": "Qian-Lulu", "orcid": "0000-0003-4115-2409", "role": "member", "display_name": "Qian, Lulu" }, { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "orcid": "0000-0002-5899-7523", "role": "member", "display_name": "Winfree, Erik" }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." } ], "option_major": [ "bioeng" ], "doi": "10.7907/qtq1-dv04", "abstract": "The DNA molecule encodes the information required for biological systems to carry out a broad range of functions. The understanding of this relationship has sparked inquiries across vast fields of biology and biological engineering as we read, edit, and write the genetic information of organisms. Great advancements have been made toward these pursuits, from revolutions in DNA reading with long read sequencing and the ability to generate terabytes of data from a single run to the breakthroughs in DNA editing with the major advancements in CRISPR/Cas technologies over the last decade. However, writing DNA, as the ability to construct DNA of any length, complexity, or diversity, lags significantly behind our capacity for reading and editing.
\r\n\r\nDNA oligo synthesis can only reach short lengths of a few hundred nucleotides of single stranded DNA. The field of DNA assembly develops the methods for stitching together DNA oligos and DNA fragments into larger constructs. The current field applies a broad range of approaches that each occupy their own niche due to their unique set of advantages and disadvantages. No existing technique is able to assemble a large number of DNA fragments simultaneously with high accuracy and without placing restrictions on the sequences being assembled. This is because all existing DNA assembly technologies rely on the information contained within the complementary sequences of the DNA molecules being constructed to direct the assembly.
\r\n\r\nTo meet the demand for robust DNA assembly, we have developed a new DNA assembly technique named Sidewinder which separates the information that guides assembly from the final assembled sequence using DNA 3-Way junctions. We demonstrate the transformative nature of the Sidewinder technique with highly robust and accurate assembly of complex DNA sequences of both high GC and high repeats, a 40-piece multi-fragment assembly, a parallel construction of multiple distinct genes in the same reaction, and construction of a combinatorial library with a large number of diversified positions across the entire length of the gene for high coverage of a library of 442,368 variants.
\r\n\r\nWhere Sidewinder excels at the assembly of oligos to the kilobase scale, we have made a series of advancements to an existing 2-Way junction assembly technique, USER cloning, for the accurate and efficient assembly of PCR-based DNA inputs. We characterize these improvements with a series of assemblies where we achieve an average accuracy over 95%, gain 3 orders of magnitude increase in yield of transformants, and conduct large multi-fragment assemblies beyond what was previously possible with the technique. We then interface these two state-of-the-art capacities for the rapid and efficient construction of a complex 10 kilobase sequence de novo and entirely cell-free.
" }, { "name": "Shivaei, Shirin", "degree": "PhD", "year": "2025", "title": "A Viral Toolkit for Ultrasound Imaging of Cellular Activity and Gene Expression", "advisor": "Shapiro, Mikhail G.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06022025-044636280", "creators": [ { "name": { "family": "Shivaei", "given": "Shirin" }, "id": "Shivaei-Shirin", "orcid": "0000-0002-6894-3289", "display_name": "Shivaei, Shirin" } ], "advisors": [ { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "advisor", "display_name": "Shapiro, Mikhail G." } ], "committee": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "chair", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Oka", "given": "Yuki" }, "id": "Oka-Yuki", "orcid": "0000-0003-2686-0677", "role": "member", "display_name": "Oka, Yuki" }, { "name": { "family": "Tanter", "given": "Mickael" }, "id": "Tanter-Mickael", "orcid": "0000-0001-7739-8051", "role": "member", "display_name": "Tanter, Mickael" }, { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "member", "display_name": "Shapiro, Mikhail G." } ], "option_major": [ "bioeng" ], "doi": "10.7907/r9gz-1482", "abstract": "Observing and manipulating cell dynamics in living organisms is essential for understanding biological processes and intervening when they malfunction. However, the lack of non-invasive, non-ionizing, and cost-effective imaging technologies limits our ability to study these processes in their native context. To address this gap, we developed a toolkit for ultrasound imaging of acoustic reporter gene expression in mammalian tissues using virally-delivered gas vesicle (GV) genes. We demonstrate the versatility of this toolkit across multiple applications, including tracking engineered cell-based therapies and imaging activity-dependent gene expression in the brain.
\r\n\r\nTo track cell-based therapies, we developed lentiviral vectors encoding the eight genes necessary for GV expression, achieving robust ultrasound contrast in both cell lines and primary human T cells. By expressing GVs downstream of activity-dependent promoters, we monitor T cell activation in cytotoxic T cells engaged with tumor cells. In a mouse xenograft model, we then image the targeted accumulation and proliferation of GV-expressing T cells within tumors. These ultrasound measurements, which closely correlate with immunohistological analysis, provide real-time, in vivo insights into the spatial dynamics of therapeutic cells. This approach offers a powerful tool to accelerate the development and clinical translation of cell-based therapies.
\r\n\r\nWe extend this technology to the brain by engineering an AAV-based system for GV expression in primary neurons. Following intracranial injection of the GV-encoding AAVs in mice, we demonstrate longitudinal imaging of in situ gene expression in the brain over several weeks. Moreover, by using immediate early gene promoters to drive GV expression, we track changes in neuronal activity in the hippocampus during seizure episodes, enabling repeated, longitudinal imaging of brain function within the same animal. Collectively, these advancements establish a robust platform for ultrasound imaging of cellular activity and gene expression in opaque tissues, with applications ranging from cancer immunotherapy to neuroscience.
" }, { "name": "Sullivan, Delaney Kalcey", "degree": "PhD", "year": "2025", "title": "Software, Tools, and Methods Development for Single-Cell Transcriptomics", "advisor": "Pachter, Lior S.; Guttman, Mitchell", "url": "https://resolver.caltech.edu/CaltechTHESIS:05232025-094543832", "creators": [ { "name": { "family": "Sullivan", "given": "Delaney Kalcey" }, "id": "Sullivan-Delaney-Kalcey", "orcid": "0000-0002-8359-6705", "display_name": "Sullivan, Delaney Kalcey" } ], "advisors": [ { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "advisor", "display_name": "Pachter, Lior S." }, { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "co-advisor", "display_name": "Guttman, Mitchell" } ], "committee": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "chair", "display_name": "Wold, Barbara J." }, { "name": { "family": "Pimentel", "given": "Harold" }, "id": "Pimentel-Harold", "orcid": "0000-0001-8556-2499", "role": "member", "display_name": "Pimentel, Harold" }, { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "member", "display_name": "Pachter, Lior S." }, { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "member", "display_name": "Guttman, Mitchell" } ], "option_major": [ "biology" ], "doi": "10.7907/kee5-ty36", "abstract": "Advances in transcriptomics have transformed the study of gene expression, enabling a shift from low-throughput bulk RNA measurements to high-resolution, large-scale single-cell RNA-sequencing (scRNA-seq). This work refines existing methodologies and introduces new strategies for achieving precise, versatile, and scalable transcriptomic analyses across a broad spectrum of assays and biological contexts.
\r\n\r\nOn the computational front, this dissertation introduces new methods for adaptable preprocessing of sequencing reads, enabling the handling of very complex read structures. It refines existing strategies for efficiently querying large-scale transcriptomic datasets and enhances approaches for quantifying nascent and mature RNA species. A general framework is introduced for discovering and organizing biologically informative sequences directly from raw sequencing data, facilitating the detection of sample-specific or condition-specific variation. On the experimental front, a novel single-cell RNA sequencing method is presented that is cost-effective, open source, and scalable, supporting large-scale studies with substantial cell numbers and high per-cell resolution.
\r\n\r\nThese developments collectively expand the toolkit for transcriptomics, enabling more efficient and comprehensive exploration of RNA biology.
" }, { "name": "Urrutia, Hugo Alexander", "degree": "PhD", "year": "2025", "title": "Understanding Cessation of Neural Crest Migration and Onset of Gangliogenesis", "advisor": "Bronner, Marianne E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04072025-220823071", "creators": [ { "name": { "family": "Urrutia", "given": "Hugo Alexander" }, "id": "Urrutia-Hugo-Alexander", "orcid": "0000-0002-2970-6918", "display_name": "Urrutia, Hugo Alexander" } ], "advisors": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "advisor", "display_name": "Bronner, Marianne E." } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "chair", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Parker", "given": "Joseph" }, "id": "Parker-J", "orcid": "0000-0001-9598-2454", "role": "member", "display_name": "Parker, Joseph" }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "orcid": "0000-0002-7371-4675", "role": "member", "display_name": "Prober, David A." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "member", "display_name": "Bronner, Marianne E." } ], "option_major": [ "biodev" ], "doi": "10.7907/b31p-rp50", "abstract": "The neural crest is a multipotent, vertebrate-specific embryonic cell population that originates at the border of the developing central nervous system. Often referred to as the \"fourth germ layer,\" the neural crest gives rise to diverse cell types, including craniofacial structures and components of the peripheral nervous system. Neural crest cells from specific axial levels in the embryo generate unique sets of progeny and migrate along distinct pathways, differing from those at other axial levels. During development, the formation of key structures within the vertebrate head, such as cranial ganglia and sense organs, requires coordinated migration and interactions between two distinct embryonic cell populations: the neural crest and the ectodermal placodes. The dual embryonic origin of cranial sensory ganglia has interested investigators for some time, yet surprisingly, little is still known about the neural crest\u2013placode relationship. Despite extensive research, the process of cranial gangliogenesis, an intriguing example of how cell\u2013cell interactions drive the assembly of complex structures in the developing embryo, remains incompletely understood. To address this gap, I aimed to advance our understanding of neural crest contributions to cranial sensory ganglia formation and investigate how interactions between these two distinct cell populations contribute to chick trigeminal gangliogenesis.\r\n\r\nTo investigate this process, I focused on the early formation of the trigeminal ganglion, emphasizing on the transcriptional regulation of neural crest-derived cells. Using a combination of lineage labeling and in situ hybridization in chick embryos, we demonstrated that the transcription factor Tlx3 is expressed in neural crest-derived cells contributing to the cranial trigeminal ganglion, coinciding with the onset of ganglion condensation. Notably, loss-of-function experiments revealed that Tlx3 deficiency results in smaller ganglia with fewer neurons. Conversely, ectopic expression in migrating cranial neural crest cells accelerates neuronal differentiation, underscoring its critical role in neural crest-derived neuronal development. Taken together, these results demonstrate a pivotal role for Tlx3 in neural crest-derived cells during chick trigeminal gangliogenesis.\r\n\r\nAs an additional candidate mediator, I investigated the potential role of Cxcl14. The concurrent expression of CXCL14 in placodal cells and its potential cognate receptor CXCR4 in neural crest cells raised the intriguing possibility that this ligand\u2013receptor pair mediates signaling from placodal to neural crest cells, representing an additional form of their cell-cell interactions. Loss of Cxcl14 disrupts gangliogenesis and axonal projections, revealing an essential role for this chemokine in guiding neural crest-placode interactions during early ganglion formation. More specifically, perturbing Cxcl14 in the placodal population resulted in increased dispersion of neural crest-derived cells in the maxillomandibular lobe but not in the ophthalmic lobe of the trigeminal ganglion, highlighting its critical role in directing neural crest-placode interactions during early ganglion formation.\r\n\r\nIn summary, the findings from my thesis advance our understanding of neural crest contributions to cranial sensory ganglia formation. By elucidating transcriptional and signaling mechanisms involved in trigeminal gangliogenesis, these results provide key insights into vertebrate neurodevelopment and lay the groundwork for further studies into neural crest biology." }, { "name": "Viloria Winnett, Alexander", "degree": "PhD", "year": "2025", "title": "Quantitative Nucleic Acid Measurements Inform Strategies to Mitigate Viral Outbreaks", "advisor": "Ismagilov, Rustem F.", "url": "https://resolver.caltech.edu/CaltechTHESIS:12092024-223834150", "creators": [ { "name": { "family": "Viloria Winnett", "given": "Alexander" }, "id": "Viloria-Winnett-Alexander", "orcid": "0000-0002-7338-5605", "display_name": "Viloria Winnett, Alexander" } ], "advisors": [ { "name": { "family": "Ismagilov", "given": "Rustem F." }, "id": "Ismagilov-R-F", "orcid": "0000-0002-3680-4399", "role": "advisor", "display_name": "Ismagilov, Rustem F." } ], "committee": [ { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "chair", "display_name": "Thomson, Matthew" }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Arboleda", "given": "Valerie" }, "id": "Aboleda-V-A", "orcid": "0000-0002-9687-9122", "role": "member", "display_name": "Arboleda, Valerie" }, { "name": { "family": "Ismagilov", "given": "Rustem F." }, "id": "Ismagilov-R-F", "orcid": "0000-0002-3680-4399", "role": "member", "display_name": "Ismagilov, Rustem F." } ], "option_major": [ "biology" ], "doi": "10.7907/qe3a-a670", "abstract": "Humans have always been and continue to be at risk of infection by pathogens that surround us. However, recent advancements in quantitative nucleic acid technologies have allowed for more detailed study of these pathogens, how they spread among individuals, and how our immune systems respond to infection. In this thesis, I describe the design and execution of the Caltech COVID-19 Study, which used quantitative nucleic acid measurements to investigate the natural history of SARS-CoV-2 infection and inform strategies for diagnostics and vaccine development to reduce viral transmission. The Caltech COVID-19 Study enrolled participants in the Los Angeles area between September 2020 and April 2022 who were at risk of SARS-CoV-2 infection due to recent exposure to a household contact with acute infection. Participants collected paired upper respiratory specimens (saliva, nasal swabs, and throat swabs) daily or twice daily for approximately two weeks. These specimens underwent SARS-CoV-2 viral load quantification to assess transmission risk and determine whether to extend or terminate study enrollment. For participants who initially tested negative for SARS-CoV-2 RNA but later developed sustained infection, we tracked viral load from the very start of infection. These measurements were then used to evaluate the performance of various COVID-19 diagnostic tests. Our findings revealed a significant advantage of high-analytical-sensitivity tests over those with lower sensitivity, as well as the benefit of testing both the throat and nose rather than just the nose. In addition to viral load quantification, we sequenced human mRNA from these specimens to assess gene expression. Analyzing these changes allowed us to study how the mucosal immune system responds to acute viral infection across multiple anatomical sites over time, providing insights that could improve mucosal vaccine design. Notably, our data showed that, contrary to current models of localized paracrine interferon signaling, distinct compartments of the upper respiratory mucosa exhibited synchronized interferon stimulation during early infection\u2014even in the absence of detectable local viral replication. Mucosal vaccines capable of triggering this coordinated interferon response, maintaining CD8+ T memory cells to rapidly execute effector functions upon viral exposure, may be key to achieving sterilizing immunity. Findings from quantitative nucleic acid measurements in this thesis inform strategies to more effectively mitigate viral outbreaks." }, { "name": "Wang, Zitong (Jerry)", "degree": "PhD", "year": "2025", "title": "Theoretical and Computational Analysis of Cell Migration in Complex Tissue Environments", "advisor": "Thomson, Matthew", "url": "https://resolver.caltech.edu/CaltechTHESIS:06152024-132652470", "creators": [ { "name": { "family": "Wang", "given": "Zitong (Jerry)" }, "id": "Wang-Zitong-Jerry", "orcid": "0000-0001-8008-7318", "display_name": "Wang, Zitong (Jerry)" } ], "advisors": [ { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "advisor", "display_name": "Thomson, Matthew" } ], "committee": [ { "name": { "family": "Cai", "given": "Long" }, "id": "Cai-Long", "orcid": "0000-0002-7154-5361", "role": "chair", "display_name": "Cai, Long" }, { "name": { "family": "Eberhardt", "given": "Frederick" }, "id": "Eberhardt-Frederick", "role": "member", "display_name": "Eberhardt, Frederick" }, { "name": { "family": "Merchant", "given": "Akil Abid" }, "id": "Merchant-Akil-Abid", "orcid": "0000-0001-7472-822X", "role": "member", "display_name": "Merchant, Akil Abid" }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" } ], "option_major": [ "sysbiol" ], "doi": "10.7907/mj08-b258", "abstract": "Cells sense and respond in spatially structured environments, including soils and tissue. My Ph.D. projects centered on developing new theoretical models and computational methods to understand how cells migrate in complex environments.
\r\n \r\nThe first project is more theoretical in nature, leveraging information theory to study how the spatial organization of cell signaling pathways are adapted to the cell's natural environment. In tissue and soil, cells must localize to their targets by navigating distributions of extracellular ligands that are spatially discontinuous, consisting of local concentration peaks, due to binding a non-uniform network of ECM fibers. It is unclear how cells navigate patchy environments while not getting trapped in local concentration peaks. To answer this question, we framed navigation as a problem of maximizing mutual information in space and developed a computational algorithm for computing signaling pathway architectures that maximize mutual information in simulated natural environments. We found that for cells in tissues and soils, dynamic localization of membrane receptors dramatically boosts sensing precision and enables cells to navigate to chemical sources 30 times faster, but this receptor localization strategy is relatively inconsequential for cells in purely diffusive environments. Further, we found that anisotropic receptor dynamics previously observed in immune cells and growth cones are nearly optimal as predicted by our model.
\r\n\r\nThe second project is more computational in nature, leveraging multiplexed tissue imaging to understand T-cell migration in tumor microenvironments. Immunotherapies can halt or slow down cancer progression by activating either endogenous or engineered T-cells to detect and kill cancer cells. T-cells must infiltrate the tumor core for immunotherapies to be effective. However, many solid tumors resist T-cell infiltration, challenging the efficacy of current therapies. In collaboration with clinician scientists at Cedars-Sinai Medical Center, we developed an integrated deep learning framework, Morpheus, that takes large-scale spatial omics profiles of patient tumors, and combines a formulation of T-cell infiltration prediction as a self-supervised machine learning problem with a counterfactual optimization strategy to generate minimal tumor perturbations predicted to boost T-cell infiltration. We applied Morpheus to 368 metastatic melanoma and colorectal cancer samples assayed using 40-plex imaging mass cytometry, discovering cohort-dependent, combinatorial perturbations, involving CXCL9, CXCL10, CCL22 and CCL18 for melanoma and CXCR4, PD-1, PD-L1 and CYR61 for colorectal cancer, predicted to support T-cell infiltration across large patient cohorts. Using only raw image data, Morpheus also identified distinct therapeutic strategies for different patient strata such as cancer stage or fatty liver presence. Our work presents a paradigm for counterfactual-based prediction and design of cancer therapeutics using spatial omics data.
" }, { "name": "Yang, Yujing", "degree": "PhD", "year": "2025", "title": "Exploring Cell Diversity in Complex Tissues through Spatial Genomics and Spatial Transcriptomics", "advisor": "Cai, Long", "url": "https://resolver.caltech.edu/CaltechTHESIS:05302025-191432965", "creators": [ { "name": { "family": "Yang", "given": "Yujing" }, "id": "Yang-Yujing", "orcid": "0000-0002-2338-6263", "display_name": "Yang, Yujing" } ], "advisors": [ { "name": { "family": "Cai", "given": "Long" }, "id": "Cai-Long", "orcid": "0000-0002-7154-5361", "role": "advisor", "display_name": "Cai, Long" } ], "committee": [ { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "chair", "display_name": "Guttman, Mitchell" }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Van Valen", "given": "David A." }, "id": "Van-Valen-D", "orcid": "0000-0001-7534-7621", "role": "member", "display_name": "Van Valen, David A." }, { "name": { "family": "Cai", "given": "Long" }, "id": "Cai-Long", "orcid": "0000-0002-7154-5361", "role": "member", "display_name": "Cai, Long" } ], "option_major": [ "biology" ], "doi": "10.7907/r85x-qs80", "abstract": "The study of cellular diversity is a fundamental requirement for understanding how multicellular organisms function. During the development of multicellular organisms, cells differentiate into various cell types with different molecular compositions, exhibit different phenotypes, and show distinct morphologies. Each single cell occupies a specific spatial location within different tissues and organs and performs a unique function. A holistic understanding of cells requires the integration of multiple \u201comics\u201d modalities, including genomics, epigenomics, transcriptomics, and proteomics. Current well-established single-cell sequencing methods have been used to build enormous single-cell transcriptomic atlases. While single-cell sequencing methods are now capable of multi-omic profiling, they all require cell dissociation, during which important spatial context information is lost. To study cellular diversity within its native spatial context, our lab has developed innovative spatial genomics and transcriptomics tools that enable multi-omics profiling at single-cell resolution while preserving intact tissue organization. This thesis presents two projects that leverage these tools to investigate cellular diversity in complex tissues across different biological scales, from subnuclear to tissue-level organization. In Chapter 2, we applied spatial multi-omics to the mouse cerebellum, achieving single-cell resolution profiling of 100,049 genomic loci, 17,856 nascent transcripts, 60 mature mRNAs, and 28 immunofluorescently labeled subnuclear structures. To achieve this, we developed innovative two-layer barcodes for DNA sequential fluorescence in situ hybridization (seqFISH). Combining cell-type information from nascent and mature transcriptomes, we captured the three-dimensional genomic architecture and its interactions with subnuclear compartments in a cell-type-specific manner. Our findings show that repressive chromatin compartments have greater cell-type specificity than active chromatin compartments in the mouse cerebellum. In Chapter 3, we integrated single-cell multiome sequencing, which profiles single-nucleus RNA and chromatin accessibility (ATAC) from the same cells, with seqFISH spatial transcriptomics. This approach was applied to the 17- to 18-week-old human fetal kidney, targeting 224 marker genes. By combining sequencing and spatial profiling data, we constructed a comprehensive developmental atlas of human kidney organogenesis, providing new insights into the tissue organization and gene expression patterns during kidney development." }, { "name": "Yang, Zheng", "degree": "PhD", "year": "2025", "title": "SpLacZ-MERCS-Coupled CRISPRi Screening Identifies Novel Mitochondria-ER Contact Sites Regulators", "advisor": "Chan, David C.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05012025-221548751", "creators": [ { "name": { "family": "Yang", "given": "Zheng" }, "id": "Yang-Zheng", "orcid": "0000-0001-8131-0868", "display_name": "Yang, Zheng" } ], "advisors": [ { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "advisor", "display_name": "Chan, David C." } ], "committee": [ { "name": { "family": "Clemons", "given": "William M." }, "id": "Clemons-W-M", "orcid": "0000-0002-0021-889X", "role": "chair", "display_name": "Clemons, William M." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "member", "display_name": "Chan, David C." } ], "option_major": [ "bioeng" ], "doi": "10.7907/r4m2-b064", "abstract": "Mitochondria-ER contact sites (MERCS) mark critical hotspots for a variety of cellular processes, including calcium homeostasis, lipid homeostasis, mitochondria dynamics, and quality control. Fluorescence-based tools have been the main approach to detect MERCS, with a large portion of studies using split fluorescent proteins, which assemble at sites of contact to yield a fluorescence signal. However, they have limitations, including little to no response to fluctuations in MERCS abundance, low sensitivity, and possible artifacts made due to reporter protein reconstitution. To overcome this, we developed the SpLacZ-MERCS sensor, the first MERCS reporter using split \u03b2-galactosidase (LacZ). Compared to using complementary GFP fragments that go to mitochondria and ER, SpLacZ-MERCS gives an integrated readout of MERCS activity for more accurate and quantitative monitoring of these contact sites in single cells over time. Our system has specific organelle targeting but does not induce artificial tethering, which allows it to be a standard tool for studying MERC dynamics in physiological and pathological conditions. Using pharmacological and genetic perturbations known to modulate mitochondria\u2013ER interactions, we validated SpLacZ-MERCS as an effective and reliable sensor of MERCS abundance.
\r\n\r\nBeyond tool development, we sought to uncover the molecular mechanisms regulating MERCS using a genome-wide CRISPR interference (CRISPRi) screen combined with SpLacZ-MERCS. This unbiased approach led to the identification of RHOA, a small GTPase known for its roles in cytoskeletal dynamics and signal transduction as a novel regulator of MERCS. We found that RHOA directly interacts with the ER-resident protein VAPB and modulates its binding to PTPIP51, a mitochondrial protein involved in forming MERCS junctions. VAPB and PTPIP51 constitute a MERCS tethering complex. RHOA depletion or overexpression of CUL3 (which promotes RHOA degradation) results in reduced MERCS levels, while RHOA overexpression enhances MERCS formation. Notably, we discovered that disease-associated mutations in RHOA, CUL3, and VAPB\u2014implicated in cancer, metabolic disorders, and neurodegeneration\u2014disrupt MERCS regulation, suggesting a potential link between MERCS dysfunction and disease pathology.
\r\n\r\nTogether, our study makes two significant contributions. SpLacZ-MERCS is a new signal-integrating MERCS reporter system that allows dynamic, cumulative tracking of mitochondria-ER interactions. RHOA has been established as a novel regulator of MERCS, providing a framework to understand how contact sites can be manipulated in a dynamic way upon cellular signals. These findings enhance the foundation of our understanding of MERCS regulation while also shedding light on new possible therapeutic targets for diseases associated with altered communication between mitochondria and the ER.
" }, { "name": "Bhamidipati, Pranav Subramanyam", "degree": "PhD", "year": "2024", "title": "Modeling and Design of Synthetic Biochemical Circuits for Biological Phenotypes", "advisor": "Thomson, Matthew", "url": "https://resolver.caltech.edu/CaltechTHESIS:06012024-054725051", "creators": [ { "name": { "family": "Bhamidipati", "given": "Pranav Subramanyam" }, "id": "Bhamidipati-Pranav-Subramanyam", "orcid": "0000-0002-6199-6505", "display_name": "Bhamidipati, Pranav Subramanyam" } ], "advisors": [ { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "advisor", "display_name": "Thomson, Matthew" } ], "committee": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "chair", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Bois", "given": "Justin S." }, "id": "Bois-J-S", "orcid": "0000-0001-7137-8746", "role": "member", "display_name": "Bois, Justin S." }, { "name": { "family": "Barr", "given": "Alan H." }, "id": "Barr-A-H", "role": "member", "display_name": "Barr, Alan H." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" } ], "option_major": [ "bioeng" ], "doi": "10.7907/gpc6-hb40", "abstract": "Biological behaviors arise from the dynamical interactions of biochemical networks. For example, the various immune responses to damage are manifestations of signaling networks between immune cell types. A central goal in systems and synthetic biology is to elucidate the design principles of these networks, or circuits, both in the sense of dissecting how function arises from structure in the natural context and in the sense of understanding the guidelines for optimal engineering of synthetic biological systems. The study of design principles in both senses is aided by mathematical modeling and simulation, which provide a self-consistent framework for evaluating the theoretical implications of biological hypotheses as well as a testbed for the development of novel circuits for desired biological phenotypes. This thesis pertains to two related challenges in this field, namely the scaling of computational design to larger circuits and the engineering of global phenotypes that emerge nonlinearly from local interactions.
\r\n \r\nThe first section of this thesis presents a novel design platform for biological circuits, called CircuiTree, that uses a game-playing paradigm to overcome the combinatorial complexity of \\textit{de novo} circuit design. This platform treats circuit design as a game of circuit assembly and traverses the tree of possible assemblies using Monte Carlo tree search (MCTS). Borrowed from artificial intelligence (AI) agents that have mastered complex games, MCTS is a reinforcement learning (RL)-based search algorithm that efficiently searches for the most effective design strategies and naturally discovers design principles in the form of network motifs, which appear as clusters of solutions in the search tree. Finally, when tasked with designing fault-tolerant oscillators with five components, CircuiTree finds a novel design strategy, which we call motif multiplexing, in which multiple sub-oscillators are interleaved so as to render the circuit highly resistant to deletions and knockdowns. This design principle, which may be responsible for the multiple oscillatory loops observed in eukaryotic circadian clocks, opens the possibility of engineering synthetic circuits at a larger scale and suggests that larger biological circuits contain yet-unknown design features that are not simply extensions of smaller circuits.
\r\n\r\nThe second section describes a novel mechanosensitive property of the SynNotch synthetic chimeric receptor and uses a multicellular modeling framework to show how it can be used to control spatiotemporal patterning \\textit{in vitro}. Modified from the endogenous juxtacrine receptor Notch, SynNotch binds to an arbitrary extracellular ligand and, in response, releases an arbitrary transcription factor, thus acting as a user-defined signal transducer. We show that, in mouse fibroblasts, a simple sender-receiver SynNotch circuit ceases to transduce a membrane-bound GFP signal at high cell densities in 2D culture. Because of this feature, a lawn of cells expressing a signal-relay circuit, which we call the transceiver circuit, can undergo spatially limited activation, where the signal propagates in a wave outward from a GFP-expressing sender cell until, due to cell division, the cell density crosses a threshold value and the signaling system shuts down. Using a multicellular lattice-based model combined with experiments, we demonstrate that perturbations of growth parameters can be used to control the size of activated spots. Finally, we achieve spatiotemporal patterns of activation by seeding the growth dish nonuniformly, creating a wave of activation at the millimeter scale that recapitulates the kinematic wave patterning phenomenon observed during vertebrate somitogenesis.
\r\n\r\nTogether, this body of work represents an advance in the use of computational methods and mathematical modeling to guide the design and control of complex biological phenotypes. Advances in these methods promise to catalyze the development of more advanced cell-based therapies and engineered tissues.
" }, { "name": "Chari, Tara Varada", "degree": "PhD", "year": "2024", "title": "Perturbing the Genome: From Bench to Biophysics", "advisor": "Pachter, Lior S.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05292024-221741183", "creators": [ { "name": { "family": "Chari", "given": "Tara Varada" }, "id": "Chari-Tara-Varada", "orcid": "0000-0002-6953-4313", "display_name": "Chari, Tara Varada" } ], "advisors": [ { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "advisor", "display_name": "Pachter, Lior S." } ], "committee": [ { "name": { "family": "Qian", "given": "Lulu" }, "id": "Qian-Lulu", "orcid": "0000-0003-4115-2409", "role": "chair", "display_name": "Qian, Lulu" }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "member", "display_name": "Pachter, Lior S." } ], "option_major": [ "bioeng" ], "doi": "10.7907/5drv-ma07", "abstract": "In single-cell genomics, we can simultaneously assay hundreds of thousands of cells, their molecular contents, and how they respond to perturbation, from genetic knockouts to environmental changes. This thesis focuses on how to merge experimental and computational techniques to generate and analyze large-scale perturbation data for high-resolution systems biology. Beginning at the bench, we demonstrate how combining large-scale cell atlas surveys with multi-condition experimentation can illuminate the diversity of cell types across whole organisms and cellular strategies in response to environmental changes and perturbations. We then investigate the limitations of current practice in exploratory analysis, and strategies for determining preservation or distortion of biological insight by these data transformation and dimensionality reduction techniques. To address these limitations, we demonstrate how stochastic biophysical models can rewrite the way we interpret complex perturbation data, taking greater advantage of the diverse molecular measurements to develop biological hypotheses about DNA and RNA regulation in cellular function, development, and disease.
" }, { "name": "Chen, Peiwei", "degree": "PhD", "year": "2024", "title": "Sexual Dimorphism and Evolutionary Innovation in piRNA-Guided Genome Defense", "advisor": "Aravin, Alexei A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06022024-075040775", "creators": [ { "name": { "family": "Chen", "given": "Peiwei" }, "id": "Chen-Peiwei", "orcid": "0000-0001-7160-6673", "display_name": "Chen, Peiwei" } ], "advisors": [ { "name": { "family": "Aravin", "given": "Alexei A." }, "id": "Aravin-A-A", "orcid": "0000-0002-6956-8257", "role": "advisor", "display_name": "Aravin, Alexei A." } ], "committee": [ { "name": { "family": "Parker", "given": "Joseph" }, "id": "Parker-J", "orcid": "0000-0001-9598-2454", "role": "chair", "display_name": "Parker, Joseph" }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "orcid": "0000-0001-6597-2036", "role": "member", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Aravin", "given": "Alexei A." }, "id": "Aravin-A-A", "orcid": "0000-0002-6956-8257", "role": "member", "display_name": "Aravin, Alexei A." } ], "option_major": [ "biology" ], "doi": "10.7907/9tme-kf22", "abstract": "The genome is a battleground, where different genetic elements vie for inheritance. In particular, selfish genetic elements enhance their own transmission at the expense of host fitness, causing intragenomic conflicts that must be resolved to protect host reproduction. To keep selfish genes in check, animals employ several genome defense mechanisms, including the PIWI-interacting RNA (piRNA) pathway, where small non-coding piRNAs guide PIWI proteins to find complementary RNAs for silencing. While selfish genes are found across the tree of life, they are often sexually dimorphic and lineage-specific. Yet, it remains poorly understood how sex- and lineage-specific selfish genes are tamed by conserved genome defense mechanisms. To address this, I used the piRNA pathway in Drosophila melanogaster as the model system to study sexual dimorphism and evolutionary innovation in genome defense.
\r\n\r\nIn this thesis, I first described my discovery of piRNA sexual dimorphism, which evolved in response to the sex-specific selfish gene landscape. Next, I dissected the genetic basis and molecular mechanisms that underpin piRNA sexual dimorphism, gaining mechanistic insights into how the biological sex modifies the piRNA pathway to tame distinct selfish genes in two sexes. Pivoting to evolutionary innovation, I discovered a novel piRNA locus on the Y chromosome, which I named petrel, that silences the expression of an X-linked host gene, which I named pirate, implicating piRNAs in resolving X-versus-Y sex chromosome conflicts. petrel piRNAs evolved very recently after the split of D. melanogaster from its sibling species, highlighting a recent piRNA innovation against a lineage-specific target. Finally, I described my discovery of a novel genome defense protein factor, which I named Trailblazer, that tames a radically expanded selfish gene family, Stellate. Mechanistically, Trailblazer is a germline transcription factor that, via recent innovation of its DNA-binding domain, up-regulates the expression of two piRNA pathway effectors to quantitatively match Stellate in abundance, indicating a new mode of defense innovation beyond target-specific repressors.
\r\n\r\nCollectively, my thesis shows that the genomic battleground against selfish genes differs substantially between sexes and across lineages, which selects for distinct innovations in the piRNA pathway to control different selfish genes, thereby safeguarding genome integrity, animal fertility, and species continuity.
" }, { "name": "Cherry, Kevin Matthew", "degree": "PhD", "year": "2024", "title": "Molecular Pattern Recognition and Supervised Learning in DNA-Based Neural Networks", "advisor": "Qian, Lulu", "url": "https://resolver.caltech.edu/CaltechTHESIS:04292024-172707491", "creators": [ { "name": { "family": "Cherry", "given": "Kevin Matthew" }, "id": "Cherry-Kevin-Matthew", "orcid": "0000-0002-2343-0754", "display_name": "Cherry, Kevin Matthew" } ], "advisors": [ { "name": { "family": "Qian", "given": "Lulu" }, "id": "Qian-Lulu", "orcid": "0000-0003-4115-2409", "role": "advisor", "display_name": "Qian, Lulu" } ], "committee": [ { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "orcid": "0000-0002-5899-7523", "role": "chair", "display_name": "Winfree, Erik" }, { "name": { "family": "Yue", "given": "Yisong" }, "id": "Yue-Yisong", "orcid": "0000-0001-9127-1989", "role": "member", "display_name": "Yue, Yisong" }, { "name": { "family": "Rothemund", "given": "Paul W. K." }, "id": "Rothemund-P-W-K", "orcid": "0000-0002-1653-3202", "role": "member", "display_name": "Rothemund, Paul W. K." }, { "name": { "family": "Qian", "given": "Lulu" }, "id": "Qian-Lulu", "orcid": "0000-0003-4115-2409", "role": "member", "display_name": "Qian, Lulu" } ], "option_major": [ "bioeng" ], "doi": "10.7907/529f-kf62", "abstract": "Adaptation in nature begins at the subcellular, molecular level with the delicate interplay of biomolecule cascades orchestrating the myriad function of cells. The intermingling activity of these cells becomes expressions of complex behavior of multi-cellular system. Nature provides a dazzling array of examples showcasing the variations of intelligent functions. However, in the realm of synthetic construction, what systems have humans managed to engineer, and what are the boundaries of our technological power? In comparison to nature's repertoire, mankind's accomplishments appear rather modest. The intricate behaviors observed in intelligent organisms emerge from the collective interactions and feedback loops among their constituent elements, resulting in the emergence of novel properties and phenomena. To develop large-scale engineered systems exhibiting ever more brain-like, intelligent behaviors, we must first devise new molecular architectures and algorithms designed for adaptation and learning at the molecular scale. My research presented here is a humble step toward those goals. I will present the design of novel molecular systems made from DNA that exhibit complex neural computation and learning behaviors.
\r\n\r\nChapter 2 covers my contribution to scaling up the computing power of DNA circuits. From bacteria following simple chemical gradients to the brain distinguishing complex odor information, the ability to recognize molecular patterns is essential for biological organisms. This type of information-processing function has been implemented using DNA-based neural networks. Winner-take-all computation has been suggested as a potential strategy for enhancing the capability of DNA-based neural networks. Compared to the linear-threshold circuits and Hopfield networks used previously, winner-take-all circuits are computationally more powerful, allow simpler molecular implementation, and are not constrained by coupling the number of patterns and their complexity, so both a large number of simple patterns and a small number of complex patterns can be recognized. Here, we report a systematic implementation of winner-take-all neural networks based on DNA-strand-displacement reactions. We use a previously developed seesaw DNA gate motif, extended to include a simple and robust component that facilitates the cooperative hybridization involved in selecting a \u2018winner.' We show that with this extended seesaw motif, DNA-based neural networks can classify patterns into up to nine categories. Each of these patterns consists of 20 distinct DNA molecules chosen from the set of 100 that represents the 100 bits in 10x10 patterns, with the 20 DNA molecules selected tracing one of the handwritten digits \u20181\u2019 to \u20189.' The network successfully classified test patterns with up to 30 of the 100 bits flipped relative to the digit patterns \u2018remembered\u2019 during training, suggesting that molecular circuits can robustly accomplish the sophisticated task of classifying highly complex and noisy information on the basis of similarity to a memory.
\r\n\r\nChapter 3 investigates the development of a computational neural network model inspired by biological learning mechanisms, particularly focusing on the new mechanisms for learning in a WTA neural network. The study incorporates novel molecular motifs used in inhibited activators and inhibited weights, designed specifically for training from environmental input patterns. These motifs emulate biological systems by facilitating memory storage and retrieval within DNA-based neural networks, similar to synaptic connections and signal processing observed in living organisms. We assess the function of the individual molecular motifs and characterize their specificity in up to 18-species cross-talk experiments. Furthermore, we characterize the network's performance across a wide array of training and test patterns, mirroring the adaptive responses and diverse conditions encountered by biological systems. Additionally, we analyze the computational efficiency and speed of the learning system, comparing it with both the previous non-learning DNA-based WTA model and a direct weight activation model. By exploring the principles of molecular learning, particularly within winner-take-all neural networks, this study aims to advance computational systems by emulating adaptability and resilience observed in biological organisms using robust, new molecular motifs.
" }, { "name": "Ciemniecki, John Alan", "degree": "PhD", "year": "2024", "title": "The Bioenergetics of a Low-Power, Phenazine-Dependent Maintenance Metabolism in Pseudomonas aeruginosa", "advisor": "Newman, Dianne K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04152024-205944516", "creators": [ { "name": { "family": "Ciemniecki", "given": "John Alan" }, "id": "Ciemniecki-John-Alanlan", "orcid": "0000-0003-2789-6700", "display_name": "Ciemniecki, John Alan" } ], "advisors": [ { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "advisor", "display_name": "Newman, Dianne K." } ], "committee": [ { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "chair", "display_name": "Phillips, Robert B." }, { "name": { "family": "Orphan", "given": "Victoria J." }, "id": "Orphan-V-J", "orcid": "0000-0002-5374-6178", "role": "member", "display_name": "Orphan, Victoria J." }, { "name": { "family": "Ruby", "given": "Edward G." }, "id": "Ruby-Edward", "orcid": "0000-0002-4112-4830", "role": "member", "display_name": "Ruby, Edward G." }, { "name": { "family": "Bois", "given": "Justin S." }, "id": "Bois-J-S", "orcid": "0000-0001-7137-8746", "role": "member", "display_name": "Bois, Justin S." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." } ], "option_major": [ "microbio" ], "doi": "10.7907/n992-ey51", "abstract": "A common feature of all life is the metabolic transformation of energy from the environment to biochemical energy in the organism. While this process is well-characterized in molecular detail for fast-growing or otherwise fast-metabolizing organisms such as humans, many microorganisms subsist in the environment around us with little to no exogenous energy for extended periods, and we have only vague ideas how. Questions about the metabolic mechanisms and rates underpinning these astounding survival capabilities speak to the fundamental question of the lower energetic limits of life. Motivated by this big-picture question in biology, this thesis represents one line of physiological inquiry into a specific anaerobic survival metabolism of Pseudomonas aeruginosa, an opportunistic bacterial pathogen. Pseudomonas is perhaps best known for its characteristic production of colorful, redox-active, secondary metabolites called phenazines that allow a metabolic process called extracellular electron transfer. Phenazine extracellular electron transfer has been previously shown to unlock a slow, anaerobic glucose catabolism that facilitates the survival of energy-limited populations of cells. My thesis work has elucidated the predominant membrane-bound protein complexes involved in phenazine reduction and the predominant subcellular location of reduction for each of the main phenazines produced by Pseudomonas. I show that the survival metabolism powered by these phenazines places them in a true maintenance state where there is no detectable growth in the population at the single-cell level. The metabolic rate of this maintenance was measured and found to be 1,000 times slower than when the cells are growing in aerobic culture, 100 times slower than estimates of maintenance rates made in continuous culture, and 10 times slower than the mean basal metabolic rate estimated across all life on the planet. These results open the door to investigations of metabolic attenuation, a physiological state that underpins microbial survival in nature and disease. In pursuit of these discoveries, various new experimental assays that allow further investigation into the bioenergetics and biochemistry of phenazine metabolism were developed. Finally, intellectual frameworks are presented that, in conjunction with the discoveries made and methods developed, collectively bring us steps closer to understanding the bioenergetic basis of microbial resiliency." }, { "name": "Duan, Mengtong (Tom)", "degree": "PhD", "year": "2024", "title": "Expanding Frontiers in Biomedical Imaging and Synthetic Biology: Dynamic Acoustic Reporter Gene Imaging and Ratio-Tuning of Mammalian mRNA Polycistronic Expression", "advisor": "Shapiro, Mikhail G.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06032024-011201674", "creators": [ { "name": { "family": "Duan", "given": "Mengtong (Tom)" }, "id": "Duan-Mengtong-Tom", "orcid": "0000-0002-1601-8876", "display_name": "Duan, Mengtong (Tom)" } ], "advisors": [ { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "advisor", "display_name": "Shapiro, Mikhail G." } ], "committee": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "chair", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Shan", "given": "Shu-ou" }, "id": "Shan-Shu-ou", "orcid": "0000-0002-6526-1733", "role": "member", "display_name": "Shan, Shu-ou" }, { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "member", "display_name": "Shapiro, Mikhail G." } ], "option_major": [ "bioeng" ], "doi": "10.7907/avgk-yc71", "abstract": "This thesis presents a comprehensive exploration of the next generation of mammalian Acoustic Reporter Genes (mARGs), unveiling a novel approach for non-invasive, real-time imaging of cellular processes and gene expression within live animals1. Building on the foundational work of first-generation ARGs2,3, which introduced the groundbreaking concept of using gas vesicle (GV) genes as genetically encoded ultrasound contrast agents, this research tackles the inherent limitations of these pioneering systems. The first segment details the development and characterization of the second-generation mARGs which significantly improve upon their predecessors by offering robust expression without the need for monoclonal screening, dynamic non-destructive imaging capabilities, and customizable acoustic properties through gene and protein level modifications. This advancement not only enhances the utility of mARGs in biomedical imaging but also paves the way for their application in novel therapeutic monitoring strategies, as exemplified by real-time tracking of tumor development and ultrasound-guided tumor biopsies that leverage gene expression information.
\r\n\r\nFurther, the thesis delves into the structural, genetic, and biochemical principles underpinning GV assembly, addressing a critical knowledge gap that has persisted despite the utility of GVs in ultrasound imaging. Understanding these assembly mechanisms is crucial for the engineering of improved ARGs.
\r\n\r\nThe exploration then extends into innovative bioengineering methodologies, specifically Stoichiometric Expression of Messenger Polycistrons by Eukaryotic Ribosomes (SEMPER), a synthetic biology breakthrough enabling the expression of multiple proteins at precise stoichiometries from single, compact transcripts.4 SEMPER represents a strategic advancement in the field, facilitating efficient formation of multi-protein complexes, minimizing cellular toxicity, and broadening the scope of potential applications in genetic engineering, including the creation of enhanced cell lines and circuits for research and therapeutic purposes.
\r\n\r\nCollectively, this work not only advances our understanding of GV-based ultrasound imaging and gene expression tracking but also introduces versatile genetic tools for the manipulation of cellular machinery. These achievements mark significant strides in the fields of synthetic biology and molecular imaging, setting the stage for future innovations in non-invasive diagnostics, cellular therapy, and cancer monitoring research. Through the integration of improved acoustic reporter genes, insights into gas vesicle assembly, and the SEMPER method for gene expression, this thesis embodies a holistic approach to overcoming current challenges and unlocking new potentials in biomedical engineering and synthetic biology.
" }, { "name": "Griffiths, Jessica Anne", "degree": "PhD", "year": "2024", "title": "Bidirectional Interactions Between the Gut Microbiome and Nervous System", "advisor": "Mazmanian, Sarkis K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02042024-025331192", "creators": [ { "name": { "family": "Griffiths", "given": "Jessica Anne" }, "id": "Griffiths-Jessica-Anne", "orcid": "0000-0002-5586-1567", "display_name": "Griffiths, Jessica Anne" } ], "advisors": [ { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "orcid": "0000-0003-2713-1513", "role": "advisor", "display_name": "Mazmanian, Sarkis K." } ], "committee": [ { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "chair", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Lois", "given": "Carlos" }, "id": "Lois-Carlos", "orcid": "0000-0002-7305-2317", "role": "member", "display_name": "Lois, Carlos" }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "orcid": "0000-0003-2713-1513", "role": "member", "display_name": "Mazmanian, Sarkis K." } ], "option_major": [ "bioeng" ], "abstract": "There is roughly one microbe for every human cell in your body. Though some are inconsequential hitchhikers, and some are potentially harmful, many perform beneficial roles. This thesis focuses on the function and interaction of resident microbes within laboratory mice, with the hope that it may translate to us as humans. Chapter (1) highlights recent findings of microbiome involvement in neurologic disorders. Each subsequent chapter presents a different interaction between the mammalian nervous system and gut microbiome. (2) Excitatory signaling in the brain is partially regulated by a genetic factor (Shank3), which is further modulated by environmental interactions through presence or absence of the gut microbiome. This genetic factor implicated in brain and behavior also affects gastrointestinal function and inflammation susceptibility. (3) Applying powerful genetic tools developed for the brain to the enteric nervous system reveals the impact of different enteric neuron populations on gut motility and fluid secretion as well as the immune system, pancreatic activity, and microbial populations. (4) Common opinion has shifted from the belief that microbes are primarily pathogens to viewing them as symbiotic organisms. With this paradigm shift, the artificially clean laboratory mouse microbiome has been found to stunt the immune system, and is being reevaluated. Male mice with natural \u201cwild\u201d microbiomes have altered behavioral and neurological profiles, which may reflect a more physiological state." }, { "name": "Guo, Jimmy Kang", "degree": "PhD", "year": "2024", "title": "Defining the Universe of Functional RNA-Protein Interactions", "advisor": "Guttman, Mitchell", "url": "https://resolver.caltech.edu/CaltechTHESIS:05012024-121056794", "creators": [ { "name": { "family": "Guo", "given": "Jimmy Kang" }, "id": "Guo-Jimmy-Kang", "orcid": "0000-0002-7211-4117", "display_name": "Guo, Jimmy Kang" } ], "advisors": [ { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "advisor", "display_name": "Guttman, Mitchell" } ], "committee": [ { "name": { "family": "Chong", "given": "Shasha" }, "id": "Chong-Shasha", "orcid": "0000-0002-5372-311X", "role": "chair", "display_name": "Chong, Shasha" }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "orcid": "0000-0001-8723-8190", "role": "member", "display_name": "Baltimore, David L." }, { "name": { "family": "Flynn", "given": "Ryan A." }, "id": "Flynn-Ryan-A", "orcid": "0000-0001-5013-0442", "role": "member", "display_name": "Flynn, Ryan A." }, { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "member", "display_name": "Guttman, Mitchell" }, { "name": { "family": "Voorhees", "given": "Rebecca M." }, "id": "Voorhees-R-M", "orcid": "0000-0003-1640-2293", "role": "member", "display_name": "Voorhees, Rebecca M." } ], "option_major": [ "biology" ], "doi": "10.7907/wbvq-bz46", "abstract": "RNA has been proposed to mediate many central mechanisms of cell biology, including protein recruitment to chromatin, genome structure organization, and gene expression. In most cases, these critical functions have been widely attributed to the proteins to which RNAs bind. One paradigm example of this is the Xist long non-coding RNA, which complexes with many distinct proteins to orchestrate X-chromosome inactivation. Beyond Xist, there are many critical non-coding RNAs (ncRNAs) that are not yet functionally characterized because we lack information on what proteins they bind to. In this thesis, Chapter 1 discusses the growing gap between the vast potential of ncRNA functions and what has been demonstrated to be functionally meaningful. We highlight critical discrepancies between biochemical evidence supporting specific RNA-protein interactions and genetic evidence demonstrating the same interactions are often dispensable for function. Chapter 2 explores previously reported RNA-protein interactions for many chromatin proteins (i.e., PRC2, CTCF, etc.), demonstrating that they do not represent bona fide interactions in cells. We present Covalent Linkage Affinity Purification (CLAP), a method that employs denaturing purification of RNA-protein complexes, showing that CLAP accurately removes false signals that do not occur in vivo, while retaining known RNA-protein interactions. Chapter 3 details a highly multiplexed method of mapping RBPs and their in vivo binding sites across dozens to hundreds of targets within a single experiment. We present Split and Pool Identification of RBP targets (SPIDR), which enables the rapid, de novo discovery of RNA-protein interactions at an unprecedented scale and separates bona fide RBPs from non-RBPs. Using SPIDR, we uncover a previously unknown LARP1 binding site on the 18S ribosomal RNA that is directly adjacent to the mRNA entry channel, which may explain how LARP1 achieves translational control of sequence-specific mRNAs. Finally, Chapter 4 proposes new experimental and analytical approaches to evaluate the potentially wide universe of ncRNA-protein functions at scale. Together, these results provide a comprehensive framework for evaluating RNA-protein interactions and underscore the growing importance of RNA-mediated functions in cell biology." }, { "name": "Hill, Andrew James", "degree": "PhD", "year": "2024", "title": "The Genetic and Neuronal Substrates of Melatonin Signaling in Zebrafish Sleep", "advisor": "Prober, David A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:09282023-003458037", "creators": [ { "name": { "family": "Hill", "given": "Andrew James" }, "id": "Hill-Andrew-James", "orcid": "0000-0002-4621-0500", "display_name": "Hill, Andrew James" } ], "advisors": [ { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "orcid": "0000-0002-7371-4675", "role": "advisor", "display_name": "Prober, David A." } ], "committee": [ { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "chair", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Hong", "given": "Elizabeth J." }, "id": "Hong-Elizabeth-J", "orcid": "0000-0003-3866-418X", "role": "member", "display_name": "Hong, Elizabeth J." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "orcid": "0000-0002-7371-4675", "role": "member", "display_name": "Prober, David A." } ], "option_major": [ "biology" ], "doi": "10.7907/4sba-6h56", "abstract": "Sleep is hypothesized to be regulated by two processes: a circadian drive, which communicates time of day to ensure that sleep is timed to the appropriate day/night phase, and a homeostatic drive, by which the propensity for sleep becomes stronger over the course of prolonged wakefulness. While studies suggest that adenosine and serotonin signaling in part mediate the homeostatic sleep drive, factors that act downstream of the circadian clock to promote sleep were unidentified until recently. Previous work in the Prober lab has shown that the nocturnal hormone melatonin acts downstream of the circadian rhythm to promote sleep in zebrafish. The downstream processes by which melatonin promotes sleep is poorly understood across all animal models. This is likely because melatonin research has been primarily conducted using nocturnal laboratory rodent models, in whom melatonin does not seem to play a role in sleep, and because of the widely held view that melatonin informs the circadian clock and does not promote sleep directly. In Chapter 1 of this thesis, I review some of the research conducted over the last 50 years that has informed our current understanding of melatonin and its role in sleep. In Chapter 2, I describe our efforts to use the zebrafish, in which melatonin is both potently sedating and essential for nightly sleep, to uncover some of the mechanisms by which melatonin might promote sleep. We found that melatonin acts through a particular melatonin receptor family called MT1, whereas melatonin receptors belonging to other families were dispensable for sleep. We show that MT1 receptors are expressed broadly throughout the zebrafish brain and are enriched in brain regions involved in sensory processing, particularly in those related to vision. We tested the hypothesis that melatonin promotes sleep, at least in part, by dampening visual responsiveness at night. We show that, separable from sleep, exogenous melatonin suppresses behavioral responses to light stimuli, and loss of endogenous melatonin results in day-like behavioral responses to light stimuli during the night. We are using whole brain imaging in live zebrafish to corroborate our behavioral results with neuronal GCaMP recordings. We hope that the findings presented here contribute to a greater understanding of melatonin\u2019s role in sleep, which may help enhance its value as a natural therapeutic aid." }, { "name": "Ivy, Tobin William", "degree": "PhD", "year": "2024", "title": "Implementing and Modeling Gene Drives for Population Modification and Suppression", "advisor": "Hay, Bruce A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06042024-163846436", "creators": [ { "name": { "family": "Ivy", "given": "Tobin William" }, "id": "Ivy-Tobin-William", "orcid": "0000-0002-9116-3854", "display_name": "Ivy, Tobin William" } ], "advisors": [ { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "advisor", "display_name": "Hay, Bruce A." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Bois", "given": "Justin S." }, "id": "Bois-J-S", "orcid": "0000-0001-7137-8746", "role": "member", "display_name": "Bois, Justin S." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "member", "display_name": "Hay, Bruce A." } ], "option_major": [ "biochem" ], "doi": "10.7907/10pa-x574", "abstract": "Gene drive as a technology has immense potential for modifying species from the local to the global population level. While the advent of the CRISPR-Cas9 system has paved the way for many previously untenable gene drives, it has also illuminated two substantial pitfalls: homing based gene drives are particularly susceptible to generating drive breaking resistance alleles and many if not most gene drives are too powerful to be regionally contained. Lack of confinability makes such gene drives impractical for real world application where international law would be violated by their usage. We developed a new general form of gene drive known as cleave and rescue (ClvR), then built and simulated the potential of multiple variants of this drive which are capable of modifying or suppressing target populations, including variants with and without introduction thresholds for drive. We also developed scripts in Python capable of generating population dynamics simulations of a user-defined gene drive, either as a deterministic, population proportion model or a stochastic, discrete individual model. This tool provides a very useful first pass answer about a given gene drive\u2019s ability to modify or suppress a population under varying fitness costs, drive activity rates, and release proportions." }, { "name": "Jiang, Jialong", "degree": "PhD", "year": "2024", "title": "Revealing Regulatory Network Organization Through Single-Cell Perturbation Profiling and Maximum Entropy Models", "advisor": "Thomson, Matthew", "url": "https://resolver.caltech.edu/CaltechTHESIS:06032024-182223499", "creators": [ { "name": { "family": "Jiang", "given": "Jialong" }, "id": "Jiang-Jialong", "orcid": "0000-0001-8560-8397", "display_name": "Jiang, Jialong" } ], "advisors": [ { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "advisor", "display_name": "Thomson, Matthew" } ], "committee": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "chair", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." }, { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "member", "display_name": "Pachter, Lior S." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" } ], "option_major": [ "sysbiol" ], "doi": "10.7907/5zta-9818", "abstract": "Gene regulatory networks within cells modulate the expression of the genome in response to signals and changing environmental conditions. Reconstructions of gene regulatory networks can reveal the information processing and control principles used by cells to maintain homeostasis and execute cell-state transitions. In this thesis, we introduce a computational framework, D-SPIN, that generates quantitative models of gene regulatory networks from single-cell mRNA-seq datasets collected across thousands of distinct perturbation conditions. D-SPIN models the cell as a collection of interacting gene-expression programs, and constructs a probabilistic model to infer regulatory interactions between gene-expression programs and external perturbations. Using large Perturb-seq and drug-response datasets, we demonstrate that D-SPIN models reveal the organization of cellular pathways, sub-functions of macromolecular complexes, and the logic of cellular regulation of transcription, translation, metabolism, and protein degradation in response to gene knockdown perturbations. D-SPIN can also be applied to dissect drug response mechanisms in heterogeneous cell populations, elucidating how combinations of immunomodulatory drugs can induce novel cell states through additive recruitment of gene expression programs. D-SPIN provides a computational framework for constructing interpretable models of gene-regulatory networks to reveal principles of cellular information processing and physiological control." }, { "name": "Johnston, Kadina Elizabeth", "degree": "PhD", "year": "2024", "title": "Acquiring Enzyme Sequence-Fitness Data at Scale Toward Predictive Methods for Enzyme Engineering", "advisor": "Arnold, Frances Hamilton", "url": "https://resolver.caltech.edu/CaltechTHESIS:09212023-182422125", "creators": [ { "name": { "family": "Johnston", "given": "Kadina Elizabeth" }, "id": "Johnston-Kadina-Elizabeth", "orcid": "0000-0002-2214-3534", "display_name": "Johnston, Kadina Elizabeth" } ], "advisors": [ { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "display_name": "Arnold, Frances Hamilton" } ], "committee": [ { "name": { "family": "Yue", "given": "Yisong" }, "id": "Yue-Yisong", "orcid": "0000-0001-9127-1989", "role": "chair", "display_name": "Yue, Yisong" }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Bois", "given": "Justin S." }, "id": "Bois-J-S", "orcid": "0000-0001-7137-8746", "role": "member", "display_name": "Bois, Justin S." }, { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "member", "display_name": "Arnold, Frances Hamilton" } ], "option_major": [ "bioeng" ], "doi": "10.7907/xjz4-k217", "abstract": "The emergence of machine learning methods for expediting directed evolution via protein fitness prediction has recently shed light on the need for more, high quality sequence-fitness data from which to learn the mapping from sequence to fitness. Enzymes specifically are highly selective catalysts and engineered enzymes are becoming increasingly important for human applications such as pharmaceutical synthesis. This thesis thus focuses on the collection of enzymatic sequence-fitness data to enable both development and validation of emerging approaches. Chapter 1 describes the process of traditional directed evolution as well as ways that machine learning methods have been used to accelerate it. It also discusses the experimental considerations for applying machine learning to the various steps of protein engineering campaigns, as the experimental constraints are not always obvious to the machine learning community. One of the major constraints for the application of machine learning methods is the requirement to sequence all variants required for model training, a step that is often skipped by traditional, lab-only directed evolution due to it not being worth the time and cost. Chapter 2 introduces a solution to this problem with \u201cevery variant sequencing\u201d (evSeq), which enables higher throughput collection of sequencing data for a similar time and cost as commonly used Sanger sequencing methods. This method not only enables implementation of ML methods such as machine learning-assisted directed evolution (MLDE) and focused training MLDE (ftMLDE) by sequencing variants during an evolution campaign, but also offers promise to fill existing protein sequence-fitness databases with protein engineering datasets. This type of data collection can enable the development of newer, more accurate ML methods, and was an inspiration for the work presented in Chapter 3, which details the collection of a combinatorially complete, epistatic sequence-fitness landscape in an enzyme active site. Oftentimes, the effects of mutations on protein fitness can be considered largely independent and laboratory recombination of them can find an optimal variant. This general principle breaks down when the effects of mutations are not independent, termed epistasis, and sequence-fitness landscapes with these interactions are difficult to traverse. Thus, collection of this dataset provides a challenging task for the development of both ML and physics-based models and pushes the boundary of predictive methods for protein engineering." }, { "name": "Lee, Justin", "degree": "PhD", "year": "2024", "title": "Ultrasound Control and Imaging of Cellular Immunotherapy", "advisor": "Shapiro, Mikhail G.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03132024-155539035", "creators": [ { "name": { "family": "Lee", "given": "Justin" }, "id": "Lee-Justin", "orcid": "0000-0002-3657-4386", "display_name": "Lee, Justin" } ], "advisors": [ { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "advisor", "display_name": "Shapiro, Mikhail G." } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "chair", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Wang", "given": "Kaihang" }, "id": "Wang-Kaihang", "orcid": "0000-0001-7657-8755", "role": "member", "display_name": "Wang, Kaihang" }, { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "member", "display_name": "Shapiro, Mikhail G." } ], "option_major": [ "bioeng" ], "doi": "10.7907/60sh-a389", "abstract": "Biomedical ultrasound-based therapeutics and diagnostics are becoming an increasingly important clinical tool. Techniques like focused ultrasound tissue heating and microbubble-enhanced ultrasound imaging have enabled new ways to noninvasively treat and detect diseases cost-effectively and safely. While these are great leaps forward in ultrasound technology, leveraging synthetic biology tools to engineer cells with the capabilities to interact with ultrasound in novel ways may enable even more avenues for ultrasound to address important clinical challenges.
\r\n\r\nIn this thesis, we explore the potential in engineering immune cells with various genetic elements which interact with either therapeutic or diagnostic ultrasound in novel ways. In Chapter 2, we engineer T-cells capable of sensing increases in temperature and responding by activating expression of therapeutic proteins to potentially increase safety of cell-based immunotherapies by controlling their spatiotemporal activation. In Chapters 3 and 4, we develop monocytes as ultrasound reporter cells for cancer detection by engineering them to express gas vesicles (GVs), a class of air-filled protein nanostructures natively found in certain aquatic microbes, which have been demonstrated to produce ultrasound contrast. We demonstrate the potential to confine GV expression to certain disease related signals to create ultrasound reporter cells. Together, these findings highlight the potential of engineering cells to activate in certain locations in response to ultrasound heating or serve as sentinel cells for disease detection.
" }, { "name": "Liu, Mengyu", "degree": "PhD", "year": "2024", "title": "Love and War: Control of Female Social Behaviors by the Hypothalamus", "advisor": "Anderson, David J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01112024-021433003", "creators": [ { "name": { "family": "Liu", "given": "Mengyu" }, "id": "Liu-Mengyu", "orcid": "0000-0003-1548-0679", "display_name": "Liu, Mengyu" } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "advisor", "display_name": "Anderson, David J." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "member", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "member", "display_name": "Adolphs, Ralph" }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." } ], "option_major": [ "neurobio" ], "abstract": "Aggressive and mating behaviors, crucial for survival, are inherently programmed in the brain and are orchestrated by genetically defined cell types within subcortical circuits. Despite the hard-wired nature, females exhibit a remarkable ability to flexibly adjust these behaviors to according to their reproductive states. The neural mechanisms governing the stable control and adaptive regulation of these behaviors remained unclear. Moreover, given the inherently complex and dynamic nature social interactions, the dynamics of the underlying motivational states and their encoding in the female brain was largely unknown.
\r\n\r\nAddressing these knowledge gaps in my thesis, I initially undertook a dissection of the subcortical circuits and genetically defined cell types involved in the control of female aggressive and mating behaviors. Using single-cell RNA sequencing and optogenetic perturbations, I identified distinct transcriptomic cell types in the ventromedial hypothalamus: \u03b1 cells governing mating and \u00df cells regulating aggression. Furthermore, longitudinal monitoring of their activity during the transition from virginity to motherhood revealed that \u00df cells became more responsive to social cues, resulting a shift from mating to aggression. In a second line of investigation, I delved into the dynamics of female mating and its neural encoding. By monitoring single-cell activity in receptive females and applying dynamical system modeling to neural activity, I uncovered that \u03b1 cell formed line attractor dynamics, encoding a sexual aroused state during mating. Additionally, longitudinal monitoring of activity across different hormonal states revealed population dynamics displaying receptivity state-dependent patterns across the estrus cycle. A third aspect of my research explored comprehensive changes in gene expression patterns in circuits influenced by hormones. Through comparative analysis of transcriptomic profiles in the ventromedial hypothalamus at different hormonal states. I identified qualitative changes in cell types within mating-activated \u03b1 cells, correlated with sexual receptivity. These studies significantly contribute to our understanding of the neural basis controlling aggressive and mating behaviors, shedding light on their flexible regulation by physiological conditions in females.
" }, { "name": "Luebbert, Laura", "degree": "PhD", "year": "2024", "title": "Complexity of Transcriptomic Data Analysis and Implications for Biological Discovery", "advisor": "Pachter, Lior S.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05042024-011724418", "creators": [ { "name": { "family": "Luebbert", "given": "Laura" }, "id": "Luebbert-Laura", "orcid": "0000-0003-1379-2927", "display_name": "Luebbert, Laura" } ], "advisors": [ { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "advisor", "display_name": "Pachter, Lior S." } ], "committee": [ { "name": { "family": "Van Valen", "given": "David A." }, "id": "Van-Valen-D", "orcid": "0000-0001-7534-7621", "role": "chair", "display_name": "Van Valen, David A." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "member", "display_name": "Pachter, Lior S." } ], "option_major": [ "biology" ], "doi": "10.7907/xnw5-v914", "abstract": "Over the past decade, the advancement of \u2018omics\u2019 technologies has ushered in a new era for the life sciences. Given the high-throughput nature of omics technologies, this era is characterized by unique computational challenges pertaining to data size and dimensionality, and technical and biological noise. Concurrently, it offers opportunities, as global, untargeted, and parallel measurement of large amounts of information often captures unexpected insights.
\r\n\r\nThis thesis describes challenges inherent to the omics era of life sciences, particularly highlighting the increasing importance of merging expertise in biology and computer science. It describes the development of multiple software tools designed to address several of these challenges, which were immediately adopted and widely implemented in transcriptomics and proteomics research. Additionally, it contains three chapters focused on unraveling previously unquantifiable information, including the interpretation of sequencing data from organisms with low-quality reference genome assemblies and workflows for identifying novel viruses using single-cell RNA sequencing data already massively generated in research, healthcare, and agriculture.
" }, { "name": "Mayfield, Acacia Michelle Hori", "degree": "PhD", "year": "2024", "title": "Customized and Modular Control of Gene Expression for Precision Gene Therapies", "advisor": "Gradinaru, Viviana", "url": "https://resolver.caltech.edu/CaltechTHESIS:05312024-185303498", "creators": [ { "name": { "family": "Mayfield", "given": "Acacia Michelle Hori" }, "id": "Mayfield-Acacia-Michelle-Hori", "orcid": "0000-0001-7308-6480", "display_name": "Mayfield, Acacia Michelle Hori" } ], "advisors": [ { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "advisor", "display_name": "Gradinaru, Viviana" } ], "committee": [ { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "chair", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "member", "display_name": "Gradinaru, Viviana" } ], "option_major": [ "bioeng" ], "doi": "10.7907/d05v-0t68", "abstract": "Genetic disorders are caused by mutations in essential genes that disturb the abundance or function of proteins, tipping cells and tissues from homeostatic harmony into disorder. Developing treatment for genetic diseases involves precision approaches, as gene therapies target the root causes of highly specific pathologic processes at the level of gene replacement, editing, or downstream compensation for a harmful genetic change. Safe access to these cell populations, and the ability to control the behavior of therapeutic cargo after delivery to target tissues, will enable the field to develop safe and effective therapies with the potential to be curative. Systemically delivered AAVs can noninvasively target therapeutic genetic cargo to diverse disease loci throughout the body, but at high doses required for therapeutic penetrance of naturally occurring serotypes, these vectors can cause severe toxicity, emphasizing the need for both targeted, efficient gene delivery vectors, and other means of transgene expression control. This work describes three examples of AAV capsid and cargo design strategies that seek to control where, when, and at what level therapeutic transgene expression can be achieved in a preclinical context. First, we utilize native putative regulatory elements to encourage physiologic level of ectopic frataxin expression in a mouse model of Friedreich\u2019s Ataxia, finding that when delivered to both the brain and peripheral nervous system, treatment prevents progression of motor and coordination deficits. Next, we utilize the genetic incoherent feedforward loop circuit motif at the RNA level to decouple vector delivery level from transgene expression level of MeCP2 in a mouse model of Rett Syndrome, finding that when regulated to near endogenous healthy levels of RNA, AAV-MeCP2-IFFL enables behavioral rescue without overexpression toxicity. Lastly, we employ the mechanism for AAV-genome stability in vivo to modulate expression using a post-hoc AAV administration. Together, these methods and applications demonstrate that modular and custom approaches can improve the precision, safety and efficacy problems that the gene therapy field needs in order to advance more treatments for rare disorders." }, { "name": "Perez, Andrew Alexander", "degree": "PhD", "year": "2024", "title": "ChIP-DIP: a Multiplexed Method for Mapping Proteins to DNA Uncovers Combinatorics Controlling Gene Expression", "advisor": "Guttman, Mitchell", "url": "https://resolver.caltech.edu/CaltechTHESIS:05262024-003033193", "creators": [ { "name": { "family": "Perez", "given": "Andrew Alexander" }, "id": "Perez-Andrew-Alexander", "orcid": "0000-0002-8723-4859", "display_name": "Perez, Andrew Alexander" } ], "advisors": [ { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "advisor", "display_name": "Guttman, Mitchell" } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Fejes T\u00f3th", "given": "Katalin" }, "id": "Fejes-T\u00f3th-K", "orcid": "0000-0001-6558-2636", "role": "member", "display_name": "Fejes T\u00f3th, Katalin" }, { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "member", "display_name": "Guttman, Mitchell" } ], "option_major": [ "biology" ], "doi": "10.7907/691f-y924", "abstract": "Gene regulation is governed by the complex interplay between thousands of regulatory proteins and chromatin states; understanding how these dynamics give rise to precisely controlled, cell type-specific gene expression has been a central goal of molecular biology. Yet, addressing this goal remains challenging because current methods for mapping proteins to DNA are labor-intensive, resource-demanding, and limited to studying a single or a small number of proteins at a time. To overcome this, we developed ChIP-DIP (ChIP Done In Parallel), a novel split-pool-based method that enables simultaneous, genome-wide mapping of hundreds of diverse regulatory proteins in a single experiment. We demonstrate that ChIP-DIP generates highly accurate maps equivalent to traditional approaches, with data quality unaffected by the number of distinct proteins or the composition of proteins measured within a single experiment. We show that, because of this multiplexed capability, ChIP-DIP enables generation of highly accurate maps using several orders of magnitude fewer cells per protein compared to traditional approaches (~30,000 fold), making it a powerful tool for studying a diverse array of proteins\u2013DNA interactions with limited cellular input. In addition, we show that ChIP-DIP can generate high-quality maps for all classes of DNA-associated proteins, including histone modifications, chromatin regulators, transcription factors, and RNA polymerases. Using these data, we explore quantitative combinations of histone modifications and integrate these signatures with RNA polymerase activity, chromatin regulatory protein binding and transcription factor binding to define distinct classes of regulatory elements (e.g., distinct types of enhancer elements), their functional activity (e.g., transcriptional activity), and their regulatory potential (e.g., poised for activation upon stimulation or differentiation). Together, our results demonstratethat ChIP-DIP enables generation of consortium-level data within a single lab and highlight the importance of this approach for studying mechanisms of gene regulation in a context and cell type-specific manner. Lastly, ChIP-DIP provides a powerful platform to multiplex protein detection and provides a unique opportunity to incorporate other split-pool-based assays such as SPRITE and single-cell SPRITE to detect protein specific nuclear structures and multiplexed single-cell chromatin profiles, respectively. This work represents a transformational framework on how to study biology in a holistic manner." }, { "name": "Ponce, Francesca V.", "degree": "PhD", "year": "2024", "title": "The Role of Integral Gain and its Neuromuscular Implementation in the Flight Control System of Drosophila", "advisor": "Dickinson, Michael H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06112024-013230923", "creators": [ { "name": { "family": "Ponce", "given": "Francesca V." }, "id": "Ponce-Francesca-V", "orcid": "0000-0002-9070-2780", "display_name": "Ponce, Francesca V." } ], "advisors": [ { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "orcid": "0000-0002-8587-9936", "role": "advisor", "display_name": "Dickinson, Michael H." } ], "committee": [ { "name": { "family": "Hong", "given": "Elizabeth J." }, "id": "Hong-Elizabeth-J", "orcid": "0000-0003-3866-418X", "role": "chair", "display_name": "Hong, Elizabeth J." }, { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "orcid": "0000-0001-8837-678X", "role": "member", "display_name": "Siapas, Athanassios G." }, { "name": { "family": "Parker", "given": "Joseph" }, "id": "Parker-J", "orcid": "0000-0001-9598-2454", "role": "member", "display_name": "Parker, Joseph" }, { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "orcid": "0000-0002-8587-9936", "role": "member", "display_name": "Dickinson, Michael H." } ], "option_major": [ "biology" ], "doi": "10.7907/j2mb-ka39", "abstract": "Small flying insects, such as the fruit fly Drosophila melanogaster, can navigate along a relatively straight path for long distances. During these journeys, they need a long-range navigational strategy to maintain a constant heading over time, as well as stabilizing behaviors to deal with perturbations, such as a sudden gust of wind or a broken wing. In this work, I characterize a behavioral strategy flies use to maintain stable flight using a combination of experimental and control theoretic approaches. I then investigate how this control strategy is implemented in the flight motor system. In Chapter 1, I describe release-and-recapture experiments performed in the Mojave Desert to investigate how flies interact with the wind to travel long distances. These experiments provide key insight into the dispersal behavior of small insects and suggest that these animals employ a single algorithm that is functionally robust in both still air and under windy conditions. In Chapter 2, I present an extensive set of behavioral experiments showing that the optomotor response, a well-described stabilizing flight reflex, can be accurately modelled by a proportional-integral controller. I also show simulations that exemplify the potential functional advantage of this controller model in natural flight conditions. In Chapter 3, I show the results from muscle imaging experiments designed to investigate how the integral gain of a proportional integral controller might be implemented within the flight motor system. Finally, in Chapter 4, I summarize the main findings, and discuss the limitations of this work and future directions.
" }, { "name": "Prabhutendolkar, Aditee Amit", "degree": "Senior Thesis", "year": "2024", "title": "The Role of Kinetochores in Mammalian Neural Development", "advisor": "Kennedy, Mary B.; Schwarz, Thomas l.; Zhao, Guoli", "url": "https://resolver.caltech.edu/CaltechTHESIS:09252024-235356573", "creators": [ { "name": { "family": "Prabhutendolkar", "given": "Aditee Amit" }, "id": "Aditee Amit", "orcid": "0009-0006-7467-6777", "display_name": "Prabhutendolkar, Aditee Amit" } ], "advisors": [ { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "advisor", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Schwarz", "given": "Thomas l." }, "id": "Schwarz-Thomas-L", "role": "co-advisor", "display_name": "Schwarz, Thomas l." }, { "name": { "family": "Zhao", "given": "Guoli" }, "id": "Zhao-Guoli", "orcid": "0000-0002-7807-5980", "role": "co-advisor", "display_name": "Zhao, Guoli" } ], "committee": [ { "name": { "family": "None", "given": "None" }, "display_name": "None, None" } ], "option_major": [ "cns" ], "doi": "10.7907/1gqg-p428", "abstract": "The kinetochore is a protein complex found at the centromere of chromosomes. It plays an essential role in mitosis, but our lab is investigating the role of the kinetochore in a surprising post-mitotic setting. Specifically, we are focusing on the presence of kinetochores in the synapses and axons of mammalian nerve cells, implying that kinetochores play a key role in neural development.
\r\n\r\nHere we have engineered strains of mouse embryonic hippocampal nerve cells with certain kinetochore-encoding genes knocked out, and then fluorescently imaged their axonal growth cones. To quantify the change in dynamicity of the growth cones after gene knockout, we used image classification and machine learning techniques and found statistically significant results between the knockout and wildtype strains of nerve cells, indicating that kinetochores are essential to functional axon growth.
" }, { "name": "Prakash, Sharan Jagdish", "degree": "PhD", "year": "2024", "title": "Experimental and Neuroinformatic Definition of Neural Circuits in Caenorhabditis elegans", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:11162023-182820794", "creators": [ { "name": { "family": "Prakash", "given": "Sharan Jagdish" }, "id": "Prakash-Sharan-Jagdish", "orcid": "0000-0003-1777-0987", "display_name": "Prakash, Sharan Jagdish" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "chair", "display_name": "Hay, Bruce A." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Hong", "given": "Elizabeth J." }, "id": "Hong-Elizabeth-J", "orcid": "0000-0003-3866-418X", "role": "member", "display_name": "Hong, Elizabeth J." } ], "option_major": [ "biology" ], "doi": "10.7907/bgvy-9p06", "abstract": "The free-living nematode Caenorhabditidis elegans is an established model organism for research in molecular genetics, cell and developmental biology, evolution, and neuroscience. This thesis describes two research projects in C. elegans neuroscience. The first project concerns the challenge of synthesizing the accumulating neurobiological literature in C. elegans. I describe how an established framework for semantic modelling of cellular pathways can be adapted for semantic modelling of neural circuits and functional annotation of the nervous system, and its potential applications for systems neuroscience. A second portion describes a series of experiments investigating a decision-making process in C. elegans larval development that is under neuronal control. C. elegans larvae have the ability to decide among alternative developmental trajectories based on environmental conditions, that are detected via its nervous system. In this work, I describe the contribution of several neurons to this decision-making process, and our discoveries about the response properties of two neurons to ethologically relevant chemical stimuli." }, { "name": "Sanfiorenzo, Charles John", "degree": "PhD", "year": "2024", "title": "Design and Construction of Bacterial Genomes at the Megabase-Scale", "advisor": "Wang, Kaihang", "url": "https://resolver.caltech.edu/CaltechTHESIS:05312024-213823439", "creators": [ { "name": { "family": "Sanfiorenzo", "given": "Charles John" }, "id": "Sanfiorenzo-Charles-John", "orcid": "0009-0004-4652-3744", "display_name": "Sanfiorenzo, Charles John" } ], "advisors": [ { "name": { "family": "Wang", "given": "Kaihang" }, "id": "Wang-Kaihang", "orcid": "0000-0001-7657-8755", "role": "advisor", "display_name": "Wang, Kaihang" } ], "committee": [ { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "orcid": "0000-0002-3904-0195", "role": "chair", "display_name": "Goentoro, Lea A." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Wang", "given": "Kaihang" }, "id": "Wang-Kaihang", "orcid": "0000-0001-7657-8755", "role": "member", "display_name": "Wang, Kaihang" } ], "option_major": [ "biology" ], "doi": "10.7907/yapk-c742", "abstract": "Building genomic chimeras would enable melding of the diverse functions and properties of life. However, prior arts in genome synthesis are limited to reconstituting functions within singular genomes rather than combining diverse genomic functions across multiple distinct genomes. Existing methods are also prohibitively expensive, laborious, time-consuming, and not scalable for creating large genomes. Addressing these limitations, the author invented Additive Conjugative-CAST Engineering (ACE) combining conjugation with CRISPR-associated transposition (CAST) to deliver and integrate up to half a genome per step from a donor into a precisely defined position in the recipient\u2019s genome. This work demonstrates ACE\u2019s engineering capacities integrating a 2-megabase donor genomic segment in a single step and at least three megabase in two steps. Importantly, ACE\u2019s generality is confirmed through the creation of genomic chimeras across species, genus, order, and class barriers. With ACE, this work further showcases that such chimeric organisms, denoted genome expanded organisms (GEOs), can be forged from at least three starting bacterial strains, and are stably maintained to express all acquired genomic parts. Principles confounding ACE are expanded onto genome editing technologies, such as Prime Editing, and further explored for the megabase-scale transfer of DNA into eukaryotes. ACE and derivative technologies thereof offer a new paradigm of creating artificial lifeforms to combine and potentially create novel functions beyond the constraints of nature, while probing and elucidating genome plasticity, architecture, and expression patterns of GEOs.
" }, { "name": "Schwartz, Morgan Sarah", "degree": "PhD", "year": "2024", "title": "Accelerating Biological Discovery with Deep Learning and Spatial Optical Barcodes", "advisor": "Van Valen, David A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05282024-221603734", "creators": [ { "name": { "family": "Schwartz", "given": "Morgan Sarah" }, "id": "Schwartz-Morgan-Sarah", "orcid": "0000-0001-8131-9125", "display_name": "Schwartz, Morgan Sarah" } ], "advisors": [ { "name": { "family": "Van Valen", "given": "David A." }, "id": "Van-Valen-D", "orcid": "0000-0001-7534-7621", "role": "advisor", "display_name": "Van Valen, David A." } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "chair", "display_name": "Thomson, Matthew" }, { "name": { "family": "Cai", "given": "Long" }, "id": "Cai-Long", "orcid": "0000-0002-7154-5361", "role": "member", "display_name": "Cai, Long" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Van Valen", "given": "David A." }, "id": "Van-Valen-D", "orcid": "0000-0001-7534-7621", "role": "member", "display_name": "Van Valen, David A." } ], "option_major": [ "biology" ], "doi": "10.7907/55c7-8142", "abstract": "Methodological advances in biology have given us a powerful suite of tools for measuring the state of the cell. Among these methods, next-generation sequencing, including single-cell methods, enables comprehensive measurement of gene expression; however, sequencing-based methods often preclude the collection of other visible phenotypic information. In contrast, light microscopy supports many different measurements that can be acquired in sequential rounds of labeling and imaging because light microscopy does not destroy the sample. Furthermore, light microscopy supports live cell imaging, including the use of fluorescent reporters to observe signaling dynamics in real time. In order to fully understand cellular function, multimodal data collection is needed that encompasses live cell response, end-point phenotypes, and finally perturbations to test the components of relevant signaling networks. In this thesis, I present key advances to create a unified experimental platform for interrogating the cell state. This platform uses light microscopy to collect multimodal measurements of cell state while supporting high-throughput perturbation screening. This platform is supported by a suite of deep learning analysis tools to enable quantitative analysis of these high-dimensional datasets. In Chapter 2, I introduce Caliban, our deep learning method for nuclear segmentation and tracking. In Chapter 3, I present a new method of optical barcodes to enable microscopy-based pooled perturbation screens. Finally, in Chapter 4, I describe preliminary work that leverages the previously described cell tracking and barcoding methodologies to explore the interdependencies of signaling pathway dynamics." }, { "name": "Strehle, Mackenzie", "degree": "PhD", "year": "2024", "title": "Mechanisms of Xist-Mediated Gene Silencing During the Initiation and Maintenance of X Chromosome Inactivation", "advisor": "Guttman, Mitchell", "url": "https://resolver.caltech.edu/CaltechTHESIS:04182024-125718373", "creators": [ { "name": { "family": "Strehle", "given": "Mackenzie" }, "id": "Strehle-Mackenzie", "orcid": "0000-0003-1410-8701", "display_name": "Strehle, Mackenzie" } ], "advisors": [ { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "advisor", "display_name": "Guttman, Mitchell" } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Zernicka-Goetz", "given": "Magdalena" }, "id": "Zernicka-Goetz-M", "orcid": "0000-0002-7004-2471", "role": "member", "display_name": "Zernicka-Goetz, Magdalena" }, { "name": { "family": "Chong", "given": "Shasha" }, "id": "Chong-Shasha", "orcid": "0000-0002-5372-311X", "role": "member", "display_name": "Chong, Shasha" }, { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "member", "display_name": "Guttman, Mitchell" } ], "option_major": [ "molbiochem" ], "doi": "10.7907/nxq5-hj97", "abstract": "X chromosome inactivation (XCI) is a critical development process during which one of the two X chromosomes in female mammals is silenced to balance gene expression with males. XCI is initiated by upregulation of the long noncoding RNA (lncRNA) Xist from the future inactive X chromosome (Xi), which recruits a variety of proteins in cis to mediate transcriptional repression that is maintained throughout the lifetime of the organism. Recent studies have demonstrated that silencing following Xist expression is dependent on direct recruitment of the transcriptional silencing protein SHARP (also known as SPEN); however, the mechanism underlying formation of the Xi silencing compartment has remained poorly defined. Similarly, it has long been thought that maintenance of XCI occurs independently of Xist and depends on differential DNA methylation enrichment on the Xi, but the evidence in support of these views is lacking. Here, we show how low copy numbers of Xist can recruit SHARP in super-stoichiometric excess to initiate gene silencing on the X and mediate formation of the silent Xi compartment. We also provide preliminary evidence suggesting that maintenance of XCI is Xist independent, but dependent on DNA methylation and histone deacetylation. Together, these results offer a more holistic view of the molecular mechanisms underlying both initiation and maintenance XCI, as well as provide a framework for further investigation into lncRNA biology and epigenetic regulation more broadly." }, { "name": "Wadia, Varun Spenta", "degree": "PhD", "year": "2024", "title": "How We Imagine: Insights from Single Neuron Recordings in the Human Brain", "advisor": "Tsao, Doris Y.; Rutishauser, Ueli", "url": "https://resolver.caltech.edu/CaltechTHESIS:09272023-165737414", "creators": [ { "name": { "family": "Wadia", "given": "Varun Spenta" }, "id": "Wadia-Varun-Spenta", "orcid": "0009-0009-5401-5367", "display_name": "Wadia, Varun Spenta" } ], "advisors": [ { "name": { "family": "Tsao", "given": "Doris Y." }, "id": "Tsao-D-Y", "orcid": "0000-0003-1083-1919", "role": "advisor", "display_name": "Tsao, Doris Y." }, { "name": { "family": "Rutishauser", "given": "Ueli" }, "id": "Rutishauser-U", "orcid": "0000-0002-9207-7069", "role": "co-advisor", "display_name": "Rutishauser, Ueli" } ], "committee": [ { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "orcid": "0000-0003-2136-6506", "role": "chair", "display_name": "Meister, Markus" }, { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "member", "display_name": "Adolphs, Ralph" }, { "name": { "family": "Rutishauser", "given": "Ueli" }, "id": "Rutishauser-U", "orcid": "0000-0002-9207-7069", "role": "member", "display_name": "Rutishauser, Ueli" }, { "name": { "family": "Tsao", "given": "Doris Y." }, "id": "Tsao-D-Y", "orcid": "0000-0003-1083-1919", "role": "member", "display_name": "Tsao, Doris Y." } ], "option_major": [ "neurobio" ], "doi": "10.7907/8nw5-1q14", "abstract": "As human beings interact with the world, we sample it, build representations of its underlying structure, and subsequently use that knowledge for future reasoning - also called modeling the world. This model is generative, allowing our knowledge of the world to influence our perception and in extreme cases induce perception in the absence of any external stimulus. This phenomenon called mental imagery is a remarkable cognitive ability that allows us to remember previous experiences, imagine new ones, make plans, and solve problems. In the visual domain, our ability to generate visual percepts without external stimulation is the basis of our memory for experiences, as episodic memories are simply a subset of all possible experiences we could imagine. Animal studies have yielded rich insight into bottom-up visual processing, from the first discovery of neurons that respond to complex objects like faces to determining the precise code for general objects in macaque inferotemporal (IT) cortex. However, the neural mechanisms of internally generated top-down processing have been much more elusive. Here we present findings on the mechanisms of visual imagery at single neuron resolution. We approached deciphering visual imagery by first laying out coding principles for object perception and then directly comparing responses during viewing to subsequent imagery of those images. We recorded 384 visually responsive neurons in inferotemporal (IT) cortex of 12 epilepsy patients as they viewed and subsequently imagined carefully parametrized visual objects. We verified that neurons in IT cortex are \u2018axis tuned\u2019, i.e. as in macaques they represent visual objects by encoding specific axes that span a high dimensional object feature space. 218/384 visually responsive neurons (~58%) were axis tuned, and the axis model explained more variance than other models tested. Armed with this code for visual objects we examined neural responses during pure imagery in the same neurons. We demonstrate robust reactivation of individual neurons across the brain (~35% of neurons across the brain and ~50% of neurons in IT cortex) and a recapitulation of viewing stimulus preference during pure visual imagery in IT. By first uncovering the code for visual objects and examining it during imagery, we demonstrate that neurons in IT cortex subserve visual imagery by reinstating visual context. This study marks the first detailed exploration of visual perception and imagery in the human brain." }, { "name": "Wagner, Julian Morgan", "degree": "PhD", "year": "2024", "title": "Uncovering Mechanisms of Host Recognition, Host Finding and Host Specificity", "advisor": "Parker, Joseph", "url": "https://resolver.caltech.edu/CaltechTHESIS:05202024-215640209", "creators": [ { "name": { "family": "Wagner", "given": "Julian Morgan" }, "id": "Wagner-Julian-Morgan", "orcid": "0000-0003-3406-0450", "display_name": "Wagner, Julian Morgan" } ], "advisors": [ { "name": { "family": "Parker", "given": "Joseph" }, "id": "Parker-J", "orcid": "0000-0001-9598-2454", "role": "advisor", "display_name": "Parker, Joseph" } ], "committee": [ { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "orcid": "0000-0002-8587-9936", "role": "chair", "display_name": "Dickinson, Michael H." }, { "name": { "family": "Hong", "given": "Elizabeth J." }, "id": "Hong-Elizabeth-J", "orcid": "0000-0003-3866-418X", "role": "member", "display_name": "Hong, Elizabeth J." }, { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "member", "display_name": "Pachter, Lior S." }, { "name": { "family": "Parker", "given": "Joseph" }, "id": "Parker-J", "orcid": "0000-0001-9598-2454", "role": "member", "display_name": "Parker, Joseph" } ], "option_major": [ "biology" ], "doi": "10.7907/k4b2-2n58", "abstract": "Insect diversification is thought to have been catalyzed by widespread specialization on novel hosts\u2014a process underlying exceptional radiations of phytophagous beetles, lepidopterans, parasitoid wasps, and inordinate lineages of symbionts, predators, and other trophic specialists. The fidelity of such interspecies partnerships is often posited to arise from sensory tuning to host-derived cues, a model supported by studies of neural function in host-specific model species. Abundant literature on parasites also suggest that extrinsic factors, namely dispersal mechanisms and aggressiveness/acceptance from novel hosts, externally enforces host specificity. Here, I first review what is known about host specificity, why it arises and how it is controlled, and then explore how these factors influence the biology of myrmecophiles, the intimate symbiotic associates of ants. I then test the mechanisms of host specificity by investigating the chemosensory basis of symbiotic interactions between a myrmecophile rove beetle and its single, natural host ant species. I show that host cues trigger analogous behaviors in both the ant and myrmecophile. Cuticular hydrocarbons\u2014the ant's nestmate recognition pheromones\u2014elicit partner recognition in the myrmecophile and execution of ant grooming behavior that achieves chemical mimicry. The myrmecophile also follows host trail pheromones, permitting inter-colony dispersal. Remarkably, however, the myrmecophile performs these same adaptive behaviors with non-host ants separated by up to ~100-million years and shows minimal preference for its natural host over non-host ant species. Experimentally validated agent-based modelling supports a scenario in which specificity is enforced by physiological constraints on dispersal, and negative fitness interactions with alternative hosts, rather than via sensory tuning. Infrequent realization of latent compatibilities of specialists with alternative hosts may facilitate host switching, and the persistence and diversification of seemingly specialized clades over deep time." }, { "name": "Zhang, Mark Guangde", "degree": "PhD", "year": "2024", "title": "Bridging Sensory Perception to Developmental Decision Making in Caenorhabditis elegans", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05212024-230922611", "creators": [ { "name": { "family": "Zhang", "given": "Mark Guangde" }, "id": "Zhang-Mark-Guangde", "orcid": "0000-0002-0802-300X", "display_name": "Zhang, Mark Guangde" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "orcid": "0000-0002-7371-4675", "role": "chair", "display_name": "Prober, David A." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Hong", "given": "Elizabeth J." }, "id": "Hong-Elizabeth-J", "orcid": "0000-0003-3866-418X", "role": "member", "display_name": "Hong, Elizabeth J." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/hf7p-aa22", "abstract": "Amidst uncertain environmental flux, organisms must be able to appropriately adapt their physiology in response to or in preparation for harsh conditions. To accomplish this task, organisms need to accurately perceive current environmental cues, make informed decisions about how the future environmental landscape might unfold, and execute the genetic programs to manifest the proper physiological changes. As a prime example, the roundworm Caenorhabditis elegans makes multiple developmental decisions during its life cycle in response to environmental cues. During larval growth, C. elegans can forego reproductive growth and instead enter diapause (also called dauer), a developmentally arrested state resistant to environmental stress, in response to unfavorable growth conditions. The decisions to enter and exit dauer involve complex neurogenetic computations that integrate environmental cues, yet despite decades of research in the field of C. elegans dauer biology, we still do not fully understand how such sensory integration occurs. Furthermore, how the dauer entry and exit decisions compare and contrast to one another remains unclear.
\r\n \r\nIn this thesis, I comprehensively analyze the C. elegans dauer exit decision using behavioral analyses, neuronal imaging, and gene reporter technologies. In Chapter Two, I I demonstrate how, during dauer exit, the ASJ chemosensory neurons integrate food availability and population density at both the levels of neuronal calcium dynamics and gene expression. I show that expression of the insulin-like peptide encoding gene ins 6 within the ASJ neurons is responsive to dauer-relevant cues, dependent on ASJ neuronal activity, and participates in an autoregulatory feedback mechanism that enforces decision commitment. In Chapter Three, I analyze how steroid hormones are essential for the dauer exit decision, and I illustrate how the spatiotemporal dynamics of steroid hormone regulation during dauer exit compares with that of dauer entry. Taken together, this thesis significantly advances our knowledge of the C. elegans dauer exit decision and helps us better understand how animals coordinate decisions over long timescales in response to changing environments.
" }, { "name": "Zhu, Wenying", "degree": "Masters", "year": "2024", "title": "Investigating the Dual Processes Underlying Human Recognition Memory", "advisor": "Adolphs, Ralph; Rutishauser, Ueli", "url": "https://resolver.caltech.edu/CaltechTHESIS:12062023-233602875", "creators": [ { "name": { "family": "Zhu", "given": "Wenying" }, "id": "Zhu-Wenying", "orcid": "0000-0003-3103-9250", "display_name": "Zhu, Wenying" } ], "advisors": [ { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "co-advisor", "display_name": "Adolphs, Ralph" }, { "name": { "family": "Rutishauser", "given": "Ueli" }, "id": "Rutishauser-U", "orcid": "0000-0002-9207-7069", "role": "co-advisor", "display_name": "Rutishauser, Ueli" } ], "committee": [ { "name": { "family": "O'Doherty", "given": "John P." }, "id": "O'Doherty-J-P", "orcid": "0000-0003-0016-3531", "role": "chair", "display_name": "O'Doherty, John P." }, { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "member", "display_name": "Adolphs, Ralph" }, { "name": { "family": "Rutishauser", "given": "Ueli" }, "id": "Rutishauser-U", "orcid": "0000-0002-9207-7069", "role": "member", "display_name": "Rutishauser, Ueli" }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "member", "display_name": "Andersen, Richard A." }, { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "orcid": "0000-0001-8837-678X", "role": "member", "display_name": "Siapas, Athanassios G." } ], "option_major": [ "cns" ], "doi": "10.7907/yfvq-8a46", "abstract": "This thesis examines two aspects of human recognition memory by using two separate behavioral paradigms. Given the dual process hypothesis of recognition memory, the first chapter investigates the correlation between encoding and retrieval of recognition and source memory for images by using a cued retrieval paradigm. Participants were shown images in a particular judgment task (source context) and later asked to retrieve them in a cued retrieval task. Recording from the human brain, I found separate cell populations to be responsive to the source context during the encoding and recognition stages of the task, suggesting a lack of single-cell level reactivation during source retrieval. In the second chapter, I examined how recognition memory signals change over time using repeated longitudinal behavioral testing in an fMRI study. Through repetitive presentation and memory tests over a period of three months, face stimuli were introduced to three participants. The behavioral outcome of the task showed that as frequency of exposure to specific faces increases, the memory performance and judged confidence increases correspondingly, supporting the hypothesis of a continuous familiarity signal." }, { "name": "Zhu, Zikun", "degree": "PhD", "year": "2024", "title": "Spatiotemporal Regulation of Nascent Protein Targeting", "advisor": "Shan, Shu-ou", "url": "https://resolver.caltech.edu/CaltechTHESIS:05302024-023258565", "creators": [ { "name": { "family": "Zhu", "given": "Zikun" }, "id": "Zhu-Zikun", "orcid": "0000-0001-5934-8368", "display_name": "Zhu, Zikun" } ], "advisors": [ { "name": { "family": "Shan", "given": "Shu-ou" }, "id": "Shan-Shu-ou", "orcid": "0000-0002-6526-1733", "role": "advisor", "display_name": "Shan, Shu-ou" } ], "committee": [ { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "chair", "display_name": "Chan, David C." }, { "name": { "family": "Voorhees", "given": "Rebecca M." }, "id": "Voorhees-R-M", "orcid": "0000-0003-1640-2293", "role": "member", "display_name": "Voorhees, Rebecca M." }, { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "member", "display_name": "Guttman, Mitchell" }, { "name": { "family": "Shan", "given": "Shu-ou" }, "id": "Shan-Shu-ou", "orcid": "0000-0002-6526-1733", "role": "member", "display_name": "Shan, Shu-ou" } ], "option_major": [ "molbiochem" ], "doi": "10.7907/ybz3-4m48", "abstract": "Proper protein targeting to the correct cellular compartments is essential for maintaining the functionality and organization of all cells. However, the mechanisms that ensure newly synthesized proteins are accurately and efficiently directed to their specific cellular destinations remain unclear. Moreover, how protein targeting is coordinated with protein folding and other cellular processes, both spatially and temporally, is largely unknown.
\r\n\r\nIn my thesis, I first demonstrated the mechanism of a nascent protein transport pathway in prokaryotes, mediated by a conserved ATPase SecA. Using a combination of ribosome profiling methods, I revealed the essential roles of SecA in recognizing and resolving the widespread accumulation of large periplasmic loops of inner membrane proteins in the cytoplasm during their cotranslational translocation, and in the cotranslational transport of secretory proteins with highly hydrophobic signal sequences. I also uncovered a function of the chaperone trigger factor (TF) in temporally regulating SecA engagement on secretory proteins. These findings elucidate the principles of SecA-driven cotranslational protein translocation and reveal a hierarchical network of protein export pathways in bacteria (Chapter 2).
\r\n\r\nThe second part of research focused on the more complex protein sorting systems of eukaryotes, where I comprehensively investigated the mitochondrial protein delivery from the cytosol using selective ribosome profiling in human cells. I found that the cotranslational protein targeting to mitochondria is initiated late during translation, directed by an N-terminal presequence and the exposure of a complex globular fold in the nascent protein. This pathway does not favor membrane proteins, but is predominantly used by large, multidomain and topologically complex proteins, whose import efficiency is enhanced when targeted cotranslationally. My results indicate that the cotranslational targeting of mitochondrial proteins is fundamentally different from that of the endoplasmic reticulum (ER) proteins, highlighting the diversity and specificity of protein targeting mechanisms across cellular systems (Chapter 3).
" }, { "name": "Bernstein, Jeremy David", "degree": "PhD", "year": "2023", "title": "Optimisation & Generalisation in Networks of Neurons", "advisor": "Yue, Yisong", "url": "https://resolver.caltech.edu/CaltechTHESIS:10132022-000100592", "creators": [ { "name": { "family": "Bernstein", "given": "Jeremy David" }, "id": "Bernstein-Jeremy-David", "orcid": "0000-0001-9110-7476", "display_name": "Bernstein, Jeremy David" } ], "advisors": [ { "name": { "family": "Yue", "given": "Yisong" }, "id": "Yue-Yisong", "orcid": "0000-0001-9127-1989", "role": "advisor", "display_name": "Yue, Yisong" } ], "committee": [ { "name": { "family": "Tropp", "given": "Joel A." }, "id": "Tropp-J-A", "orcid": "0000-0003-1024-1791", "role": "chair", "display_name": "Tropp, Joel A." }, { "name": { "family": "Liu", "given": "Ming-Yu" }, "id": "Liu-Ming-Yu", "orcid": "0000-0002-2951-2398", "role": "member", "display_name": "Liu, Ming-Yu" }, { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "orcid": "0000-0003-2136-6506", "role": "member", "display_name": "Meister, Markus" }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Yue", "given": "Yisong" }, "id": "Yue-Yisong", "orcid": "0000-0001-9127-1989", "role": "member", "display_name": "Yue, Yisong" } ], "option_major": [ "cns" ], "doi": "10.7907/1jz8-5t85", "abstract": "The goal of this thesis is to develop the optimisation and generalisation theoretic foundations of learning in artificial neural networks. The thesis tackles two central questions. Given training data and a network architecture:
\r\n\r\nOn optimisation, an essential feature of neural network training is that the network weights affect the loss function only indirectly through their appearance in the network architecture. This thesis proposes a three-step framework for deriving novel \u201carchitecture aware\u201d optimisation algorithms. The first step\u2014termed functional majorisation\u2014is to majorise a series expansion of the loss function in terms of functional perturbations. The second step is to derive architectural perturbation bounds that relate the size of functional perturbations to the size of weight perturbations. The third step is to substitute these architectural perturbation bounds into the functional majorisation of the loss and to obtain an optimisation algorithm via minimisation. This constitutes an application of the majorise-minimise meta-algorithm to neural networks.
\r\n\r\nOn generalisation, a promising recent line of work has applied PAC-Bayes theory to derive non-vacuous generalisation guarantees for neural networks. Since these guarantees control the average risk of ensembles of networks, they do not address which individual network should generalise best. To close this gap, the thesis rekindles an old idea from the kernels literature: the Bayes point machine. A Bayes point machine is a single classifier that approximates the aggregate prediction of an ensemble of classifiers. Since aggregation reduces the variance of ensemble predictions, Bayes point machines tend to generalise better than other ensemble members. The thesis shows that the space of neural networks consistent with a training set concentrates on a Bayes point machine if both the network width and normalised margin are sent to infinity. This motivates the practice of returning a wide network of large normalised margin.
\r\n\r\nPotential applications of these ideas include novel methods for uncertainty quantification, more efficient numerical representations for neural hardware, and optimisers that transfer hyperparameters across learning problems.
" }, { "name": "Bhat, Prashant", "degree": "PhD", "year": "2023", "title": "On the Role of Three-Dimensional Genome Organization in Gene Regulation and mRNA Splicing", "advisor": "Guttman, Mitchell", "url": "https://resolver.caltech.edu/CaltechTHESIS:05302023-040856946", "creators": [ { "name": { "family": "Bhat", "given": "Prashant" }, "id": "Bhat-Prashant", "orcid": "0000-0003-3832-4871", "display_name": "Bhat, Prashant" } ], "advisors": [ { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "advisor", "display_name": "Guttman, Mitchell" } ], "committee": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "chair", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "orcid": "0000-0001-8723-8190", "role": "member", "display_name": "Baltimore, David L." }, { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "member", "display_name": "Guttman, Mitchell" }, { "name": { "family": "Chang", "given": "Howard Y." }, "id": "Chang-Howard-Y", "orcid": "0000-0002-9459-4393", "role": "member", "display_name": "Chang, Howard Y." }, { "name": { "family": "Black", "given": "Douglas L." }, "id": "Black-Douglas-L", "orcid": "0000-0002-2705-8187", "role": "member", "display_name": "Black, Douglas L." } ], "option_major": [ "molbio" ], "doi": "10.7907/vg8a-y851", "abstract": "The nucleus is spatially organized such that DNA, RNA, and protein molecules involved in shared functional and regulatory processes are compartmentalized in three-dimensional (3D) structures. These structures are emerging as a paradigm for gene regulation, a highly complex process that requires the dynamic coordination of hundreds of regulatory factors around precise targets in different cell states. We describe the discovery of hundreds of RNA-DNA hubs throughout the nucleus that are organized around essential nuclear functions such as RNA processing, centromeric heterochromatin organization, and gene regulation. Focusing on RNA processing, specifically co-transcriptional splicing, we find that genome-wide organization of active genes near nuclear speckles drives the efficiency of pre-mRNA splicing in a cell-type specific manner. The results of this thesis illustrate how spatial compartmentalization of biomolecules increases the local concentration of reactants and enzymes such that greater efficiency is achieved in scenarios where rapid responses are required for cell survival." }, { "name": "Blumenfeld, Zachary", "degree": "PhD", "year": "2023", "title": "Genetically Encoded Biosensors for Ketamine and Other Rapidly Acting Antidepressants in Zebrafish and Cell Culture", "advisor": "Lester, Henry A.; Prober, David A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05292023-213541270", "creators": [ { "name": { "family": "Blumenfeld", "given": "Zachary" }, "id": "Blumenfeld-Zachary", "orcid": "0000-0002-4627-5582", "display_name": "Blumenfeld, Zachary" } ], "advisors": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "advisor", "display_name": "Lester, Henry A." }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "orcid": "0000-0002-7371-4675", "role": "co-advisor", "display_name": "Prober, David A." } ], "committee": [ { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "chair", "display_name": "Adolphs, Ralph" }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "orcid": "0000-0002-7371-4675", "role": "member", "display_name": "Prober, David A." }, { "name": { "family": "Hong", "given": "Elizabeth J." }, "id": "Hong-Elizabeth-J", "orcid": "0000-0003-3866-418X", "role": "member", "display_name": "Hong, Elizabeth J." } ], "option_major": [ "biology" ], "doi": "10.7907/4wtg-qb69", "abstract": "Over the past century, the development and use of treatments for depression has been one of the most important projects in both neuroscience and medicine. Not only is relatively little known about the underlying pathophysiology of major depressive disorder (MDD), a mechanistic understanding of the ways in which common antidepressants \u2014 such as selective serotonin reuptake inhibitors (SSRIs) \u2014 contribute to symptomatic relief remains elusive. Furthermore, the delay until typical antidepressant treatments take effect (a 'therapeutic lag' of weeks to months) presents a series of challenges to researchers in chemistry, neuroscience, pharmacology, and medicine, as the connection between apparent physiological changes and clinical benefit has yet to be established. The recent advent of a new class of drugs \u2014 rapidly acting antidepressants (RAADs), including the multi-purpose compound ketamine \u2014 which ameliorate symptoms within hours to days provides a crucial (if perplexing) perspective on the treatment of MDD and neuropsychiatric disorders more broadly. To answer questions concerning how various kinds of antidepressants might exert their effects, where those interactions take place, and what sorts of physiological changes drive clinical response, we have designed genetically encoded drug-specific intensity-based sensing fluorescent reporters (iDrugSnFRs) which are engineered to detect drugs of interest in both in vitro and in vivo applications. We have successfully evolved iDrugSnFRs for an array of RAADs (iRAADSnFRs) which detect pharmacologically relevant concentrations of their target drugs sensitively and specifically in both cell culture as well as in the nervous tissue of larval zebrafish. Another set of iDrugSnFRs for SSRIs has provided novel insights into the potential reasons for the aforementioned 'therapeutic lag' as well as side effects, while yet another set has provided a pharmacokinetic basis for the evaluation of smoking cessation drugs. In all, our findings lead us to posit that iDrugSnFRs can aid in the elucidation of mechanisms by which a wide variety of orally active pharmaceutical compounds operate as well as provide a crucial basis for the development of better medicines.
" }, { "name": "Chen, Xinhong", "degree": "PhD", "year": "2023", "title": "Non-Invasive Functional Gene Delivery to the Central and Peripheral Nervous System Across Species", "advisor": "Gradinaru, Viviana", "url": "https://resolver.caltech.edu/CaltechTHESIS:04132023-174709152", "creators": [ { "name": { "family": "Chen", "given": "Xinhong" }, "id": "Chen-Xinhong", "orcid": "0000-0003-0408-0813", "display_name": "Chen, Xinhong" } ], "advisors": [ { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "advisor", "display_name": "Gradinaru, Viviana" } ], "committee": [ { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "orcid": "0000-0003-2713-1513", "role": "chair", "display_name": "Mazmanian, Sarkis K." }, { "name": { "family": "Oka", "given": "Yuki" }, "id": "Oka-Yuki", "orcid": "0000-0003-2686-0677", "role": "member", "display_name": "Oka, Yuki" }, { "name": { "family": "Lois", "given": "Carlos" }, "id": "Lois-Carlos", "orcid": "0000-0002-7305-2317", "role": "member", "display_name": "Lois, Carlos" }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "member", "display_name": "Gradinaru, Viviana" } ], "option_major": [ "neurobio" ], "doi": "10.7907/48a2-0y07", "abstract": "The normal function of the central nervous system (CNS) and peripheral nervous system (PNS) relies on precise regulation. When this regulation breaks down in diseases, genetic access to the nervous system is critical for therapeutic intervention. However, access to the nervous system remains difficult, reflecting the critical need for development of effective and non-invasive gene delivery vectors across species. By applying directed evolution approach, we identified 2 capsids, AAV-MaCPNS1 and AAV-MaCPNS2, which efficiently transduced the PNS in rodents following intravenous administration. Combining with rational optimization, we also identified AAV-X1 capsid family, which transduce brain endothelial cells specifically and efficiently following systemic administration in wild-type mice with diverse genetic backgrounds and rats. Some previously-engineered AAVs that target the nervous system fail to translate across non-human primate (NHP). We thus also further tested our novel vectors across species and showed that AAV-MaCPNS1/2 efficiently transduced both the PNS and CNS in NHPs. AAV-X1.1 also exhibit superior transduction of the CNS in rhesus macaques and ex vivo human brain slices although the endothelial tropism is not conserved across species.
\r\n\r\nWith these enhanced systemic AAVs, we wanted to explore whether they could enable neuronal recording and modulation which has been challenging with the nature AAV serotypes. We used AAV-MaCPNS1 to systemically deliver the neuronal sensor jGCaMP8s to record calcium signal dynamics in nodose ganglia. We observed specific nodose neuronal response to physiological modulation in the gut. Furthermore, we showed that the MaCPNS1-delivered neuronal actuator DREADD to dorsal root ganglia could enable non-invasive neuronal modulation and create a model of pain. The functional utility of the novel systemic vectors demonstrated here provide a non-invasive approach to better explore the nervous system, which would lead to better therapeutic intervention. To this end, we also demonstrated that the X1 capsids can be used to genetically engineer the blood-brain barrier by transforming the mouse brain vasculature into a functional biofactory for production of therapeutic agents for CNS. We showed that vasculature-secreted Hevin (a synaptogenic protein), whose coding sequence is delivered by X1 vectors, rescued synaptic deficits in a mouse model.
\r\n\r\nAAV repeated dosing could be favorable for certain therapeutic applications, however, neutralizing antibody generated following the first injection creates major obstacle for second injection. We explored whether serotype switching with 2 AAV capsids that have a distinguished neutralizing antibody profile could be a potential solution. To this end, we firstly showed that the X1 capsid modifications translate from AAV9 to other serotypes such as AAV1 and AAV-DJ. We then combined the different engineered serotype to enable serotype switching for sequential AAV administration in mice, showing the first AAV-delivered receptor for the second AAV could boost its CNS targeting.
\r\n\r\nIn general, we developed strategies to enable non-invasive functional gene delivery to the central and peripheral nervous system across species, which would be incremental for both basic neuroscience research and gene therapies for neurological disorders.
" }, { "name": "Chen, Zhewei", "degree": "PhD", "year": "2023", "title": "Engineering Conditional Guide RNAs for Cell-Selective Regulation of CRISPR/Cas9", "advisor": "Pierce, Niles A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:12022022-010002116", "creators": [ { "name": { "family": "Chen", "given": "Zhewei" }, "id": "Chen-Zhewei", "orcid": "0000-0002-7422-095X", "display_name": "Chen, Zhewei" } ], "advisors": [ { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "advisor", "display_name": "Pierce, Niles A." } ], "committee": [ { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "chair", "display_name": "Murray, Richard M." }, { "name": { "family": "Rothemund", "given": "Paul W. K." }, "id": "Rothemund-P-W-K", "orcid": "0000-0002-1653-3202", "role": "member", "display_name": "Rothemund, Paul W. K." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "member", "display_name": "Pierce, Niles A." } ], "option_major": [ "bioeng" ], "doi": "10.7907/cajs-d417", "abstract": "CRISPR/Cas9 is a versatile platform for implementing diverse modes of genetic perturbation such as gene silencing, induction, deletion, or replacement. This technology is popularly used in developmental biology to probe genetic circuitry via constitutive gene knockdown. Global gene silencing could introduce artifacts in the study of developmental regulatory pathways, and this motivates the development of cell-selective gene editing. Our lab has recently created conditional guide RNAs (cgRNA) that enable CRISPR/Cas9 systems to silence a desired gene Y conditioned on the detection of an RNA transcript X inside of a cell. cgRNA systems were discovered via insertion and deletion mutations that systematically explored the structure function of the guide RNA. Nucleic acid engineering software (NUPACK) was used to generate orthogonal libraries of cgRNA molecules that executed both ON \u2192 OFF logic (conditional inactivation by an RNA trigger) and OFF \u2192 ON logic (conditional activation by an RNA trigger). A dCas9-based RFP silencing assay in bacteria was developed and used to show these cgRNA sequences were functional and could detect short exogenous trigger sequences in an orthogonal and doseresponsive manner. Subsequent studies on cgRNA structure and function enabled us to engineer next-generation systems that have fewer constraints on the trigger sequence or structure. These next-generation cgRNAs were tested against short synthetic mRNA transcripts, truncated sub-sequences of endogenous mRNAs, and full-length endogenous mRNAs. Synthetic mRNA transcripts were used to study the effect of protein translation on trigger RNA binding. cgRNAs were capable of detecting synthetic sequences embedded in the 3\u2032 UTR of fluorescent protein mRNAs. cgRNAs could also detect short synthetic mRNAs or truncated subsequences from endogenous mRNAs. However, the detection of native full-length endogenous mRNAs remained challenging because we cannot reliably predict the local structure of sub-sequences within a long RNA transcript. High-throughput cgRNAscreening may prove necessary for finding accessible binding sites onmRNA transcripts. Nevertheless, cgRNA functionalities could be useful in developmental biology by enabling precision perturbation of regulatory events, linking guide RNA activity to an RNA marker X correlated to a specific cell type or temporal expression pattern. This work opens the possibility for future applications such as cell-selective gene therapies.
" }, { "name": "Chuapoco, Miguel Roberto Estella", "degree": "PhD", "year": "2023", "title": "Adeno-Associated Viral Vectors for Gene Delivery to the Non-Human Primate Brain", "advisor": "Gradinaru, Viviana", "url": "https://resolver.caltech.edu/CaltechTHESIS:06012023-221050920", "creators": [ { "name": { "family": "Chuapoco", "given": "Miguel Roberto Estella" }, "id": "Chuapoco-Miguel-Roberto-Estella", "orcid": "0000-0001-5397-996X", "display_name": "Chuapoco, Miguel Roberto Estella" } ], "advisors": [ { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "advisor", "display_name": "Gradinaru, Viviana" } ], "committee": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "chair", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Van Valen", "given": "David A." }, "id": "Van-Valen-D", "orcid": "0000-0001-7534-7621", "role": "member", "display_name": "Van Valen, David A." }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "member", "display_name": "Gradinaru, Viviana" } ], "option_major": [ "bioeng" ], "doi": "10.7907/gngs-q727", "abstract": "Viral vectors are efficient gene-delivery carriers for somatic cell gene therapy, and replication-deficient vectors are actively being used in the clinic to replace and correct disease-causing genes and mutations. Mirroring their therapeutic effectiveness, viral vectors are also powerful in vivo gene-delivery tools in basic research. In the field of neuroscience, adeno-associated viruses (AAVs) in particular have emerged as a major workhorse that enable efficient in vivo expression of opsins for optogenetics, designer GPCRs for chemogenetics, and GCaMP for calcium imaging. Recently, AAV engineering efforts by our group and others have expanded the toolbox of AAV vectors to include capsid variants that traverse the blood-brain-barrier (BBB) in rodents. However, not all engineered AAV capsids translate from mice to non-human primates (NHPs). This is especially true for translation to the rhesus macaque (Macaca mulatta), an Old World primate that is the predominant NHP model and shares a more recent common ancestor with humans (~25 million years ago) compared to rodents (~75 million years ago) and New World primates, such as the common marmoset (Callithrix jacchus; ~35 million years ago). The primary contents of this dissertation will focus on the development of AAV.CAP-Mac as a vector for non-invasive gene-transfer to the NHP brain and demonstrations of its utility to interrogate neuronal morphology and physiology in the macaque central nervous system. We identified and selected CAP-Mac using a multi-species selection strategy in adult marmosets and infant macaques, where it demonstrated improved delivery efficiency compared to AAV9 and other engineered variants. In individual characterization, CAP-Mac was biased towards neurons in two infant Old World primate species, the rhesus macaque and the green monkey (Chlorocebus sabaeus). Given this neuronal tropism in Old World primates, we demonstrated how CAP-Mac can be readily used for non-invasive, Brainbow-like labeling of macaque neurons and calcium imaging of GCaMP ex vivo. In closing, we describe CAP-Mac tropism across multiple developmental states, species, and routes of administration. Additionally, we present preliminary data on \u201corphan capsids,\u201d capsid variants that were engineered to be non-infective, but can be readily re-functionalized using known receptor-ligand pairs. Collectively, the work covered in this dissertation disseminates non-invasive, gene-delivery tools for NHP researchers, and lays the groundwork for further development of more specific and efficacious AAVs that access the NHP brain." }, { "name": "Dam, Kim-Marie Anh", "degree": "PhD", "year": "2023", "title": "Structural Insights into the Conformational Plasticity and Antibody Recognition of HIV-1 Env", "advisor": "Bjorkman, Pamela J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06022023-003659646", "creators": [ { "name": { "family": "Dam", "given": "Kim-Marie Anh" }, "id": "Dam-Kim-Marie-Anh", "orcid": "0000-0002-1416-4757", "display_name": "Dam, Kim-Marie Anh" } ], "advisors": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "advisor", "display_name": "Bjorkman, Pamela J." } ], "committee": [ { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "orcid": "0000-0003-2713-1513", "role": "chair", "display_name": "Mazmanian, Sarkis K." }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "member", "display_name": "Chan, David C." }, { "name": { "family": "Voorhees", "given": "Rebecca M." }, "id": "Voorhees-R-M", "orcid": "0000-0003-1640-2293", "role": "member", "display_name": "Voorhees, Rebecca M." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." } ], "option_major": [ "biology" ], "doi": "10.7907/8pvb-dx31", "abstract": "Acquired immunodeficiency syndrome (AIDS) and its causal agent, the human immunodeficiency virus 1 (HIV-1), remain a global public health concern since they were first identified in the early 1980s. Diligent research and gradual scientific advances have led to innovative strategies in HIV-1/AIDS prevention and treatment, transforming an obscure and deadly disease into a manageable condition with a normal life expectancy. Despite this progress, researchers have yet to develop a safe and effective vaccine against HIV-1. The work presented here describes a structural perspective related to the HIV-1 Envelope (Env) glycoprotein, the sole viral target of vaccines that seek to elicit neutralizing antibodies.
\r\n\r\nEnv is the only viral protein on the surface of HIV-1 virions and is composed of a homotrimer of gp120/gp41 heterodimers. Env mediates entry into target cells by engaging the host receptor, CD4. CD4 triggers conformational changes in gp120, thereby enabling coreceptor recognition. Interactions with the host coreceptor trigger structural rearrangements in gp41 that facilitate fusion of host and viral membranes leading to infection. Our work builds upon our understanding of Env structural plasticity. First, we evaluated the conformational plasticity of soluble Env constructs using double electron-electron resonance (DEER) spectroscopy. This method measured distances between probes in Env subunits, allowing us to interpret the distribution in distances as Env flexibility. Our findings captured previously unseen nuances in static Env structures including gp41 elasticity and conformational heterogeneity associated with CD4-receptor binding. Although our work gave a new perspective on Env flexibility, it largely corroborated observations from static Env structures. Importantly, this suggested that soluble versions of Env, which serve as templates for immunogen design, retain favorable structural properties.
\r\n\r\nInformed with these insights in Env structure, we then sought to address a prevailing question related to receptor engagement: how many CD4 receptors are needed to induce gp120 conformation changes that lead to coreceptor binding followed by fusion? Prior work only characterized CD4-induced Env structural changes in Envs complexed with three soluble CD4 proteins. In our work, we designed and structurally characterized Envs bound to only one or two CD4 receptors. We found that Env engagement of one CD4 resulted in minor changes to the prefusion, closed Env conformation while Env bound to two CD4 molecules led to CD4-induced opening in the CD4-bound gp120s and a partially open conformation in the unliganded gp120.
\r\n\r\nStructural biology has also been leveraged to characterize the mechanism by which broadly neutralizing antibodies (bNAbs) recognize HIV-1 Env. We include an extensive review of how structural observations from antibodies bound to viral proteins contribute to our understanding of antibody-mediated viral neutralization. We also present a technical evaluation of bNAb binding assays that revealed how Env conformations can be unintentionally altered resulting in misleading antibody binding results and identified ideal methods to ensure reliable data.
\r\n\r\nAdditionally, we report on projects related to bNAbs that target the CD4 binding site (CD4bs) epitope of Env. In the first, we characterized the inferred germline (iGL) precursor of BG24, a VRC01-class bNAb with features that make it a promising target for vaccine design. We solved four cryo-EM structures of BG24iGL constructs complexed with different Envs and provided insight on the mode of iGL accommodation. The second project centers around the IOMA-class of CD4bs bNAbs. We characterized features of IOMA-class bNAbs and measured how different features contribute to neutralization breadth and potency. Taken together, the conclusions from our work provide guidance for the next generation of structure-based, CD4bs-targeting immunogen design.
" }, { "name": "Ding, Xiaozhe", "degree": "PhD", "year": "2023", "title": "Computation-Aided Protein Engineering for Targeted Therapeutic Delivery", "advisor": "Gradinaru, Viviana", "url": "https://resolver.caltech.edu/CaltechTHESIS:05052023-192721059", "creators": [ { "name": { "family": "Ding", "given": "Xiaozhe" }, "id": "Ding-Xiaozhe", "orcid": "0000-0002-0267-0791", "display_name": "Ding, Xiaozhe" } ], "advisors": [ { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "advisor", "display_name": "Gradinaru, Viviana" } ], "committee": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "chair", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "member", "display_name": "Shapiro, Mikhail G." }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "member", "display_name": "Gradinaru, Viviana" } ], "option_major": [ "bioeng" ], "doi": "10.7907/7n15-3076", "abstract": "My Ph.D. projects centered on using computational structural biology tools to develop protein engineering methods for targeted therapeutic delivery, emphasizing delivering molecules to the brain. In this thesis, I focus on three main projects. First, utilizing computational structural biology techniques, I investigate the molecular mechanism that enables engineered adeno-associated viral (AAV) capsids to cross the blood-brain barrier (BBB). I develop a pipeline to model the vast and dynamic complex between engineered AAV capsids and their BBB receptors. I also apply a tool, recently developed by myself and discussed in Chapter 3, to distinguish capsids that bind to different receptors. The findings of this study can lead to novel approaches for developing chemicals and biologicals that can penetrate the human brain (Chapter 2). Second, I describe the development of Automated Pairwise Peptide-Receptor AnalysIs for Screening Engineered proteins (APPRAISE). This computational pipeline predicts the receptor binding propensity of engineered proteins based on competitive modeling and physics-grounded analysis. I show that APPRAISE is capable of distinguishing between receptor-dependent and receptor-independent adeno-associated viral vectors and ranking various engineered proteins, such as miniproteins binding to the SARS-CoV-2 spike and nanobodies binding to a G-protein-coupled receptor. A top performer in an in silico screening using APPRAISE was validated experimentally (Chapter 3). Third, I show an example to engineer a genetically encoded transmitter indicator (GETI), which may eventually be a cargo delivered to the brain. The GETI has a novel scaffold based on bacterial repressors, a class of transcriptional regulators that are critical for bacteria to respond to environmental chemicals. I repurposed an antibiotic-sensing repressor protein to bind a neurotransmitter, melatonin, using machine-learning-guided directed evolution. A melatonin indicator was then created by integrating the repurposed receptor with a fluorescent protein. This engineering platform may be adapted to create bio-orthogonal GETIs for various neurotransmitters (Chapter 4)." }, { "name": "Galton, Riley", "degree": "PhD", "year": "2023", "title": "Co-Option of the piRNA Pathway to Regulate Neural Crest Specification", "advisor": "Bronner, Marianne E.; Fejes T\u00f3th, Katalin", "url": "https://resolver.caltech.edu/CaltechTHESIS:01232023-192745187", "creators": [ { "name": { "family": "Galton", "given": "Riley" }, "id": "Galton-Riley", "orcid": "0000-0001-6777-2177", "display_name": "Galton, Riley" } ], "advisors": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "co-advisor", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Fejes T\u00f3th", "given": "Katalin" }, "id": "Fejes-T\u00f3th-K", "orcid": "0000-0001-6558-2636", "role": "co-advisor", "display_name": "Fejes T\u00f3th, Katalin" } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "chair", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Fejes-T\u00f3th", "given": "Katalin" }, "id": "Fejes-T\u00f3th-K", "orcid": "0000-0001-6558-2636", "role": "member", "display_name": "Fejes-T\u00f3th, Katalin" }, { "name": { "family": "Aravin", "given": "Alexei A." }, "id": "Aravin-A-A", "orcid": "0000-0002-6956-8257", "role": "member", "display_name": "Aravin, Alexei A." }, { "name": { "family": "Zernicka-Goetz", "given": "Magdalena" }, "id": "Zernicka-Goetz-M", "orcid": "0000-0002-7004-2471", "role": "member", "display_name": "Zernicka-Goetz, Magdalena" } ], "option_major": [ "biodev" ], "doi": "10.7907/9rvr-rr55", "abstract": "The piRNA pathway has persisted throughout evolution as an essential regulatory pathway to protect genomic integrity in the metazoan germline. It achieves this through the repression of transposable elements, or \u201cselfish genes,\u201d which would otherwise jump throughout the genome unchecked, causing genomic instability and infertility. While transposable elements are generally deleterious in nature, their persistence in our genome remains an important driver of evolution, both as an agent of mutation and source of raw genetic material. Thus, a delicate balance must be struck to both maintain genomic integrity for the next generation but still enable enough mutation to allow for adaptation. The arms race between the ever-adapting piRNA pathway and its transposon targets provides this balance, and for a long time the piRNA pathway was considered to be germline specific, since that is where both transposon and piRNA pathway activity is highest. It has since become clear that the piRNA pathway is also active in somatic tissue of several invertebrate species, and may even target host genes in some. Whether the piRNA pathway plays a role outside of the germline in vertebrates, however, has remained elusive.
\r\n\r\nIn this thesis, we demonstrate that the piRNA pathway is active in a vertebrate somatic cell type, the chick neural crest, where it has been co-opted into the gene regulatory network to repress the transposon-derived gene, ERNI. ERNI, in turn, supresses Sox2 when piRNA pathway protein Piwil1 is downregulated upon neural crest specification. Thus, the piRNA pathway functions to maintain Sox2 expression in the neural plate border stem cell niche, protecting its proliferative abilities and setting the timing of neural crest specification. We also provide preliminary evidence that the neural crest piRNA pathway might be conserved in other vertebrate species, and that two highly conserved transcription factors regulate its expression in the chick neural crest. Our work provides mechanistic insight into a novel function of the piRNA pathway as a regulator of somatic development in vertebrates, and raises the possibility that this ancient pathway may play a more significant role in evolution and transposon co-option by host genomes than previously thought.
" }, { "name": "Gholamin, Sharareh", "degree": "PhD", "year": "2023", "title": "Mechanism of Response and Resistance to CAR T Cell Therapies", "advisor": "Bronner, Marianne E.; Brown, Christine; Forman, Stephen", "url": "https://resolver.caltech.edu/CaltechTHESIS:06132023-010825748", "creators": [ { "name": { "family": "Gholamin", "given": "Sharareh" }, "id": "Gholamin-Sharareh", "orcid": "0000-0001-7425-6074", "display_name": "Gholamin, Sharareh" } ], "advisors": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "advisor", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Brown", "given": "Christine" }, "id": "Brown-Christine", "role": "co-advisor", "display_name": "Brown, Christine" }, { "name": { "family": "Forman", "given": "Stephen" }, "id": "Forman-Stephen", "role": "co-advisor", "display_name": "Forman, Stephen" } ], "committee": [ { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "chair", "display_name": "Shapiro, Mikhail G." }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "orcid": "0000-0001-8723-8190", "role": "member", "display_name": "Baltimore, David L." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Heath", "given": "James R." }, "id": "Heath-J-R", "orcid": "0000-0001-5356-4385", "role": "member", "display_name": "Heath, James R." }, { "name": { "family": "Brown", "given": "Christine" }, "id": "Brown-Christine", "role": "member", "display_name": "Brown, Christine" }, { "name": { "family": "Ribas", "given": "Antoni" }, "id": "Ribas-Antoni", "role": "member", "display_name": "Ribas, Antoni" }, { "name": { "family": "Forman", "given": "Stephen" }, "id": "Forman-Stephen", "role": "member", "display_name": "Forman, Stephen" } ], "option_major": [ "biology" ], "doi": "10.7907/jf79-pv58", "abstract": "While chimeric antigen T (CAR) T cell therapy has shown remarkable success in leukemia, lymphoma, and multiple myeloma, its effectiveness in solid tumors including glioblastoma (GBM) remains limited. It is crucial to understand mechanisms that reduce the efficacy of CAR T cell therapies and develop strategies to prevent tumor resistance. In this study, we conjectured that alterations in tumor cell-intrinsic interferon (IFN) signaling pathways contribute to establishment of immunosuppressive tumor microenvironment in solid tumors, leading to resistance of solid tumor cells to CAR T cell-mediated killing. We established syngeneic IFN signaling-deficient tumor models for murine IL-13Ra2 targeted CAR T cell therapy and showed that these models modulate the tumor microenvironment (TME), leading to resistance to CAR T cell therapy. We identified variations in gene expression associated with IFN signaling components and cytokines between IFN signaling-deficient tumor cells and wild type (WT) tumor cells after CAR T cell treatment. Furthermore, single-cell RNA sequencing and mass cytometry analysis of the tumor immune cell infiltrates in IFN-signaling deficient tumors compared to WT controls identified the immune-mediated causal components for the resistance of Janus Kinase1 knockout (JAK1/KO) tumors to CAR T cell therapy. CAR T cell-treated IFN signaling-deficient tumors presented decreased T-cell transcripts, with decreased frequency of CD8-early active, CD8-naive like T cells. Conversely, there were more regulatory and follicular T cells, exhausted endogenous T cells , and exhausted CAR T cells in treated IFN signaling-deficient tumors compared to treated WT tumors. The analyses also showed the superior enrichment and crosstalk of genes that identified fibroblasts, neutrophils, and myeloid cells in IFN signaling-deficient tumors compared to those of WT tumors. Mass cytometry analysis on the immune cells infiltrates of JAK1/KO and WT tumors post CAR T cell treatment corroborated the results from gene expression analysis. The potential cause of immune suppressive crosstalk in IFN signaling-deficient tumor niches could be attributed to the varied enhancement of receptor-ligand interactions such as SPP1+ tumor-associated macrophages (TAMs) and CD44+ cancer-associated fibroblasts (CAFs), as well as SPP1+ TAMs and integrins present on other cell lineages. To overcome resistance to CAR T cell therapies, we employed two distinct actionable approaches: triggering the immune microenvironment and disrupting the extracellular matrix. Unconjugated interferon signaling gene-15 (ISG-15) enhanced CAR T cell efficacy in an INF-signaling deficient model, increasing the recruitment of endogenous T cells and reshaping the TME. Anti-SPP1 blocking antibody was used to prime the JAK1/KO tumors prior to the treatment with CAR T cell therapy potentially via enhancing the persistence and trafficking of CAR T cells in the TME.
\r\n\r\nWe next identified immune signatures of 32 GBM patients who had progressive disease after CAR T cell treatment compared to those who had relatively stable disease or showed improvement. We identified the presence of fibroblasts and SPP1+ APOE+ C1QA+ C1QC+ myeloid cells in GBM signatures that are associated with immune suppression and resistance to therapy. Patients with GBM who exhibited a relatively stable response to treatment and increased T cell recruitment had differential expression of interferon regulatory factors (IRFs) and ISGs compared to patients with less response to the treatment. Our findings uncover a correlation between tumor-intrinsic driver mutations, the composition of the TME, and the responsiveness of solid tumors to CAR T cell therapy, providing insights into potential approaches to address resistance in IFN non-responsive tumors.
" }, { "name": "Goertsen, David Gerald", "degree": "PhD", "year": "2023", "title": "Expanding Adeno-Associated Viral Capsid Engineering to Multiple Variable Regions for Diversified Tropism", "advisor": "Gradinaru, Viviana", "url": "https://resolver.caltech.edu/CaltechTHESIS:05172023-011031610", "creators": [ { "name": { "family": "Goertsen", "given": "David Gerald" }, "id": "Goertsen-David-Gerald", "orcid": "0000-0001-7138-1697", "display_name": "Goertsen, David Gerald" } ], "advisors": [ { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "advisor", "display_name": "Gradinaru, Viviana" } ], "committee": [ { "name": { "family": "Van Valen", "given": "David A." }, "id": "Van-Valen-D", "orcid": "0000-0001-7534-7621", "role": "chair", "display_name": "Van Valen, David A." }, { "name": { "family": "Bois", "given": "Justin S." }, "id": "Bois-J-S", "orcid": "0000-0001-7137-8746", "role": "member", "display_name": "Bois, Justin S." }, { "name": { "family": "Wang", "given": "Kaihang" }, "id": "Wang-Kaihang", "orcid": "0000-0001-7657-8755", "role": "member", "display_name": "Wang, Kaihang" }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "member", "display_name": "Gradinaru, Viviana" } ], "option_major": [ "bioeng" ], "doi": "10.7907/7x3a-g504", "abstract": "Adeno-associated virus research is critical for the advancement of gene therapy and treatment of myriad debilitating genetic disorders. Targeted delivery of genetic components to a tissue or cell population remains a bottleneck for gene therapy, but the selection of AAV capsids through directed evolution can yield vectors that target desired tissues or cells. This thesis details the engineering of the AAV capsid to acquire desired tropism, namely reduction in liver transduction or increased transduction of the lung. Chapter I chronicles the history of AAV engineering, provides useful information about the AAV capsid proteins, and describes how AAV has been engineered for altered tropism in works preceding this thesis. Chapter II describes the development of AAV9.452sub.LUNG1, an AAV variant that is enriched in the lung of mice after systemic injection. Chapter III details the engineering of variants with attenuated tropism in the liver while maintaining previously acquired brain transduction after systemic injection. Two of these variants, AAV.CAP-B10 and AAV.CAP-B22, display similar tropism in the marmoset after systemic injection. Chapter IV describes the parallel engineering of prominent variable regions of the AAV capsid. Overall, the work presented in this thesis expands the toolbox available for gene therapy and represents an advancement of methods for AAV capsid engineering.
" }, { "name": "Griggs, Whitney Scott", "degree": "PhD", "year": "2023", "title": "Listening to the Internal Representation of Actions Within the Posterior Parietal Cortex", "advisor": "Andersen, Richard A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01192023-014456964", "creators": [ { "name": { "family": "Griggs", "given": "Whitney Scott" }, "id": "Griggs-Whitney-Scott", "orcid": "0000-0003-2941-6803", "display_name": "Griggs, Whitney Scott" } ], "advisors": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "advisor", "display_name": "Andersen, Richard A." } ], "committee": [ { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "chair", "display_name": "Shapiro, Mikhail G." }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "member", "display_name": "Andersen, Richard A." }, { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "member", "display_name": "Adolphs, Ralph" }, { "name": { "family": "O'Doherty", "given": "John P." }, "id": "O'Doherty-J-P", "orcid": "0000-0003-0016-3531", "role": "member", "display_name": "O'Doherty, John P." }, { "name": { "family": "Rutishauser", "given": "Ueli" }, "id": "Rutishauser-U", "orcid": "0000-0002-9207-7069", "role": "member", "display_name": "Rutishauser, Ueli" } ], "option_major": [ "biology" ], "doi": "10.7907/0eae-hk18", "abstract": "More than 5.4 million people in the United States live with chronic paralysis and roughly 20 million people worldwide live with spinal cord injuries. Brain-machine interfaces (BMIs) can be transformative for these people, enabling them to control computers, robots, and more with only thought. State-of-the-art BMIs have already made this future a reality in limited clinical trials. However, these state-of-the-art BMIs have shortcomings that limit user adoption; high-performance BMIs currently require highly invasive electrodes above or in the brain; device degradation limits longevity to about 5 years; and their field of view is small, restricting the number, and type, of applications possible. This illustrates the need for a new generation of BMIs with a brain recording modality that is longer lasting, less invasive, and scalable to sense activity from large regions of the brain.
\r\n\r\nFunctional ultrasound imaging (fUSI) is a recently developed technique that meets these criteria. fUSI measures cerebral hemodynamics with exceptional spatiotemporal resolution (<100 \u00b5m; ~100 ms) and a large field of view (several cm)\u2014specifications ideally suited to recording detailed activity of entire cortical regions in parallel. In a series of novel results, we work towards developing the first high-performance ultrasonic BMI for human use. We first demonstrate that posterior parietal cortex (PPC), an area important for sensorimotor transformation, contains mesoscopic populations tuned to the intended movement direction. Using offline recorded data from several rhesus macaque monkeys, we can decode intended movement direction, task state, and expected action reward magnitude on a single trial basis. Having demonstrated that we could decode a variety of motor and cognitive variables using offline data, we developed a real-time, closed-loop ultrasonic BMI capable of decoding up to eight directions of intended movement with high accuracy. Finally, we began to translate these results into human applications and demonstrate the ability to measure changes in cerebral hemodynamics with high sensitivity through an acoustically transparent skull replacement in human subjects.
\r\n \r\nTaken together, our work is a novel characterization of how functional ultrasound neuroimaging may enable a new generation of BMIs. Additionally, this work reinforces the validity of fUSI as a robust and accessible neuroimaging technique for future neuroscience questions about mesoscopic populations and their interrelationships throughout the brain.
" }, { "name": "Guan, Charles", "degree": "PhD", "year": "2023", "title": "Neural Coding of Finger Movements in Human Posterior Parietal Cortex and Motor Cortex", "advisor": "Andersen, Richard A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04092023-200347393", "creators": [ { "name": { "family": "Guan", "given": "Charles" }, "id": "Guan-Charles", "orcid": "0000-0002-8040-8844", "display_name": "Guan, Charles" } ], "advisors": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "advisor", "display_name": "Andersen, Richard A." } ], "committee": [ { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "orcid": "0000-0003-2136-6506", "role": "chair", "display_name": "Meister, Markus" }, { "name": { "family": "Rutishauser", "given": "Ueli" }, "id": "Rutishauser-U", "orcid": "0000-0002-9207-7069", "role": "member", "display_name": "Rutishauser, Ueli" }, { "name": { "family": "Yue", "given": "Yisong" }, "id": "Yue-Yisong", "orcid": "0000-0001-9127-1989", "role": "member", "display_name": "Yue, Yisong" }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "member", "display_name": "Andersen, Richard A." } ], "option_major": [ "bioeng" ], "doi": "10.7907/31rt-cy14", "abstract": "We use our hands constantly in our everyday lives. This seemingly simple ability is disrupted in individuals with cervical spinal cord injuries. By circumventing injured signal pathways, brain-computer interfaces (BCIs) promise to enable such individuals to control artificial limbs for everyday use. However, existing BCI limb control remains coarse and inflexible, because we do not understand how the recorded neural activity relates to dexterous movement. As a result, BCI control in physical settings remains frustratingly difficult for paralyzed users. To improve dexterous BCI control, I studied the neural coding of individual finger movements in the posterior parietal cortex and motor cortex of tetraplegic participants. These regions are directly involved in dexterous hand movements and are candidates for BCI recording implants. Finger coding matched the correlation structure and dynamics of able-bodied usage, reflecting preserved motor circuits even after paralysis. Individual finger movements of each hand were coded in a factorized, correlated manner that still allowed decoding. Participants controlled artificial fingers with state-of-the-art accuracy. Finally, we studied the temporal dynamics of neural control to understand how existing models of neural activity extend to BCI control. These findings contribute to the understanding of human hand movements and advance the development of dexterous BCIs." }, { "name": "G\u00e1lvez Merch\u00e1n, \u00c1ngel", "degree": "PhD", "year": "2023", "title": "Studies of mRNA Expression and Degradation", "advisor": "Pachter, Lior S.; Voorhees, Rebecca M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06042023-195408313", "creators": [ { "name": { "family": "G\u00e1lvez Merch\u00e1n", "given": "\u00c1ngel" }, "id": "G\u00e1lvez-Merch\u00e1n-\u00c1ngel", "orcid": "0000-0001-7420-8697", "display_name": "G\u00e1lvez Merch\u00e1n, \u00c1ngel" } ], "advisors": [ { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "co-advisor", "display_name": "Pachter, Lior S." }, { "name": { "family": "Voorhees", "given": "Rebecca M." }, "id": "Voorhees-R-M", "orcid": "0000-0003-1640-2293", "role": "co-advisor", "display_name": "Voorhees, Rebecca M." } ], "committee": [ { "name": { "family": "Aravin", "given": "Alexei A." }, "id": "Aravin-A-A", "orcid": "0000-0002-6956-8257", "role": "chair", "display_name": "Aravin, Alexei A." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "member", "display_name": "Pachter, Lior S." }, { "name": { "family": "Voorhees", "given": "Rebecca M." }, "id": "Voorhees-R-M", "orcid": "0000-0003-1640-2293", "role": "member", "display_name": "Voorhees, Rebecca M." } ], "option_major": [ "biology" ], "doi": "10.7907/esxk-ch24", "abstract": "Part 1: Protein degradation coupled to Nonsense-mediated mRNA decay
\r\n\r\nTranslation of mRNAs containing premature termination codons (PTCs) results in truncated protein products with deleterious effects. Nonsense-mediated decay (NMD) is a surveillance pathway responsible for detecting PTC containing transcripts. While the molecular mechanisms governing mRNA degradation have been extensively studied, the fate of the nascent protein product remains largely uncharacterized. In part 1 of this thesis, we use a fluorescent reporter system in mammalian cells to reveal a selective degradation pathway specifically targeting the protein product of an NMD mRNA. We show that this process is post-translational, and dependent on the ubiquitin proteasome system. To systematically uncover factors involved in NMD-linked protein quality control, we conducted genome-wide flow cytometry-based screens. Our screens recovered known NMD factors, but suggested protein degradation did not depend on the canonical ribosome-quality control (RQC) pathway. A subsequent arrayed screen demonstrated that protein and mRNA branches of NMD rely on a shared recognition event. Our results establish the existence of a targeted pathway for nascent protein degradation from PTC containing mRNAs, and provides a reference for the field to identify and characterize required factors.
\r\n\r\nPart 2: The Commons Cell Atlas
\r\n\r\nCurrent cell atlas projects aim to curate representative datasets, cell-types, and marker genes for tissues across an organism. Despite their ubiquity, atlas projects rely on duplicated and manual effort to curate marker genes and annotate cell-types. Importantly, the lack of data-compatible tools and a fixed representation of the atlas make their reanalysis near-impossible. To overcome these challenges, we present a collection of data, algorithms, and tools to automate cataloging and analyzing cell-types across all tissues in an organism. We leveraged this work to build a Human Commons Cell Atlas comprising 2.9 million cells across 27 tissues that can be easily updated and that is structured to facilitate custom analyses. To showcase the flexibility of the atlas, we demonstrate that it can be used for isoform analyses. In particular, we study cell-type specificity of isoforms of OAS1, which has recently been shown to offer SARS-CoV-2 protection in certain individuals that display higher expression of the p46 isoform. Using our Commons Cell Atlas, we localize the OAS1 p44b isoform to the testis, and find that it is specific to germ line cells. By virtue of enabling customized analyses via a modular and dynamic atlas structure, the Commons Cell Atlas should be useful for exploratory analyses that are intractable within the rigid framework of current gene-centric static atlases.
" }, { "name": "Hsu, Alice", "degree": "PhD", "year": "2023", "title": "Neurotechnology for Multiplexed Interrogation of Brain Circuits and Synaptic Activity", "advisor": "Roukes, Michael Lee", "url": "https://resolver.caltech.edu/CaltechTHESIS:06032023-030758184", "creators": [ { "name": { "family": "Hsu", "given": "Alice" }, "id": "Hsu-Alice", "orcid": "0000-0001-6609-2559", "display_name": "Hsu, Alice" } ], "advisors": [ { "name": { "family": "Roukes", "given": "Michael Lee" }, "id": "Roukes-M-L", "orcid": "0000-0002-2916-6026", "role": "advisor", "display_name": "Roukes, Michael Lee" } ], "committee": [ { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "chair", "display_name": "Zinn, Kai George" }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Tian", "given": "Lin" }, "id": "Tian Lin", "orcid": "0000-0001-7012-6926", "role": "member", "display_name": "Tian, Lin" }, { "name": { "family": "Shepard", "given": "Kenneth L." }, "id": "Shepard-Kenneth-L", "orcid": "0000-0003-0665-6775", "role": "member", "display_name": "Shepard, Kenneth L." }, { "name": { "family": "Moreaux", "given": "Laurent C." }, "id": "Moreaux-Laurent-C", "orcid": "0000-0003-1276-5062", "role": "member", "display_name": "Moreaux, Laurent C." }, { "name": { "family": "Roukes", "given": "Michael Lee" }, "id": "Roukes-M-L", "orcid": "0000-0002-2916-6026", "role": "member", "display_name": "Roukes, Michael Lee" } ], "option_major": [ "bioeng" ], "doi": "10.7907/tba9-sb03", "abstract": "This thesis describes the development of neural technologies for 1) multiplexed brain circuit electrophysiology (ephys) recordings and control of activity in optogenetic mice lines with concurrent recording paired with two-photon imaging and 2) multiplexed measurements of synaptic release events in microfluidic platforms. The first part of this thesis describes efforts to provide deterministic correlation of excited neuron action potential with resulting ephys recordings in vivo. This consisted of technological development of novel, high density multisite silicon probes for electrophysiology recordings in vivo. The probes consist of four columns of electrodes densely packed at the shank tip. This density of electrode arrays allowed for higher resolution isolation of more distinct waveforms than previous ephys probes and benchmarking measurements to triangulate the locations of emitting neurons. These measurements help benchmark the ability of existing silicon extracellular probes to capture surrounding extracellular activity. When combined with two-photon imaging, we can simultaneously record ephys activity, image the probe and surrounding brain, quantify brain damage during probe implantation, and control neural activity using optogenetic mouse lines.
\r\n\r\nThe second project described development of a microfluidic platform to monitor synaptic release of the neurotransmitter glutamate. Microfluidic devices were used to isolate synaptic processes expressing synaptic reporters and provide targeted recording of glutamate activity across the synapse. Synaptic glutamate release was monitored with a two part genetically encoded fluorescent reporter that detects glutamate released at the synapse, called split-iGluSnFR, developed in Professor Lin Tian\u2019s lab at UC Davis. We designed new microfluidic devices to better isolate neuron processes with split-iGluSnFR and be compatible with existing fluorescent complementary metal\u2013oxide\u2013semiconductor (CMOS) contact imagers. Using computational fluid dynamic simulations, we demonstrate efficient perfusion in the device. The form factor of this new device is designed to be compatible with CMOS contact imagers, and that when combined will help us achieve our ultimate goal to monitor the kinetics of simultaneous synaptic release events modulated by perfused neuromodulating drugs.
" }, { "name": "Huang, Jining", "degree": "PhD", "year": "2023", "title": "Applications of Dynamic Nucleic Acid Nanotechnology in Closed-Loop Genetic Circuits and Detection of Viral Pathogens", "advisor": "Pierce, Niles A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01172023-195222304", "creators": [ { "name": { "family": "Huang", "given": "Jining" }, "id": "Huang-Jining", "orcid": "0000-0002-3798-4790", "display_name": "Huang, Jining" } ], "advisors": [ { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "advisor", "display_name": "Pierce, Niles A." } ], "committee": [ { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "orcid": "0000-0002-5899-7523", "role": "chair", "display_name": "Winfree, Erik" }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "member", "display_name": "Pierce, Niles A." } ], "option_major": [ "bioeng" ], "doi": "10.7907/54tw-ym95", "abstract": "Nucleic acid nanotechnologies have provided a platform where biologically relevant molecules can be engineered to perform programmable functions. Relative to proteins, complex nucleic acid-based systems can be designed more readily due to the countable nature of base-pairing interactions and readily available physical models. These features of nucleic acids enable us to design novel interaction pathways and functions by providing well-behaved molecular mechanisms. Two examples of these mechanisms are the conditional guide RNA (cgRNA) and the hybridization chain reaction (HCR). A cgRNA is a conditional programmable regulator where an expressed RNA trigger can conditionally turn on or off transcriptional regulation. HCR is a molecular mechanism for in vitro and in situ amplification of signals to spatially identify proteins, RNA, or DNA in a sample. This thesis will first demonstrate the use of these nucleic acid molecular mechanisms in closed-loop genetic circuits and infectious disease testing using cgRNAs and HCR, respectively, then provide updated tools for the nucleic acid design community to exploit the programmable nature of nucleic acids.
\r\n\r\nWe begin by demonstrating the use of conditional programmable cgRNAs in closed-loop genetic circuits. Synthetic genetic circuits allow scientists to engineer arbitrary molecular interactions in living organisms. Feedback circuits in particular are recurrently found in nature and enable useful functionalities. However, protein components of genetic circuits cannot be designed scalably, are often mined from preexisting genomes, and present difficulties in being biologically orthogonal to themselves or the host organism. We are motivated to address these limitations by using orthogonal nucleic acid circuits created de novo. One potential component of these circuits are conditional guide RNAs (cgRNAs). cgRNAs are switchable transcriptional regulators, and this allows gene expression to be modulated through the expression of a small RNA trigger. Here we assess cgRNAs as a component for feedback genetic circuits. As an initial demonstration of cgRNA synthetic circuits, we built and validated a simple threshold circuit and demonstrated its orthogonality and scalability by showing independent circuit functions of two switches in a single cell. We also created a larger toggle switch that is made from the same components as the previous switches. These experiments show the orthogonality and feedback capabilities of cgRNAs will position them as a composable component for scalable synthetic biology.
\r\n\r\nWe then used the hybridization chain reaction mechanism to develop an adaptable and sensitive test for the detection of SARS-CoV-2. The lateral flow assay format enables rapid, instrument-free, at-home testing for SARS-CoV-2. Due to the absence of signal amplification, this simplicity comes at a cost in sensitivity. Here, we enhance sensitivity by developing an amplified lateral flow assay that incorporates isothermal, enzyme-free signal amplification based on the mechanism of hybridization chain reaction (HCR). The simplicity of the user experience after the test begins is maintained by using a disposable 3-channel lateral flow device to automatically deliver reagents to the test region in three successive stages without user interaction. Prior to starting the test, a 15-minute heat step is required. Detecting gamma-irradiated SARS-CoV-2 virions in an extraction buffer, the current amplified HCR lateral flow assay achieves a limit of detection of 200 copies/\u00b5L using nucleic acid probes to target the SARS-CoV-2 RNA genome. By comparison, five commercial unamplified lateral flow assays that use proprietary antibodies to target the viral nucleocapsid protein exhibit limits of detection of 500 copies/\u00b5L, 1000 copies/\u00b5L, 2000 copies/\u00b5L, 2000 copies/\u00b5L, and 20,000 copies/\u00b5L. By swapping out nucleic acid probes to target different pathogens, amplified HCR lateral flow assays offer a platform for adaptable and sensitive at-home testing for emergent diseases.
\r\n\r\nComponents for the previous two projects are designed and analyzed with NUPACK. NUPACK is a growing software suite for the analysis and design of nucleic acid structures, devices, and systems serving the needs of researchers in the fields of nucleic acid nanotechnology, molecular programming, synthetic biology, and across the life sciences. NUPACK algorithms are unique in treating complex and test tube ensembles containing arbitrary numbers of interacting strand species, providing crucial tools for capturing concentration effects essential to analyzing and designing the intermolecular interactions that are a hallmark of these fields. The all-new NUPACK web app (nupack.org) has been re-architected for the cloud, leveraging a cluster that scales dynamically in response to user demand to enable rapid job submission and result inspection even at times of peak user demand. The web app exploits the all-new NUPACK 4 scientific code base as its backend, offering enhanced physical models (coaxial and dangle stacking sub-ensembles), dramatic speedups (20-120\u00d7 for test tube analysis), and increased scalability for large complexes.
" }, { "name": "Hurt, Robert Cooper", "degree": "PhD", "year": "2023", "title": "Engineering of Second-Generation Acoustic Reporter Genes", "advisor": "Shapiro, Mikhail G.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05192023-001330664", "creators": [ { "name": { "family": "Hurt", "given": "Robert Cooper" }, "id": "Hurt-Robert-Cooper", "orcid": "0000-0002-4347-6901", "display_name": "Hurt, Robert Cooper" } ], "advisors": [ { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "advisor", "display_name": "Shapiro, Mikhail G." } ], "committee": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "chair", "display_name": "Lester, Henry A." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "member", "display_name": "Shapiro, Mikhail G." } ], "option_major": [ "neurobio" ], "doi": "10.7907/qs6v-5d67", "abstract": "A major outstanding challenge in the fields of biological research, synthetic biology, and cell-based medicine is visualizing the functions of natural and engineered cells noninvasively inside opaque organisms. Ultrasound imaging has the potential to address this challenge as a widely available technique with a tissue penetration of several centimeters and spatial resolution below 100 \u00b5m. Recently, the first genetically encoded acoustic reporters were developed based on bacterial gas vesicles (GVs) to link ultrasound signals to molecular and cellular function. However, the properties of these first-generation acoustic reporter genes (ARGs) resulted in limited sensitivity and specificity for imaging gene expression in vivo.
\r\n\r\nThe goal of my thesis work has been to engineer second-generation ARGs with improved acoustic and expression phenotypes compared to the existing first-generation constructs. I took two complementary engineering approaches to developing these constructs: homolog screening and directed evolution, sometimes referred to as the \u201cnature and nurture\u201d of protein engineering. The resulting constructs offer major qualitative and quantitative improvements, including much stronger ultrasound contrast, the ability to produce nonlinear signals distinguishable from background tissue in vivo, stable long-term expression, and compatibility with in vitro multiplexed imaging. In collaboration with others in the lab, we demonstrate the capabilities of these next-generation ARGs by imaging in situ gene expression in mouse models of breast cancer and tumor-homing therapeutic bacteria, noninvasively revealing the unique spatial distributions of tumor growth and colonization by therapeutic cells in living subjects and providing real-time guidance for interventions such as needle biopsies.
\r\n\r\nThis thesis is organized as follows: in the first two chapters, I introduce the key background needed to understand both the importance and properties of ARGS, and how they have been and could be engineered. In the next two chapters, I detail specific efforts to engineer them\u2014one involving the construction of a high-throughput, semi-automated setup for acoustic phenotyping of cells and its application to ARG directed evolution, and another involving the screening of several GV cluster homologs to identify ones suitable for use as improved ARGs. Finally, I conclude with insights gleaned from these two ARG engineering projects and suggestions for future ones.
\r\n\r\nThe approaches, results, and ideas presented in this thesis represent the current state-of-the-art in ARG engineering and application. While recent technology development in this field has unlocked exciting new use cases for ARGs in noninvasive biological imaging, most of their potential for basic science and disease diagnosis and treatment has yet to be realized.
" }, { "name": "Jorgensen, Victoria Lynn", "degree": "PhD", "year": "2023", "title": "Stem Cell-Derived Embryo Models in Mouse and Human to Illuminate the \u201cBlack Box\u201d of Pre- to Post-Implantation Development", "advisor": "Zernicka-Goetz, Magdalena", "url": "https://resolver.caltech.edu/CaltechTHESIS:06112023-211027828", "creators": [ { "name": { "family": "Jorgensen", "given": "Victoria Lynn" }, "id": "Jorgensen-Victoria-Lynn", "orcid": "0000-0002-4205-6198", "display_name": "Jorgensen, Victoria Lynn" } ], "advisors": [ { "name": { "family": "Zernicka-Goetz", "given": "Magdalena" }, "id": "Zernicka-Goetz-M", "orcid": "0000-0002-7004-2471", "role": "advisor", "display_name": "Zernicka-Goetz, Magdalena" } ], "committee": [ { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "chair", "display_name": "Guttman, Mitchell" }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Parker", "given": "Joseph" }, "id": "Parker-J", "orcid": "0000-0001-9598-2454", "role": "member", "display_name": "Parker, Joseph" }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Zernicka-Goetz", "given": "Magdalena" }, "id": "Zernicka-Goetz-M", "orcid": "0000-0002-7004-2471", "role": "member", "display_name": "Zernicka-Goetz, Magdalena" } ], "option_major": [ "biodev" ], "doi": "10.7907/t1fe-3915", "abstract": "Mammalian development is a complex and highly regulated process by which a single cell, the totipotent zygote, gives rise to all lineages of the future organism. While incredible advancements have been made to study and understand the earliest events of our life, many questions are still unanswered. Moreover, the most precarious stage of development, implantation, remains a \u201cblack box\u201d to researchers due to inaccessibility of the embryo within the uterus of the mother. In the last decade, however, the emergence of stem cell derived embryos represents an exciting alternative avenue to study these dynamic stages.
\r\n\r\nDuring my PhD, I worked to establish two pre-implantation stem cell models, one in human and one in mouse, to better understand the earliest days of mammalian development. These models replicate the blastocyst stage of development; at this point in time the embryo is ready to implant into the uterus and contains all embryonic and extra-embryonic tissues needed to form the future organism: the epiblast, the hypoblast, and the trophectoderm. Beginning with my human model, I demonstrate the ability of a single cell type, expanded potential stem cells (EPSCs), to give rise to structures that replicate the natural blastocyst in size, morphology, and initiation of lineage segregation. Furthermore, these human blastocyst-like structures can undergo the very beginning of post-implantation remodeling by forming an epiblast rosette and initiating lumenogenesis. Nevertheless, single cell RNA-seq (scRNA-seq) analysis reveals that lineages are not fully committed in this model, perhaps explaining why development is limited in these structures up to about Day 7/8. In the context of my mouse model, I combine not one but three distinct cell types to generate blastocyst-like structures: 1) wildtype embryonic stem cells (ESCs) to form the epiblast, 2) trophoblast stem cells (TSCs) to form the trophectoderm, and 3) Gata4-inducible ESCs to form the primitive endoderm. Again, these structures mimic the natural mouse blastocyst in morphology and lineage segregation and demonstrate the ability to transition to post-implantation stages. Development of the three blastocyst lineages was further confirmed via global scRNA-seq analysis comparing our Gata4i-Blastoids to natural embryos; importantly, however, this analysis also showed that differentiation of the mural trophectoderm, the tissue responsible for uterine invasion, is lacking in our stem cell model and likely explains the inability for these blastoids to implant in vivo.
\r\n\r\nAltogether, this dissertation explains key aspects of pre- to post-implantation development and highlights the incredible power of stem cell-derived embryos to self-organize into structures that closely mimic the natural embryo.
" }, { "name": "Liaw, Eric Jer-Jiun", "degree": "PhD", "year": "2023", "title": "A Novel, Rapid Phenotypic Assay for a Beta-Lactam Antibiotic Susceptibility and an Analysis of its Theoretical Limits", "advisor": "Ismagilov, Rustem F.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05112023-130637882", "creators": [ { "name": { "family": "Liaw", "given": "Eric Jer-Jiun" }, "id": "Liaw-Eric-Jer-Jiun", "orcid": "0000-0003-2244-8335", "display_name": "Liaw, Eric Jer-Jiun" } ], "advisors": [ { "name": { "family": "Ismagilov", "given": "Rustem F." }, "id": "Ismagilov-R-F", "orcid": "0000-0002-3680-4399", "role": "advisor", "display_name": "Ismagilov, Rustem F." } ], "committee": [ { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "chair", "display_name": "Murray, Richard M." }, { "name": { "family": "Ismagilov", "given": "Rustem F." }, "id": "Ismagilov-R-F", "orcid": "0000-0002-3680-4399", "role": "member", "display_name": "Ismagilov, Rustem F." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." }, { "name": { "family": "Cai", "given": "Long" }, "id": "Cai-Long", "orcid": "0000-0002-7154-5361", "role": "member", "display_name": "Cai, Long" } ], "option_major": [ "biology" ], "doi": "10.7907/qhvg-7q92", "abstract": "Current management of bacterial infections is limited by the slow turnaround time of culture-based antibiotic susceptibility testing (AST). Culture-free phenotypic AST methods, though faster, are limited not only by analytical sensitivity but also by the low number, density, and purity of live pathogens present in clinical specimens before culturing. Separating and concentrating pathogens from clinical specimen matrices and improving the analytic sensitivity of phenotypic measurement technologies remain active areas of research. However, to date, the literature lacks consensus over what is a reasonable goal for the minimum number of pathogens in a clinical specimen needed to accurately perform phenotypic AST.
\r\n\r\nI describe \"bulk filtration AST\" and \"digital filtration AST,\" two new filtration-based AST methods that improve an AST method previously published by others and myself. These methods use nucleic acid quantification to assess the activity of antibiotic classes (and only those classes) targeting peptidoglycan turnover, specifically the beta-lactams, which are the most frequently prescribed class of antibiotics. I use filtration AST to quantify the in vitro pharmacodynamics of beta-lactam antibiotics over time scales shorter than two hours, and I simultaneously validate the methods' accuracies on clinical isolates of Enterobacteriaceae. To analyze filtration AST results, either for fitting parameter values or for predicting susceptibility, I derive probabilistic models for the outcomes of each of the two filtration AST methods, then perform Bayesian parameter inference from my data.
\r\n\r\nI then propose a general mathematical framework for defining the concepts of the phenotypic assay and the ideal phenotypic assay. Within this framework, I calculate the ideal filtration AST performance as a function of the number of cells assayed, my fitted pharmacodynamic parameters, and other variables. Interestingly, the observed performance of my implementation of digital filtration AST is consistent with the implementation's approaching the ideal performance. I hope my demonstration of these new methods and my theoretical framework will help guide future research into rapid phenotypic AST.
" }, { "name": "Liu, Grace", "degree": "Senior Thesis", "year": "2023", "title": "\u201cIt\u2019s Our War Too\u201d: Barriers to Authorship by Women Writing Vietnam War Poetry", "advisor": "Jurca, Catherine", "url": "https://resolver.caltech.edu/CaltechTHESIS:04262023-070455932", "creators": [ { "name": { "family": "Liu", "given": "Grace" }, "id": "Liu-Grace", "display_name": "Liu, Grace" } ], "advisors": [ { "name": { "family": "Jurca", "given": "Catherine" }, "id": "Jurca-C", "role": "advisor", "display_name": "Jurca, Catherine" } ], "committee": [ { "name": { "family": "None", "given": "None" }, "display_name": "None, None" } ], "option_major": [ "biology", "english" ], "doi": "10.7907/r3ec-ev97", "abstract": "Even though American women had higher rates of involvement in the Vietnam War than any previous war, poems about their experiences were extremely scarce until over a decade after American troops withdrew. A major contributor to the lack of literary representation is the critical dismissal of women\u2019s war poetry as being unable to teach readers meaningful \u201ctruths\u201d about war. This thesis examines two collections of female-authored poems, Visions of War, Dreams of Peace and Shallow Graves, which were published in 1991 and 1986 respectively. The former contains poems from 40 women, most of whom served as army nurses; the latter combines the experiences of Wendy Wilder Larsen, an American woman who lived in Vietnam for two years, and Tran Thi Nga, a Vietnamese woman who immigrated to America. The collections reveal that most American women responded to critical expectations either through self-erasure or active rebellion. In contrast to the American women, Nga is granted authority by critics because her Vietnamese perspective is unique in English literature, but her authorship is instead challenged during the process of adapting her story for an American audience." }, { "name": "Luo, Yicheng", "degree": "PhD", "year": "2023", "title": "Maternally Inherited siRNAs Initiate piRNA Cluster Formation", "advisor": "Aravin, Alexei A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:11042022-055503832", "creators": [ { "name": { "family": "Luo", "given": "Yicheng" }, "id": "Luo-Yicheng", "orcid": "0000-0003-3704-2389", "display_name": "Luo, Yicheng" } ], "advisors": [ { "name": { "family": "Aravin", "given": "Alexei A." }, "id": "Aravin-A-A", "orcid": "0000-0002-6956-8257", "role": "advisor", "display_name": "Aravin, Alexei A." } ], "committee": [ { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "chair", "display_name": "Guttman, Mitchell" }, { "name": { "family": "Shan", "given": "Shu-ou" }, "id": "Shan-Shu-ou", "orcid": "0000-0002-6526-1733", "role": "member", "display_name": "Shan, Shu-ou" }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "member", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Aravin", "given": "Alexei A." }, "id": "Aravin-A-A", "orcid": "0000-0002-6956-8257", "role": "member", "display_name": "Aravin, Alexei A." } ], "option_major": [ "biology" ], "doi": "10.7907/f2nh-8w08", "abstract": "PIWI-interacting RNAs (piRNAs) guide repression of transposable elements in germlines of animals to protect genome integrity. In Drosophila, the majority of piRNAs are produced from heterochromatic genomic loci, called piRNA clusters, that act as repositories of information about genome invaders. piRNA generation by dual-strand clusters depends on the chromatin-bound Rhino-Deadlock-Cutoff (RDC) complex, a complex specifically enriched on a dual-strand cluster, which is deposited on clusters guided by piRNAs, forming a feed-forward loop in which piRNAs promote their own biogenesis. However, this rises a fundamental question about how piRNA clusters are formed initially before cognate piRNAs are present.
\r\n\r\nHere we report the spontaneous de novo formation of a Rhino-dependent piRNA cluster from repetitive transgenic sequences. We show that cluster formation occurs gradually over several generations and requires continuous trans-generational transmission of small RNAs from mothers to their progeny. Importantly, we discovered that maternally-supplied siRNAs are responsible for triggering de novo cluster activation in progeny. In contrast, the siRNA pathway is dispensable for piRNA cluster function and maintenance after its establishment. These results revealed an unexpected cross-talk between the siRNA and piRNA pathways and suggested a mechanism for de novo formation of piRNA clusters triggered by production of siRNAs.
" }, { "name": "Ma, Yitong", "degree": "PhD", "year": "2023", "title": "Multicellular Synthetic Biology in Mammalian Systems", "advisor": "Elowitz, Michael B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04132023-015900885", "creators": [ { "name": { "family": "Ma", "given": "Yitong" }, "id": "Ma-Yitong", "orcid": "0000-0003-4446-7326", "display_name": "Ma, Yitong" } ], "advisors": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "advisor", "display_name": "Elowitz, Michael B." } ], "committee": [ { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "chair", "display_name": "Guttman, Mitchell" }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." } ], "option_major": [ "biology" ], "doi": "10.7907/w0q1-7s17", "abstract": "In multicellular organisms, different types of cells use intercellular signals to communicate and regulate population dynamics, and further coordinate complex behaviors. This presents a rarely tapped into potential for mammalian synthetic biology, which was largely restricted to engineering a single cell type in the past to mimic and use similar multicellular designs to achieve more functionalities. However, with current synthetic biology tools and designs, there are several major challenges to achieve a multicellular circuit. Challenges include precise and tunable control over cell type switching, having an orthogonal cell-cell communication signal, and robust control of cell populations.
\r\n\r\nTo address these challenges, this thesis presents a system for tunable regulating of gene expression with DNA methylation, an auxin-based module for mammalian cell-cell communication, and a robust circuit for population control in mammalian cells. I further applied these work to engineering immune cells to show the potential of multicellular circuits in immunotherapies. Together, these works demonstrated the possibility of constructing multicellular circuits in mammalian systems, and that multicellular circuit can further extend the scope of synthetic biology to achieve more complex functions.
" }, { "name": "Marken, John Paul", "degree": "PhD", "year": "2023", "title": "Experimental and Theoretical Frameworks for Enabling Environmental Synthetic Biology", "advisor": "Murray, Richard M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05292023-181810775", "creators": [ { "name": { "family": "Marken", "given": "John Paul" }, "id": "Marken-John-Paul", "orcid": "0000-0001-9696-088X", "display_name": "Marken, John Paul" } ], "advisors": [ { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "advisor", "display_name": "Murray, Richard M." } ], "committee": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "chair", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." } ], "option_major": [ "bioeng" ], "doi": "10.7907/h50w-p058", "abstract": "Although the field of synthetic biology has made great advances toward becoming a mature engineering discipline over its first quarter-century, the vast majority of these efforts have focused on improving the design and performance of genetic circuits intended to operate in well-controlled, laboratory settings. The goal of safely deploying engineered microbes to reliably perform their programmed functions in natural, uncontrolled environments begets its own set of foundational challenges that will require new frameworks that shift our existing mindsets about the way we engineer biological systems.
\r\n\r\nThese frameworks, because they focus on enabling system properties that were not priorities for conventional synthetic biology research, can constitute a new field of research which I refer to as environmental synthetic biology. The central priorities of environmental synthetic biology include (1) developing and characterizing effective ways to introduce engineered biological systems into natural environments, (2) ensuring that the performance of these systems can remain robust and predictable in the face of environmental variability, (3) developing and characterizing ways to control and monitor the behavior of an engineered system after deployment in an inaccessible environment, and (4) developing fundamental architectures to enable autonomous system operation and adaptation within environmental contexts.
\r\n\r\nIn this thesis, I present the initial steps towards the development of three frame- works that address these priorities of environmental synthetic biology. The first framework, described in Chapter 2, demonstrates the potential of using DNA as the substrate for addressable and adaptable intercellular communication in engineered populations. This enables the ability to one day create multicellular systems that can autonomously reconfigure their own architecture in the face of changing environmental conditions. The second framework, described in Chapters 3 and 4, presents a new mathematical representation of biomolecular reaction systems that enables geometric bounds on the space of possible behaviors under all possible configurations for a particular system architecture. The third, ongoing framework emphasizes the importance of explicitly incorporating the physiological state of the host cell into the assessment of a genetic circuit\u2019s behavior by exploring the impact of cellular growth arrest on transcriptional response curves. The preliminary results of this work are presented in Chapter 5.
" }, { "name": "McGehee, James Mitchell", "degree": "PhD", "year": "2023", "title": "Optogenetic Approaches for Determining the Temporal Role of Morphogen Inputs on Target Gene Expression", "advisor": "Stathopoulos, Angelike", "url": "https://resolver.caltech.edu/CaltechTHESIS:05302023-162053583", "creators": [ { "name": { "family": "McGehee", "given": "James Mitchell" }, "id": "McGehee-James-Mitchell", "orcid": "0000-0002-9353-1235", "display_name": "McGehee, James Mitchell" } ], "advisors": [ { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "orcid": "0000-0001-6597-2036", "role": "advisor", "display_name": "Stathopoulos, Angelike" } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Bois", "given": "Justin S." }, "id": "Bois-J-S", "orcid": "0000-0001-7137-8746", "role": "member", "display_name": "Bois, Justin S." }, { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "orcid": "0000-0002-3904-0195", "role": "member", "display_name": "Goentoro, Lea A." }, { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "orcid": "0000-0001-6597-2036", "role": "member", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "member", "display_name": "Zinn, Kai George" } ], "option_major": [ "biology" ], "doi": "10.7907/c610-za20", "abstract": "The Dorsal transcription factor and morphogen is important for patterning the Dorsal- Ventral axis of Drosophila melanogaster and while it has been extensively studied, the temporal dynamics of Dorsal are not well understood. There are many processes that contribute to Dorsal nuclear concentration levels, including Toll signaling and Cactus degradation, interactions with other proteins, shuttling of Dorsal to the ventral side, DNA binding, and nuclear spacing. Dorsal nuclear levels are known to activate or repress target gene expression in a concentration or threshold dependent manner. To test how Dorsal dynamics and changes to the Dorsal gradient over time affect target gene expression, we added two optogenetic tags to Dorsal at the endogenous locus to control Dorsal nuclear levels: Blue Light Inducible Degradation (BLID) and Light Inducible Nuclear Export System (LEXY). We found that upon degradation of Dorsal using blue light and BLID that a downstream ratchet was able to maintain the expression of high threshold target genes. Using blue light and LEXY to export Dorsal, we identified an important window where Dorsal activity is required to allow activation of high threshold target genes at later stages. In comparing BLID and LEXY in conjunction with mutations to a nuclear export sequence, we also identified how rapid nuclear import and export of Dorsal is sufficient for low threshold target gene expression but actively disrupts high threshold target gene expression. We conclude that not only are final concentration levels, but also the dynamics leading to those levels are important for proper gene expression." }, { "name": "Melis, Johan Matthijs", "degree": "PhD", "year": "2023", "title": "A Neural Network Model of an Insect's Wing Hinge Reveals How Steering Muscles Control Flight", "advisor": "Dickinson, Michael H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02272023-213525351", "creators": [ { "name": { "family": "Melis", "given": "Johan Matthijs" }, "id": "Melis-Johan-Matthijs", "orcid": "0000-0001-8966-9496", "display_name": "Melis, Johan Matthijs" } ], "advisors": [ { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "orcid": "0000-0002-8587-9936", "role": "advisor", "display_name": "Dickinson, Michael H." } ], "committee": [ { "name": { "family": "Burdick", "given": "Joel Wakeman" }, "id": "Burdick-J-W", "orcid": "0000-0002-3091-540X", "role": "chair", "display_name": "Burdick, Joel Wakeman" }, { "name": { "family": "Gharib", "given": "Morteza" }, "id": "Gharib-M", "orcid": "0000-0003-0754-4193", "role": "member", "display_name": "Gharib, Morteza" }, { "name": { "family": "Colonius", "given": "Tim" }, "id": "Colonius-T", "orcid": "0000-0003-0326-3909", "role": "member", "display_name": "Colonius, Tim" }, { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "orcid": "0000-0002-8587-9936", "role": "member", "display_name": "Dickinson, Michael H." } ], "option_major": [ "bioeng" ], "doi": "10.7907/teej-tb66", "abstract": "The flight system of the fly is remarkable. A fly can execute an escape maneuver in milliseconds, compensate for wing damage when half of the wing is missing, fly in turbulent conditions, and migrate over large distances. While there are many factors that contribute to the robustness and versatility of insect flight, it is the mechanical encoding of wing motion in the wing hinge that allows flies to rapidly and accurately change wing motion over a large dynamic range. The wing hinge consists of several hardened skeletal elements, named sclerites, and a set of twelve steering muscles are attached to some of these components within the exoskeleton. Due to the anatomical complexity and minute size of the sclerites, the way in which the steering muscles alter the mechanical encoding of wing motion in the hinge is poorly understood.
\r\n\r\nUsing genetically encoded calcium indicators and high-speed videography, is is possible to simultaneously image steering muscle activity and wing motion. In order to extract wing pose from the high-speed video frames, an automated tracking algorithm was developed, that used a neural network and model fitting to accurately reconstruct the wing kinematics. The synchronous recordings of wing motion and steering muscle activity were used to train a convolutional neural network that learned to accurately predict the wing kinematics from muscle activity patterns. After training, the convolutional neural network was used to perform virtual experiments, revealing how the steering muscles regulate wing motion. Correlation analysis revealed that the 12 steering muscles have highly correlated activity. The correlation of muscle activity can be approximated well by a 12D-plane, in which all activity has to reside.
\r\n\r\nTo study the function of the sclerites, a bottleneck was introduced in the convolutional neural network. The bottleneck consists of five neurons, or latent parameters, four parameters corresponding to the state of the different sclerites, on which the steering muscles act, and one parameter representing the wingbeat frequency. This so called latent network predicts both the changes in wing motion and muscle activity patterns as a function of sclerite state. The predicted wing motion as a function of sclerite state matches with previous anatomy and electrophysiology studies for the basalare, first axillary and third axillary sclerites. The fourth axillary sclerite has not been studied before, but shows an antagonistic relationship between the hg1,2 and hg3,4 muscles, resulting in a strong decrease and increase, respectively, of stroke amplitude, deviation and wing pitch angles.
\r\n\r\nBy replaying the wing kinematics of the virtual experiments on a dynamically scaled robotic fly, a model of the aerodynamic and inertial control forces as a function of steering muscle activity was constructed. This control force model was subsequently integrated in a state-space system of fly flight, which in turn was integrated in a model predictive control simulation that was used to simulate free flight maneuvers. The body motion, steering muscle activity, and wing kinematics of the model predictive control simulations were strikingly similar to the recorded maneuvers of free-flying flies.
\r\n\r\nThe integrative, multi-disciplinary approach that was used to reveal the mechanical logic of the wing hinge, and the control problem that a fly needs to solve to stay airborne, are both unprecedented in prior literature. The methodologies and models of this study will be a valuable resource in future research on how the fly's nervous system controls the complex behavior that is flight.
" }, { "name": "Miller, David Ryan", "degree": "PhD", "year": "2023", "title": "Developing Dalotia coriaria, the Greenhouse Rove Beetle, as a Novel Model Organism", "advisor": "Parker, Joseph", "url": "https://resolver.caltech.edu/CaltechTHESIS:08172022-204326931", "creators": [ { "name": { "family": "Miller", "given": "David Ryan" }, "id": "Miller-David-Ryan", "orcid": "0000-0002-7186-2623", "display_name": "Miller, David Ryan" } ], "advisors": [ { "name": { "family": "Parker", "given": "Joseph" }, "id": "Parker-J", "orcid": "0000-0001-9598-2454", "role": "advisor", "display_name": "Parker, Joseph" } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Parker", "given": "Joseph" }, "id": "Parker-J", "orcid": "0000-0001-9598-2454", "role": "member", "display_name": "Parker, Joseph" }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/wwp6-xq35", "abstract": "This thesis deals with the development of Dalotia coriaria, the greenhouse rove beetle, as a novel model organism. A fundamental characteristic of metazoan life is inter-species interactions. Chapter 1 explores why there is a need for Dalotia as a new model organism to study these interspecies interactions in ways that are intractable with current established models. It also explores the life history characteristics of Dalotia that make it amenable to development as a novel model organism as well as the need for genetic access in order to successfully make Dalotia an established laboratory model organism.
\r\n\r\nChapter 2 explores how I have solved the husbandry techniques required for genetic manipulations in Dalotia. These include the ability to collect large amounts of early embryos, mount embryos on slides, micro inject embryos, raise single housed larvae to adulthood, and set up one-on-one adult crosses.
\r\n\r\nChapter 3 explores how I have developed the Piggybac transposon system to successfully knock in trans-genes into the Dalotia genome. It also shows how I have developed the UAS Gal4\u0394 binary expression system to work in Dalotia, allowing for controlled high expression of inserted trans-genes.
\r\n\r\nChapter 4 explores how I have developed the CRISPR Cas9 system to successfully perform targeted germline point mutations in the Dalotia genome. It also shows how I have developed fast and accurate genotyping techniques for producing and maintaining homozygous stocks of mutant Dalotia for long periods of time. The appendices of this thesis include step-by-step protocols that allow for reproduction of all of the husbandry and genetic manipulation techniques covered in Chapters 2-4.
\r\n\r\nLastly, Chapter 5 of this thesis explores olfactory receptor guided behaviors in Dalotia. I use RNA Smartseq techniques to produce a map of chemoreceptors across the Dalotia body. I also use the Dalotia CRISPR Cas9 protocol I developed to produce a homozygous line of olfactory receptor-deficient Dalotia by knocking out the olfactory receptor co-receptor. I show that the line of olfactory receptor-deficient Dalotia are incapable of olfaction, and then explore how it affects their defensive behavior when interacting with ants. These interactions are studied in a free moving arena and in a tethered beetle on the ball setup using machine learning to analyze pose.
" }, { "name": "Moses, Lambda", "degree": "PhD", "year": "2023", "title": "Computation Foundations of Spatial Transcriptomics", "advisor": "Pachter, Lior S.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05312023-213322223", "creators": [ { "name": { "family": "Moses", "given": "Lambda" }, "id": "Moses-Lambda", "orcid": "0000-0002-7092-9427", "display_name": "Moses, Lambda" } ], "advisors": [ { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "advisor", "display_name": "Pachter, Lior S." } ], "committee": [ { "name": { "family": "Van Valen", "given": "David A." }, "id": "Van-Valen-D", "orcid": "0000-0001-7534-7621", "role": "chair", "display_name": "Van Valen, David A." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Pimentel", "given": "Harold" }, "id": "Pimentel-Harold", "orcid": "0000-0001-8556-2499", "role": "member", "display_name": "Pimentel, Harold" }, { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "member", "display_name": "Pachter, Lior S." } ], "option_major": [ "biology" ], "doi": "10.7907/rt24-pq60", "abstract": "Single-cell and spatial transcriptomics have come of age in the past few years; datasets and data analysis software packages have proliferated. With the increasing sizes of datasets, proliferating new data collection technologies, and mainstreaming of high-throughput technologies, the software can be improved for better speed and memory efficiency, standardized and consistent user interface for multiple technologies, and in documentation to onboard new users. First, I collected a database of spatial transcriptomics literature and analyzed the data on trends and sociology in this field. Based on the database and data analyses, I wrote a comprehensive book both qualitatively and quantitatively documenting the history of the field since the 1960s and reviewing more recent developments, which informed the software and methods I later developed. Then, to address the challenges with the pre-processing large datasets, we developed \\texttt{kallisto} \\texttt{bustools} for fast and modular pseudoalignment of sequencing reads to the transcriptome in single-cell RNA-seq (scRNA-seq), giving consistent results with the established and much more computationally demanding alignment method Cell Ranger. Briefly summarized are my attempt to map dissociated cells in scRNA-seq to a spatial gene expression reference and to build a image processing pipeline for image based spatial transcriptomics data analysis. Finally, to address the challenges in downstream analyses of spatial -omics data, I first wrote the new \\texttt{SpatialFeatureExperiment} (SFE) data structure to represent and operate on geometries in spatial transcriptomics data and to organize results from spatial analyses. Based on SFE, I wrote Voyager, which brings decades of research in geospatial data analysis to spatial transcriptomics, to better utilize the opportunities from spatial information to gain novel biological insights. To reduce user learning curve, Voyager conforms to SCE styles and conventions and has a comprehensive documentation website and consistent user interface to many geospatial methods.
" }, { "name": "Ni, Yu-Li", "degree": "PhD", "year": "2023", "title": "Developing Tools for Neurobiology: The Retina as a\u00a0Neuropharmacology Testbed & Electrode Pooling\u00a0to Boost Extracellular Array Recording", "advisor": "Meister, Markus", "url": "https://resolver.caltech.edu/CaltechTHESIS:10112022-213905012", "creators": [ { "name": { "family": "Ni", "given": "Yu-Li" }, "id": "Ni-Yu-Li", "orcid": "0000-0003-1600-9854", "display_name": "Ni, Yu-Li" } ], "advisors": [ { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "orcid": "0000-0003-2136-6506", "role": "advisor", "display_name": "Meister, Markus" } ], "committee": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "chair", "display_name": "Lester, Henry A." }, { "name": { "family": "Lois", "given": "Carlos" }, "id": "Lois-Carlos", "orcid": "0000-0002-7305-2317", "role": "member", "display_name": "Lois, Carlos" }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." }, { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "orcid": "0000-0003-2136-6506", "role": "member", "display_name": "Meister, Markus" } ], "option_major": [ "neurobio" ], "doi": "10.7907/jeh6-w316", "abstract": "This thesis presents two tool development projects for neurobiology and one explorative project to find organizing principles for autism.
\r\n\r\nThe first project (Chap 2, Retina as Probe) was conceived to tackle the problem that there hasn\u2019t been a reliable model system for system-level neuropharmacology. We introduce a testbed for this: the mammalian retina. The retina involves many of the known neurotransmitters and modulators. Yet its synaptic wiring is well understood, and quantitative models exist to explain its input-output functions. One can connect the systems-level effects to the underlying cellular and molecular causes. To demonstrate the retina\u2019s use, we explored the effects of a range of general anesthetics on the light responses of the mouse retina. At sub-anesthetic doses, we found that certain anesthetics exert a paradoxical effect: they increase the light response of some retinal neurons and suppress the response of others. Notably, this occurred for alcohols and ketamine but not for isoflurane. We traced these effects to transmitter release at a specific synapse and, in one case, to a specific presynaptic ion channel. All the anesthetics silenced the output of the retina completely at concentrations similar to their effective dose for anesthesia in humans. Sedatives reduced retinal sensitivity but did not silence it. Finally, we used specific drugs that target hypothesized molecular mechanisms to probe how much they each contribute to anesthesia of the retina.
\r\n\r\nThe second project which attempted to probe the principles of autism (Chap 3) was conceptually a direct extension of the retina as a testbed. Similar to the situation in seeking for what the mechanism of general anesthesia is, the field of autism research also lacks a good testbed but for systemically comparing gene mutation - circuit defect - behavior outcomes. Similarly, we utilized the retina as a platform to identify circuit defects in four different autism model mice and followed through the different mouse line\u2019s behavior readouts using our lab\u2019s maze navigation paradigm. We discovered that the different autism mouse lines varied in the retinal circuits and varied in their navigation preferences. Nevertheless, unlike the anesthetic project, there wasn\u2019t a simple mechanism to explain why or how these differences are coupled together.
\r\n\r\nThe last project, Electrode Pooling, (Chap 4) aimed to boost the yield of extracellular recording electrode arrays with a novel method we named electrode pooling. The per-implant yield of extracellular recording leaped significantly from the order of tens to the order of hundreds when engineers built multiple electrode arrays based on silicon technology to replace tetrode wires. Unfortunately, this yield-per-site is already maxed out with modern silicon technology. The constraint of the yield is mainly biological, as explained in the chapter, and thus could not be further advanced by improving the manufacturing processes of semiconductors. Our solution utilized an approach that multiplexed the array recording sites (not the bottleneck) onto the readout wires with accompanying filters (the actual bottleneck). Specifically, the method proposes intelligently choosing many recording sites that carry signals and connecting them to a single wire via manipulating the switches and later un-mixed with a spike-sorting algorithm. We demonstrated the first proof-of-principle study that shows that one could get more single-neuron recordings per implant site with electrode pooling, and made recommendations on the hardware design that could facilitate the advancement of probes that use pooling algorithms.
" }, { "name": "Raghavan, Guruprasad", "degree": "PhD", "year": "2023", "title": "Engineering Artificial Systems with Natural Intelligence", "advisor": "Thomson, Matthew", "url": "https://resolver.caltech.edu/CaltechTHESIS:03172023-050019811", "creators": [ { "name": { "family": "Raghavan", "given": "Guruprasad" }, "id": "Raghavan-Guruprasad", "orcid": "0000-0002-1970-9963", "display_name": "Raghavan, Guruprasad" } ], "advisors": [ { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "advisor", "display_name": "Thomson, Matthew" } ], "committee": [ { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "orcid": "0000-0002-5899-7523", "role": "chair", "display_name": "Winfree, Erik" }, { "name": { "family": "Rutishauser", "given": "Ueli" }, "id": "Rutishauser-U", "orcid": "0000-0002-9207-7069", "role": "member", "display_name": "Rutishauser, Ueli" }, { "name": { "family": "Lois", "given": "Carlos" }, "id": "Lois-Carlos", "orcid": "0000-0002-7305-2317", "role": "member", "display_name": "Lois, Carlos" }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" } ], "option_major": [ "bioeng" ], "doi": "10.7907/374f-1202", "abstract": "Although Deep neural networks achieve human-like performance on a variety of perceptual and decision-making tasks, they perform poorly when confronted with changing tasks or goals, and broadly fail to match the flexibility and robustness of human intelligence. Additionally, artificial neural networks rely heavily on human-designed, hand-programmed architectures for their remarkable performance. In this thesis, I work towards achieving two goals: (i) development of a set of mathematical frameworks inspired by facets of natural intelligence, to endow artificial networks with flexibility and robustness, two key traits of natural intelligence; and (ii) inspired by the development of the biological vision system, I propose an algorithm that can \u2018grow\u2019 a functional, layered neural network from a single initial cell, with the aim of enabling autonomous development of artificial networks akin to living neural networks.
\r\n\r\nFor the first goal of endowing networks with flexibility and robustness, I propose a mathematical framework to enable continuous training of neural networks on a range of objectives by constructing path connected sets of networks, resulting in the discovery of a series of networks with equivalent functional performance on a given machine learning task. In this framework, I view the weight space of a neural network as a curved Riemannian manifold and move a network along a functionally invariant path in weight space while searching for networks that satisfy secondary objectives. A path-sampling algorithm trains computer vision and natural language processing networks with millions of weight parameters to learn a series of classification tasks without performance loss while accommodating secondary objectives including network sparsification, incremental task learning, and increased adversarial robustness. Broadly, for achieving this goal, I conceptualize a neural network as a mathematical object that can be iteratively transformed into distinct configurations by the path- sampling algorithm to define a sub-manifold of networks that can be harnessed to achieve user goals.
\r\n\r\nFor the second goal of \u2018growing\u2019 artificial neural networks in a manner similar to living neural networks, I develop an approach inspired by the mechanisms employed by the early visual system to wire the retina to the lateral geniculate nucleus (LGN), days before animals open their eyes. I find that the key ingredients for robust self- organization are (a) an emergent spontaneous spatiotemporal activity wave in the first layer and (b) a local learning rule in the second layer that \u2018learns\u2019 the underlying activity pattern in the first layer. As the bio-inspired developmental rule is adapt- able to a wide-range of input-layer geometries and robust to malfunctioning units in the first layer, it can be used to successfully grow and self-organize pooling architectures of different pool-sizes and shapes. The algorithm provides a primitive procedure for constructing layered neural networks through growth and self-organization. Finally, I also demonstrate that networks grown from a single unit perform as well as hand-crafted networks on a wide variety of static (MNIST recognition) and dynamic (gesture-recognition) tasks. Broadly, the work in the second section of this thesis shows that biologically inspired developmental algorithms can be applied to autonomously grow functional \u2018brains\u2019 in-silico.
" }, { "name": "Rosenthal, Isabelle Anna", "degree": "PhD", "year": "2023", "title": "The Representation of Multimodal Tactile Sensations in the Human Somatosensory System", "advisor": "Andersen, Richard A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05162023-172436741", "creators": [ { "name": { "family": "Rosenthal", "given": "Isabelle Anna" }, "id": "Rosenthal-Isabelle-Anna", "orcid": "0000-0002-9791-3820", "display_name": "Rosenthal, Isabelle Anna" } ], "advisors": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "advisor", "display_name": "Andersen, Richard A." } ], "committee": [ { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "chair", "display_name": "Adolphs, Ralph" }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "member", "display_name": "Andersen, Richard A." }, { "name": { "family": "Rutishauser", "given": "Ueli" }, "id": "Rutishauser-U", "orcid": "0000-0002-9207-7069", "role": "member", "display_name": "Rutishauser, Ueli" }, { "name": { "family": "Eberhardt", "given": "Frederick D." }, "id": "Eberhardt-F-D", "role": "member", "display_name": "Eberhardt, Frederick D." } ], "option_major": [ "cns" ], "doi": "10.7907/yxtg-sr73", "abstract": "The sense of touch is critical to executing basic motor tasks and generating a feeling of embodiment. To construct touch percepts, the brain integrates information from tactile mechanoreceptors with inputs from other senses and top-down variables such as attention and task context. In this thesis, we investigate how these factors influence neural activity within the somatosensory system at different stages of tactile processing, using electrophysiological and behavioral data from a human tetraplegic participant implanted with microelectrode arrays. First, we find that neural responses to imagined touches of different types are decodable in the primary somatosensory cortex, ventral premotor cortex, and the supra-marginal gyrus, and these responses remain stable over many months. Following this analysis, the primary somatosensory cortex is explored in greater depth to better characterize early-stage cortical tactile processing. Touches to the arm and finger are examined during a passive task, in a variety of conditions including visually observed physical touches, physical touches without vision, and visual touches without physical contact. Analysis of the two touch locations suggests that touch encoding in primary somatosensory cortex may be less rigid than in the classical topographic view. Additionally, this experiment uncovers a modulatory effect of vision in the primary somatosensory cortex when it is paired with a physical touch, but no effect of vision alone. Finally, we investigate how visual information impacts artificial tactile sensations, which can be elicited using intra-cortical microstimulation to the primary somatosensory cortex. The ability to elicit reliable, naturalistic artificial touch sensations is vital to the implementation of a tactile brain-machine interface, which would benefit patients with spinal cord injury and others with somatosensory impairments. We find that visual information biases the qualitative percept of artificial stimulation towards an interpretation that is visually plausible. The temporal binding window between vision and stimulation is found to be larger when visual information is biologically relevant, suggesting that the brain\u2019s ability to causally relate artificial stimulation to visual cues depends on visual context. Additionally, recordings from the primary somatosensory cortex indicate that visual information relevant to artificial stimulation is represented across contexts, during an active task. The effect of task on the responsiveness of the primary somatosensory cortex to visual information points to a role of attention in mediating early cortical tactile processing. In combination, the findings presented in this thesis provide insight into the basic neuroscience of how tactile experiences are constructed by the brain, suggesting that early tactile processing is influenced by multisensory, contextual factors. These findings also have clinical applications to developing a brain-machine interface capable of providing naturalistic sensations within a complex real world environment." }, { "name": "Sarma, Anish Anandsai", "degree": "PhD", "year": "2023", "title": "Multicellular Control", "advisor": "Doyle, John Comstock; Goentoro, Lea A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:07052022-033543855", "creators": [ { "name": { "family": "Sarma", "given": "Anish Anandsai" }, "id": "Sarma-Anish-Anandsai", "orcid": "0000-0003-1261-0589", "display_name": "Sarma, Anish Anandsai" } ], "advisors": [ { "name": { "family": "Doyle", "given": "John Comstock" }, "id": "Doyle-J-C", "orcid": "0000-0002-1828-2486", "role": "advisor", "display_name": "Doyle, John Comstock" }, { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "orcid": "0000-0002-3904-0195", "role": "co-advisor", "display_name": "Goentoro, Lea A." } ], "committee": [ { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "orcid": "0000-0002-3904-0195", "role": "chair", "display_name": "Goentoro, Lea A." }, { "name": { "family": "Doyle", "given": "John Comstock" }, "id": "Doyle-J-C", "orcid": "0000-0002-1828-2486", "role": "member", "display_name": "Doyle, John Comstock" }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Brandman", "given": "David M." }, "id": "Brandman-D-M", "role": "member", "display_name": "Brandman, David M." } ], "option_major": [ "cns" ], "doi": "10.7907/kf54-fr89", "abstract": "Robust control theory was developed in the late twentieth century as a mathematical framework to enable the principled incorporation of uncertainty into engineering design in applications like aerospace. However, engineered technologies that interface with living systems in applications like medicine and ecology must accommodate uncertainties and unmodeled dynamics far beyond what robust control theory has historically achieved. This thesis develops a robust control foundation for overcoming large-scale uncertainty and designing interfaces with living systems, through formal theory and three case studies: neural control of movement, immune control of viruses, and homeostatic control of neoplasia in the moon jellyfish.
\r\n \r\nThe central argument of this thesis is that these three systems, along with many others, have two key properties that enable new approaches to the uncertainty intrinsic to their study: they are themselves control systems, and they are multicellular systems. These properties motivate new work in control theory, blending recent results in localized and distributed control with older results from robust and modern control. The resulting theory framework answers domain-specific questions, guides the design of new experiments and technologies, and enables a conceptual synthesis. By leveraging the fact that these are multicellular control systems, we are able to make progress in theory, basic science, and engineering.
" }, { "name": "Sarraf, Namita", "degree": "PhD", "year": "2023", "title": "Towards Integrated Molecular Machines: Structural, Mechanical, and Computational Motifs", "advisor": "Qian, Lulu", "url": "https://resolver.caltech.edu/CaltechTHESIS:01272023-184413283", "creators": [ { "name": { "family": "Sarraf", "given": "Namita" }, "id": "Sarraf-Namita", "orcid": "0000-0001-8692-7429", "display_name": "Sarraf, Namita" } ], "advisors": [ { "name": { "family": "Qian", "given": "Lulu" }, "id": "Qian-Lulu", "orcid": "0000-0003-4115-2409", "role": "advisor", "display_name": "Qian, Lulu" } ], "committee": [ { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "chair", "display_name": "Shapiro, Mikhail G." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Rothemund", "given": "Paul W. K." }, "id": "Rothemund-P-W-K", "orcid": "0000-0002-1653-3202", "role": "member", "display_name": "Rothemund, Paul W. K." }, { "name": { "family": "Qian", "given": "Lulu" }, "id": "Qian-Lulu", "orcid": "0000-0003-4115-2409", "role": "member", "display_name": "Qian, Lulu" } ], "option_major": [ "bioeng" ], "doi": "10.7907/cdwp-c709", "abstract": "The programmability of DNA has made it well-suited for building molecular machines, performing nanoscale self-assembly, and computing via biochemical circuits. In the last few decades, great strides have been made in characterizing the interactions between DNA molecules such that they can be predicted and engineered.
\r\n\r\nThe development of frameworks for those interactions has enabled the construction of more complex molecular systems that can execute specified programs. Such programs have included mechanical tasks, like walking and sorting cargo; assembly and reconfiguration of 2D and 3D shapes; and computation, like Boolean logic and pattern recognition.
\r\n\r\nHowever, the continuing development of more complex molecular programs relies upon expanding the modules available for molecular systems to use to execute them. Expanded functionality of mechanical, structural, and computation modules are required in order to build compound systems that can interact with the physical world, reconfigure, and analyze signals in a variety of interesting ways. In this dissertation, we will discuss our contributions to this effort, which include exploring a motif for molecular robotic behavior, characterizing tile-tile interactions, and developing new capabilities for bimolecular circuits.
\r\n\r\nWithin the framework of a maze-solving molecular robot, we aim to implement walking behavior on DNA origami that introduces a surface modification via a four-way strand displacement reaction. Surprisingly, our experiments suggest that the walking behavior is at least two orders of magnitude slower than expected. To understand why, we quantitatively explore to what extent the speed and completion level of the robot can be modulated by design considerations such as toehold lengths, track redundancy, and strand purity. Another factor affecting the reaction rate is the number of tethering points, and we demonstrate an order of magnitude speed up in the four-way strand displacement reaction when we remove one tethering point. The characterization of a surface-modifying four-way strand displacement reaction is a useful tool for the continued development of molecular robots with more complex functionality.
\r\n\r\nFree-floating DNA origami tiles, called invaders here, can swap out DNA origami tiles within larger assemblies via a technique called tile displacement, which has previously been demonstrated using single tile and dimer invaders with 4- and 9-tile arrays. We introduce initial structures and invading assemblies with more complex shapes. We explore the robustness of this reaction by testing a variety of edge configurations and comparing their reaction rates. We demonstrate tunable growth of one of the invaders, which can grow into polymers of arbitrary length or close into 3D structures. By a tile displacement reaction, we reconfigure the 3D structures into 2D. The invaders with complex shapes are able to reconfigure the original tile assembly at rates comparable to simpler tile displacement reactions, and two reconfiguration events can take place sequentially or simultaneously.
\r\n\r\nFinally, we build two new modules for use with biochemical circuits. The first, a loser-take-all circuit, yields binary outputs indicating which analog signal is the smallest among all inputs. We implement a signal reversal function that converts the smallest input to the largest output, which can then be composed with a previously developed winner-take-all function to achieve loser-take-all. By making concentration adjustments, we can mitigate biases in the circuit that are a result of sequence-dependent different in reaction rates. We experimentally demonstrate a three-input loser-take-all circuit with nine input combinations. With further development, this circuit could be used to implement the activation function in neural networks that perform pattern classification according to which memory an input pattern is least similar to.
\r\n\r\nThe second circuit processes information using temporary memory. We design and implement a circuit that outputs distinct logic decisions based on relative timing information of a pair inputs and their logic values. We show that we can mitigate crosstalk in the circuit by utilizing mismatches and adjusting toehold lengths. The circuit is able to display clear ON-OFF separation at time intervals as short as one minute between the two inputs arriving.
" }, { "name": "Schulte, Samuel Jordan", "degree": "PhD", "year": "2023", "title": "New HCR Technologies: 10-Plex Quantitative Spectral Imaging of RNAs and Proteins; Multiplexed Quantitative Imaging of Protein:Protein Complexes; and Sensitive, Instrument-Free, At-Home Pathogen Detection", "advisor": "Pierce, Niles A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06092023-193714022", "creators": [ { "name": { "family": "Schulte", "given": "Samuel Jordan" }, "id": "Schulte-Samuel-Jordan", "orcid": "0000-0001-9982-6504", "display_name": "Schulte, Samuel Jordan" } ], "advisors": [ { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "advisor", "display_name": "Pierce, Niles A." } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Qian", "given": "Lulu" }, "id": "Qian-Lulu", "orcid": "0000-0003-4115-2409", "role": "member", "display_name": "Qian, Lulu" }, { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "member", "display_name": "Pierce, Niles A." } ], "option_major": [ "biology" ], "doi": "10.7907/nzk8-2d38", "abstract": "Signal amplification based on the mechanism of hybridization chain reaction (HCR) enables researchers to quantitatively image RNA and protein expression in highly autofluorescent biological samples. This thesis extends the capabilities of HCR to three new domains: spectral HCR imaging for quantitative 10-plex immunofluorescence and in situ hybridization in highly autofluorescent samples; imaging of protein:protein complexes using cooperative probes for logical control over HCR signal amplification; and HCR lateral flow tests for sensitive, instrument-free, at-home testing for infectious diseases.
\r\n\r\nWhile 4- or 5-plex imaging is readily achieved using orthogonal HCR systems labeled with spectrally distinct fluorophores, higher levels of multiplexing are challenging due to overlap in the broad excitation and emission spectra of commonly used fluorophores. In Chapter 2, we simultaneously image a combination of 10 protein and RNA targets via spectral imaging with linear unmixing. A combination of 10 reference spectra for 10 fluorophores chosen for optimal unmixing, 10 orthogonal HCR systems, and 11 optimized excitation and emission settings enable robust, user-friendly performance, which is demonstrated in whole-mount zebrafish embryos and mouse brain sections. We validate that unmixed subcellular voxel intensities enable accurate and precise relative target quantitation with subcellular resolution across all 10 channels and demonstrate single-molecule sensitivity and resolution for absolute RNA quantitation.
\r\n\r\nIn Chapter 3, we introduce an enzyme-free method for multiplexed imaging of protein:protein complexes using split-initiator HCR signal amplification. Antibodies specific to each protein of the complex carry fractional initiators that become colocalized upon introduction of a DNA ruler strand to form a full HCR initiator and trigger growth of a tethered amplification polymer. Automatic background suppression is present throughout the protocol, as split-initiator antibody probes that bind to the sample nonspecifically or to isolated protein targets are too far apart to become colocalized by the ruler strand, precluding colocalization of a full initiator and preventing HCR signal amplification. We demonstrate the technique with high signal-to-background in adherent mammalian cells, pro-T cells, and highly autofluorescent formalin-fixed paraffin-embedded human breast tissue sections. Leveraging existing orthogonal HCR amplifiers, we design three orthogonal cooperative junctions for simultaneous 3-plex detection of protein:protein complexes. We validate that quantitative subcellular voxel intensities are generated, allowing for built-in relative quantitation of protein:protein complexes within the spatial context of the sample. Lastly, we demonstrate simultaneous detection of protein targets, RNA targets, and protein:protein complexes via a unified protocol for HCR immunofluorescence, in situ hybridization, and protein:protein complex imaging.
\r\n\r\nIn Chapter 4, we enhance the sensitivity of conventional unamplified lateral flow tests for at-home infectious disease testing by developing an amplified assay with isothermal, enzyme-free signal amplification based on the mechanism of HCR. Traditional lateral flow tests are amenable to at-home testing and return a result within 10\u201315 minutes but demonstrate a high false-negative rate (e.g., 25-50% for SARS-CoV-2) due to the absence of signal amplification. The HCR lateral flow assay we develop maintains the simplicity of the conventional lateral flow assay user experience via a disposable 3-channel lateral flow device to automatically deliver reagents to the test region in three successive stages without user interaction. To perform a test, the user loads the sample, closes the device, and reads the result by eye after 60 minutes. Detecting gamma-irradiated SARS-CoV-2 virions in a mixture of saliva and extraction buffer, the current amplified HCR lateral flow assay achieves a limit of detection of 200 copies/\u03bcL using available antibodies to target the SARS-CoV-2 nucleocapsid protein. By comparison, five commercial unamplified lateral flow assays that use proprietary antibodies exhibit limits of detection of 500 copies/\u03bcL, 1000 copies/\u03bcL, 2000 copies/\u03bcL, 2000 copies/\u03bcL, and 20,000 copies/\u03bcL. By swapping out antibody probes to target different pathogens, amplified HCR lateral flow assays offer a platform for simple, rapid, and sensitive at-home testing for infectious diseases.
" }, { "name": "Tan, Fayth Hui", "degree": "PhD", "year": "2023", "title": "Animal Regeneration and its Loss: the Mouse as a Model of Limited Regeneration", "advisor": "Bronner, Marianne E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05292023-182121722", "creators": [ { "name": { "family": "Tan", "given": "Fayth Hui" }, "id": "Tan-Fayth-Hui", "orcid": "0000-0002-2160-5311", "display_name": "Tan, Fayth Hui" } ], "advisors": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "advisor", "display_name": "Bronner, Marianne E." } ], "committee": [ { "name": { "family": "Parker", "given": "Joseph" }, "id": "Parker-J", "orcid": "0000-0001-9598-2454", "role": "chair", "display_name": "Parker, Joseph" }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "member", "display_name": "Chan, David C." }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "member", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "member", "display_name": "Bronner, Marianne E." } ], "option_major": [ "biology" ], "doi": "10.7907/wedb-2f55", "abstract": "In this dissertation, we explore animal regeneration through a comparative evolutionary-developmental framework. In Chapter 1, we review animal regeneration and its loss through examining broad developmental and physiological factors that correlate with regenerative ability. We also highlight the mouse as a model of regeneration loss, examining the limited regeneration of the digit tip and the heart. For each context, we discuss how these regenerative processes occur, the physiological and molecular factors involved, and previous attempts to induce or improve the regenerative response.
\r\n\r\nIn Chapter 2, we explore the possibility of inducing regeneration in non-regenerating systems. Along with experiments in the jellyfish Aurelia coerulea, (formerly A. aurita sp. 1 strain) and the fruit fly Drosophila melanogaster, we find that supplementation with the amino acid L-leucine and sucrose induce appendage regeneration across these highly evolutionarily-diverged organisms. We discuss how this intervention highlights the conserved role of energetic parameters in regeneration, and how surpassing nutrient-based limitations may unlock regenerative responses in diverse contexts.
\r\n\r\nIn Chapter 3, we characterize the derivatives of cardiac neural crest cells (CNCCs) in the hearts of neonatal mice at P1 and >P7. We confirm previous work on the diverse cardiac derivatives resulting from CNCCs, and provide additional evidence for CNCC-derived cardiomyocytes, a contribution still contested in current literature. We also demonstrate how CNCC derivatives form a distinct age-related developmental trajectory in the heart, and discuss how these changes may affect cardiac physiology and relate to the loss of neonatal heart regeneration.
\r\n\r\nFinally, in Chapter 4, we propose future directions based on the work carried out in Chapter 3. While CNCCs have explicitly been studied in the context of heart regeneration in zebrafish, their role in neonatal mouse heart regeneration has not been explored. First, we suggest further investigation into the differences between CNCC-derived and non-CNCC derived CMs, to see if their proliferative ability and molecular profile show different temporal dynamics over the course of embryonic and early postnatal stages. Then, to examine all CNCC-derived cell types in regenerating P1 and non-regenerating P8 hearts, we propose a single-nuclei RNA-sequencing experiment to better resolve questions about how the myocardial lineage interacts with nonmyocytes, and to capture the full extent of potential cardiomyocyte decline postnatally. Lastly, to understand how CNCC derivatives influence endogenous heart regeneration, we design a dual Cre and (r)tTA driver system in transgenic mice to perform a conditional ablation experiment of CNCCs in a cryoinjury model of neonatal heart regeneration.
" }, { "name": "Torok, Zsofia Erzsebet", "degree": "PhD", "year": "2023", "title": "Resilience of a Precise Motor Behavior", "advisor": "Lois, Carlos", "url": "https://resolver.caltech.edu/CaltechTHESIS:10122022-003401041", "creators": [ { "name": { "family": "Torok", "given": "Zsofia Erzsebet" }, "id": "Torok-Zsofia-Erzsebet", "orcid": "ORCID: 0000-0001-6298-9940", "display_name": "Torok, Zsofia Erzsebet" } ], "advisors": [ { "name": { "family": "Lois", "given": "Carlos" }, "id": "Lois-Carlos", "orcid": "0000-0002-7305-2317", "role": "advisor", "display_name": "Lois, Carlos" } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "member", "display_name": "Pachter, Lior S." }, { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "orcid": "0000-0001-8837-678X", "role": "member", "display_name": "Siapas, Athanassios G." }, { "name": { "family": "Lois", "given": "Carlos" }, "id": "Lois-Carlos", "orcid": "0000-0002-7305-2317", "role": "member", "display_name": "Lois, Carlos" } ], "option_major": [ "neurobio" ], "doi": "10.7907/2ff5-e145", "abstract": "Motor memory retention is an essential part of survival and reproduction of most species. However, these behaviors are variable and hard to measure. The zebra finch provides a great model organism to study motor behavior on a fine scale and ask fundamentally important questions. Zebra finch males learn their song from their father and once learnt this song remains unchanged for the remainder of the animals\u2019 life. This highly stereotypic and precise motor function engages a handful of motor nuclei organized in a spatially spread out manner that allows for precise targeting of each key circuit participant for the production of the behavior. In my studies, I focus on better understanding the role of excitatory and inhibitory neurons in the pre-motor nucleus of the song production system. The goal was to perturb the precision of behavioral execution by collapsing the neuronal circuit responsible for sequential activity. Then, to study if the behavior could re-establish in an adult less plastic state of neuronal organization. After I have shown that motor function recovers to produce the same song post disruption, I investigated the large and small scale changes in neuronal activity and transcriptomics accompanying this degradation and recovery trajectory. I have learned that loss of inhibition leads to hyperactivation which eventually leads to a circuit level homeostatic compensation to shut down the pathological activity level. In addition, the upregulation of MHC1 receptors and microglia points to a homeostatic mechanism for synaptic reorganization and re-establishment. Now that we have the means to execute precise cell-type specific manipulations that are reversible and that we understand the underlying phenomenology of perturbation and recovery, we can ask many questions about the architecture of a highly resilient motor pathway. This could shine light on specific electrophysiological and molecular candidates to study for brain damage repair and neurodegenerative research." }, { "name": "Tsypin, Lev Maximovich", "degree": "PhD", "year": "2023", "title": "The Discovery and Biological Mechanisms of a Widespread Phenazine's Oxidation", "advisor": "Newman, Dianne K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01202023-073514292", "creators": [ { "name": { "family": "Tsypin", "given": "Lev Maximovich" }, "id": "Tsypin-Lev-Maximovich", "orcid": "0000-0002-0642-8468", "display_name": "Tsypin, Lev Maximovich" } ], "advisors": [ { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "advisor", "display_name": "Newman, Dianne K." } ], "committee": [ { "name": { "family": "Parker", "given": "Joseph" }, "id": "Parker-J", "orcid": "0000-0001-9598-2454", "role": "chair", "display_name": "Parker, Joseph" }, { "name": { "family": "Orphan", "given": "Victoria J." }, "id": "Orphan-V-J", "orcid": "0000-0002-5374-6178", "role": "member", "display_name": "Orphan, Victoria J." }, { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "orcid": "0000-0002-7033-0844", "role": "member", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Bois", "given": "Justin S." }, "id": "Bois-J-S", "orcid": "0000-0001-7137-8746", "role": "member", "display_name": "Bois, Justin S." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." } ], "option_major": [ "microbio" ], "doi": "10.7907/rmsf-e465", "abstract": "During the 2017 Microbial Diversity course at the Marine Biological Laboratory in Woods Hole, MA, Scott Saunders and Yinon Bar-On started enrichment cultures in hopes of dis-covering biological oxidation of phenazine-1-carboxylic acid (PCA). I took these enrich-ment cultures and described their PCA oxidation activity. From one of the mixed cultures, I isolated a bacterial strain that recapitulated the behavior of the enrichment. I identified it as a strain of Citrobacter portucalensis via a whole-genome analysis and called the strain \"MBL\" in reference to the Marine Biological Laboratory. Using a combination of analytical chemistry, quantitative fluorescence measurements, and genetic engineering, I showed that C. portucalensis MBL couples PCA oxidation to each mode of anaerobic respiration it employs with nitrate, fumarate, dimethyl sulfoxide (DMSO), and trimethylamine-N-oxide (TMAO) as terminal electron acceptors (TEAs). I further found that most of the PCA oxidation activi-ty depends on electron flux through the quinone/quinol pool but can be driven by certain terminal reductase complexes when no quinones are available, particularly in the case of ni-trate reductases. Every bacterial strain I tested catalyzed PCA oxidation when provided the appropriate TEA. My described mechanism for bacterial PCA oxidation is generalizable and implies that this previously undocumented phenomenon should occur wherever PCA is produced in rhizosphere environments.
" }, { "name": "Wandelt, Sarah Kim", "degree": "PhD", "year": "2023", "title": "Grasp, Speech, and Internal Speech Representation in the Human Cortical Grasp Circuit", "advisor": "Andersen, Richard A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05122023-085319625", "creators": [ { "name": { "family": "Wandelt", "given": "Sarah Kim" }, "id": "Wandelt-Sarah-Kim", "orcid": "0000-0001-9551-8491", "display_name": "Wandelt, Sarah Kim" } ], "advisors": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "advisor", "display_name": "Andersen, Richard A." } ], "committee": [ { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "chair", "display_name": "Adolphs, Ralph" }, { "name": { "family": "Rutishauser", "given": "Ueli" }, "id": "Rutishauser-U", "orcid": "0000-0002-9207-7069", "role": "member", "display_name": "Rutishauser, Ueli" }, { "name": { "family": "Yue", "given": "Yisong" }, "id": "Yue-Yisong", "orcid": "0000-0001-9127-1989", "role": "member", "display_name": "Yue, Yisong" }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "member", "display_name": "Andersen, Richard A." } ], "option_major": [ "cns" ], "doi": "10.7907/0461-g107", "abstract": "The ability to move freely and to connect with others through communication is invaluable for human independence. In this thesis, we explore how brain-machine interfaces (BMIs) can help patients affected by movement or speech deficits to recover lost human experiences. This work builds on previous findings indicating that premotor and posterior parietal areas are involved in movement generation and language processes. These higher-level brain areas do not only engage in movement execution, but also during planning, representing rich behavioral patterns that can be leveraged for BMI applications. In this work, we investigated how the ventral premotor cortex (PMv), the posterior parietal cortex (PPC), and the sensorimotor cortex (S1) represent grasp and speech processes at a single-neuron level. Using multielectrode Utah arrays, neuronal populations were recorded in tetraplegic human participants. We found that the supramarginal gyrus (SMG), PMv and S1 significantly encode motor imagery of grasping. By studying the cognitive processes underlying neural activity during the cue phase of grasping, we found a transition from cue-modality dependent to cue-modality independent grasp representation in SMG, the anterior intraparietal cortex (AIP) and PMv. Our findings suggest SMG integrates audio, written, and image cue modalities, but more similarly represents audio and written cues, indicating language involvement. We confirmed this hypothesis by demonstrating that SMG encodes spoken words, engaging different motor plans for speech compared to grasping even when the semantic content remained unchanged. These results suggest a BMI could be trained to decode both grasp motor imagery and speech from one brain area. Lastly, we showed that SMG is highly involved in language processes, modulating for written word recognition, auditory tones, vocalized speech, and internal speech. As a proof-of-concept, we built a real-time internal speech BMI from signals recorded in SMG that can decode eight words with high accuracy. This work is the first of its kind, demonstrating internal speech can be robustly decoded from an implant in a single brain area. We find high neural SMG generalization between seeing a written word, saying it internally and vocalizing the word, suggesting shared cognitive functions between different language processes. Furthermore, words in different languages are represented in SMG. This thesis advances the BMI field by providing a better understanding of the neural processes that underly grasp motor imagery and language. To summarize, our findings suggest that studying higher-level brain areas can lead to the development of more effective and versatile brain-machine interfaces." }, { "name": "Wang, Sheng", "degree": "PhD", "year": "2023", "title": "Synthetic Circuits for Multicellular Spatial Patterning", "advisor": "Elowitz, Michael B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06022023-192547723", "creators": [ { "name": { "family": "Wang", "given": "Sheng" }, "id": "Wang-Sheng", "orcid": "0000-0002-4070-7313", "display_name": "Wang, Sheng" } ], "advisors": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "advisor", "display_name": "Elowitz, Michael B." } ], "committee": [ { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "orcid": "0000-0002-3904-0195", "role": "chair", "display_name": "Goentoro, Lea A." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "orcid": "0000-0003-2136-6506", "role": "member", "display_name": "Meister, Markus" }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." } ], "option_major": [ "bioeng" ], "doi": "10.7907/3rbk-g805", "abstract": "Self-organized spatial periodic patterning mechanisms are responsible for the generation of repetitive structures, such as digits, vertebrae, and teeth, during multicellular development. Adopting a synthetic biology approach, we aim to unravel the core principles of multicellular spatial patterning by designing and reconstituting it in tissue-cultured cell lines.
\r\n\r\nThe reaction-diffusion mechanism, as an established paradigm, has successfully elucidated and forecasted pattern formation across varying scales and species. However, the potential for reconstituting synthetic reaction-diffusion patterns using unconventional reaction-diffusion elements within mammalian cell cultures has been insufficiently explored, thus leaving a gap in our comprehension of how spatial periodic patterns could be generated.
\r\n\r\nThe simplest reaction-diffusion systems are thought to necessitate a minimum of two morphogens to generate periodic patterns. In contrast, with the help of mathematical modeling, we illustrate that a simpler circuit, comprising only a single diffusible morphogen, can adequately produce long-range, spatially periodic patterns. These patterns propagate outward from transient initiating perturbations and remain stable after the disturbance is removed. Moreover, introducing an additional bistable intracellular feedback or operation on a growing cell lattice can enhance the robustness of the patterning against noise.
\r\n\r\nConcurrently, we reconstruct the Turing pattern in mammalian cell culture utilizing a bottom-up approach. We construct a synthetic circuit based on two signaling pathways. After validation of each circuit component, we exhibit the spatial pattern formation driven by a synthetic reaction-diffusion circuit within the mammalian cell line. This adaptable circuit facilitates us to adjust circuit parameters or implement various boundary conditions, thereby revealing the impact of these alterations on patterning dynamics.
\r\n\r\nCollectively, these findings lay the groundwork for the engineering of pattern formation in the nascent field of synthetic developmental biology.
" }, { "name": "Wilbert, Steven Alexander", "degree": "PhD", "year": "2023", "title": "The Role of Context-Dependent Metabolic Interactions in Organizing Microbial Communities", "advisor": "Newman, Dianne K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06122023-184806431", "creators": [ { "name": { "family": "Wilbert", "given": "Steven Alexander" }, "id": "Wilbert-Steven-Alexander", "orcid": "0009-0008-4409-8974", "display_name": "Wilbert, Steven Alexander" } ], "advisors": [ { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "advisor", "display_name": "Newman, Dianne K." } ], "committee": [ { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "chair", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Orphan", "given": "Victoria J." }, "id": "Orphan-V-J", "orcid": "0000-0002-5374-6178", "role": "member", "display_name": "Orphan, Victoria J." }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "orcid": "0000-0003-2713-1513", "role": "member", "display_name": "Mazmanian, Sarkis K." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." } ], "option_major": [ "microbio" ], "doi": "10.7907/7sv2-gj10", "abstract": "We can image the strikingly beautiful compositions of natural microbial communities, but we still lack an understanding of the factors that shape their organization. Understanding the drivers of these structures at the microscale may allow us to better predict and control large-scale community functions in dynamic environments. In this thesis, I developed quantitative image analysis pipelines for uncovering the spatiotemporal growth of aggregate biofilms within a developing oxygen gradient by expanding upon the Agar Block Biofilm Assay (ABBA). I then developed the Agar Disk Biofilm Assay (ADBA) for improved imaging resolution. These tools push the bounders of laboratory experiments to better capture the complexity of natural environments. Next, I built a synthetic microbial community reflecting a metabolic pathway often partitioned between members found in nature: Pseudomonas aeruginosa (PA) strains with a denitrification pathway genetically split at the nitric oxide (NO) node. I characterized the growth of a strict consumer and a strict producer of NO and found that PA metabolizes NO in a manner that supports growth, a previously underappreciated energy conservation strategy. Local oxygen flips this interaction from beneficial to detrimental by increasing toxicity. I found these principles drive context-dependent cellular organization. This work underscores the contributions of partitioned metabolic pathways, redox-active metabolites, and dynamic micro-niches to the organization of microbial communities. Finally, combining my efforts towards method development and an appreciation for how redox-active metabolites drive context-dependent microbial interactions, I show how phenazines promote a previously unrecognized form of slow growth under nutrient limited environments. Taken together, this thesis highlights the importance of understanding dynamic micron-scale microbial interactions and presents several methodological improvements to capture it." }, { "name": "Zhu, Ronghui", "degree": "PhD", "year": "2023", "title": "Multicellular Circuit Design in Mammalian Cells", "advisor": "Elowitz, Michael B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:07252022-061122576", "creators": [ { "name": { "family": "Zhu", "given": "Ronghui" }, "id": "Zhu-Ronghui", "orcid": "0000-0001-8171-482X", "display_name": "Zhu, Ronghui" } ], "advisors": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "advisor", "display_name": "Elowitz, Michael B." } ], "committee": [ { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "chair", "display_name": "Hay, Bruce A." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." } ], "option_major": [ "biology" ], "doi": "10.7907/p0fn-qa56", "abstract": "Multicellular circuits control the development of multicellular organisms, through programming processes such as cell proliferation, cell differentiation, cell movement, and cell signaling. A fundamental goal of biology is to understand the design principles of these multicellular circuits, and use these principles to design synthetic multicellular systems for therapeutic purposes. Top-down approaches, for example analyzing embryos bearing genetic mutations, have identified key genes in many multicellular circuits, but are challenging to study these circuits in an isolated context and in a quantitative and systematic manner. An alternative, complementary approach is to engineer or reconstitute multicellular circuits from bottom-up, which allows us to overcome the limitations of top-down approach and gain quantitative insights into multicellular circuit design. In this thesis, we use this bottom-up approach to explore the design principles of two multicellular circuits. In the first project, we took inspiration from two prevalent features from natural multistable circuits, namely competitive protein-protein interactions and positive autoregulation, to design a synthetic multistable circuit architecture called MultiFate. Both in the model and in the experiment, MultiFate circuits generate multiple cellular states, each stable for weeks, allow control over state-switching and state stability, and can be easily expanded to generate more states. In the second project, we use a gradient reconstitution system to systematically analyze a gradient modulation circuit consisting of BMP4 and its modulators, Chordin, Twsg and BMP-1. We found that the circuit can give rise to diverse gradient modulation capabilities. In particular, the full circuit is sufficient for active ligand shuttling and generation of non-monotonic displaced gradient. These multicellular circuits could provide a foundation for engineering synthetic multicellular systems in mammalian cells.
" }, { "name": "Abdel-Haq, Reem", "degree": "PhD", "year": "2022", "title": "Gut Microbiome Modulates Microglia Physiology in Homeostatic and Disease States", "advisor": "Mazmanian, Sarkis K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03162022-173632582", "creators": [ { "name": { "family": "Abdel-Haq", "given": "Reem" }, "id": "Abdel-Haq-Reem", "orcid": "0000-0002-7418-5736", "display_name": "Abdel-Haq, Reem" } ], "advisors": [ { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "orcid": "0000-0003-2713-1513", "role": "advisor", "display_name": "Mazmanian, Sarkis K." } ], "committee": [ { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "chair", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "orcid": "0000-0003-2713-1513", "role": "member", "display_name": "Mazmanian, Sarkis K." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "member", "display_name": "Chan, David C." } ], "option_major": [ "biology" ], "doi": "10.7907/ht1j-2461", "abstract": "The gastrointestinal tract (GI) harbors a complex community of ~100 trillion bacteria, fungi, and viruses collectively referred to as the gut microbiome. Through direct and indirect signaling mechanisms, the gut microbiome exerts its effects on almost every organ system, including the brain. Constant, bi-directional communication along the gut-brain axis is required for the normal and healthy development of the host Central Nervous System (CNS). One of the cells in the CNS shaped by microbial-derived cues is microglia, the resident immune cells in the brain. Aberrant microglia activity is a driving force of several neurological diseases in which the gut microbiome plays a role, including Parkinson\u2019s disease (PD). \r\n\r\nIn this thesis, we explore the interplay between gut microbiota signaling and microglia physiology during homeostatic and disease states. We first detail how microbial signaling along the gut-brain axis shapes microglial development and function. Next, we explore how the gut microbiome composition influences microglial activation states in the context of disease. Leveraging a preclinical mouse model of PD, we show that dietary-driven changes to the gut microbiome through the use of prebiotics attenuates motor deficits and \u03b1-synuclein aggregation. These effects result from changes in microglial gene expression and activation status. Collectively, these findings have broad implications for the gut microbiome research community and highlight potential for development of microbiome-based therapies for diseases of the brain." }, { "name": "Aceves, Aiden Joseph", "degree": "PhD", "year": "2022", "title": "Machine Learning and Modeling Methods for Protein Engineering", "advisor": "Mayo, Stephen L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:09092021-024951318", "creators": [ { "name": { "family": "Aceves", "given": "Aiden Joseph" }, "id": "Aceves-Aiden-Joseph", "orcid": "0000-0001-6583-5373", "display_name": "Aceves, Aiden Joseph" } ], "advisors": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "advisor", "display_name": "Mayo, Stephen L." } ], "committee": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "chair", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Miller", "given": "Thomas F." }, "id": "Miller-T-F", "orcid": "0000-0002-1882-5380", "role": "member", "display_name": "Miller, Thomas F." }, { "name": { "family": "Yue", "given": "Yisong" }, "id": "Yue-Yisong", "orcid": "0000-0001-9127-1989", "role": "member", "display_name": "Yue, Yisong" }, { "name": { "family": "Van Valen", "given": "David A." }, "id": "Van-Valen-D", "orcid": "0000-0001-7534-7621", "role": "member", "display_name": "Van Valen, David A." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." } ], "option_major": [ "bioeng" ], "doi": "10.7907/5j34-0d55", "abstract": "Computation has been an integral part of structural biology, ever since the first protein macromolecular structure was solved via Fourier Synthesis on the EDSAC Mark I electronic computer in 1958 (Kendrew et al., 1958). Throughout my time at Caltech, I have endeavored to develop new methods to apply machine learning and molecular modeling to the study of biological macromolecules. These efforts have taken two distinct tracks, but are unified by a focus on studying proteins on a structural level.
\r\n\r\nThrough the application of molecular dynamics and modeling, I have studied insulin from several angles, including the incorporation of non-canonical amino acids, and how these modifications might be responsible for the modification of critical properties such as hexamer dissociation and fibrillation formation. Additionally, I have probed how insulin behaves at the interface of water and silica, a property which is critical for the effective dissemination and administration of this therapeutic molecule. I have helped to develop a novel computationally guided workflow for integrating drug conjugates into antibody CDRs. This technique yields molecules which exhibit synergistic binding and an enhanced ability for selective binding.
\r\n\r\nThe second major thrust of my research has focused on applying machine learning to protein engineering problems, particularly developing tools for working with structural data, and for making efficient re-use of data which has already been laboriously collected by other groups. The basic data parsing and processing tools which were created and refined over the course of my time at Caltech has enabled many other projects, both of my own and of collaborators. Studies into the use of generative networks for protein-protein docking have been conducted which lend useful insights for network architecture, the inclusion of intermediate learning objectives, and overcoming sparsity. The technique introduced in our ICLR 2021 paper demonstrates a regularization method which enables data from past protein engineering campaigns to be leveraged to learn policies which optimally select molecules to synthesize in unrelated engineering efforts, to potentially save a significant amount of time and money for future projects.
\r\n\r\nReference
\r\n\r\nKendrew, J. C.; Bodo, G.; Dintzis, H. M.; Parrish, R. G.; Wyckoff, H.; Phillips, D. C. A. \"Three-Dimensional Model of the Myoglobin Molecule Obtained by X-Ray Analysis\". Nature 1958, 181 (4610), 662\u2013666.
" }, { "name": "Andrade Meirelles, Lucas", "degree": "PhD", "year": "2022", "title": "The Nuanced Effects of Redox-Active Metabolites on Bacterial Physiology and Antibiotic Susceptibility", "advisor": "Newman, Dianne K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02152022-081613451", "creators": [ { "name": { "family": "Andrade Meirelles", "given": "Lucas" }, "id": "Andrade-Meirelles-Lucas", "orcid": "0000-0003-3194-7136", "display_name": "Andrade Meirelles, Lucas" } ], "advisors": [ { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "advisor", "display_name": "Newman, Dianne K." } ], "committee": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "chair", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "orcid": "0000-0002-7033-0844", "role": "member", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." } ], "option_major": [ "biology" ], "doi": "10.7907/67p2-q992", "abstract": "The production of secondary metabolites is widespread throughout the tree of life. Bacteria, including many relevant opportunistic pathogens, can make redox-active secondary metabolites, both in the environment and while causing infections. Yet, their physiological consequences for the microbial communities exposed to them are much less understood. This thesis investigates the multifaceted and nuanced effects that such metabolites can have on their producers and other bacteria found in the producer's vicinity, focusing on the role these molecules play as modulators of antibiotic susceptibility. I start by presenting a literature review addressing the link between secondary metabolite production and resilience to clinical antibiotics in diverse opportunistic and enteric bacterial pathogens.
\r\n\r\nNext, using Pseudomonas aeruginosa (a widespread opportunistic pathogen) and its endogenously produced metabolite called pyocyanin, I explore the nuanced effects of the metabolite's production throughout the producer's lifecycle. Pyocyanin is part of a class of redox-active molecules made by P. aeruginosa called phenazines. I show that the production of pyocyanin, due to its self-poisoning effects, is a \"double-edged sword,\" where the ultimate consequences for the producer are directly dependent on the physiological and environmental conditions. Carbon source limitation plays a major role in the self-poisoning effect of pyocyanin, a process responsible for killing a subpopulation of cells that, through extracellular DNA release, seems critical for proper biofilm development.
\r\n\r\nDespite pyocyanin's toxicity, P. aeruginosa is remarkably tolerant to its harmful effects. For this reason, I then explore how P. aeruginosa handles the stress caused by the metabolite. I present results using a functional genomics approach (transposon-sequencing) to screen for genes involved in P. aeruginosa tolerance to pyocyanin. Defenses involved in pyocyanin tolerance are similar to ones involved in tolerance to clinical antibiotics. These shared mechanisms lead to testing the hypothesis that defenses induced by the production of or exposure to \"natural antibiotics\" (such as pyocyanin) may affect the efficacy of treatments with clinical antibiotics. Supporting this hypothesis, exposure to pyocyanin significantly induces tolerance and resistance to certain clinical drugs, both in P. aeruginosa and other opportunistic pathogens within the Burkholderia cepacia complex (Bcc). Pyocyanin and the drugs affected, such as fluoroquinolones, share molecular structure similarities, which is likely responsible for the shared protection.
\r\n\r\nFinally, based on these results, I explore the broader role of redox-active metabolites as modulators of antibiotic resilience in opportunistic pathogens. I show that pyocyanin, another phenazine called phenazine-1-carboxylic acid, and a non-phenazine redox-active molecule called toxoflavin can all modulate antibiotic susceptibility in Bcc species. Depending on the antibiotic's class, the metabolites' presence can either antagonize or potentiate the drug's efficacy. All the studied metabolites are produced by clinical isolates that infect cystic fibrosis and other immunocompromised patients. I demonstrate that the modulator effect of redox-active molecules in the pathogens is dependent on the transcription factor SoxR, which senses the presence of the metabolites and induces specific redox-regulated efflux systems that are effective in transporting both the metabolites and the structurally related drugs. To end, I provide a proof-of-principle that including such metabolites during clinical drug susceptibility tests may lead to a more accurate assessment of pathogens' resistance profile.
\r\n\r\nTaken together, the findings presented in this thesis demonstrate that redox-active secondary metabolites have profound effects on the physiology and antibiotic sensitivity levels of opportunistic pathogens. Their modulator effect on antibiotic susceptibility is likely a widespread phenomenon in polymicrobial communities that has been overlooked and may have direct consequences for the evolution of antibiotic resistance. Understanding the physiological roles of these metabolites at the molecular level is essential for accurate predictions of the drugs and pathogens affected, which may lead to more effective treatment strategies.
" }, { "name": "Barlow, Jacob T.", "degree": "PhD", "year": "2022", "title": "Quantitative Sequencing and its Application to Studies of the Human Small-Intestine Microbiota", "advisor": "Ismagilov, Rustem F.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10282021-191743624", "creators": [ { "name": { "family": "Barlow", "given": "Jacob T." }, "id": "Barlow-Jacob-T", "orcid": "0000-0002-1842-4835", "display_name": "Barlow, Jacob T." } ], "advisors": [ { "name": { "family": "Ismagilov", "given": "Rustem F." }, "id": "Ismagilov-R-F", "orcid": "0000-0002-3680-4399", "role": "advisor", "display_name": "Ismagilov, Rustem F." } ], "committee": [ { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "orcid": "0000-0003-2713-1513", "role": "chair", "display_name": "Mazmanian, Sarkis K." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Cai", "given": "Long" }, "id": "Cai-Long", "orcid": "0000-0002-7154-5361", "role": "member", "display_name": "Cai, Long" }, { "name": { "family": "Ismagilov", "given": "Rustem F." }, "id": "Ismagilov-R-F", "orcid": "0000-0002-3680-4399", "role": "member", "display_name": "Ismagilov, Rustem F." } ], "option_major": [ "bioeng" ], "doi": "10.7907/ca28-fk21", "abstract": "Our understanding of the interplay between microbial species and the hosts they live on and in is continually expanding. New insights have focused not only microorganisms that drive specific disease states but also those that help maintain human health. As research drives towards mechanistic understanding of host-microbe relationships new quantitative tools are needed to help interrogate these complex interactions. Chapter I of this thesis discusses formulation of a method for rapid detection of antibiotic resistance in Neisseria gonorrhoeae. Our approach identified RNA signatures from transcriptional profiling of Neisseria gonorrhoeae after 10-minute antibiotic exposure. Utilization of these RNA markers allowed for rapid identification of antibiotic susceptibility or resistance to the antibiotic ciprofloxacin. Chapter II shifts focus to the development of a quantitative sequencing technique for the measurement of absolute taxon abundances in complex microbial communities. Combining the precision of digital PCR with the high-throughput nature of 16S rRNA gene amplicon sequencing allowed for simultaneous quantitative profiling of all bacterial taxa in host-associated microbial communities. We extensively characterized our quantitative sequencing methodology in the presence of high host nucleic acid levels and low microbial loads to understand the limits of quantification and detection in complex sample types. Last, Chapter III applies the quantitative sequencing technology from Chapter II to investigate the microbial community of the human small intestine, specifically the duodenum. Data from the duodenum of 250 individuals revealed a wide range of total microbial loads and a distinct subset of microbes, termed disruptor taxa, that were associated with small intestinal bacterial overgrowth (SIBO) and GI symptom severity.
" }, { "name": "Beeler, Suzannah Michelle", "degree": "PhD", "year": "2022", "title": "Deciphering Regulation in Escherichia coli: From Genes to Genomes", "advisor": "Phillips, Robert B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08182021-053622635", "creators": [ { "name": { "family": "Beeler", "given": "Suzannah Michelle" }, "id": "Beeler-Suzannah-Michelle", "orcid": "0000-0002-1930-4827", "display_name": "Beeler, Suzannah Michelle" } ], "advisors": [ { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "advisor", "display_name": "Phillips, Robert B." } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "chair", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "orcid": "0000-0002-3904-0195", "role": "member", "display_name": "Goentoro, Lea A." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." } ], "option_major": [ "sysbiol" ], "doi": "10.7907/p3rg-m937", "abstract": "Advances in DNA sequencing have revolutionized our ability to read genomes. However, even in the most well-studied of organisms, the bacterium Escherichia coli, for \u2248 65% of promoters we remain ignorant of their regulation. Until we crack this regulatory Rosetta Stone, efforts to read and write genomes will remain haphazard. We introduce a new method, Reg-Seq, that links massively-parallel reporter assays with mass spectrometry to produce a base pair resolution dissection of more than 100 E. coli promoters in 12 growth conditions. We demonstrate that the method recapitulates known regulatory information. Then, we examine regulatory architectures for more than 80 promoters which previously had no known regulatory information. In many cases, we also identify which transcription factors mediate their regulation. This method clears a path for highly multiplexed investigations of the regulatory genome of model organisms, with the potential of moving to an array of microbes of ecological and medical relevance.
" }, { "name": "Brown, David", "degree": "PhD", "year": "2022", "title": "Principles of Massively Parallel Sequencing for Engineering and Characterizing Gene Delivery", "advisor": "Gradinaru, Viviana; Thomson, Matthew", "url": "https://resolver.caltech.edu/CaltechTHESIS:02132022-064810187", "creators": [ { "name": { "family": "Brown", "given": "David" }, "id": "Brown-David", "orcid": "0000-0002-9757-1744", "display_name": "Brown, David" } ], "advisors": [ { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "advisor", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "co-advisor", "display_name": "Thomson, Matthew" } ], "committee": [ { "name": { "family": "Yue", "given": "Yisong" }, "id": "Yue-Yisong", "orcid": "0000-0001-9127-1989", "role": "chair", "display_name": "Yue, Yisong" }, { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "member", "display_name": "Arnold, Frances Hamilton" }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "member", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" } ], "option_major": [ "cns" ], "doi": "10.7907/yqjm-6609", "abstract": "The advent of massively parallel sequencing and synthesis technologies have ushered in a new paradigm of biology, where high throughput screening of billions of nucleid acid molecules and production of libraries of millions of genetic mutants are now routine in labs and clinics. During my Ph.D., I worked to develop data analysis and experimental methods that take advantage of the scale of this data, while making the minimal assumptions necessary for deriving value from their application. My Ph.D. work began with the development of software and principles for analyzing deep mutational scanning data of libraries of engineered AAV capsids. By looking at not only the top variant in a round of directed evolution, but instead a broad distribution of the variants and their phenotypes, we were able to identify AAV variants with enhanced ability to transduce specific cells in the brain after intravenous injection. I then shifted to better understand the phenotypic profile of these engineered variants. To that end, I turned to single-cell RNA sequencing to seek to identify, with high resolution, the delivery profile of these variants in all cell types present in the cortex of a mouse brain. I began by developing infrastructure and tools for dealing with the data analysis demands of these experiments. Then, by delivering an engineered variant to the animal, I was able to use the single-cell RNA sequencing profile, coupled with a sequencing readout of the delivered genetic cargo present in each cell type, to define the variant\u2019s tropism across the full spectrum of cell types in a single step. To increase the throughput of this experimental paradigm, I then worked to develop a multiplexing strategy for delivering up to 7 engineered variants in a single animal, and obtain the same high resolution readout for each variant in a single experiment. Finally, to take a step towards translation to human diagnostics, I leveraged the tools I built for scaling single-cell RNA sequencing studies and worked to develop a protocol for obtaining single-cell immune profiles of low volumes of self-collected blood. This study enabled repeat sampling in a short period of time, and revealed an incredible richness in individual variability and time-of-day dependence of human immune gene expression. Together, my Ph.D. work provides strategies for employing massively parallel sequencing and synthesis for new biological applications, and builds towards a future paradigm where personalized, high-resolution sequencing might be coupled with modular, customized gene therapy delivery.
" }, { "name": "Chivukula, Srinivas", "degree": "PhD", "year": "2022", "title": "Felt, Imagined, and Seen Touch Share a Substrate in Human Posterior Parietal Cortex", "advisor": "Andersen, Richard A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05022022-175609842", "creators": [ { "name": { "family": "Chivukula", "given": "Srinivas" }, "id": "Chivukula-Srinivas", "orcid": "0000-0002-3570-162X", "display_name": "Chivukula, Srinivas" } ], "advisors": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "advisor", "display_name": "Andersen, Richard A." } ], "committee": [ { "name": { "family": "Burdick", "given": "Joel Wakeman" }, "id": "Burdick-J-W", "orcid": "0000-0002-3091-540X", "role": "chair", "display_name": "Burdick, Joel Wakeman" }, { "name": { "family": "Aflalo", "given": "Tyson" }, "id": "Aflalo-Tyson", "orcid": "0000-0002-0101-2455", "role": "member", "display_name": "Aflalo, Tyson" }, { "name": { "family": "Pouratian", "given": "Nader" }, "id": "Pouratian-Nader", "role": "member", "display_name": "Pouratian, Nader" }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "orcid": "0000-0002-7371-4675", "role": "member", "display_name": "Prober, David A." }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "member", "display_name": "Andersen, Richard A." } ], "option_major": [ "biology" ], "doi": "10.7907/0tgv-t705", "abstract": "One of the most remarkable aspects of human cognition is its flexibility. We can think new thoughts, infer meaning, plan actions, predict, extrapolate, and so much more. How do our brains enable this versatility? A growing ability to simultaneously record from large populations of single neurons in human cortex has begun to provide insight. Recent studies have identified that shared populations of neurons in posterior parietal cortex (PPC) of a human subject (involved in a brain-machine interface (BMI) clinical trial) encode many aspects of motor cognition: attempted and imagined actions, observed actions and the semantic processing of action verbs. Individual units are complex, but population representations manifest rich associations across neurons, supporting diverse behavioral contexts. Here, in novel work, we establish that the same PPC substrate also encodes aspects of sensory cognition, and unpack the functional organization of information that enables this versatility. We record populations of neurons in PPC of the same human subject, a tetraplegic trial participant implanted with a 4x4 mm microelectrode array. In a series of novel results, we first establish that neurons in this PPC substrate encode actual (or felt) touch to oneself, at short latency, with bilateral receptive fields, organized by body-part. We show that imagined touch to oneself and observed touch to others engage the same substrate. To understand coding mechanisms further, we manipulated the touch location (cheek, shoulder), and the touch type (pinch, press, rub, tap). As in the motor domain, individual neurons exhibit highly variable responses. At the population-level, however, we find that the diverse touch conditions are explained by a small number of subspaces (meaningful groupings of neurons) that encode basic-level, elemental information such as touch location, and touch type. This suggests a compositional basis in PPC, such that various touch conditions are encoded through diverse combinations of common primitive elements. Moreover, these subspaces are generalizable, able to explain novel (held out) data. These principles of compositionality and generalizability suggest a basis by which PPC may support cognitive behaviors such as comprehension, in situations that extend beyond our experiences. In support of this interpretation, we show finally that this PPC substrate encodes seen touch universally \u2013 not only to insensate arm regions on the tetraplegic human subject, and to other human individuals, but also to a wide sampling of inanimate objects. As predicted, neural information combines and generalizes across conditions such that touch to objects with more similar features, is more similarly encoded. Taken together, our work is a novel, neuron-level characterization of how high-level cortex in humans may support diverse sensory, motor, and cognitive behaviors. We speculate that populations of neurons in PPC encode rich internal models of the world that can be flexibly repurposed for diverse (and novel) behavioral contexts." }, { "name": "Clamons, Samuel Eric", "degree": "PhD", "year": "2022", "title": "Three Problems in the Design and Specification of Biomolecular Circuits", "advisor": "Murray, Richard M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:09262021-022402778", "creators": [ { "name": { "family": "Clamons", "given": "Samuel Eric" }, "id": "Clamons-Samuel-Eric", "orcid": "0000-0002-7993-2278", "display_name": "Clamons, Samuel Eric" } ], "advisors": [ { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "advisor", "display_name": "Murray, Richard M." } ], "committee": [ { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "orcid": "0000-0002-5899-7523", "role": "chair", "display_name": "Winfree, Erik" }, { "name": { "family": "Rothemund", "given": "Paul W. K." }, "id": "Rothemund-P-W-K", "orcid": "0000-0002-1653-3202", "role": "member", "display_name": "Rothemund, Paul W. K." }, { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "orcid": "0000-0002-3904-0195", "role": "member", "display_name": "Goentoro, Lea A." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." } ], "option_major": [ "bioeng" ], "doi": "10.7907/9b4h-8f95", "abstract": "Programming biological materials is a daunting challenge. Although part of this challenge is practical -- cloning is difficult, synthesizing DNA is expensive at scale, etc. -- a number of the challenges of bioengineering (and synthetic biology in particular) are problems of design and specification. If we could place arbitrary molecules on a surface with perfect precision, what should we place and where? If we could arbitrarily change the genetic content of a cell, even with perfect knowledge of the function and action of every component, what changes would actually enact the functions we want that cell to have? In this thesis, we explore three specific design and specification challenges at three different levels of abstraction, and demonstrate methods for overcoming them. On the level of design language, we use a specialized class of cellular automaton to probe what chemistry can do when restricted to a surface. On the level of \\textbf{part specification}, we use several models of CRISPR/Cas9-based transcriptional regulators to understand what dynamic functions those regulators can perform and why, and provide some some suggestions for how to engineer such regulators to more robustly perform those functions. On the level of module design, we consider an easy-to-encounter trap in when modeling a replicating DNA species in a CRN-based biocircuit simulation, for which we suggest a simple, flexible, biologically-plausible workaround.
" }, { "name": "Cross, Logan Matthew", "degree": "PhD", "year": "2022", "title": "The Neural Mechanisms of Value Construction", "advisor": "O'Doherty, John P.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04012022-011925533", "creators": [ { "name": { "family": "Cross", "given": "Logan Matthew" }, "id": "Cross-Logan-Matthew", "orcid": "0000-0002-5248-9499", "display_name": "Cross, Logan Matthew" } ], "advisors": [ { "name": { "family": "O'Doherty", "given": "John P." }, "id": "O'Doherty-J-P", "orcid": "0000-0003-0016-3531", "role": "advisor", "display_name": "O'Doherty, John P." } ], "committee": [ { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "chair", "display_name": "Adolphs, Ralph" }, { "name": { "family": "Yue", "given": "Yisong" }, "id": "Yue-Yisong", "orcid": "0000-0001-9127-1989", "role": "member", "display_name": "Yue, Yisong" }, { "name": { "family": "Mobbs", "given": "Dean" }, "id": "Mobbs-Dean", "orcid": "0000-0003-1175-3772", "role": "member", "display_name": "Mobbs, Dean" }, { "name": { "family": "O'Doherty", "given": "John P." }, "id": "O'Doherty-J-P", "orcid": "0000-0003-0016-3531", "role": "member", "display_name": "O'Doherty, John P." } ], "option_major": [ "cns" ], "doi": "10.7907/x5kk-8h27", "abstract": "Research in decision neuroscience has characterized how the brain makes decisions by assessing the expected utility of each option in an abstract value space that affords the ability to compare dissimilar options. Experiments at multiple levels of analysis in multiple species have localized the ventromedial prefrontal cortex (vmPFC) and nearby orbitofrontal cortex (OFC) as the main nexus where this abstract value space is represented. However, much less is known about how this value code is constructed by the brain in the first place. By using a combination of behavioral modeling and cutting edge tools to analyze functional magnetic resonance imaging (fMRI) data, the work of this thesis proposes that the brain decomposes stimuli into their constituent attributes and integrates across them to construct value. These stimulus features embody appetitive or aversive properties that are either learned from experience or evaluated online by comparing them to previously experienced stimuli with similar features. Stimulus features are processed by cortical areas specialized for the perception of a particular stimulus type and then integrated into a value signal in vmPFC/OFC.
\r\n\r\nThe project presented in Chapter 2 examines how food items are evaluated by their constituent attributes, namely their nutrient makeup. A linear attribute integration model succinctly captures how subjective values can be computed from a weighted combination of the constituent nutritive attributes of the food. Multivariate analysis methods revealed that these nutrient attributes are represented in the lateral OFC, while food value is encoded both in medial and lateral OFC. Connectivity between lateral and medial OFC allows this nutrient attribute information to be integrated into a value representation in medial OFC.
\r\n\r\nIn Chapter 3, I show that this value construction process can operate over higher-level abstractions when the context requires bundles of items to be valued, rather than isolated items. When valuing bundles of items, the constituent items themselves become the features, and their values are integrated with a subadditive function to construct the value of the bundle. Multiple subregions of PFC including but not limited to vmPFC compute the value of a bundle with the same value code used to evaluate individual items, suggesting that these general value regions contextually adapt within this hierarchy. When valuing bundles and single items in interleaved trials, the value code rapidly switches between levels in this hierarchy by normalizing to the distribution of values in the current context rather than representing all options on an absolute scale.
\r\n\r\nAlthough the attribute integration model of value construction characterizes human behavior on simple decision-making tasks, it is unclear how it can scale up to environments of real-world complexity. Taking inspiration from modern advances in artificial intelligence, and deep reinforcement learning in particular, in Chapter 4 I outline how connectionist models generalize the attribute integration model to naturalistic tasks by decomposing sensory input into a high dimensional set of nonlinear features that are encoded with hierarchical and distributed processing. Participants freely played Atari video games during fMRI scanning, and a deep reinforcement learning algorithm trained on the games was used as an end-to-end model for how humans evaluate actions in these high-dimensional tasks. The features represented in the intermediate layers of the artificial neural network were found to also be encoded in a distributed fashion throughout the cortex, specifically in the dorsal visual stream and posterior parietal cortex. These features emerge from nonlinear transformations of the sensory input that connect perception to action and reward. In contrast to the stimulus attributes used to evaluate the stimuli presented in the preceding chapters, these features become highly complex and inscrutable as they are driven by the statistical properties of high-dimensional data. However, they do not solely reflect a set of features that can be identified by applying common dimensionality reduction techniques to the input, as task-irrelevant sensory features are stripped away and task-relevant high-level features are magnified.
" }, { "name": "Ding, Ke", "degree": "PhD", "year": "2022", "title": "Imaging Neuropeptide Release and Localization with Genetically Engineered Reporters", "advisor": "Anderson, David J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06012022-062105760", "creators": [ { "name": { "family": "Ding", "given": "Ke" }, "id": "Ding-Ke", "orcid": "0000-0002-5261-4843", "display_name": "Ding, Ke" } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "advisor", "display_name": "Anderson, David J." } ], "committee": [ { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "orcid": "0000-0002-7371-4675", "role": "chair", "display_name": "Prober, David A." }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "member", "display_name": "Chan, David C." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." } ], "option_major": [ "neurobio" ], "doi": "10.7907/3ey5-9p35", "abstract": "Neuropeptides are a class of neural signaling molecules that play a pivotal role in brain function and human health through neuromodulatory influences. There are over 100 types of neuropeptides identified and characterized, yet genomic analysis suggests that it is only the tip of the iceberg, with extra hundreds of putative neuropeptides awaiting further investigation. Neuropeptides collectively regulate a variety of developmental, physiological, and behavioral functions. While each neuropeptide is idiosyncratic in regard to its molecular structure, chemical properties, and anatomical distribution, they impinge on the nervous system in a similar fashion.
\r\n\r\nSurprisingly, despite their fundamental importance, techniques for measuring the localization, expression and release of neuropeptides, at large scale and with high spatio\u00adtemporal resolution, have lagged far behind. Microdialysis and fast-scanning cyclic voltammetry are useful primarily for measuring \"volume transmission,\" but are invasive, and have poor spatial resolution and limited general applicability. FP-tagged vesicle reporters are mainly tested and used in limited cell types. Little is characterized about their functional universality and specificity. GPCR-based sensors are designed to visualize the binding, instead of expression and release, of a neuropeptide.
\r\n\r\nTherefore, I aim to develop new methods for visualizing, detecting, and inhibiting NP expression and release in vivo. The long-term goal is to apply these methods to understanding the dynamics of neuromodulation of specific, behaviorally relevant neural circuits, and to providing a dynamic, high-resolution view of chemical modulation of circuit function.
\r\n\r\nIn Chapter 2, I will describe the design, screening, and proof-of-concept validation of novel genetically engineered neuropeptide release reporters (NPRR) in Drosophila. I further demonstrated the idiosyncrasy of neuropeptide release dynamics, as well as cell-type specific release properties of a neuropeptide. In Chapter 3, I conceived and constructed a neuropeptide imaging platform that exploits the discoveries and strategies from Drosophila NPRRs. Besides a series of redesign of mammalian NPRRs, a collection of sister reporters to visualize localization and expression (Neuropeptide Localization and Expression Reporter, NPLER) were built in parallel. I also established a prototypical pipeline to systematically screen for appropriate cell lines for the purpose of NPRR/NPLER applications.
\r\n\r\nMalfunctioning of neuropeptide pathways can potentially result in a variety of mental illnesses triggered by stress, and metabolic disorders including obesity. Drugs targeting neuropeptide signaling have received heavy investment, but most have failed in the clinical trials. We therefore propose alternative strategies to target the processing/release of the neuropeptide from neurons, rather than blocking its receptor. In Chapter 4, I describe the ongoing process of adapting modern biotechnologies to the imaging platform to explore novel therapeutic strategies for neuropeptide- relevant disorders and abnormalities.
\r\n\r\nThe Appendix includes a serendipitous finding from our attempt to generalize NPRR to Caenorhabditis elegans.
" }, { "name": "Edmonds, KeHuan Kuo", "degree": "PhD", "year": "2022", "title": "Imaging Cell Lineage with a Synthetic Digital Recording System", "advisor": "Elowitz, Michael B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:11272021-193744399", "creators": [ { "name": { "family": "Edmonds", "given": "KeHuan Kuo" }, "id": "Edmonds-KeHuan-Kuo", "orcid": "0000-0002-7317-2669", "display_name": "Edmonds, KeHuan Kuo" } ], "advisors": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "advisor", "display_name": "Elowitz, Michael B." } ], "committee": [ { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "chair", "display_name": "Hay, Bruce A." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Lois", "given": "Carlos" }, "id": "Lois-Carlos", "orcid": "0000-0002-7305-2317", "role": "member", "display_name": "Lois, Carlos" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Cai", "given": "Long" }, "id": "Cai-Long", "orcid": "0000-0002-7154-5361", "role": "member", "display_name": "Cai, Long" } ], "option_major": [ "biology" ], "doi": "10.7907/a4m3-m603", "abstract": "In multicellular organisms, the lineage history and spatial organization of cells both play pivotal roles in cell fate determination during development, homeostasis, and disease. Investigating lineage relationships alongside cell state and space would provide a fundamental understanding of these biological processes. Current lineage tracking approaches rely on the progressive accumulation of either naturally-occurring somatic mutations or experimentally introduced markers. In most cases, these marks are then read out by sequencing, discarding the spatial information of the cells. To address this vital gap in our toolkit, we developed a new synthetic lineage tracking system that allows us to image single-cell lineage history. This system, termed integrase-editable memory by engineered mutagenesis with optical in situ readout (intMEMOIR), uses serine integrases to stochastically and irreversibly edit a synthetic memory array, generating up to 59,049 different outcomes that can be unambiguously distinguished by fluorescence in situ hybridization (FISH). We evaluated the reconstruction accuracy of our system in mouse embryonic stem (mES) cells and disentangled the relative contribution of lineage and space to cell fate determination in Drosophila brain development, establishing the foundation for an expandable synthetic microscopy-readable system. In this thesis, Chapter 1 introduces the importance of cell lineage and spatial organization to cell fate determination, and includes a brief history of the existing technologies of the lineage tracking field. Chapter 2 describes our characterization and demonstration of the intMEMOIR system. Finally, Chapter 3 discusses design principles for robust, serine-integrase-based recording systems and suggests future directions for intMEMOIR." }, { "name": "Gallo Aquino, Tomas", "degree": "PhD", "year": "2022", "title": "Single Neuron Correlates of Learning, Value, and Decision in the Human Brain", "advisor": "O'Doherty, John P.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03042022-195852401", "creators": [ { "name": { "family": "Gallo Aquino", "given": "Tomas" }, "id": "Gallo-Aquino-Tomas", "orcid": "0000-0002-6944-1053", "display_name": "Gallo Aquino, Tomas" } ], "advisors": [ { "name": { "family": "O'Doherty", "given": "John P." }, "id": "O'Doherty-J-P", "orcid": "0000-0003-0016-3531", "role": "advisor", "display_name": "O'Doherty, John P." } ], "committee": [ { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "chair", "display_name": "Adolphs, Ralph" }, { "name": { "family": "O'Doherty", "given": "John P." }, "id": "O'Doherty-J-P", "orcid": "0000-0003-0016-3531", "role": "member", "display_name": "O'Doherty, John P." }, { "name": { "family": "Rutishauser", "given": "Ueli" }, "id": "Rutishauser-U", "orcid": "0000-0002-9207-7069", "role": "member", "display_name": "Rutishauser, Ueli" }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" } ], "option_major": [ "cns" ], "doi": "10.7907/has2-gk35", "abstract": "In this thesis, I present several new results on how the human brain performs value-based learning and decision-making, leveraging rare single neuron recordings from epilepsy patients in vmPFC, preSMA, dACC, amygdala, and hippocampus, as well as reinforcement learning models of behavior. With a probabilistic gambling task we determined that human preSMA neurons integrate computational components of stimulus value such as expected values, uncertainty, and novelty, to encode an utility value and, subsequently, decisions themselves. Additionally, we found that post-decision related encoding of variables for the chosen option was more widely distributed and especially prominent in vmPFC. Additionally, with a Pavlovian conditioning task we found evidence of stimulus-stimulus associations in vmPFC, while both vmPFC and amygdala performed predictive value coding, establishing direct evidence for model-based Pavlovian conditioning in human vmPFC neurons. Finally, in a Pavlovian observational learning paradigm, we found a significant proportion of amygdala neurons whose activity correlated with both expected rewards for oneself and others, and in tracking outcome values received by oneself or other agents, further establishing amygdala as an important center in social cognition. Taken together, our findings expand our understanding of the role of several human cortical brain regions in creating and updating value representations which are leveraged during decision-making.
" }, { "name": "Gong, Mengsha", "degree": "PhD", "year": "2022", "title": "Remodeling Jellyfish", "advisor": "Goentoro, Lea A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01032022-225455509", "creators": [ { "name": { "family": "Gong", "given": "Mengsha" }, "id": "Gong-Mengsha", "orcid": "0000-0003-3940-9869", "display_name": "Gong, Mengsha" } ], "advisors": [ { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "orcid": "0000-0002-3904-0195", "role": "advisor", "display_name": "Goentoro, Lea A." } ], "committee": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "chair", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." }, { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "orcid": "0000-0002-8587-9936", "role": "member", "display_name": "Dickinson, Michael H." }, { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "orcid": "0000-0002-3904-0195", "role": "member", "display_name": "Goentoro, Lea A." } ], "option_major": [ "bioeng" ], "doi": "10.7907/pfn8-aw15", "abstract": "Why are jellyfish round? Circularity facilitates many physiological functions in jellyfish like the moon jelly Aurelia aurita, including swimming and feeding. Previous work suggests that Aurelia might maintain its circularity through its muscle contractions. We use grafting experiments to investigate how these muscle contractions regulate shape in Aurelia and find that the same mechanism Aurelia uses to quickly recover circularity after it is injured can also produce square, oval, and triangular jellyfish. We then turn to modeling to ask what characteristics of the jellyfish muscle contractions and body materials give Aurelia the capability to reorganize its shape. Our simulations suggest that Aurelia body shape is a dynamic equilibrium that is not only reorganized by periodic muscle contractions when it is disrupted, but is also reinforced by the same muscle contractions over the course of normal physiological function.
" }, { "name": "Han, Yanting", "degree": "PhD", "year": "2022", "title": "Emotion Experience from Stories, Videos and Everyday Life: Structure and Individual Differences", "advisor": "Adolphs, Ralph", "url": "https://resolver.caltech.edu/CaltechTHESIS:05272022-181044242", "creators": [ { "name": { "family": "Han", "given": "Yanting" }, "id": "Han-Yanting", "orcid": "0000-0003-3381-2059", "display_name": "Han, Yanting" } ], "advisors": [ { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "advisor", "display_name": "Adolphs, Ralph" } ], "committee": [ { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "orcid": "0000-0003-2136-6506", "role": "chair", "display_name": "Meister, Markus" }, { "name": { "family": "Eberhardt", "given": "Frederick D." }, "id": "Eberhardt-F-D", "role": "member", "display_name": "Eberhardt, Frederick D." }, { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "orcid": "0000-0002-1290-5232", "role": "member", "display_name": "Shimojo, Shinsuke" }, { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "member", "display_name": "Adolphs, Ralph" } ], "option_major": [ "neurobio" ], "doi": "10.7907/yhmt-9t69", "abstract": "Most studies of emotion have as their subject matter the emotion experiences that people can describe and rate. By contrast to this approach from psychology, studies in animals, and some biological studies in humans, focus on behavior and its adaptive function. These two literatures typically use very different corresponding features by which to characterize emotion: categories or dimensions describing feelings for which we have convenient words, for the former (e.g., happiness, pleasantness), and functional properties for the latter (e.g., persistence, generalizability, approachabil- ity). In this thesis I use both sets of ratings, and I ask whether the latter, biologically inspired features could also be used to characterize people\u2019s emotion experiences, and might reveal novel dimensions of variability. They also typically use different sets of stimuli to induce the emotions: lexical stimuli in which participants are asked to imagine something hypothetical are common in human studies; ecologically valid stimuli that at least the subjects cannot distinguish from the real world are common in animal studies. Here I used three domains of stimuli: stories, videos, and real-life experiences, in the same set of participants, permitting a unique comparison.
\r\n\r\nI took advantage of a sample of approximately 1000 Americans who were surveyed longitudinally over the internet during the COVID-19 pandemic. I collected ratings of emotion experiences evoked by three classes of stimuli: a validated set of short stories, a validated set of short videos, and actual experiences in real life across multiple waves. I found that all three types of emotion experiences could be characterized by low dimensional spaces, with the first two factors that accounted for most of the variance in people\u2019s ratings corresponding to the dimensions of valence and arousal, in line with prior work. However, I discovered additional novel factors related to generalizability (the extent to which an emotion experience is shared across many different situations and occurrences) or modularity (the extent to which an emotion experience is unique to specific situations). The findings show that emotion features not usually assessed in humans can be recovered from subjective ratings of their experiences. I argue for a revision of current dimensional theories of emotion: they have been incomplete because they were restricted to ratings entrenched in how we think of our conscious experience, and the typical English words we use to describe it. The new dimensions validate some theories of emotion and offer hope for linking psychological studies in humans with behavioral or neurobiological work across species. I also characterized the distributions of the three types of emotion experiences and found that emotions were distributed along continuous gradients, with no well-separated clusters even for emotions belonging to the six basic emotion categories.
\r\n\r\nMy thesis presents two additional topics that capitalize on my unique sample: the emotions experienced during the COVID pandemic, and individual differences. For example, I also found that resilience buffered individuals against the effect of loneliness on depression, and that people who had tested positive for COVID felt more morally disgusted towards acts of violating social norms. I also explored the association between psychological traits and differences in emotion experiences both in terms of the magnitudes of the ratings and the overall correlation structure across scales. Again, the richness of my dataset reveals a number of associations that are theoretically interesting and that will be of relevance to understanding mood and anxiety disorders as well.
\r\n\r\nAll of the data will be made publicly available, and the core parts of many of the investigations were pre-registered.
" }, { "name": "Hoffmann, Magnus Adrian Gero", "degree": "PhD", "year": "2022", "title": "Nanoparticle Technologies to Cure and Prevent Infectious Diseases", "advisor": "Bjorkman, Pamela J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10302021-184745797", "creators": [ { "name": { "family": "Hoffmann", "given": "Magnus Adrian Gero" }, "id": "Hoffmann-Magnus-Adrian-Gero", "orcid": "0000-0003-4923-9568", "display_name": "Hoffmann, Magnus Adrian Gero" } ], "advisors": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "advisor", "display_name": "Bjorkman, Pamela J." } ], "committee": [ { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "orcid": "0000-0003-2713-1513", "role": "chair", "display_name": "Mazmanian, Sarkis K." }, { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "member", "display_name": "Pierce, Niles A." }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "orcid": "0000-0001-8723-8190", "role": "member", "display_name": "Baltimore, David L." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." } ], "option_major": [ "biology" ], "doi": "10.7907/g0w1-rc77", "abstract": "Despite almost 40 years of intensive research, there is still no curative treatment for HIV-1/AIDS. Anti-retroviral therapy (ART) prolongs the life expectancy of HIV-1-infected individuals but is associated with side effects, and multiple drugs need to be given in combination to prevent the development of viral resistance. In addition, treatment must continue for the lifetime of the individual due to the existence of a long-lived latent proviral reservoir. While a \"sterilizing\" cure remains difficult to achieve due to difficulties associated with identifying and clearing latently-infected cells, recent research has focused on designing a \"functional\" cure, i.e., a therapeutic strategy that enables long-term suppression of HIV-1 replication and remission of symptoms in the absence of ART. The work presented here describes a new therapeutic direction for the development of a functional cure against HIV-1. This approach is based on the hypothesis that HIV-1 is unable to escape from a nanoparticle (NP)-based decoy that presents clusters of the HIV-1 receptor CD4, because CD4-NPs mimic viral target cells more accurately than soluble CD4-based inhibitors and permit high-avidity interactions with trimeric HIV-1 Env proteins. We demonstrate that CD4-NPs are >10,000-fold more potent than soluble CD4 (sCD4) and prevent viral escape in vitro. AAV-mediated delivery of self-assembling CD4-NPs produced stable CD4-NP serum concentrations in mice that were almost 1,000-fold higher than concentrations required to neutralize HIV-1 in vitro, suggesting that these concentrations could be therapeutic. Viral challenge studies in non-human primates are underway to evaluate the potential of this therapeutic strategy.
\r\n\r\nAs an alternative approach to generate decoys against HIV-1, we generated engineered red blood cells (RBCs) that expressed viral receptors and potently inhibited HIV-1 infection of target cells in vitro. Because RBCs do not contain nuclei or functional organelles required for protein translation, infection of engineered RBCs represents a dead-end for a lentivirus such as HIV-1, which must integrate into the host cell genome as part of its lifecycle. We generated stable erythroid progenitor cell lines that continuously produced HIV-1 receptor-expressing RBCs that could be administered to HIV-1-infected individuals. As RBCs vastly outnumber CD4+ T-cells, HIV-1\u2019s main target cells, and have extended lifetimes, only a fraction of an individual\u2019s RBCs would need to be replaced with the engineered RBC viral traps in order to suppress HIV-1 infection in vivo.
\r\n\r\nMy work on CD4-NP therapeutics against HIV-1 also led to the invention and development of the EBR NP technology that is ideally suited for vaccine design applications. This technology can be used to modify any type of membrane protein to self-assemble into enveloped virus-like NPs without the need for additional proteins. EBR NP assembly is induced by inserting a short amino acid sequence into the cytoplasmic tail of the membrane protein, which was designed to recruit host proteins from the endosomal sorting complex required for transport (ESCRT) pathway. We applied this technology to design protein NP-based vaccines against Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), which elicited potent serum neutralizing antibody responses in mice. The EBR NP technology is also ideally suited for the development of hybrid vaccine approaches that allow genetic encoding of protein-based NPs, thereby combining attributes of mRNA and protein-based NP vaccines. Pilot studies demonstrated that mRNA and DNA vaccines encoding the self-assembling SARS-CoV-2 spike-EBR construct elicited ~10-fold higher neutralizing antibody responses than mRNA and DNA vaccines encoding the unmodified spike protein. This hybrid approach has the potential to substantially enhance the potency of mRNA vaccines and could become a leading vaccine platform technology. Future applications for the EBR NP technology are discussed, including the development of a universal coronavirus vaccine to prevent future pandemics, and engineering EBR NPs to mRNA vaccines or therapeutic cargoes for efficient and targeted delivery.
" }, { "name": "Jo, HyeongChan", "degree": "PhD", "year": "2022", "title": "Bidirectional Brain-Machine Interfaces for Modulating Stimulation and Neural Plasticity", "advisor": "Andersen, Richard A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08272021-174548241", "creators": [ { "name": { "family": "Jo", "given": "HyeongChan" }, "id": "Jo-HyeongChan", "orcid": "0000-0002-1435-9124", "display_name": "Jo, HyeongChan" } ], "advisors": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "advisor", "display_name": "Andersen, Richard A." } ], "committee": [ { "name": { "family": "Burdick", "given": "Joel Wakeman" }, "id": "Burdick-J-W", "orcid": "0000-0002-3091-540X", "role": "chair", "display_name": "Burdick, Joel Wakeman" }, { "name": { "family": "Rutishauser", "given": "Ueli" }, "id": "Rutishauser-U", "orcid": "0000-0002-9207-7069", "role": "member", "display_name": "Rutishauser, Ueli" }, { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "orcid": "0000-0001-8837-678X", "role": "member", "display_name": "Siapas, Athanassios G." }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "member", "display_name": "Andersen, Richard A." } ], "option_major": [ "bioeng" ], "doi": "10.7907/df0t-1t79", "abstract": "In prosthetics, tactile feedback can let us feel how we interact with the environment. Without this, it is extremely difficult to perform a motor task with fine control. The same idea can be applied in the brain-machine interface (BMI), which is an interface that directly connects external devices such as prosthetic limbs to the brain. Bidirectional BMI can deliver a stimulation to the brain as a sensory feedback, which can improve the performance of motor tasks. Such a bidirectional BMI can also serve a different role, if the stimulation encodes different information: if it encodes neural activity from another brain area, for example, then bidirectional BMI can provide a bypass for a damaged neural circuit. This may also affect the neural connectivity, strengthening or weakening the underlying neural connections. In this thesis, we present experiments that explore such applications of bidirectional BMI. First, we describe an experiment for characterizing neural connectivity between different brain areas. We found neural connectivity between supramarginal gyrus (SMG) and PMv (ventral premotor area), and also between anterior intraparietal (AIP) and Brodmann\u2019s area 5 (BA5), characterized by field-field, spike-field, and partial spike-field coherence. Through partial spike-field coherence, we also revealed that the spikes in PMv may drive the activity in SMG, which is obscured in ordinary spike-field coherence. Next, we provide evidence of changes in neural connectivity caused by stimulation in S1. With spike-triggered stimulation, which delivers stimulation in S1 in response to spikes recorded in a selected channel in SMG, we could significantly increase the correlation between SMG and S1, measured by the spike time tilling coefficient (STTC) to avoid dependencies of the correlation on firing rates. Furthermore, we found that not only spike-triggered stimulations, but also random stimulations on multiple channels in S1, can vary partial spike-field coherence in theta and alpha bands within S1; such changes mostly occurred in channel pairs with zero phase difference in partial spike-field coherence. Finally, we demonstrate the possibility of volitional control on stimulation pattern in bidirectional BMI. It is shown that the participants could not only increase or decrease a single-channel firing rate, but also hold the firing rate in a given range, demonstrating a fine control over firing rate. These findings would begin to establish a framework for closed-loop modulation of neural activity with bidirectional BMI and could be used to develop new treatments for neurological damage, such as to promote plasticity in or bridge brain areas affected by stroke.
" }, { "name": "Kajikawa, Koichiro", "degree": "PhD", "year": "2022", "title": "Inhibition is the Hallmark of CA3 Intracellular Dynamics Around Awake Ripples", "advisor": "Siapas, Athanassios G.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05272022-195420095", "creators": [ { "name": { "family": "Kajikawa", "given": "Koichiro" }, "id": "Kajikawa-Koichiro", "orcid": "0000-0002-8626-4407", "display_name": "Kajikawa, Koichiro" } ], "advisors": [ { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "orcid": "0000-0001-8837-678X", "role": "advisor", "display_name": "Siapas, Athanassios G." } ], "committee": [ { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "orcid": "0000-0002-8587-9936", "role": "chair", "display_name": "Dickinson, Michael H." }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "member", "display_name": "Andersen, Richard A." }, { "name": { "family": "Hong", "given": "Elizabeth J." }, "id": "Hong-Elizabeth-J", "role": "member", "display_name": "Hong, Elizabeth J." }, { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "orcid": "0000-0001-8837-678X", "role": "member", "display_name": "Siapas, Athanassios G." } ], "option_major": [ "cns" ], "doi": "10.7907/pwh5-7229", "abstract": "Hippocampal ripples are transient population bursts that structure cortico-hippocampal communication and play a central role in memory processing. However, the mechanisms controlling ripple initiation in behaving animals remain poorly understood. Here we combine multisite extracellular and whole cell recordings in awake mice to contrast the brain state and ripple modulation of subthreshold dynamics across hippocampal subfields. We find that entorhinal input to DG exhibits UP and DOWN dynamics with ripples occurring exclusively in UP states. While elevated cortical input in UP states generates depolarization in DG and CA1, it produces persistent hyperpolarization in CA3 neurons. Furthermore, growing inhibition is evident in CA3 throughout the course of the ripple buildup, while DG and CA1 neurons exhibit depolarization transients 100 ms before and during ripples. These observations highlight the importance of CA3 inhibition for ripple generation, while pre-ripple responses indicate a long and orchestrated ripple initiation process in the awake state.
" }, { "name": "Koontz, Alison Lindsay", "degree": "PhD", "year": "2022", "title": "Neural Crest and Placodal Cells Contributions to Cranial Sensory Development", "advisor": "Bronner, Marianne E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:07072021-193559924", "creators": [ { "name": { "family": "Koontz", "given": "Alison Lindsay" }, "id": "Koontz-Alison-Lindsay", "display_name": "Koontz, Alison Lindsay" } ], "advisors": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "advisor", "display_name": "Bronner, Marianne E." } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "chair", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Lois", "given": "Carlos" }, "id": "Lois-Carlos", "orcid": "0000-0002-7305-2317", "role": "member", "display_name": "Lois, Carlos" }, { "name": { "family": "Parker", "given": "Joseph" }, "id": "Parker-J", "orcid": "0000-0001-9598-2454", "role": "member", "display_name": "Parker, Joseph" }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "member", "display_name": "Bronner, Marianne E." } ], "option_major": [ "biology" ], "doi": "10.7907/30rt-s038", "abstract": "The sensory system of vertebrates is incredibly complex. Many important components of the sensory system are located within the cranial region, including the sense organs and cranial sensory ganglia. Early in development two progenitor populations, the neural crest and the cranial placodes, arise at the neural plate border and throughout vertebrate development contribute to the developing vertebrate peripheral sensory system. The interactions and contributions of both of these cell populations to the development of the pituitary system, the eyes, the nose, the ears, and the cranial ganglia of the head and neck are vital for the appropriate development of an embryo\u2019s nervous system.
\r\n\r\nIn this dissertation we explore the contributions of both the neural crest and placodal cells to the sensory system of the developing embryo. In Chapter 1 we review the origin of these two cell populations at the neural plate border and give an overview of the development of the various cranial peripheral sensory systems and their placode and neural crest contributions.
\r\n\r\nIn Chapter 2 we use replication incompetent avian retroviruses to lineage trace both the olfactory placode and the neural crest to their respective cellular contributions in the olfactory system. We confirm previous studies which showed that GnRH neurons of the nose receive contributions from both the olfactory placode and the neural crest and we show that both the olfactory placode and the neural crest contribute to the olfactory neurons of the olfactory epithelium. However, neural crest alone gives rise to the olfactory ensheathing cells which are critical for neuronal migration from the olfactory epithelium to the forebrain. We also show for the first time that the neural crest gives rise to the p63 positive horizontal basal stem cell population of the olfactory epithelium.
\r\n\r\nIn Chapter 3, along with collaborators from SUNY Buffalo, we show that multipotent and functional NC cells can be derived by induction with a growth factor cocktail containing FGF2 and IGF1 from cultures of human inter-follicular keratinocytes (KC) isolated from elderly donors. They also maintained their multipotency, as evidenced by their ability to differentiate into all NC-specific lineages including neurons, Schwann cells, melanocytes, and smooth muscle cells (SMC). Notably, upon implantation into chick embryos, adult NC cells behaved similar to their embryonic counterparts, migrated along stereotypical pathways, and contributed to multiple NC derivatives in ovo. These results suggest that KC-derived NC cells may provide an easily accessible, autologous source of stem cells that can be used for treatment of neurodegenerative diseases or as a model system for studying disease pathophysiology and drug development.
\r\n\r\nFinally, in Chapter 4 we discuss future directions and experiments that I plan to pursue post-graduation. I propose to conduct a closer examination of the variants of GnRH neurons across developmental time in various representative taxa of cartilaginous fish and reptiles. Furthermore, I intend to identify and experimentally confirm a molecular regulatory region for GnRH2, the most highly conserved variant across vertebrates, within the chicken embryo. Once this regulatory region is identified, the sequence can also be used to probe the genomes of other non-model taxa. Finally, I would like to perform lineage analysis using DiI in a non-model system to probe the embryonic origins (neural crest vs. placode) of the GnRH neurons in more ancient taxa.
" }, { "name": "Ortiz-Mu\u00f1oz, Andr\u00e9s", "degree": "PhD", "year": "2022", "title": "Combinatorics and Stochasticity for Chemical Reaction Networks", "advisor": "Winfree, Erik; Fontana, Walter", "url": "https://resolver.caltech.edu/CaltechTHESIS:04182022-222416729", "creators": [ { "name": { "family": "Ortiz-Mu\u00f1oz", "given": "Andr\u00e9s" }, "id": "Ortiz-Mu\u00f1oz-Andr\u00e9s", "orcid": "0000-0003-1824-3230", "display_name": "Ortiz-Mu\u00f1oz, Andr\u00e9s" } ], "advisors": [ { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "orcid": "0000-0002-5899-7523", "role": "advisor", "display_name": "Winfree, Erik" }, { "name": { "family": "Fontana", "given": "Walter" }, "id": "Fontana-Walter", "orcid": "0000-0003-4062-9957", "role": "co-advisor", "display_name": "Fontana, Walter" } ], "committee": [ { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "chair", "display_name": "Pierce, Niles A." }, { "name": { "family": "Bois", "given": "Justin S." }, "id": "Bois-J-S", "orcid": "0000-0001-7137-8746", "role": "member", "display_name": "Bois, Justin S." }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." }, { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "orcid": "0000-0002-5899-7523", "role": "member", "display_name": "Winfree, Erik" } ], "option_major": [ "sysbiol" ], "doi": "10.7907/s9mc-3d59", "abstract": "Stochastic chemical reaction networks (SCRNs) are a mathematical model which serves as a first approximation to ensembles of interacting molecules. SCRNs approximate such mixtures as always being well-mixed and consisting of a finite number of molecules, and describe their probabilistic evolution according to the law of mass-action. In this thesis, we attempt to develop a mathematical formalism based on formal power series for defining and analyzing SCRNs that was inspired by two different questions. The first question relates to the equilibrium states of systems of polymerization. Formal power series methods in this case allow us to tame the combinatorial complexity of polymer configurations as well as the infinite state space of possible mixture states. Chapter 1 presents an application of these methods to a model of polymerizing scaffolds. The second question relates to the expressive power of SCRNs as generators of stochasticity. In Chapter 2, we show that SCRNs are universal approximators of discrete distributions, even when only allowing for systems with detailed-balance. We further show that SCRNs can exactly simulate Boltzmann machines. In Chapter 3, we develop a formalism for defining the semantics of SCRNs in terms of formal power series which grew as a result of work included in the previous chapters. We use that formulation to derive expressions for the dynamics and stationary states of SCRNs. Finally, we focus on systems that satisfy complex balance and conservation of mass and derive a general expressions for their factorial moments using generating function methods." }, { "name": "Poole, William", "degree": "PhD", "year": "2022", "title": "Compilation and Inference with Chemical Reaction Networks", "advisor": "Winfree, Erik; Murray, Richard M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:11102021-210013472", "creators": [ { "name": { "family": "Poole", "given": "William" }, "id": "Poole-William", "orcid": "0000-0002-2958-6776", "display_name": "Poole, William" } ], "advisors": [ { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "orcid": "0000-0002-5899-7523", "role": "advisor", "display_name": "Winfree, Erik" }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "co-advisor", "display_name": "Murray, Richard M." } ], "committee": [ { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "chair", "display_name": "Thomson, Matthew" }, { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "orcid": "0000-0002-5899-7523", "role": "member", "display_name": "Winfree, Erik" }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." } ], "option_major": [ "cns" ], "doi": "10.7907/x3qc-je74", "abstract": "The successful advancement and deployment of technologies in the field of synthetic biology will require sophisticated computational infrastructure coupled with new theoretical ideas in order to more effectively engineer and reverse engineer biochemical networks. This thesis argues that the field of machine learning can inform the development of these underlying principles and techniques. First, software for compiling diverse chemical reaction network models of biological circuits from simple specifications is described. Second, three chemical reaction network implementations of a powerful machine learning model called a Boltzmann machine are analyzed and compared. Third, the class of detailed balanced chemical reaction networks are proven to be capable of probabilistic inference and, when coupled to a driven chemical system, autonomous learning. Finally, the use of machine learning to interpret and understand biological systems is explored in an experimental case study modeling E. coli cell extract metabolism.
" }, { "name": "Rosenberg, Matthew Hutson", "degree": "PhD", "year": "2022", "title": "Innate Navigation: Magnetic Sensation and Maze Learning", "advisor": "Meister, Markus", "url": "https://resolver.caltech.edu/CaltechTHESIS:06072022-184731596", "creators": [ { "name": { "family": "Rosenberg", "given": "Matthew Hutson" }, "id": "Rosenberg-Matthew- Hutson", "orcid": "0000-0002-6728-915", "display_name": "Rosenberg, Matthew Hutson" } ], "advisors": [ { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "orcid": "0000-0003-2136-6506", "role": "advisor", "display_name": "Meister, Markus" } ], "committee": [ { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "orcid": "0000-0002-8587-9936", "role": "chair", "display_name": "Dickinson, Michael H." }, { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "orcid": "0000-0001-8837-678X", "role": "member", "display_name": "Siapas, Athanassios G." }, { "name": { "family": "Rutishauser", "given": "Ueli" }, "id": "Rutishauser-U", "orcid": "0000-0002-9207-7069", "role": "member", "display_name": "Rutishauser, Ueli" }, { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "orcid": "0000-0003-2136-6506", "role": "member", "display_name": "Meister, Markus" } ], "option_major": [ "cns" ], "doi": "10.7907/hmyr-3d58", "abstract": "This thesis aims to advance the understanding of the neurobiology of navigation through the investigation of two topics: magnetic sensation and maze navigation. The central question of this work may be framed as follows: how do animals find their way to key resources that are necessary for survival? Three projects are presented to address this.
\r\n\r\nChapter II explores a sensory hypothesis that some animals may navigate long distances by directly sensing the earth\u2019s magnetic field. Awake zebra finches were stimulated with magnetic fields that varied sinusoidally in time while electrical recordings were collected via multi-channel electrodes. Preliminary negative results are presented, along with a detailed statistical treatment indicating no significant effect of magnetic stimulation on neural activity.
\r\n\r\nChapter III presents a novel approach to studying learning and navigation in animal subjects. Mice are allowed free passage between a normal home cage and a complex maze environment, coming and going as they please. Sated animals, with free access to food and water, spend significant portions of a given multi-hour experiment in the maze and display efficient exploration. Water-restricted animals show three additional phenomena: immediate knowledge of the route home, rapid learning of the location of a single water port among 64 similar locations, and a moment of \"sudden insight\" in which the rate at which long, direct routes to the water source, beginning from many locations, increases discontinuously.
\r\n\r\nChapter IV offers a simple, biologically feasible circuit model that recapitulates and explains some of the rapid learning behaviors we observe in mice. This model suggests a mechanism that might allow mice to flexibly store and recall direct routes to different resources that are activated by different internal drives.
\r\n\r\nThe final chapter outlines some potential directions for future inquiry, including potential maze experiments to conduct with wireless electrophysiology and expansion of the range of species tested for magnetic perception. The Appendix briefly describes some follow-up experiments and intriguing preliminary results. Similarities in the navigation deficit displayed by mice that have been experimentally perturbed in several disparate ways is noted briefly. These perturbations include whisker trimming, olfactory neuron ablation, genetic ablation of cortex and hippocampus, and opiate intoxication.
" }, { "name": "Savela, Emily Sue", "degree": "PhD", "year": "2022", "title": "Nucleic Acid Measurements for Antibiotic Susceptibility Testing and Early Detection of SARS-CoV-2", "advisor": "Ismagilov, Rustem F.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10072021-173853251", "creators": [ { "name": { "family": "Savela", "given": "Emily Sue" }, "id": "Savela-Emily-Sue", "orcid": "0000-0001-9614-4276", "display_name": "Savela, Emily Sue" } ], "advisors": [ { "name": { "family": "Ismagilov", "given": "Rustem F." }, "id": "Ismagilov-R-F", "orcid": "0000-0002-3680-4399", "role": "advisor", "display_name": "Ismagilov, Rustem F." } ], "committee": [ { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "chair", "display_name": "Pierce, Niles A." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." }, { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "orcid": "0000-0002-7033-0844", "role": "member", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Ismagilov", "given": "Rustem F." }, "id": "Ismagilov-R-F", "orcid": "0000-0002-3680-4399", "role": "member", "display_name": "Ismagilov, Rustem F." } ], "option_major": [ "bioeng" ], "doi": "10.7907/vp9a-n206", "abstract": "Nucleic-acid-amplification tests (NAATs) are widely used in microbial detection both in environmental characterization and human diagnostics. NAATs offer highly sensitive and specific detection of target molecules among the noise of complex samples. This thesis covers two important applications of nucleic-acid quantification techniques in human clinical samples. First, I co-developed a new phenotypic antibiotic susceptibility test that uses species-specific DNA detection to detect bacterial cell-wall damage following incubation with beta-lactam antibiotics. Second, I helped compile a longitudinal dataset of SARS-CoV-2 viral loads during a community-based COVID-19 study run by the Ismagilov Lab through October 2020 \u2013 April 2021 in the greater Los Angeles County area, USA. Sensitive and specific nucleic-acid tests allowed for robust detection of pathogenic microbes in both these applications. Designing and implementing NAATs for these applications required consideration of biological constraints of the microorganisms, molecular stability over the time of quantification, and the practical constraints of acquiring and transporting samples. Continued innovation of NAAT technologies will be critical to contain present and future pandemics and empower medical professionals with data to inform treatment options.
" }, { "name": "Shur, Andrey Sergeyvich", "degree": "PhD", "year": "2022", "title": "Serine Integrase-Based Event Recording in E. coli", "advisor": "Murray, Richard M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:12212021-193826426", "creators": [ { "name": { "family": "Shur", "given": "Andrey Sergeyvich" }, "id": "Shur-Andrey-Sergeyvich", "orcid": "0000-0001-9372-6713", "display_name": "Shur, Andrey Sergeyvich" } ], "advisors": [ { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "advisor", "display_name": "Murray, Richard M." } ], "committee": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "chair", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "member", "display_name": "Pierce, Niles A." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." } ], "option_major": [ "bioeng" ], "doi": "10.7907/x4q0-nx18", "abstract": "DNA is a unique molecule that has evolved to serve as the genetic material for life. It seems straightforward to consider this molecule not only as a wonder of the natural world but as a tool for information storage and retrieval. Bacteria have evolved to conserve DNA, but bacteriophages have evolved to specifically integrate their genomes using integrases. In response to viruses, bacteria have evolved the RNA-guided nuclease Cas9 to destroy viral DNA before it can be integrated. The fruits of these evolutionary pressures prove useful to the researcher interested in easily editing DNA. In this work, we have engineered a genetic circuit that can enact specific and controlled genetic changes in response to changing small molecule concentrations. Known DNA sequences can be repeatedly integrated into a synthetic array such that their identity and order encodes information about past small molecule concentrations that the cell has experienced. To accomplish this, we use catalytically inactive CRISPR-Cas9 (dCas9) to bind to and block attachment sites for the integrase Bxb1. Through the co-expression of dCas9 and guide RNA, Bxb1 can be directed to integrate one of two engineered \"ink\" plasmids, which correspond to two orthogonal small molecule inducers whose presence or absence as a function of time can be recorded with this system. Integrase sites present on these plasmids are found to not participate in intramolecular \"deletion\" reactions if closer than 100 bp. Guide RNAs overlapping integrase attachment sites are found to effectively block integrase activity at those sites if the overlap is equal to 9 or 19 base pairs. Other overlap values, including forward or reverse binding result in ineffective integrase activity repression. We develop 8 orthogonal guide RNA sequences capable of binding to and repressing integrase activity at the attP site. Plasmid multimers are sequenced using Oxford Nanopore sequencing and found to follow population-level predictions of event record identity. Single DNA states are found insufficient for identifying past history of events; an ensemble of DNA states at the population level must be used. A modular modeling framework is developed (Global enumeration) to describe this system, and integrated with the existing chemical reaction network creation automation software BioCRNpyler. The modeling framework developed here automatically creates chemical reaction networks based on typical linear DNA-based synthetic biology \"genetic constructs\" and predicts transcripts and proteins produced based on simple transcription/translation rules. Integrase-based recombination events can also be predicted in a recursive way.
" }, { "name": "Su, Christina Janet", "degree": "PhD", "year": "2022", "title": "Principles of Addressing Specificity in Promiscuous Ligand-Receptor Systems", "advisor": "Elowitz, Michael B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06022022-032024376", "creators": [ { "name": { "family": "Su", "given": "Christina Janet" }, "id": "Su-Christina-Janet", "orcid": "0000-0002-9223-9777", "display_name": "Su, Christina Janet" } ], "advisors": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "advisor", "display_name": "Elowitz, Michael B." } ], "committee": [ { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "chair", "display_name": "Chan, David C." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "orcid": "0000-0002-3904-0195", "role": "member", "display_name": "Goentoro, Lea A." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" } ], "option_major": [ "sysbiol" ], "doi": "10.7907/z7dv-m192", "abstract": "In multicellular organisms, a relatively small number of highly conserved signaling pathways are used to enable intercellular communication. While the underlying molecular components and interactions are increasingly well understood, a fundamental mystery is how the diverse cell types of the body can be so precisely coordinated by so few pathways. It has long been known that different cell types exhibit varied responses to molecular signals, and it is unclear how this cell type specificity arises. In this work, we take a different perspective on this question and explore how cell type specificity can be generated at the level of intracellular signal. We refer to this ability to selectively activate different cell types as \"addressing.\" By eliminating the complexity of considering downstream pathway effectors, we are able to more comprehensively understand how cell type specificity can arise in spite of\u2014or because of\u2014promiscuity in ligand-receptor interactions. We focus on the bone morphogenetic protein (BMP) pathway as an ideal example. This pathway is essential in development, is of therapeutic interest in an array of pathologies, and has proven amenable to theoretical and experimental analysis. We first describe a minimal model of the pathway and identify what types of response functions can be achieved. We show that each layer of computation, from the formation of signaling complexes to the activation of downstream second messenger, can provide nontrivial integrations of ligand inputs. We then extend this analysis to systems with multiple cell types that may vary in receptor expression profile. The diverse response functions of this pathway enable systems in which different cell types or sets of cell types may be addressed with high specificity. In particular, the BMP pathway can address multiple cell types with high capacity, flexibility, and robustness. Taken together, these results provide a framework for understanding how molecular promiscuity in signaling pathways can, in fact, enable cellular specificity in pathway responses.
" }, { "name": "Tang, Weiyi", "degree": "PhD", "year": "2022", "title": "Retroviral Lineage Analysis of the Vagal Neural Crest Reveals Multipotency Towards the Cardiac and Enteric Fates", "advisor": "Bronner, Marianne E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04162022-233242577", "creators": [ { "name": { "family": "Tang", "given": "Weiyi" }, "id": "Tang-Weiyi", "orcid": "0000-0002-1279-1001", "display_name": "Tang, Weiyi" } ], "advisors": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "advisor", "display_name": "Bronner, Marianne E." } ], "committee": [ { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "orcid": "0000-0001-6597-2036", "role": "chair", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "member", "display_name": "Bronner, Marianne E." } ], "option_major": [ "biology" ], "doi": "10.7907/qakz-vm04", "abstract": "The neural crest is a migratory stem cell population that gives rise to the craniofacial skeleton, heart septa, pigment cells, and peripheral nervous system. Defects in neural crest development can lead to a broad range of congenital diseases, e.g., persistent truncus arteriosus, characterized by a mixture of oxygenated and deoxygenated blood, is related to the absence of the neural crest-derived outflow tract septum. Thus, a thorough understanding about neural crest migration, differentiation, and cell fate can shine lights on diagnosis and treatment of many congenital defects. A long-standing question is whether neural crest cells are composed of multipotent cells capable of giving rise to a wide range of cell types, or a mixture of fate-determined cells migrating to their destinations. Avian embryos resemble humans during neural crest development, but are more accessible to experimental manipulations than mammalian models, making them an ideal model to study the neural crest. Despite the abundance of information obtained from elegant experiments through interspecies grafting, the avian model lacks a direct tool to determine whether these cells are multipotent in vivo.
\r\n\r\nHere, we present a new clonal analysis tool that takes advantage of Replication Incompetent Avian retroviruses (RIAs). We validate the method in vitro and present the potential application in the chick embryo to test the multipotency of the trunk neural crest. Next, we perform RIA-mediated lineage tracing at a population level and uncover cardiomyocytes as a previously unknown cardiac neural crest derivative in both chicken and mouse. Furthermore, we utilize RIA-mediated clonal analysis to identify individual premigratory vagal neural crest cells as a multipotent stem cell that forms cell types in both the heart and the gut. We then confirm the results by single-cell photoconversion assay that further confirms that migrating neural crest cells are also multipotent. Time-lapse imaging shows that stochastic post-mitotic migration is a cellular mechanism underlying multipotency. Finally, molecular perturbation experiments show that CXCR4 and RET are essential guidance cues for migratory neural crest cells to enter the heart and the gut, respectively. Together, these results demonstrate the utility of using RIA viruses to tackle questions regarding the lineage, developmental potential, and migratory pathways followed by neural crest cells in avian embryos.
" }, { "name": "Watkins-Dulaney, Ella Jenn\u00e1", "degree": "PhD", "year": "2022", "title": "Engineering the Tryptophan Synthase \u03b2-Subunit for Synthesis of Noncanonical Amino Acids", "advisor": "Arnold, Frances Hamilton", "url": "https://resolver.caltech.edu/CaltechTHESIS:07062021-155047044", "creators": [ { "name": { "family": "Watkins-Dulaney", "given": "Ella Jenn\u00e1" }, "id": "Watkins-Dulaney-Ella-Jenn\u00e1", "orcid": "0000-0002-0585-1598", "display_name": "Watkins-Dulaney, Ella Jenn\u00e1" } ], "advisors": [ { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "advisor", "display_name": "Arnold, Frances Hamilton" } ], "committee": [ { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "chair", "display_name": "Shapiro, Mikhail G." }, { "name": { "family": "Ismagilov", "given": "Rustem F." }, "id": "Ismagilov-R-F", "orcid": "0000-0002-3680-4399", "role": "member", "display_name": "Ismagilov, Rustem F." }, { "name": { "family": "Tirrell", "given": "David A." }, "id": "Tirrell-D-A", "orcid": "0000-0003-3175-4596", "role": "member", "display_name": "Tirrell, David A." }, { "name": { "family": "Buller", "given": "Andrew R." }, "id": "Buller-A-R", "orcid": "0000-0002-9635-4844", "role": "member", "display_name": "Buller, Andrew R." }, { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "member", "display_name": "Arnold, Frances Hamilton" } ], "option_major": [ "bioeng" ], "doi": "10.7907/yekm-y267", "abstract": "The tryptophan synthase \u03b2-subunit (TrpB) naturally catalyzes a pyridoxal phosphate cofactor-mediated \u03b2-substitution reaction between indole and serine to form L-tryptophan. Almost half a century ago, it was realized that TrpB could accept nucleophiles other than indole to synthesize noncanonical amino acids (ncAAs), which are highly useful small-molecule building blocks that are found in many bioactive molecules. Since then, TrpB has been applied to synthesize a wide range of ncAAs. This thesis details the engineering of TrpB for synthesis of new and useful ncAAs and the application of TrpB as a model to study the principles that govern intra-protein interactions. Chapter I chronicles the history of tryptophan synthase, provides useful information about the enzyme\u2019s catalytic cycle, and describes how TrpB has been used to synthesize ncAAs in works preceding this thesis. Chapter II describes the evolution, application, and characterization of TrpB for the synthesis of a blue, fluorescent noncanonical amino acid \u03b2-(1-azulenyl)-L-alanine (AzAla). Chapter III details the engineering and mechanistic characterization of TrpB to asymmetrically catalyze C\u2013C bond formation with an entirely new class of nucleophile: ketones. Chapter IV describes the in vivo continuous evolution of TrpB which resulted in sequence-diverse TrpB orthologs that have been adapted to function at lower temperatures and display a range of substrate-selectivity profiles. Chapter V describes the development of a deep mutational scanning experiment of combinatorial site-saturation mutagenesis (SSM) libraries for generating a large dataset that maps enzyme sequence to function for the purpose of studying epistasis with machine learning. Overall, the work presented in this thesis expands the repertoire of ncAAs that can be synthesized by TrpB and demonstrates unique applications of TrpB as a model enzyme for continuous in vivo directed evolution and for generating a dataset that will be useful to the protein machine learning community.
" }, { "name": "Williams, Rory Logan", "degree": "PhD", "year": "2022", "title": "Development and Scaling of Differentiation Circuit Architectures for Improving the Evolutionary Stability of Burdensome Functions in E. coli", "advisor": "Murray, Richard M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01042022-184525578", "creators": [ { "name": { "family": "Williams", "given": "Rory Logan" }, "id": "Williams-Rory-Logan", "orcid": "0000-0003-2605-5790", "display_name": "Williams, Rory Logan" } ], "advisors": [ { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "advisor", "display_name": "Murray, Richard M." } ], "committee": [ { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "orcid": "0000-0002-7033-0844", "role": "chair", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "orcid": "0000-0002-3904-0195", "role": "member", "display_name": "Goentoro, Lea A." }, { "name": { "family": "Ismagilov", "given": "Rustem F." }, "id": "Ismagilov-R-F", "orcid": "0000-0002-3680-4399", "role": "member", "display_name": "Ismagilov, Rustem F." }, { "name": { "family": "Bois", "given": "Justin S." }, "id": "Bois-J-S", "orcid": "0000-0001-7137-8746", "role": "member", "display_name": "Bois, Justin S." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." } ], "option_major": [ "bioeng" ], "doi": "10.7907/5k67-b636", "abstract": "With advances in the sequencing and synthesis of DNA, automation, and computation, we are increasingly able to rapidly and reliably program functions into cells. However, because the functions we engineer cells to perform are often both unnecessary for the cell\u2019s survival and burdensome to cell growth, mutation and natural selection can rapidly lead to loss of function. Though numerous strategies have made headway, improving the evolutionary stability of engineered functions remains a goal of the synthetic biology community. To address this problem generally, we developed a strategy relying on integrase-mediated recombination which allows non-producing progenitor cells to differentiate at a tunable rate, thereby continuously replenishing producer cells expressing the orthogonal T7 RNAP. While this strategy removes selective pressure for mutations inactivating the function of interest in the progenitor cell population, a strategy of terminal differentiation, in which the capacity of differentiated cells to grow is limited, was necessary to prevent the expansion of such mutations in the differentiated cell population. To experimentally implement terminal differentiation, we co-opted the R6K plasmid system, using differentiation to simultaneously activate expression of T7 RNAP, and inactivate expression of \u03c0 protein (an essential factor for R6K plasmid replication), thereby allowing limitation of differentiated cell growth through antibiotic selection. Critically, we demonstrated computationally that terminal differentiation endows the circuit with robustness to mutations which disrupt T7 RNAP driven expression, and to plasmid instability effects that result in decreased expression. Intuitively and computationally identifying the category of mutations which disrupt the differentiation process as the Achilles's heel of terminal differentiation, we developed a redundant architecture using a novel split-\u03c0 protein system which required 2 mutations to break the circuit. We experimentally demonstrated a trade-off between rate of production and duration of function as the differentiation rate is tuned, an increased benefit of terminal differentiation with higher-burden expression, and that redundancy improves the evolutionary stability of the terminal differentiation architecture. Specifically we achieve a maximum of ~2.8x (single-cassette terminal differentiation) and ~4.2x (redundant terminal differentiation) the total fluorescent protein production achieved from comparable high-burden naive expression in which all cells inducibly express T7 RNAP. We further demonstrate differentiation can enable the expression of even toxic functions, and develop a terminal differentiation circuit architecture which will allow the degree of redundancy and therefore the evolutionary stability of the architecture to be scaled to arbitrary degrees.
" }, { "name": "Wittmann, Bruce James", "degree": "PhD", "year": "2022", "title": "Strategies and Tools for Machine Learning-Assisted Protein Engineering", "advisor": "Arnold, Frances Hamilton", "url": "https://resolver.caltech.edu/CaltechTHESIS:05262022-234214451", "creators": [ { "name": { "family": "Wittmann", "given": "Bruce James" }, "id": "Wittmann-Bruce-James", "orcid": "0000-0001-8144-9157", "display_name": "Wittmann, Bruce James" } ], "advisors": [ { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "advisor", "display_name": "Arnold, Frances Hamilton" } ], "committee": [ { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "chair", "display_name": "Pachter, Lior S." }, { "name": { "family": "Reisman", "given": "Sarah E." }, "id": "Reisman-S-E", "orcid": "0000-0001-8244-9300", "role": "member", "display_name": "Reisman, Sarah E." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "member", "display_name": "Arnold, Frances Hamilton" } ], "option_major": [ "bioeng" ], "doi": "10.7907/azzt-0q97", "abstract": "Proteins perform critical roles in a growing list of human-devised applications, and as demands for new applications arise, new proteins must be engineered to meet them. Machine learning-assisted protein engineering (MLPE) has recently arisen as a new philosophy of protein engineering, promising to overcome many of the limitations of existing engineering strategies. Despite its promise, however, as a relatively new approach to protein engineering, MLPE faces many challenges that hinder its routine application. This thesis is focused on addressing a number of them. Chapter 1 provides a theoretical overview of protein engineering, introduces the core steps of a typical MLPE pipeline, and discusses the challenges that currently hinder MLPE\u2019s advancement. This chapter is written to be accessible to all members of the highly multidisciplinary audience that either use or develop MLPE tools, in turn providing a resource that eliminates the steep barrier to entry that can hinder broader participation in the field. Chapter 2 provides a solution to the challenge of applying MLPE to proteins whose fitness landscapes are dominated by \u201choles\u201d (protein variants with zero or extremely low fitness). Using my development of the strategy \u201cfocused training machine learning-assisted directed evolution (ftMLDE)\u201d as an example, I demonstrate how auxiliary information from protein sequence and structure can be used to navigate landscapes despite holes, in turn dramatically improving the efficiency of MLPE. Chapter 3 explores strategies for reducing the amount of sequence-fitness data needed for building MLPE models. Specifically, I detail the motivation behind and development of a new model designed to augment limited protein sequence-fitness datasets with information extracted from raw protein sequence and structure data. Finally, chapter 4 introduces \u201cevery variant sequencing\u201d (evSeq), a collection of tools and protocols that enables extremely low-cost, routine collection of large protein sequence-fitness datasets. Not only does this technology drastically improve the financial feasibility of numerous MLPE applications, but it also potentiates the construction of a massive database of diverse protein sequence-fitness data, the likes of which would revolutionize our ability to engineer proteins with data-driven methods. Overall, the work described in this thesis advances both our understanding of MLPE and our ability to engineer proteins using it." }, { "name": "Xiao, Fangzhou", "degree": "PhD", "year": "2022", "title": "Biocontrol of Biomolecular Systems: Polyhedral Constraints on Binding's Regulation of Catalysis from Biocircuits to Metabolism", "advisor": "Doyle, John Comstock", "url": "https://resolver.caltech.edu/CaltechTHESIS:05302022-235732314", "creators": [ { "name": { "family": "Xiao", "given": "Fangzhou" }, "id": "Xiao-Fangzhou", "orcid": "0000-0002-5001-5644", "display_name": "Xiao, Fangzhou" } ], "advisors": [ { "name": { "family": "Doyle", "given": "John Comstock" }, "id": "Doyle-J-C", "orcid": "0000-0002-1828-2486", "role": "advisor", "display_name": "Doyle, John Comstock" } ], "committee": [ { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "chair", "display_name": "Murray, Richard M." }, { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "orcid": "0000-0002-5899-7523", "role": "member", "display_name": "Winfree, Erik" }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." }, { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "member", "display_name": "Pachter, Lior S." }, { "name": { "family": "Doyle", "given": "John Comstock" }, "id": "Doyle-J-C", "orcid": "0000-0002-1828-2486", "role": "chair", "display_name": "Doyle, John Comstock" } ], "option_major": [ "bioeng" ], "doi": "10.7907/rtwq-v497", "abstract": "One eventual goal of bioengineering is to build complex biological machines that fully realize the unique potential of biotechnology, namely adaptation, survival, growth, and dominance. In order to do so, not only do we need theoretical understanding and reliable manufacturing of biological parts and components, we also need a systems theory that captures fundamental structures to obtain insight about the space of all possible behaviors when parts are put together. This enables us to understand what can and cannot be achieved. Examples from other engineering disciplines are Turing machines for computers, information channels for communication networks, linear input output systems for electrical circuits, and thermodynamics for heat engines. This work is an attempt at developing a systems theory tailored to biomolecular systems in cells. The results form the following statements.
\r\n\r\nBiomolecular systems are binding and catalysis reactions. Catalysis determines the direction of change, while binding regulates how the catalysis rates vary with reactant concentrations. Given a binding reaction network, the full range of regulatory profiles can be captured by the reaction orders of catalysis, which in turn is constrained in polyhedral sets determined by the stoichiometry of binding. This constitute a rule, that since cells control catalysis by binding, cells control catalysis rates by regulating reaction orders constrained in polyhedral sets. This rule has ramifications in several directions. On metabolism, by incorporating the constraint that reaction orders of metabolic fluxes, not the fluxes themselves, are controlled, we can predict metabolism dynamics directly from network stoichiometry, e.g. glycolytic oscillations and growth arrests. This is a fully dynamic upgrade of flux balance analysis, a popular constraint-based method to model metabolism. On systems biology, this rule derives a method of biocircuit analysis based on the full range of values that reaction orders can take. This allows discovery of necessary and sufficient conditions for a circuit to achieve a certain function, thus revealing regimes hidden by traditional methods of analysis. It also promotes holistic comparisons of different circuit implementations, e.g. activating versus repressing, thur enabling biocircuit design where we know when a design will work, and when a design will fail. On dynamics and control of biocircuits, reaction order can work as a robust basis for stability, perfect adaptation, multistability, and oscillations. Lyapunov functions and dissipative control theory tailored for biomolecular systems are constructed based on reaction orders. On the mathematics of biology, it relates bioregulation to convex polyhedra, log derivative operator decompositions, and fundamental rules of calculus for positive variables.
" }, { "name": "Yang, Bin", "degree": "PhD", "year": "2022", "title": "Transformations and Functions of Neural Representations in a Subcortical Social Behavior Network", "advisor": "Anderson, David J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05232022-073842501", "creators": [ { "name": { "family": "Yang", "given": "Bin" }, "id": "Yang-Bin", "orcid": "0000-0002-3878-1530", "display_name": "Yang, Bin" } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "advisor", "display_name": "Anderson, David J." } ], "committee": [ { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "orcid": "0000-0003-2136-6506", "role": "chair", "display_name": "Meister, Markus" }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "member", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Oka", "given": "Yuki" }, "id": "Oka-Yuki", "orcid": "0000-0003-2686-0677", "role": "member", "display_name": "Oka, Yuki" }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." } ], "option_major": [ "neurobio" ], "doi": "10.7907/v13r-yt57", "abstract": "The brain functions by processing sensory information such as vision, smell, and touch, integrating it with internal states (hunger, fear, aggression) and memory to produce relevant motor outputs (eating, fleeing, or fighting). To understand the brain, neuroscientists study neural representations (patterns of neural activity that correlate with features of the outside world) to and perform perturbations (activate or silence groups of neurons) to determine its function. Past studies on neural representations gave us insights into how sensory regions filter complex inputs to retain relevant information and how coordinated activity in the motor regions produce complex motor actions. However, little is known about how information is processed in the inner brain (between sensory and motor) and how behaviors are controlled. Mating and aggression are innate social behaviors that are essential for animals\u2019 survival. During social interactions, such as those preceding mating or fighting, the brain must determine the sex of a conspecific to produce sex-appropriate behaviors that are conducive to its survival. Functional studies demonstrated that they are controlled by deep subcortical circuits in the extended amygdala and hypothalamus. My thesis attempts to understand how the inner brain works by 1) showing that chemosensory cues encoding conspecific\u2019s sex are transformed to neural representations of mating and aggression during social interactions by recording from a genetically defined group of neurons in different regions of the extended amygdala and hypothalamus. 2) Demonstrating that the neural activity representing conspecific\u2019s sex is necessary for the emergence of behavioral representations in the hypothalamus.
" }, { "name": "Yang, Zhi", "degree": "PhD", "year": "2022", "title": "Conformational Plasticity of HIV-1 Env and Implications for Vaccine Design", "advisor": "Bjorkman, Pamela J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01222022-001845303", "creators": [ { "name": { "family": "Yang", "given": "Zhi" }, "id": "Yang-Zhi", "orcid": "0000-0001-8680-3784", "display_name": "Yang, Zhi" } ], "advisors": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "advisor", "display_name": "Bjorkman, Pamela J." } ], "committee": [ { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "chair", "display_name": "Rees, Douglas C." }, { "name": { "family": "Voorhees", "given": "Rebecca M." }, "id": "Voorhees-R-M", "orcid": "0000-0003-1640-2293", "role": "member", "display_name": "Voorhees, Rebecca M." }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "member", "display_name": "Chan, David C." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." } ], "option_major": [ "biochem" ], "doi": "10.7907/99hp-y284", "abstract": "The human immunodeficiency virus (HIV) envelope glycoprotein (Env), a (gp120/gp41)\u2083 trimer, is present on the surface of the viral envelope membrane. Env binding to the host cell receptor, CD4, and the co-receptor, CCR5 or CXCR4, triggers a cascade of Env conformational changes and structural rearrangements which ultimately leads to the viral and host cell membrane fusion, marking the initiation of a viral infection. In this work, we present findings of the conformational changes of an Env trimer from a closed, pre-fusion state to an asymmetrically open state when bound to receptor CD4 and a co-receptor mimicking antibody, E51. We showed the importance of tyrosine sulfation in gp120 binding. The EM structures also indicate the existence of Env\u2019s multiple conformational states. Based on the structural information, we modeled the order of conformations on the path to co-receptor binding and viral-host cell membrane fusion.
\r\n\r\nAs the sole viral protein present on the virion surface, the Env acts as the target for anti-HIV antibodies. Using various types of engineered Env as the immunogen, researchers made attempts to elicit anti-HIV neutralizing antibodies in animals. In this work, we identified and analyzed two neutralizing antibodies, Ab1303 and Ab1573, that target the Env CD4 binding site (CD4bs), one of the conserved epitopes on the Env gp120 surface. Using biophysical and structural methods, we described a novel recognition mechanism of these antibodies and proposed a model about the unique behavior of Env under physiological conditions. This study proved that CD4bs Abs that recognize an \"occluded open\" Env can be raised by sequential animal immunizations, thereby guiding the future immunogen design and therapeutic applications.
" }, { "name": "Zhang, Tony (Haoyu)", "degree": "PhD", "year": "2022", "title": "Biological Intelligence: from Behavior to Learning Theory", "advisor": "Perona, Pietro", "url": "https://resolver.caltech.edu/CaltechTHESIS:12152021-005204320", "creators": [ { "name": { "family": "Zhang", "given": "Tony (Haoyu)" }, "id": "Zhang-Tony-Haoyu", "orcid": "0000-0002-5198-499X", "display_name": "Zhang, Tony (Haoyu)" } ], "advisors": [ { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "advisor", "display_name": "Perona, Pietro" } ], "committee": [ { "name": { "family": "Yue", "given": "Yisong" }, "id": "Yue-Yisong", "orcid": "0000-0001-9127-1989", "role": "chair", "display_name": "Yue, Yisong" }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "member", "display_name": "Perona, Pietro" }, { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "orcid": "0000-0003-2136-6506", "role": "member", "display_name": "Meister, Markus" }, { "name": { "family": "Bouman", "given": "Katherine L." }, "id": "Bouman-K-L", "orcid": "0000-0003-0077-4367", "role": "member", "display_name": "Bouman, Katherine L." } ], "option_major": [ "cns" ], "doi": "10.7907/8z4q-8g15", "abstract": "Knowing how to learn, think, and act is not just a hallmark of intelligence, but a necessity of survival for many organisms. Behavior, the complete set of actions of species, allows us to glimpse into the minds of humans and animals, and by extension, intelligence itself. Biological intelligence is characterized by fast adaptation to changes and challenges, which is what allows species to survive in natural environments from starvation and predation. To study learning in a controlled setting, we can observe the behavior evoked through decision-making tasks that make it possible to quantify and analyze learning. By modeling the extracted behavioral features, we could start to understand the possible underlying mechanisms by proposing neural theory models, and look for those signals in the brain. Understanding the neural mechanisms of learning also strengthens the basis for building intelligent machines that are flexible and adaptive to the nonstationary world we live in. In this thesis, I present works in (1) automating behavioral setups and modeling suboptimal behavior in a traditional decision-making task, (2) using an ethological navigation task to characterize fast-sequence learning, and (3) how neural theory can explain some core behavioral phenomena in (2), and be used to solve a central problem in graph search.
" }, { "name": "da Veiga Beltrame, Eduardo", "degree": "PhD", "year": "2022", "title": "Stories in Single Cell RNA Sequencing", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06022022-201232129", "creators": [ { "name": { "family": "da Veiga Beltrame", "given": "Eduardo" }, "id": "da-Veiga-Beltrame-Eduardo", "orcid": "0000-0002-1529-9207", "display_name": "da Veiga Beltrame, Eduardo" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "chair", "display_name": "Thomson, Matthew" }, { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "member", "display_name": "Pachter, Lior S." }, { "name": { "family": "Van Valen", "given": "David A." }, "id": "Van-Valen-D", "orcid": "0000-0001-7534-7621", "role": "member", "display_name": "Van Valen, David A." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "bioeng" ], "doi": "10.7907/4kgh-8420", "abstract": "This thesis describes the projects I have worked on since starting the Caltech bioengineering program in fall 2017. The general theme of my projects is that they are all about single cell RNA sequencing (scRNA-seq), spanning the experimental and computational realms.
\r\n\r\nChapter 1 is an introduction explaining the essential concepts and is meant to be readable by a wide audience. For the other chapters, each one describes a separate project in a succinct manner, including links to the related preprint, published paper or code repositories at the start of each chapter.
\r\n\r\nChapter 2 describes the scVI generative model for scRNA-seq data and the scvi-tools framework, which forms the basis of many of my computational projects.
\r\n\r\nChapter 3 describes an open source 3D printable syringe pump system that was developed envisioning facilitating many kinds of experiments, in particular droplet based scRNA-seq.
\r\n\r\nChapter 4 describes a new way of fabricating hydrogel beads with unique DNA barcodes that are used for scRNA-seq experiments.
\r\n\r\nChapter 5 describes a database listing most published scRNA-seq studies that I helped create, and provides a useful overview of the state of the field.
\r\n\r\nChapter 6 describes the kallisto bus workflow, which is used for pre-processing scRNA-seq data, going from FASTQ file to gene count matrix in a very efficient manner.
\r\n\r\nChapter 7 describes a new way of using scVI to quantify the trade- off in the quality of scRNA-seq of a given dataset when surveying more cells or sequencing more reads per cell.
\r\n\r\nChapter 8 describes tools developed for the WormBase users to leverage scRNA-seq data on C. elegans, and which can be deployed with any other scRNA-seq dataset.
\r\n\r\nChapter 9 describes a remarkably successful offshoot of the devel- opment of these tools: a simple scVI based analysis and visualization strategy for finding candidate marker genes using C. elegans scRNA-seq data, which was experimentally validated by members of the Sternberg lab.
" }, { "name": "de Souza, Alysha Maria", "degree": "PhD", "year": "2022", "title": "Sparse Neural and Motor Networks Underlying Control in the Drosophila Flight System", "advisor": "Dickinson, Michael H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10312021-193119771", "creators": [ { "name": { "family": "de Souza", "given": "Alysha Maria" }, "id": "de-Souza-Alysha-Maria", "display_name": "de Souza, Alysha Maria" } ], "advisors": [ { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "orcid": "0000-0002-8587-9936", "role": "advisor", "display_name": "Dickinson, Michael H." } ], "committee": [ { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "orcid": "0000-0001-8837-678X", "role": "chair", "display_name": "Siapas, Athanassios G." }, { "name": { "family": "Hong", "given": "Elizabeth J." }, "id": "Hong-Elizabeth-J", "role": "member", "display_name": "Hong, Elizabeth J." }, { "name": { "family": "Parker", "given": "Joseph" }, "id": "Parker-J", "orcid": "0000-0001-9598-2454", "role": "member", "display_name": "Parker, Joseph" }, { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "orcid": "0000-0002-8587-9936", "role": "member", "display_name": "Dickinson, Michael H." } ], "option_major": [ "biology" ], "doi": "10.7907/part-mf19", "abstract": "We often look to the natural world for inspiration in design and engineering. The fruit fly, Drosophila melanogaster, with approximately 100,000 neurons in its central nervous system (CNS) versus the roughly 100 billion neurons of the human brain, is relatively uncompromising in the richness of behaviors it is capable of performing given its comparatively sparse nervous system. It exhibits exceptional aerial agility, despite the steep aerodynamic constraints of miniaturization thanks to unique physiological and biomechanical thoracic adaptations. However, the mechanisms governing its sparse and precise flight control have remained largely inaccessible due to technological and geometric limitations, leaving many long-standing questions in the field of insect flight control unexplored. Recent advances in the field of molecular biology have created a vast toolkit for both optical imaging and genetic manipulation of cellular function. This revolution of genetic advances allows us to visualize changes in muscle activity in situ as fluorescent signals, to record from fluorescently targeted cells via electrophysiology or 2-photon imaging, and to optogenetically activate or silence the activity of targeted cells. This thesis utilizes recent technological and molecular advances to probe three key aspects of fly flight control: 1) the dynamic interactions of flight steering muscles to produce flight maneuvers, 2) the source of timing information for the structuring of the the motor phase code, an extremely temporally precise wingbeat-synchronous aspect neural firing, and 3) the mechanisms by which slow, graded descending visual process recruit the flight muscles.
\r\n\r\nIn the contents of the ensuing chapters I propose mechanisms for flight control pertaining to the wing muscles as well as their inputs. First, I describe the activities of each of the flight steering muscles in response to visual motion to generate movement in yaw, pitch, and roll (Chapter II). I then characterize the flexible individual dynamics and combinatorial timing of the system, and propose specific mechanisms by which interneurons rather than muscle physiology govern these adaptable firing patterns according to sensory inputs(Chapter II). Sensory inputs within this thesis take two forms: thoracic mechanosensory and timing information as well as descending visual input. I characterize mechanosensory and timing adaptations of an evolutionarily evolved hind wing, as well as the impact of haltere feedback to flight control (Chapter III). Lastly, I propose a mechanism by which descending visual commands produce graded outputs of the muscles.
" }, { "name": "Abedi, Mohamad", "degree": "PhD", "year": "2021", "title": "Thermal Bioswitches for Non-Invasive Control of Cellular Therapies", "advisor": "Shapiro, Mikhail G.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03032021-224823348", "creators": [ { "name": { "family": "Abedi", "given": "Mohamad" }, "id": "Abedi-Mohamad", "orcid": "0000-0001-9717-6288", "display_name": "Abedi, Mohamad" } ], "advisors": [ { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "advisor", "display_name": "Shapiro, Mikhail G." } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "chair", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "orcid": "0000-0001-8723-8190", "role": "member", "display_name": "Baltimore, David L." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "member", "display_name": "Shapiro, Mikhail G." } ], "option_major": [ "bioeng" ], "doi": "10.7907/z7ac-2g66", "abstract": "Temperature is a unique input signal that could be used by engineered therapeutic cells to sense and respond to host conditions or spatially targeted external triggers such as focused ultrasound. To enable these possibilities, I present here a new class of thermal bioswitches that enables thermal control over bacterial and mammalian cells. For bacterial applications, we developed two new families of tunable, orthogonal, temperature-dependent transcriptional repressors providing switch-like control of bacterial gene expression at thresholds spanning the biomedically relevant range of 32\u201346 \u00b0C. We integrated these molecular bioswitches into thermal logic circuits and demonstrated their utility in three in vivo microbial therapy scenarios, including spatially precise activation using focused ultrasound, modulation of activity in response to a host fever, and self-destruction after fecal elimination to prevent environmental escape. This technology provides a critical capability for coupling endogenous or applied thermal signals to cellular function in basic research, biomedical and industrial applications.
\r\n\r\nTo apply this technology in a relevant clinical scenario, we sought to engineer microbial immunotherapies that can be thermally controlled with focused ultrasound. This technology was enabled by rapid advances in synthetic biology that are driving the development of genetically modified microbes as therapeutic agents for a multitude of human diseases, including cancer. In particular, the reduced immune surveillance within the core of some solid tumors creates an ideal environment for microbes to engraft and release therapeutic payloads. However, these therapeutic payloads could be harmful if released in healthy tissues where microbes tend to also engraft in smaller numbers. As described in Chapter 2, my colleagues and I introduced a temperature-actuated state switch that enables tight spatiotemporal control over the activity of therapeutic microbes when combined with focused ultrasound hyperthermia. Through a combination of rational design and high throughput screening, we optimized the behavior of this switch to minimize leakage and maximize inducibility. When tested in a clinically relevant in vivo model, engineered microbes, successfully switched states, and induced a marked suppression of tumor growth upon focal activation. This bioswitch provides a critical tool to attain selective and sustained activity of therapeutic microbes in vivo.
\r\n\r\nEncouraged by the successful development of thermally actuated circuits in microbes, we aimed to establish equivalent technologies for thermal control of human T cells. Genetically engineered T cells are actively being developed to perform a variety of therapeutic functions with great clinical promise. However, no robust mechanisms exist to externally control the activity of T cells at specific locations within the body. Such spatiotemporal control could help mitigate potential off-target toxicity due to incomplete molecular specificity in applications such as T-cell immunotherapy against solid tumors. In Chapter 4, my colleagues and I tested the ability of heat shock promoters to mediate thermal actuation of genetic circuits in primary human T cells in the well-tolerated temperature range of 37\u221242 \u00b0C, and we introduced genetic architectures enabling the tuning of the amplitude and duration of thermal activation. We demonstrated the use of these circuits to control the expression of chimeric antigen receptors and cytokines, and the killing of target tumor cells. Overall, the technologies developed here provide critical tools to direct control therapeutic cells after they have been deployed deep inside the body.
" }, { "name": "Altermatt, Michael", "degree": "PhD", "year": "2021", "title": "Serotonergic Circuits: Role in Sleep and Enhanced Genetic Tools for Access and Optical Recording", "advisor": "Gradinaru, Viviana", "url": "https://resolver.caltech.edu/CaltechTHESIS:01202021-155015733", "creators": [ { "name": { "family": "Altermatt", "given": "Michael" }, "id": "Altermatt-Michael", "orcid": "0000-0003-2841-5374", "display_name": "Altermatt, Michael" } ], "advisors": [ { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "advisor", "display_name": "Gradinaru, Viviana" } ], "committee": [ { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "orcid": "0000-0002-7371-4675", "role": "chair", "display_name": "Prober, David A." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Hong", "given": "Elizabeth J." }, "id": "Hong-Elizabeth-J", "role": "member", "display_name": "Hong, Elizabeth J." }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "member", "display_name": "Gradinaru, Viviana" } ], "option_major": [ "neurobio" ], "doi": "10.7907/gfe2-w578", "abstract": "Overall, this thesis encompasses three main directions: the study of neural circuits in sleep (Chapter 2), the development and testing of tools for measuring neuromodulator release (Chapter 3), and methods for in vivo characterization of gene delivery vehicles (Chapter 5).
\r\n\r\nThe role of the neuromodulator serotonin in sleep has been debated for over 60 years. Until recently, the serotonergic system was widely thought to be part of the arousal system and promote wakefulness. In Chapter 2, we investigate the function of serotonin-producing neurons in murine and zebrafish sleep with tools featuring superior specificity and precision compared to previously employed techniques. Our results demonstrate that the serotonergic raphe are sleep-promoting and required for sleep homeostasis. Intriguingly, serotonergic neurons in mice can have opposing effects on sleep depending on the firing mode.
\r\n\r\nThe release of serotonin from neurons can be regulated by the frequency of neuronal firing and can occur at classical synapses, varicosities, soma, and dendrites. Further examination of the complex signaling mechanism of serotonin would benefit from tools capable of measuring the release of serotonin in vivo with long-term stability and high spatiotemporal resolution. To this end, we developed and characterized iSeroSnFR, an intensity-based genetically encoded serotonin indicator. In Chapter 3, we demonstrate that iSeroSnFR can detect serotonin release in freely behaving mice during fear conditioning, social interaction, and sleep-wake transitions.
\r\n\r\nAdeno-associated viruses (AAVs) have been extensively used as gene delivery vehicles in basic neuroscience and gene therapy. However, optimization of transduction efficiency and target specificity remain a key challenge to overcome. Several AAV vector engineering approaches have been devised for this purpose and yield large collections of candidates that require further in vivo characterization. However, conventional characterization methods fall short with regard to in-depth cell type tropism analysis and/or high-throughput capabilities. In Chapter 5, we address this shortcoming with single-cell RNA sequencing technologies based on the Drop-seq method. We established an experimental and computational pipeline that allows us to profile the viral tropism of multiple AAV variants in parallel across numerous complex cell types.
" }, { "name": "Anaya, Michael Aurelio", "degree": "PhD", "year": "2021", "title": "Modernization of Monoclonal Antibody Screening and Protein-interaction Assays", "advisor": "Zinn, Kai George", "url": "https://resolver.caltech.edu/CaltechTHESIS:06072021-233800139", "creators": [ { "name": { "family": "Anaya", "given": "Michael Aurelio" }, "id": "Anaya-Michael-Aurelio", "orcid": "0000-0002-6944-3614", "display_name": "Anaya, Michael Aurelio" } ], "advisors": [ { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "advisor", "display_name": "Zinn, Kai George" } ], "committee": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "chair", "display_name": "Wold, Barbara J." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." } ], "option_major": [ "biology" ], "doi": "10.7907/xy05-bb64", "abstract": "In this research, multiplexed bead-based technology was employed to develop a high-throughput monoclonal antibody production method and to refine a protein-protein interaction (PPI) assay for interactome screening. Hybridoma supernatants produced from mice injected with multiple antigens were screened with color-coded, antigen-coupled beads in a semi-automated workflow. Two monoclonal antibodies, each demonstrating high specificity and strong binding, were produced. To our knowledge, these results are the first demonstrated usage of multiplexed suspension bead-based screening as a critical component of high-throughput antibody production. The technology was also utilized as a PPI assay due to its numerous advantages over ELISA-based screens. We studied interactions of the extracellular domains of the Beat and Side protein families, whose members control neuromuscular specificity in Drosophila melanogaster and form a highly-connected interaction network. We demonstrated that a screen utilizing avidin-captured bait was superior to a screen utilizing Protein A-captured bait and deorphanized five proteins within the network, namely Beat 1b, Beat 3a, Beat 3c, Side 5, and Side 8.
" }, { "name": "Bagherian, Dawna Paria", "degree": "PhD", "year": "2021", "title": "Artificial Neural Networks for Nonlinear System Identification of Neuronal Microcircuits", "advisor": "Meister, Markus", "url": "https://resolver.caltech.edu/CaltechTHESIS:05282021-174607976", "creators": [ { "name": { "family": "Bagherian", "given": "Dawna Paria" }, "id": "Bagherian-Dawna-Paria", "orcid": "0000-0003-4465-552X", "display_name": "Bagherian, Dawna Paria" } ], "advisors": [ { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "orcid": "0000-0003-2136-6506", "display_name": "Meister, Markus" } ], "committee": [ { "name": { "family": "Yue", "given": "Yisong" }, "id": "Yue-Yisong", "orcid": "0000-0001-9127-1989", "role": "member", "display_name": "Yue, Yisong" }, { "name": { "family": "Tsao", "given": "Doris Y." }, "id": "Tsao-D-Y", "orcid": "0000-0003-1083-1919", "role": "member", "display_name": "Tsao, Doris Y." }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "chair", "display_name": "Perona, Pietro" }, { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "orcid": "0000-0003-2136-6506", "role": "member", "display_name": "Meister, Markus" } ], "option_major": [ "bioeng" ], "doi": "10.7907/rj2p-8g11", "abstract": "This thesis explores the application of artificial neural networks (ANNs) to nonlinear system identification. We use neuronal microcircuits in the retina as a testbed for our technique, which relies upon the marriage of partial anatomical information with large electrophysiological datasets. Rather than a typical application of machine learning, our primary goal is not to predict the output of retinal circuits, but rather to uncover their structure. We begin with a theoretical exploration in a toy problem and provide a proof of unique identifiability under a specific set of conditions. We then perform empirical simulations in a number of different circuit architectures and explore the space of constraints and regularizers to demonstrate that this technique is feasible in a hyperparametric regime that lends itself well to neuroscience datasets. We then apply the technique to mouse retinal datasets and show that we can both recover known biological information as well as discover new hypotheses for biological exploration. We end with an exploration of active stimulus design algorithms to distinguish between circuit hypotheses.
" }, { "name": "Banerjee, Abhik Kumar", "degree": "PhD", "year": "2021", "title": "Diverse Roles of RNA-protein Interactions: From Viral Antagonism to Mammalian Development", "advisor": "Guttman, Mitchell; Rothenberg, Ellen V.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02032021-180657616", "creators": [ { "name": { "family": "Banerjee", "given": "Abhik Kumar" }, "id": "Banerjee-Abhik-Kumar", "orcid": "0000-0002-9797-0104", "display_name": "Banerjee, Abhik Kumar" } ], "advisors": [ { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "advisor", "display_name": "Guttman, Mitchell" }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "co-advisor", "display_name": "Rothenberg, Ellen V." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "orcid": "0000-0001-8723-8190", "role": "member", "display_name": "Baltimore, David L." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "member", "display_name": "Guttman, Mitchell" }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." } ], "option_major": [ "biology" ], "doi": "10.7907/tfb9-n887", "abstract": "RNA is a widely utilized and integrated component of core cellular function because of its abilities to recognize and hybridize to nucleic acid templates, spatially localize to different compartments within the cell, bind combinatorially to effector molecules, and in some cases directly catalyze chemical reactions. In this thesis, I describe three cases, illustrating the biomolecule\u2019s unique importance in several different aspects of cellular homeostasis. Chapter 1 provides historical context for studying RNA-protein interactions within RNA biology and Virology. Chapter 2 details experiments in which we explored RNA as a central target of host cell takeover by SARS-CoV-2. In the process, we highlight the importance of RNA in many integral complexes within the cell, including components of the spliceosome, the eukaryotic ribosome, and signal recognition particle. Chapter 3 presents data from our consideration of RNA within the context of cis gene regulation. We specifically focus on a model RNA-binding protein, SMRT/HDAC1 Associated Repressor Protein (SHARP), and the paternally imprinted long non-coding RNA, Kcnq1ot1, as case studies. Chapter 4 describes our dissection of a transcriptional circuit involving SHARP and discusses implications of RNA-binding to developmentally sensitive circuits and processes. Finally, Chapter 5 poses new questions raised by these studies. Together these data emphasize the diverse and unique role RNA plays in cellular homeostasis and suggest additional roles in nuclear compartment stabilization and crosstalk.
" }, { "name": "Chai, Cynthia Mei-Ling", "degree": "PhD", "year": "2021", "title": "Neurogenetic Analysis of C. elegans Developmental Decision-making", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06012021-063721816", "creators": [ { "name": { "family": "Chai", "given": "Cynthia Mei-Ling" }, "id": "Chai-Cynthia-Mei-Ling", "display_name": "Chai, Cynthia Mei-Ling" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "chair", "display_name": "Anderson, David J." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Parker", "given": "Joseph" }, "id": "Parker-J", "orcid": "0000-0001-9598-2454", "role": "member", "display_name": "Parker, Joseph" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "neurobio" ], "doi": "10.7907/r71f-nz93", "abstract": "How does the neuronal genome dictate cellular physiology and function, which in turn impacts organismal development? This is the central question driving all the projects in this thesis. Here, we leveraged the well-defined nervous system and genetics of Caenorhabditis elegans to apply this genes to neurons to phenotype experimental approach. In Chapter 2, we delineate how a pair of first order amphid interneurons integrates conspecific cues and propagates this information via neuropeptidergic pathways to influence larval developmental fate. In Chapter 3, we identify and characterize a role for the evolutionarily-conserved forkhead transcription factor FKH-7/FOXP in regulating sensory neuron function during developmental decision-making. Our results show that perturbations of single genes encoding transcription factors, neuropeptides, and receptors can significantly alter neuronal function and ultimately have profound effects on an organism\u2019s life history.
" }, { "name": "Chiu, Hui", "degree": "PhD", "year": "2021", "title": "Neural Control of Male and Female Aggression in Drosophila", "advisor": "Anderson, David J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05222021-190729800", "creators": [ { "name": { "family": "Chiu", "given": "Hui" }, "id": "Chiu-Hui", "orcid": "0000-0002-1820-8411", "display_name": "Chiu, Hui" } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "advisor", "display_name": "Anderson, David J." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Hong", "given": "Elizabeth J." }, "id": "Hong-Elizabeth-J", "role": "member", "display_name": "Hong, Elizabeth J." }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "member", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "orcid": "0000-0003-2136-6506", "role": "member", "display_name": "Meister, Markus" }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." } ], "option_major": [ "biology" ], "doi": "10.7907/9rf6-5727", "abstract": "Aggression is essential for an individual\u2019s survival, but it can also lead to unfavorable consequences when misregulated. It is thus important to study the neural basis of this behavior not only for learning how the nervous system is constructed to generate an innate behavior but also for finding the causality of misregulation. Although many circuit and molecular mechanisms underlying aggression have been revealed, our knowledge is mostly restricted to males. Given that sexual differences in aggression are seen in most if not all species, the mechanisms that we learned in one sex may not be directly applied to the other. Therefore, studying the neural basis of aggression in both sexes is necessary for gaining a full understanding of this behavior. Drosophila serves as a unique model for such studies because males and females differ not only in the level of aggressiveness but also in the motor patterns. Interestingly, the aggression-promoting neurons that have been identified so far are mostly sex-specific, raising the possibility that males and females adopt distinct circuits for controlling aggression. However, many sexually shared features of aggression also imply the existence of common circuit elements. My thesis work investigated whether any aggression circuit modules are shared by the two sexes and how the circuit is organized to generate sexually shared and dimorphic motor patterns. Through a behavioral screen and the genetic intersection approach, we identified a pair of sexually shared neurons, CAP, that regulates aggressive approach in both sexes, as well as a pair of male-specific neurons, MAP, whose activation promotes the transition from approach to male-specific attack. We subsequently identified the female homologue, fpC1 neurons, whose activation induces female aggression. Supported by the in vivo imaging and the behavioral epistasis results, we confirmed the functional connectivity between CAP and MAP/fpC1 in males and females, respectively. Lastly, we showed that the connectivity between CAP and MAP/fpC1 is strengthened in socially isolated flies, which exemplifies how circuits can be modified by social isolation to enhance aggression in both sexes. The connectivity between CAP and MAP/fpC1 provides a circuit logic for the control of sexually shared and dimorphic aggressive behaviors. It can be used as an entry point for circuit mapping as well as for further investigation of mechanisms underlying sexual differences in aggression.
" }, { "name": "Chong, Lucy Shin", "degree": "PhD", "year": "2021", "title": "Engineering and Delivery of Programmable Protein Circuits as Potential Therapeutic Devices", "advisor": "Elowitz, Michael B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06072021-211522720", "creators": [ { "name": { "family": "Chong", "given": "Lucy Shin" }, "id": "Chong-Lucy-Shin", "orcid": "0000-0002-5858-9984", "display_name": "Chong, Lucy Shin" } ], "advisors": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "advisor", "display_name": "Elowitz, Michael B." } ], "committee": [ { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "chair", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "member", "display_name": "Shapiro, Mikhail G." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." } ], "option_major": [ "sysbiol" ], "doi": "10.7907/jdhe-by95", "abstract": "Cell-specific targeting of therapeutics is a fundamental challenge in biomedicine. The use of engineered proteins that interact with one another as designed, synthetic circuits represents a promising solution to this challenge. These circuits can be constructed to directly sense endogenous cell signals, act on these signals to classify cellular state, and produce a specific response such as conditional triggering of cell death or targeted expression of a reporter. Synthetic protein circuits can also be delivered in mRNA vectors transiently to avoid permanent gene modification.
\r\n\r\nWe recently showed viral proteases can be engineered to regulate one another in a composable manner, permitting the construction of diverse protein-level circuits (Circuits of Hacked Orthogonal Modular Proteases). CHOMP could perform a wide range of computations including Boolean logic, analogue signal processing, and dynamic signal processing. Using this system we were also able to directly sense key cellular pathways and conditionally respond to trigger apoptosis in cancer-like cells. Further expansion of synthetic protein circuits to include nonlinear signal processing enables new system-level behaviors.
\r\n\r\nProtein-based circuits are compatible with innovative delivery methods including mRNA encapsulated in lipid-nanoparticle formulations and engineered viruses. As a proof of principle, we were able to develop a controllable, transient RNA-virus delivery system that allowed for targeted delivery to defined cell populations. This paradigm requires control over multiple aspects of the viral delivery system, including (1) production and release of viral particles, (2) target cell entry based on cell-surface proteins, (3) replication within the cell depending on intracellular proteins, and (4) drug-dependent elimination of the virus. Here, we integrate each of these distinct levels of control can into a single system based on the well-characterized negative stranded RNA virus. This RNA-virus platform will enable synthetic protein circuit delivery.
\r\n\r\nCombining viral engineering and protein circuit construction, the work described here suggests a roadmap towards \u201csmarter\u201d circuit-based therapies that can integrate multiple cues to maximize therapeutic specificity and establishes a role for post-translational circuits as future therapeutic devices.
" }, { "name": "Chour, William", "degree": "PhD", "year": "2021", "title": "Molecular Technologies for Antigen-Based Immunity", "advisor": "Heath, James R.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02192021-010538691", "creators": [ { "name": { "family": "Chour", "given": "William" }, "id": "Chour-William", "orcid": "0000-0003-1817-0123", "display_name": "Chour, William" } ], "advisors": [ { "name": { "family": "Heath", "given": "James R." }, "id": "Heath-J-R", "orcid": "0000-0001-5356-4385", "role": "advisor", "display_name": "Heath, James R." } ], "committee": [ { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "chair", "display_name": "Shapiro, Mikhail G." }, { "name": { "family": "Heath", "given": "James R." }, "id": "Heath-J-R", "orcid": "0000-0001-5356-4385", "role": "co-chair", "display_name": "Heath, James R." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Yang", "given": "Changhuei" }, "id": "Yang-Changhuei", "orcid": "0000-0001-8791-0354", "role": "member", "display_name": "Yang, Changhuei" }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" } ], "option_major": [ "bioeng" ], "doi": "10.7907/z20t-nq62", "abstract": "The presence and proliferation antigen-specific T cells is a defining characteristic of an adaptive immune response against various disease types (autoimmune, cancer, and infectious). The use of Class I and Class II peptide-major histocompatibility complex (pMHC) reagents to identify such cells, however, is technically difficult and expensive, and it has been challenging to refine synthesis protocols for higher yield and more efficient assembly to accommodate large-scale applications. This achievement would enable high-throughput capture of corresponding T cell receptors (TCR), which may be further used in clinical applications such as adoptive cell transfer therapies. Overcoming this hurdle requires the development and integration of various molecular technologies and analytical methods.
\r\n\r\nToward this end, the bulk of my thesis work, covered in Chapter 2, introduces these developments in the context of pMHCs, where the three subunits of each reagent are covalent linked together and expressed as a single protein. These single-chain trimer (SCT) technologies primarily consist of traditional DNA cloning and protein production techniques which have been streamlined for applications requiring output on the scale of 102-103 of reagents. This chapter serves as the foundation for much of the methodology discussed throughout the rest of my thesis, and thus should serve as a reference point. The generated constructs are also functionally validated here, and potential future research directions are outlined.
\r\n\r\nIn Chapter 3, I explore the use of this technology in the context of COVID-19 to enumerate antigen specificity of the CD8+ T cell immune response. Class I SCTs were constructed to present peptides across several SARS-CoV-2 protein domains, using various HLA alleles to match haplotyped participant blood samples. These reagents were then used to capture SARS-CoV-2-specific T cells through flow and nanoparticle cytometry to demonstrate HLA-dependent, domain-dependent immune responses. Identified TCRs were cloned into T cells for confirmation of antigen specificity and functional cytotoxicity.
\r\n\r\nIn Chapters 4 and 5, I explore potential pMHC applications in cancer antigen contexts, covering both tumor-associated and tumor-specific antigens. Through various collaborations across the west coast (UCLA, Parker Institute, Fred Hutchinson Cancer Research Center), I make use of the SCT platform to showcase new assays to discover and rank key tumor targets (Chapter 4). Finally, Chapter 5 is a reproduction of our lab\u2019s published work concerning identification of antigen-specific CD8+ T cells from melanoma cancer patients.
\r\n\r\nIn summary, the adaptation of SCTs in a high-throughput format allows for the rapid enumeration of antigen-specific T-cell receptor sequences. As demonstrated in the contexts of COVID-19 and cancer, this SCT platform enables subsequent downstream applications, such as single-cell, antigen-specific immunophenotypic mapping/analysis and target discovery for personalized immunotherapies.
" }, { "name": "Cohen, Alexander Armand", "degree": "PhD", "year": "2021", "title": "Developing Multivalent Nanoparticle Vaccines Against Current and Future Viruses", "advisor": "Bjorkman, Pamela J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06082021-015935441", "creators": [ { "name": { "family": "Cohen", "given": "Alexander Armand" }, "id": "Cohen-Alexander-Armand", "orcid": "0000-0002-2818-656X", "display_name": "Cohen, Alexander Armand" } ], "advisors": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "advisor", "display_name": "Bjorkman, Pamela J." } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "chair", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Clemons", "given": "William M." }, "id": "Clemons-W-M", "orcid": "0000-0002-0021-889X", "role": "member", "display_name": "Clemons, William M." }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "orcid": "0000-0003-2713-1513", "role": "member", "display_name": "Mazmanian, Sarkis K." }, { "name": { "family": "Scott", "given": "David W." }, "id": "Scott D-W", "orcid": "0000-0003-2708-4251", "role": "member", "display_name": "Scott, David W." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." } ], "option_major": [ "biochem" ], "doi": "10.7907/r33z-kj46", "abstract": "The 1918-1919 flu pandemic resulted in an estimated 50 to 100 million deaths worldwide, making it the deadliest pandemic in modern history. It was caused by a new influenza virus that likely spilled over from birds and reassorted with a human influenza virus. Since the human population was immunologically na\u00efve to this virus, transmission and lethality was much higher than for seasonal influenza outbreaks. Numerous pandemic influenza viruses emerged within the next century, with none causing the same amount of carnage. There is likely to be future influenza pandemics, with wild migratory birds being carriers of a wide swath of different influenza A viruses. Zoonotic transmission of Avian influenza has taken place with limited human to human transmission. There is evidence showing that the barrier of human transmissibility by some of these avian viruses is not very high, and therefore emergence into humans is possible, with most if not all of the population immunologically na\u00efve. The humoral immune response to influenza is defined by the imprinting of the antibody response to immunodominant epitopes. Such responses can impair immunity, providing less adequate protection against seasonal and pandemic infections, as well as poorer immunity induced by seasonal vaccines. There are instances where imprinting can be advantageous and even offer protection against pandemic or avian viruses, particularly when conserved epitopes to the HA stalk are exploited. Manipulating the antibody response to recognizing conserved stalk epitopes on influenza HA is therefore a strategy being used for universal influenza vaccines. In the second Chapter of this thesis, a mosaic nanoparticle immunization strategy for inducing breadth of antibody responses against HA will be described. This strategy involves the co-display of HAs from up to eight different strains on a particle platform. Although the breadth of antibody responses elicited by immunization of these particles was limited, this work provides insight into the antigenicity of such particles, and a possible alternative to current influenza vaccines.
\r\n \r\nApproximately 100 years after the 1918-1919 flu pandemic, a deadly SARS-like coronavirus, known as SARS-CoV-2, emerged in the human population resulting in a currently ongoing pandemic. This came less than two decades after the small but deadly SARS outbreak, essentially a warning call for this class of coronaviruses. Other SARS-like coronavirus strains in bats have been identified and shown to be human tropic, though resulting in an attenuated infection. Some of these viruses can infect via hACE2 but there are others that may use an unknown receptor for entry into VERO cells as well as human cell lines. There is evidence that the major barrier to zoonosis is protease compatibility, which could be gained through recombination events or errors during replication. Therefore, future SARS-like coronaviruses (sarbecovirus) may emerge in humans, seeding future outbreaks. The antibody response to SARS-CoV-2 is robust and protective. Furthermore, there is the presence of conserved epitopes particularly on the RBD that can be targeted by antibodies that are cross-neutralizing against many SARS-like coronaviruses. Exploiting these cross-reactive epitopes is one strategy that can be used for developing a universal coronavirus vaccine. In Chapter 3 of this thesis, a similar mosaic nanoparticle immunization strategy will be described, that attempts to elicit cross-reactive antibodies against the SARS-like coronavirus family. The mosaic nanoparticles co-display the RBDs of eight different sarbecovirus strains including SARS-CoV-2. Immunization with these mosaic-RBD nanoparticles elicited polyclonal antibody responses that were cross-reactive as well as cross-neutralizing against sarbecoviruses strains both present and not present on the particles.
" }, { "name": "Cohen, Sarah Michelle", "degree": "PhD", "year": "2021", "title": "Formation and Function of Ascarosides in the Nematodes C. elegans and C. briggsae", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05252021-185133192", "creators": [ { "name": { "family": "Cohen", "given": "Sarah Michelle" }, "id": "Cohen-Sarah-Michelle", "orcid": "0000-0003-4469-2670", "display_name": "Cohen, Sarah Michelle" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "chair", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "orcid": "0000-0002-7371-4675", "role": "member", "display_name": "Prober, David A." }, { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "orcid": "0000-0002-7033-0844", "role": "member", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/84ck-kn25", "abstract": "As an easily culturable, hermaphroditic, and short-lived species with a fully annotated genome and neural connectome, the nematode Caenorhabditis elegans is used as a model organism to study many different biological problems. Examining the communication systems among these worms is important not only to understand how they control and affect one another's behavior, but also gives us clues to the communications systems of closely related parasitic worms.
\r\n\r\nNematode worms use small-molecule signaling to send messages about their environments in order to influence behavioral decisions of other animals in their vicinity. A main type of pheromone signaling uses a group of stable small molecules, collectively called ascarosides, that are built modularly from common waste products in cells such as sugars, fatty acids, and amino acid derivatives. Ascarosides are synthesized by C. elegans in precise concentrations and combinations to produce finely-tuned messages which control major behaviors such as mating and entry into dauer, an alternative lifestage that allows worms to survive adverse conditions. We are still unraveling exactly how ascarosides are produced and how they affect behaviors in C. elegans and other worms species.
\r\n\r\nTo further understanding of the formation of ascarosides in C. elegans, I studied the O-acyltransferase gene class to see if they helped catalyze the 4' modifications of ascarosides, as predicted based on their chemistry. Surprisingly, oac genes were found to be uninvolved in the biosynthesis of ascarosides; but they do affect ascaroside production and secretion. To understand the underlying mechanisms of formation and function of ascarosides across worm species, I also studied ascarosides in the closely-related species Caenorhabditis briggsae. First, I developed an efficient CRISPR/Cas9 method for use in C. briggsae. From there, I was able to make the C. briggsae mutants Cbr-glo-1 and Cbr-daf-22, genes that we showed were one-to-one orthologs of their C. elegans counterparts. I then showed that ascr#2 was the main component of daumone in C. briggsae. Additionally, I found that there is an anti-dauer signal, hypothesized to be another type of small signaling molecule \u2013 a glucoside. These findings further our understanding of the formation and function of ascaroside signaling molecules in nematode worms.
" }, { "name": "Gandhi, Shashank", "degree": "PhD", "year": "2021", "title": "Molecular Mechanisms Underlying Cardiac Neural Crest Development in Avian Embryos", "advisor": "Bronner, Marianne E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06012021-203020365", "creators": [ { "name": { "family": "Gandhi", "given": "Shashank" }, "id": "Gandhi-Shashank", "orcid": "0000-0002-4081-4338", "display_name": "Gandhi, Shashank" } ], "advisors": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "advisor", "display_name": "Bronner, Marianne E." } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "chair", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Parker", "given": "Joseph" }, "id": "Parker-J", "orcid": "0000-0001-9598-2454", "role": "member", "display_name": "Parker, Joseph" }, { "name": { "family": "Zernicka-Goetz", "given": "Magdalena" }, "id": "Zernicka-Goetz-M", "orcid": "0000-0002-7004-2471", "role": "member", "display_name": "Zernicka-Goetz, Magdalena" }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "member", "display_name": "Bronner, Marianne E." } ], "option_major": [ "biodev" ], "doi": "10.7907/y1e4-d090", "abstract": "The neural crest is a multipotent, vertebrate-specific stem cell population that gives rise to diverse cell types in the developing embryo, including craniofacial cartilage, enteric ganglia, and cardiac septa. Neural crest cells that originate from a given axial level in the embryo give rise to a characteristic array of progeny and follow distinct pathways from those arising at other levels. One of these subpopulations, called the cardiac neural crest, originates in the dorsal hindbrain and migrates into the developing heart, where it forms the aorticopulmonary septum, cardiac ganglion, and part of the interventricular septum. Mutations in or loss of these cells causes heart defects that are among the most common birth defects in the general population. For my thesis, I sought to identify the mechanisms that underlie the formation of neural crest cells, and confer cardiac neural crest cells with their unique developmental potential.
\r\n\r\nTo enable interrogation of epistatic relationships between key neural crest genes during neural crest induction and crest specification, I first optimized the CRISPR-Cas9 system for genome editing in gastrula and neurula-stage chicken embryos. I then further improved the CRISPR toolbox by devising an all-in-one single-plasmid strategy that harnesses the self-cleavage properties of ribozymes for the simultaneous delivery of Cas9, gRNAs, and fluorescent reporters in transfected cells. This has enabled live tracking of wildtype and mutant neural crest cells as they migrate to their terminal locations.
\r\n\r\nPrior to their induction at the neural plate border, precursors in the neural plate border are transcriptionally primed toward multiple cell fates, including neural tube, neural crest, epidermis, and placode. While this priming has been thought to involve epigenetic regulation, chromatin remodeler genes have been overlooked in the context of neural crest formation given their concomitant expression in surrounding cell types. By combining single-cell transcriptional profiling of the early chick embryonic hindbrain with temporally-controlled knockouts, I uncovered a novel bimodal mechanism whereby the chromatin remodeler gene Hmga1 first regulates Pax7-dependent neural crest induction at the neural plate border, and later modulates Wnt signaling in the dorsal neural tube to control neural crest delamination. These results established Hmga1 as a direct regulator of neural crest induction and emigration.
\r\n\r\nFinally, given that amongst distinct neural crest subpopulations designated as cranial, cardiac/vagal, and trunk, only cardiac crest has the ability to contribute to heart development, and that neither trunk nor cranial neural crest subpopulations can rescue the loss of cardiac crest, I investigated the genetic logic that imbues cardiac crest with its unique ability to form cardiovascular derivatives. To this end, I combined surgical ablations, bulk and single-cell transcriptional profiling, RNA labeling, CRISPR-Cas9-mediated gene editing, transcription factor binding motif mutation analysis, and transgenic tissue grafting approaches to uncover and characterize a cardiac-neural-crest-specific subcircuit comprised of the transcription factors Sox8, Tgif1, and Ets1. I demonstrated that ectopic expression of this subcircuit in trunk neural crest cells reprogrammed them towards a cardiac-crest-like fate, and transplanting these reprogrammed cells in place of ablated cardiac crest restored cardiac-crest-like migration patterns and rescued outflow tract septation defects.
\r\n\r\nTaken together, my thesis work has not only built a genome engineering toolbox for a key model system in developmental biology, but has also expanded our understanding of the genetic circuits that govern the formation of the cardiac neural crest and underlie its unique ability to contribute to the heart.
" }, { "name": "Huang, Xiawei", "degree": "PhD", "year": "2021", "title": "Molecular Function and Regulation of Aub Arginine Methylation in the piRNA Pathway", "advisor": "Aravin, Alexei A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05192021-055633048", "creators": [ { "name": { "family": "Huang", "given": "Xiawei" }, "id": "Huang-Xiawei", "orcid": "0000-0001-9084-0510", "display_name": "Huang, Xiawei" } ], "advisors": [ { "name": { "family": "Aravin", "given": "Alexei A." }, "id": "Aravin-A-A", "orcid": "0000-0002-6956-8257", "role": "advisor", "display_name": "Aravin, Alexei A." } ], "committee": [ { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "chair", "display_name": "Chan, David C." }, { "name": { "family": "Shan", "given": "Shu-ou" }, "id": "Shan-Shu-ou", "orcid": "0000-0002-6526-1733", "role": "member", "display_name": "Shan, Shu-ou" }, { "name": { "family": "Voorhees", "given": "Rebecca M." }, "id": "Voorhees-R-M", "orcid": "0000-0003-1640-2293", "role": "member", "display_name": "Voorhees, Rebecca M." }, { "name": { "family": "Aravin", "given": "Alexei A." }, "id": "Aravin-A-A", "orcid": "0000-0002-6956-8257", "role": "member", "display_name": "Aravin, Alexei A." } ], "option_major": [ "biology" ], "doi": "10.7907/5734-db78", "abstract": "Transposon elements (TEs, Transposons) are DNA sequences that can change their position within the genome. TEs, so-called 'jump genes', sometimes create mutation which will disrupt genes or damage the genome integrity by causing double-stranded DNA breaks and germ cell death. It is important for living animals to maintain the integrity of genetic information during reproduction. In Metazoa germline, cells use the piwi-interacting RNA (piRNA) pathway, which is an RNA \u2013 interference (RNAi) based defense strategy to protects the genome from the attacking of the \"selfish\" transposons. The core unit of the piRNA pathway is the RNA-induced silencing complex (RISC), a conserved family of Argonaute protein that interacts with small (19\u201333 nt) RNA guides in eukaryotic species. In Drosophila melanogaster, three PIWI-clade Argonaute proteins are present in the germline \u2013 Aubergine (Aub), Argonaute 3 (Ago3) and Piwi. PIWI proteins together with their substrate piRNAs forming the RISC to suppress the TE activity. Arginine (Arg) methylation is an important post-translational modification among Argonaute proteins. Defects of Arginine methylation cause the de-repression of deleterious TE.
\r\n\r\nThe work present in this thesis examines the molecular function and regulation mechanism of Aub Arginine methylation in Drosophila germline cells. Chapter I presents a general introduction to the TEs, key components of the piRNA pathway, potential piRNA processing site, \"nuage\", and the correlation between arginine methylation and its interaction partner, Tudor domain-containing proteins. Chapter II presents the piRNA biogenesis in Drosophila germline and somatic cells, dividing the piRNA pathway into cytoplasmic and nuclear branches. We describe the mechanism of piRNA 5' end and 3' end formations. Chapter III explores the specific molecular function of Aub arginine methylation in the piRNA ping-pong cycle. Further, we decipher the regulation mechanism of Aub Arginine methylation, addressing its biological meaning for the piRNA biogenesis. In chapter 4, we developed a heterologous two-hybrid system to identify factors that directly interact with Piwi, which can further be applied to elucidate the interaction network of the piRNA pathway. In chapter 5, we discuss the potential role of phase separation in the assembly of ping-pong processing granule and the biological meaning in the piRNA biogenesis. We also propose the future plan and the protocol to examine the hypothesis in the future.
" }, { "name": "Irizarry, Jihyun", "degree": "PhD", "year": "2021", "title": "Temporally Changing Roles of Morphogen Dorsal in the Drosophila Early Embryo", "advisor": "Stathopoulos, Angelike", "url": "https://resolver.caltech.edu/CaltechTHESIS:09052020-090207113", "creators": [ { "name": { "family": "Irizarry", "given": "Jihyun" }, "id": "Irizarry-Jihyun", "orcid": "0000-0003-3967-2288", "display_name": "Irizarry, Jihyun" } ], "advisors": [ { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "orcid": "0000-0001-6597-2036", "role": "advisor", "display_name": "Stathopoulos, Angelike" } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "member", "display_name": "Guttman, Mitchell" }, { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "orcid": "0000-0001-6597-2036", "role": "member", "display_name": "Stathopoulos, Angelike" } ], "option_major": [ "biology" ], "doi": "10.7907/t53b-vc87", "abstract": "Morphogen gradients provide positional cues during development, with cell fate specification proceeding in a morphogen concentration-dependent manner during patterning. However, morphogens also are dynamic as their concentrations change not only in space but also in time, but how these dynamics are translated into cell fate specification over time is not well understood. To provide a better understanding of morphogens\u2019 temporal roles, we studied how Drosophila dorsal-ventral body patterning is controlled by the dynamic morphogen Dorsal (Dl). Dl is present in a nuclear-cytoplasmic gradient along the dorsal-ventral (DV) axis, but Dl levels also continuously increase between and within nuclear cell divisions associated with the early syncytial embryo. To experimentally manipulate Dl levels in time in order to determine whether these dynamics are important, we developed a light-activated degradation system. The blue light inducible degron domain, BLID, was fused to the C-terminus of Dl by genomic editing using CRISPR-Cas9. To assay effects on temporally manipulated Dl levels, we combined this light-inducible degradation system with the MS2-MCP.GFP nascent transcript imaging system, and used to monitor transcription changes in vivo at the snail (sna) locus, a gene requiring high Dl levels. We found that while high Dl levels are required for sna activation at early nuclear cycle 14, late expression can be supported even if Dl levels are extinguished. Twist, an early Dl target gene, is later auto-activating and can support the later sna expression without Dl. Surprisingly, we found that peak levels of Dl, present at late nuclear cycle 14, are required only to fine tune, in particular to decrease, sna levels. This differential action of Dl, first functioning as an activator and next as a damper of expression, is manifest by the coordinate action of two enhancers acting at the sna locus. Here, we highlight how morphogen roles change in time, and suggest that this may be a general characteristic of dynamic morphogens that allows them to control developmental patterning.
" }, { "name": "Knight, Anders Matthew", "degree": "PhD", "year": "2021", "title": "Expanding the Scope of Metalloprotein Families and Substrate Classes in New-to-Nature Reactions", "advisor": "Arnold, Frances Hamilton", "url": "https://resolver.caltech.edu/CaltechTHESIS:06212020-111155395", "creators": [ { "name": { "family": "Knight", "given": "Anders Matthew" }, "id": "Knight-Anders-Matthew", "orcid": "0000-0001-9665-8197", "display_name": "Knight, Anders Matthew" } ], "advisors": [ { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "advisor", "display_name": "Arnold, Frances Hamilton" } ], "committee": [ { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "chair", "display_name": "Shapiro, Mikhail G." }, { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "member", "display_name": "Arnold, Frances Hamilton" }, { "name": { "family": "Clemons", "given": "William M." }, "id": "Clemons-W-M", "orcid": "0000-0002-0021-889X", "role": "member", "display_name": "Clemons, William M." }, { "name": { "family": "Reisman", "given": "Sarah E." }, "id": "Reisman-S-E", "orcid": "0000-0001-8244-9300", "role": "member", "display_name": "Reisman, Sarah E." }, { "name": { "family": "Bois", "given": "Justin S." }, "id": "Bois-J-S", "orcid": "0000-0001-7137-8746", "role": "member", "display_name": "Bois, Justin S." } ], "option_major": [ "bioeng" ], "doi": "10.7907/7qh5-5130", "abstract": "Heme proteins, in particular cytochromes P450, have been extensively used in biocatalytic applications due to their high degree of regio-, chemo-, and stereoselectivity in oxene-transfer reactions. In 2013, it was shown for the first time that engineered heme proteins can also catalyze analogous carbene- and nitrene-transfer reactions. Research in this field has since grown dramatically, with emphasis on developing new heme protein variants to increase the scope of biotransformations accessible through these new transfer reactions. This thesis details the expansion of these new-to-nature carbene and nitrene-transfer reactions to include new substrate classes previously unexplored with iron-porphyrin proteins, the use of non-heme metalloproteins for these transformations, and steps toward improving the robustness of the new-to-nature biocatalytic platform. Chapter 1 introduces the steps the field of biocatalysis has taken toward engineering enzymes with new catalytic functions and the process by which these activities are discovered and enhanced. Chapter 2 details the discovery and engineering of heme proteins which catalyze the stereodivergent cyclopropanation of unactivated and electron-deficient alkenes via carbene transfer, expanding the substrate classes beyond styrenyl alkenes. Chapter 3 shows the development of engineered variants of a heme protein (Rhodothermus marinus nitric oxide dioxygenase) for the diastereodivergent synthesis of cyclopropanes functionalized with a pinacolborane moiety, enabling product diversification through standard cross-coupling reactions. In Chapter 4, a collection of non-heme metalloproteins is curated, and a non-heme iron enzyme (Pseudomonas savastanoi ethylene-forming enzyme) is shown to be both amenable to directed evolution and non-native ligand substitution to enhance its nitrene-transfer activity. Chapter 5 describes the expansion of sequence space targeted for screening in the serine-ligated cytochrome P411 from Bacillus megaterium (P411BM3) biocatalytic platform to enhance the mutational robustness of these remarkable enzymes. Overall, this work provides a framework for bringing model new-to-nature reactions to their full potential in synthetic biocatalytic reactions.
" }, { "name": "Marino, Joseph Louis", "degree": "PhD", "year": "2021", "title": "Learned Feedback & Feedforward Perception & Control", "advisor": "Yue, Yisong; Perona, Pietro", "url": "https://resolver.caltech.edu/CaltechTHESIS:05272021-042158260", "creators": [ { "name": { "family": "Marino", "given": "Joseph Louis" }, "id": "Marino-Joseph-Louis", "orcid": "0000-0001-6387-8062", "display_name": "Marino, Joseph Louis" } ], "advisors": [ { "name": { "family": "Yue", "given": "Yisong" }, "id": "Yue-Yisong", "orcid": "0000-0001-9127-1989", "role": "co-advisor", "display_name": "Yue, Yisong" }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "co-advisor", "display_name": "Perona, Pietro" } ], "committee": [ { "name": { "family": "Yue", "given": "Yisong" }, "id": "Yue-Yisong", "orcid": "0000-0001-9127-1989", "role": "member", "display_name": "Yue, Yisong" }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "member", "display_name": "Perona, Pietro" }, { "name": { "family": "O'Doherty", "given": "John P." }, "id": "O'Doherty-J-P", "orcid": "0000-0003-0016-3531", "role": "member", "display_name": "O'Doherty, John P." }, { "name": { "family": "Tsao", "given": "Doris Y." }, "id": "Tsao-D-Y", "orcid": "0000-0003-1083-1919", "role": "chair", "display_name": "Tsao, Doris Y." }, { "name": { "family": "Rao", "given": "Rajesh P. N." }, "id": "Rao-Rajesh-P-N", "role": "member", "display_name": "Rao, Rajesh P. N." } ], "option_major": [ "cns" ], "doi": "10.7907/4mjd-ce53", "abstract": "The notions of feedback and feedforward information processing gained prominence under cybernetics, an early movement at the dawn of computer science and theoretical neuroscience. Negative feedback processing corrects errors, whereas feedforward processing makes predictions, thereby preemptively reducing errors. A key insight of cybernetics was that such processes can be applied to both perception, or state estimation, and control, or action selection. The remnants of this insight are found in many modern areas, including predictive coding in neuroscience and deep latent variable models in machine learning. This thesis draws on feedback and feedforward ideas developed within predictive coding, adapting them to improve machine learning techniques for perception (Part II) and control (Part III). Upon establishing these conceptual connections, in Part IV, we traverse this bridge, from machine learning back to neuroscience, arriving at new perspectives on the correspondences between these fields.
" }, { "name": "McCardell, Reed Dillard", "degree": "PhD", "year": "2021", "title": "Genetic Circuits for the Control of Multi-Strain Bacterial Populations", "advisor": "Murray, Richard M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05082021-185615529", "creators": [ { "name": { "family": "McCardell", "given": "Reed Dillard" }, "id": "McCardell-Reed-Dillard", "orcid": "0000-0002-0955-3133", "display_name": "McCardell, Reed Dillard" } ], "advisors": [ { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "advisor", "display_name": "Murray, Richard M." } ], "committee": [ { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "chair", "display_name": "Pierce, Niles A." }, { "name": { "family": "Orphan", "given": "Victoria J." }, "id": "Orphan-V-J", "orcid": "0000-0002-5374-6178", "role": "member", "display_name": "Orphan, Victoria J." }, { "name": { "family": "Ismagilov", "given": "Rustem F." }, "id": "Ismagilov-R-F", "orcid": "0000-0002-3680-4399", "role": "member", "display_name": "Ismagilov, Rustem F." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." } ], "option_major": [ "bioeng" ], "doi": "10.7907/wgpp-vj97", "abstract": "Microbial species rarely exist alone. Nearly everywhere you could think to look, microorganisms of various species live together in harmony. Microbes together in their communities are incredibly powerful actors wherever they are found; they perform small miracles---the conversion of milk into yogurt---and large ones---production of most of the planet's oxygen and organic carbon.
\r\n\r\nOur burgeoning knowledge of microbial life combined with modern technologies to manipulate it create a critical, exciting opportunity to harness microbial power for the betterment of technology, people, and the planet. This thesis presents a body of work which explores the manipulation of microbial communities using the intersectional bio-engineering approach of synthetic biology. We demonstrate how molecular tools evolved by bacteria can be repurposed to create rationally designed systems for controlling features of bacterial populations.
\r\n\r\nWe begin by examining a genetic circuit that caps the size of a bacterial population by coordinating the deaths of population members -- the population capping or \"pop cap\" circuit. Briefly, E. coli cells in the pop cap circuit are engineered to synthesize a chemical -- a quorum sensing (QS) signal -- that reports the density of the population, sense this chemical, and produce the ccdB toxin to destroy themselves in response. The molecular tools that make up this circuit are drawn from organisms across the spectrum of bacterial diversity. Brought together, they create a feedback control circuit that controls population size by causing member cells to die when a target population size has been reached. To improve the performance of this population controller and reduce the influence of the environment on the circuit, we add the aiiA quorum sensing signal degradase to allow the experimenter control over the degradation rate of the QS density signal. Additionally, we explore RNA and protein mechanisms to sequester the death-causing toxin---inactivating it---allowing us to release a population cap. The resulting \"cap and release\" circuit is a flexible motif that can be scaled to control multi-strain populations, expanding the scope of control beyond the single-strain populations regulated by the base pop cap circuit.
\r\n\r\nUsing the scalable cap and release motif, we design a genetic circuit to regulate a multi-strain community. Two different cell strains expressing symmetric, interconnected cap and release systems form the \"A=B\" circuit, so named for its ability to control the composition of the community to a target ratio of A cells to B cells, or Apopulation = \u03b1Bpopulation. Through dynamical system models of the system, we explore the effects of active QS signal degradation on composition control performance and perform a parameter sensitivity analysis of the system to help determine the best method for building a functioning A=B system in the laboratory. We use a high throughput construction and screening protocol to create variants of the A=B system with identical architectures, but slightly differing component production rates. We crown the most successful variant with a series of experiments to determine if it indeed recapitulates our model's predictions for its performance. Our implementation of the A=B circuit can successfully regulate the composition of a community, with interesting additional effects on total population density.
\r\n\r\nThe cap and release and A=B circuits need parts that can do three things: 1) send a signal between cells to communicate information, 2) compare two signals, 3) regulate cell growth or death. We highlight bacteriocins, bacterial protein exotoxins that are released from a producer cell to kill other cells of similar species, as attractive tools for bacterial community engineering both for their multi-functionality and modular protein structure. By themselves, bacteriocins can perform all the functions needed for population control: they transmit themselves between cells, have unique high-affinity sequestering antitoxin proteins, and are toxins to receiver cells. We begin the process of their characterization and usage as synthetic biological \"parts\" by creating non-native expression systems that match native expression strengths. Using these experimenter-controlled systems we design preliminarily test a bacteriocin-based bacterial community control circuit. Additionally, given the E. coli colicin bacteriocins' unique, nearly plug-and-play modular domain structure, we explore possibilities for engineering colicin proteins themselves for increased functional diversity or uses outside of growth regulation.
" }, { "name": "Parkin, James Michael", "degree": "PhD", "year": "2021", "title": "Signal Amplification in Synthetic Bacterial Communication", "advisor": "Murray, Richard M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03262021-160841703", "creators": [ { "name": { "family": "Parkin", "given": "James Michael" }, "id": "Parkin-James-Michael", "orcid": "0000-0002-4058-2338", "display_name": "Parkin, James Michael" } ], "advisors": [ { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "advisor", "display_name": "Murray, Richard M." } ], "committee": [ { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "orcid": "0000-0002-5899-7523", "role": "chair", "display_name": "Winfree, Erik" }, { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "orcid": "0000-0002-7033-0844", "role": "member", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Bois", "given": "Justin S." }, "id": "Bois-J-S", "orcid": "0000-0001-7137-8746", "role": "member", "display_name": "Bois, Justin S." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." } ], "option_major": [ "bioeng" ], "doi": "10.7907/50p8-bd89", "abstract": "Synthetic biology will one day enable embedded control of a variety of chemical and biological contexts, from the human gastrointestinal tract to crop roots. Groups of engineered organisms, also known as synthetic consortia, can inhabit niches of interest while monitoring and intervening according to their genetic design. However, the spatial structure of the deployment environments can obstruct coordination between cosortia members. The mechanisms engineered bacteria use to communicate must contend with these adversarial conditions to maximize group performance.
\r\n\r\nCoordination between synthetic bacteria is typically achieved using small molecules that can traverse cell membranes through passive transport. Cell communicate by producing and sensing these small molecules. In cell-cell signaling relationships composed of a sender population and a receiver population, the concentration of signaling molecule sensed by the receiver cells depends on the spatial patterning of the two groups, the geometry of the diffusive environment, and the sender population\u2019s signal secretion rate.
\r\n\r\nTo make sender-receiver communication more robust to these environmental features, we introduce a third consortium strain that transiently amplifies local signaling molecule concentrations. These amplifier cells employ a synchronized pulse-generating circuit built using Lux-type quorum sensing components and an IFFL transcriptional architecture. When applied to sender-receiver consortia growing on semi-solid media, these amplifier cells respond to sender-secreted signaling molecules by contributing a small amount themselves. The support of amplifier cells enables communication over longer distances than can be achieved by sender cells alone and can partially recover coordination in small consortia where the sender population is too small to successfully signal its receiver population alone. We extend these results using simulation to investigate the benefit that amplifier cells confer to consortia of varying complexity.
" }, { "name": "Perry, Elena Kim", "degree": "PhD", "year": "2021", "title": "Mechanisms and Consequences of Bacterial Resistance to Natural Antibiotics", "advisor": "Newman, Dianne K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04152021-173245433", "creators": [ { "name": { "family": "Perry", "given": "Elena Kim" }, "id": "Perry-Elena-Kim", "orcid": "0000-0002-7151-1479", "display_name": "Perry, Elena Kim" } ], "advisors": [ { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "advisor", "display_name": "Newman, Dianne K." } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Ismagilov", "given": "Rustem F." }, "id": "Ismagilov-R-F", "orcid": "0000-0002-3680-4399", "role": "member", "display_name": "Ismagilov, Rustem F." }, { "name": { "family": "Parker", "given": "Joseph" }, "id": "Parker-J", "orcid": "0000-0001-9598-2454", "role": "member", "display_name": "Parker, Joseph" }, { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "orcid": "0000-0002-7033-0844", "role": "member", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." } ], "option_major": [ "biology" ], "doi": "10.7907/tv8n-kr43", "abstract": "Many bacteria secrete natural antibiotics\u2014toxic small molecules that can kill or inhibit the growth of other microorganisms. Several of these compounds have been commercialized as antimicrobial drugs, and the mechanisms and public health consequences of bacterial resistance to clinically-used antibiotics are well understood. By contrast, the role of bacterially-produced antibiotics in natural environments, where they have existed for millions of years, remains an open question. Besides potentially serving as tools of warfare between competing microbes, natural antibiotics have been proposed to serve less antagonistic functions ranging from the acquisition of nutrients to the transmission of signals between cells. Indeed, despite evidence that natural antibiotics can suppress sensitive microbes in environments such as the soil surrounding plant roots, the ecological significance of the toxicity of these molecules has sometimes been questioned. At the same time, for most natural antibiotics, the mechanisms and prevalence of resistance are either poorly characterized or entirely unknown.
\r\n\r\nThis thesis addresses the molecular mechanisms and consequences of bacterial resistance to a particular class of redox-active natural antibiotics called phenazines. Phenazines are produced by a major opportunistic human pathogen, Pseudomonas aeruginosa, during infections, as well as by several bacterial species that associate with the roots of crops such as wheat, where they serve to protect their plant hosts against fungal pathogens. Resistance to this family of natural antibiotics is therefore potentially relevant to multiple sectors of human society. I begin by investigating the intrinsic phenazine resistance of a common soil bacterium, Agrobacterium tumefaciens, that does not itself produce phenazines. Using a functional genetics approach, I find that the composition of the respiratory electron transport chain plays a critical role in mitigating phenazine toxicity, one that cannot be compensated by increased expression of efflux pumps that transport phenazines out of the cell or oxidative stress responses that neutralize the toxic byproducts of phenazine redox-cycling. Subsequently, we turn to P. aeruginosa, the phenazine-producing opportunistic pathogen, and demonstrate that the defenses it activates against its own toxic phenazine, pyocyanin, collaterally accelerate the acquisition of resistance to certain clinical antibiotics. Other bacteria known to form multispecies infections with P. aeruginosa can also benefit from exposure to pyocyanin in the presence of these clinical antibiotics; we show that in at least one strain isolated from a patient, the effect of pyocyanin on the frequency of spontaneous antibiotic-resistant mutants rivals that of disruptions in DNA repair machinery. Importantly, a growing body of reports suggests that, besides pyocyanin, other metabolites produced by bacterial pathogens can also affect the efficacy of clinical antibiotics. We review the evidence for which types of bacterial metabolites alter susceptibility to antimicrobial drugs, as well as the mechanisms underlying this phenomenon. Finally, I examine the prevalence of bacterial resistance to an agriculturally-relevant phenazine in a wheat field where the use of native phenazine producers to control crop diseases has been studied for decades. I discover that while Gram-positive bacteria are generally more susceptible to this phenazine compared to Gram-negative bacteria, the sharpness of this distinction is pH-dependent; moreover, I uncover surprising heterogeneity in phenazine resistance within certain taxonomic groups. Taken together, these findings illuminate recurring themes in mechanisms of phenazine resistance and point to an underappreciated role for natural antibiotics in the resilience of opportunistic pathogens to clinical antibiotics. This thesis also lays the groundwork for developing a predictive model of phenazine resistance across diverse bacteria, with potential implications for optimizing the use of clinical antibiotics and improving agricultural sustainability.
" }, { "name": "Quintero Cadena, Porfirio", "degree": "PhD", "year": "2021", "title": "Mechanism and Scaling of Eukaryotic Transcription Activation", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:07072020-154545363", "creators": [ { "name": { "family": "Quintero Cadena", "given": "Porfirio" }, "id": "Quintero-Cadena-Porfirio", "orcid": "0000-0003-0067-5844", "display_name": "Quintero Cadena, Porfirio" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "chair", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "member", "display_name": "Guttman, Mitchell" }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "sysbiol" ], "doi": "10.7907/m21w-8461", "abstract": "Transcription activation is a universal process by which living cells adapt. Decades of work in this field have produced an intelligible paradigm of transcription activation that provides fundamental insights into its underlying molecular mechanisms. This thesis attempts to extend such paradigm to explain how transcription activation can be implemented across the diversity of molecular environments found in eukaryotic nuclei. Specifically, this diversity calls for an explanation of how this process scales throughout a range of genome sizes that spans five orders of magnitude, and of how to think about this subject in the increasingly relevant context of liquid-liquid phase-separation. We leverage data from RNA-seq, smFISH, growth-rate, fluorescence microscopy, computer simulations and literature to identify an appropriate and useful level of abstraction in which to grow our current paradigm. We propose scaling and phase-separation, two seemingly disparate aspects of transcription, are explained and intrinsically linked by a novel molecular state in which multiple RNA polymerases can bind the transcription complex. We provide support and rationale for this addition to the transcription model, and generate testable hypotheses that may further clarify the mechanism and evolution of eukaryotic transcription activation.
" }, { "name": "Ross, Tyler David", "degree": "PhD", "year": "2021", "title": "Guiding Self-Organization in Active Matter with Spatiotemporal Boundary Conditions", "advisor": "Thomson, Matthew", "url": "https://resolver.caltech.edu/CaltechTHESIS:07082020-113341068", "creators": [ { "name": { "family": "Ross", "given": "Tyler David" }, "id": "Ross-Tyler-David", "orcid": "0000-0002-7872-3992", "display_name": "Ross, Tyler David" } ], "advisors": [ { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "advisor", "display_name": "Thomson, Matthew" } ], "committee": [ { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "orcid": "0000-0002-5899-7523", "role": "chair", "display_name": "Winfree, Erik" }, { "name": { "family": "Rothemund", "given": "Paul W. K." }, "id": "Rothemund-P-W-K", "orcid": "0000-0002-1653-3202", "role": "member", "display_name": "Rothemund, Paul W. K." }, { "name": { "family": "Qian", "given": "Lulu" }, "id": "Qian-Lulu", "orcid": "0000-0003-4115-2409", "role": "member", "display_name": "Qian, Lulu" }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." }, { "name": { "family": "Brady", "given": "John F." }, "id": "Brady-J-F", "orcid": "0000-0001-5817-9128", "role": "member", "display_name": "Brady, John F." }, { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "member", "display_name": "Shapiro, Mikhail G." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" } ], "option_major": [ "biology" ], "doi": "10.7907/q85h-j730", "abstract": "In this thesis, I demonstrate that self-organized structures and forces can be guided by modulating the interactions between force-generating molecules in space and time. The physics of self-organizing systems is an open frontier. We do not have a complete set of principles that can describe how a dynamic structure forms based on the non-equilibrium dynamics of its constituent components. Yet, living systems appear to depend on some set of rules of self-organization in order to reliably carry out their mechanical functions. Force-generating, active, molecules in the form of motor proteins and filamentous polymers are responsible for performing fundamental tasks in living matter, such as locomotion and division. While it is known that the regulation of motor-filament interactions is necessary to achieve the dynamic structures that drive movement and propagation, the role of spatial and temporal patterning in self-organizing systems has not been explored. I design a artificial system of purified molecules where the interactions between motors and filaments are toggled with light. By patterning molecular interactions in space and time, I show that it is possible to localize the formation of spherically symmetric asters, which can be moved, merged, and used to generate advective fluid flows. The ability to pattern molecular interactions in space and time offers a new perspective in the search for principles of active self-organization. Spatial and temporal control makes it possible to start distilling how the interactions between active molecules determine the mesoscopic behaviors of self-organized structures. These rules ultimately govern the physics of living matter and may eventually be harnessed to build new materials and cell-like machines.
" }, { "name": "Sawyer, Daniel Patrick", "degree": "PhD", "year": "2021", "title": "Enhanced Noninvasive Imaging of Acoustic Biomolecules", "advisor": "Shapiro, Mikhail G.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06022021-043318684", "creators": [ { "name": { "family": "Sawyer", "given": "Daniel Patrick" }, "id": "Sawyer-Daniel-Patrick", "orcid": "0000-0003-2926-191X", "display_name": "Sawyer, Daniel Patrick" } ], "advisors": [ { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "advisor", "display_name": "Shapiro, Mikhail G." } ], "committee": [ { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "chair", "display_name": "Phillips, Robert B." }, { "name": { "family": "Roukes", "given": "Michael Lee" }, "id": "Roukes-M-L", "orcid": "0000-0002-2916-6026", "role": "member", "display_name": "Roukes, Michael Lee" }, { "name": { "family": "Qian", "given": "Lulu" }, "id": "Qian-Lulu", "orcid": "0000-0003-4115-2409", "role": "member", "display_name": "Qian, Lulu" }, { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "member", "display_name": "Shapiro, Mikhail G." } ], "option_major": [ "bioeng" ], "doi": "10.7907/p52e-qv56", "abstract": "The extensive scientific interest in cellular and biomolecular processes is due in large part to the importance of such processes deep inside living organisms, in the context of both health and disease. However, most methods for imaging cellular processes such as gene expression have relied on fluorescent proteins and other optical reporters that, while providing a direct optical readout of the biomolecular environment in cells readily exposed to light, have greatly limited performance in large animals due to the poor penetration of visible light beyond 1 mm of biological tissue. In contrast, ultrasound is widely used to noninvasively image tissue deep inside living organisms but has rarely been used to investigate cellular function due a lack of acoustic reporters whose production and properties are coupled to biomolecular events. Recently, the first acoustic reporter genes (ARGs) were developed for ultrasound imaging of a unique class of air-filled protein nanostructures known as gas vesicles, or GVs, which scatter sound waves when expressed in bacterial and mammalian cells. ARGs allow gene expression to be visualized with ultrasound similar to how green fluorescent protein (GFP) allowed gene expression to be visualized with light. However, ARGs will have limited utility in practical applications involving living organisms without ultrasound imaging methods providing the specificity to reliably distinguish GVs from surrounding tissue and the sensitivity to detect GVs at low concentrations.
\r\n\r\nIn this thesis, we present two novel ultrasound imaging methods that exploit the unique nonlinear physical properties of gas vesicles to enhance image quality in situations that pose challenges for conventional imaging methods. In Chapter 1, we address the problem of distinguishing GVs from tissue with cross-Amplitude Modulation (xAM), an ultrasound pulse sequence that uses X-waves to isolate the signal generated by reversible buckling of the GV shell while cancelling scattering and artifacts from tissue. In Chapter 2, we present an application of xAM to imaging of dynamic biomolecular processes. We show that, when GVs are engineered such that buckling is induced by enzyme activity, xAM can visualize enzymatic processes deep inside living animals. In Chapter 3, we address the problem of detecting very low concentrations of ARG-expressing cells with Burst Ultrasound Reconstructed with Signal Templates (BURST), an imaging method that exploits the strong, transient signals generated during sudden GV collapse under acoustic pressure by unmixing the temporal dynamics of such signals from background scattering. BURST imaging improves cellular sensitivity by more than 1000-fold and, in dilute cell suspensions, enables the detection of gene expression in individual bacteria and mammalian cells. In Chapter 4, we present an application of an early formulation of BURST to imaging gene expression in mammalian cells. We use this imaging method to visualize vascularization patterns in tumors containing mammalian cells expressing acoustic reporter genes.
" }, { "name": "Takei, Yodai", "degree": "PhD", "year": "2021", "title": "Integrated Spatial Genomics Reveals Organizational Principles of Single-Cell Nuclear Architecture", "advisor": "Cai, Long", "url": "https://resolver.caltech.edu/CaltechTHESIS:06022021-012404326", "creators": [ { "name": { "family": "Takei", "given": "Yodai" }, "id": "Takei-Yodai", "orcid": "0000-0002-7226-5185", "display_name": "Takei, Yodai" } ], "advisors": [ { "name": { "family": "Cai", "given": "Long" }, "id": "Cai-Long", "orcid": "0000-0002-7154-5361", "role": "advisor", "display_name": "Cai, Long" } ], "committee": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "chair", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "member", "display_name": "Guttman, Mitchell" }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Cai", "given": "Long" }, "id": "Cai-Long", "orcid": "0000-0002-7154-5361", "role": "member", "display_name": "Cai, Long" } ], "option_major": [ "biology" ], "doi": "10.7907/4ces-zm75", "abstract": "Three-dimensional (3D) nuclear architecture plays key roles in many cellular processes such as gene regulation and genome replication. Recent sequencing-based and imaging-based single-cell studies have characterized a high variability of nuclear features in individual cells from a wide-range of measurement modalities, such as chromosome structures, subnuclear structures, chromatin states, and nascent transcription. However, the lack of technologies that allow us to interrelate those nuclear features simultaneously in the same single cells limits our understanding of nuclear architecture. To overcome this limitation, a technology that can examine 3D nuclear features across modalities from the same single cells is required. Here, we demonstrate integrated spatial genomics approaches, which enable genome-wide investigation of chromosome structures, subnuclear structures, chromatin states, and transcriptional states in individual cells. In Chapter 2, we introduce the \"track first and identify later\" approach, which enables multiplexed tracking of genomic loci in live cells by combining CRISPR/Cas9 live imaging and DNA sequential fluorescence in situ hybridization (DNA seqFISH) technologies. We demonstrate our approach by resolving the dynamics of 12 unique subtelomeric loci in mouse embryonic stem (ES) cells. In Chapter 3, we present the intron seqFISH technology, which enables transcriptome-scale gene expression profiling at their nascent transcription active sites in individual nuclei in mouse ES cells and fibroblasts, along with mRNA and lncRNA seqFISH and immunofluorescence. We show the transcription active sites position at the surfaces of chromosome territories with variable inter-chromosomal organization in individual nuclei. By building upon those technologies, in Chapter 4, we demonstrate integrated spatial genomics in mouse ES cells, which enables to image thousands of genomic loci by DNA seqFISH+, along with sequential immunofluorescence and RNA seqFISH in individual cells. We show \"fixed loci\" that are invariably associated with specific subnuclear structures across hundreds of single cells that can constrain nuclear architecture in individual nuclei. In addition, we find individual genomic loci appear to be pre-positioned to specific nuclear compartments with different frequencies, which are independent from nascent transcriptional states of single cells. Lastly, in Chapter 5, we demonstrate the integrated spatial genomics technology in the mouse brain cortex, enabling the investigation of single-cell nuclear architecture in a cell-type specific fashion as well as the exploration of common organizational principles of nuclear architecture across cell types. We reveal that inter-chromosomal organization and radial positioning of chromosomes are arranged with cell-type specific chromatin fixed loci and subnuclear structure organization in diverse cell types. We also uncover the variable organization of chromosome domain structures at the sub-megabase scale in individual cells, which can be obscured with bulk measurements. Together, these results demonstrate the ability of integrated spatial genomics to advance our overall understanding of single-cell nuclear architecture in various biological systems.
" }, { "name": "Turan, Zeynep", "degree": "PhD", "year": "2021", "title": "Life Without Cortex: Subcortical Circuits in Naturalistic Behaviors", "advisor": "Meister, Markus", "url": "https://resolver.caltech.edu/CaltechTHESIS:06082021-032229529", "creators": [ { "name": { "family": "Turan", "given": "Zeynep" }, "id": "Turan-Zeynep", "orcid": "0000-0003-0704-5116", "display_name": "Turan, Zeynep" } ], "advisors": [ { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "orcid": "0000-0003-2136-6506", "role": "advisor", "display_name": "Meister, Markus" } ], "committee": [ { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "chair", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Lois", "given": "Carlos" }, "id": "Lois-Carlos", "orcid": "0000-0002-7305-2317", "role": "member", "display_name": "Lois, Carlos" }, { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "orcid": "0000-0003-2136-6506", "role": "member", "display_name": "Meister, Markus" } ], "option_major": [ "neurobio" ], "doi": "10.7907/czd1-dp02", "abstract": "A major goal of neuroscience is to understand the neural circuits underlying animal behavior. Many contemporary studies focus on behavioral tasks which do not reflect realistic conditions, such as mapping an arbitrary sensory stimulus to motor output. Given that the brain evolved within the context of the natural environment, it is more likely that these circuits were optimized for naturalistic behaviors such as avoiding predators, hunting, and social interactions with conspecifics. Many of these naturalistic behaviors predate the great expansion of the neocortex in mammals, as they are crucial for the survival of any animal. Using a mutant mouse model and surgical techniques, we show that the evolutionarily ancient subcortical circuits of mice are sufficient for sensory processing, stimulus discrimination, and exhibiting robust innate defensive behaviors in a predator avoidance assay. Furthermore, these animals are capable of navigating a complex labyrinth, which challenges long-held beliefs that learning and memory require the neocortex and the hippocampus. Our results emphasize the significant capacity of subcortical circuits in behaviors necessary for survival and illustrate the importance of using naturalistic behaviors to probe brain function." }, { "name": "Varuzhanyan, Grigor", "degree": "PhD", "year": "2021", "title": "Mitochondrial Dynamics and Mitophagy during Male Germline Development", "advisor": "Chan, David C.", "url": "https://resolver.caltech.edu/CaltechTHESIS:09232020-201548312", "creators": [ { "name": { "family": "Varuzhanyan", "given": "Grigor" }, "id": "Varuzhanyan-Grigor", "orcid": "0000-0001-6165-0857", "display_name": "Varuzhanyan, Grigor" } ], "advisors": [ { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "advisor", "display_name": "Chan, David C." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Aravin", "given": "Alexei A." }, "id": "Aravin-A-A", "orcid": "0000-0002-6956-8257", "role": "member", "display_name": "Aravin, Alexei A." }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "member", "display_name": "Chan, David C." } ], "option_major": [ "biology" ], "doi": "10.7907/1mes-yw82", "abstract": "Mitochondrial fusion and fission (mitochondrial dynamics) and mitophagy are well-established mitochondrial quality control mechanisms that safeguard cellular homeostasis. However, their role during development remains poorly understood. In this thesis, we establish the role of mitochondrial dynamics and mitophagy during the development of the male germline (spermatogenesis).
\r\n\r\nSpermatogenesis is one of biology\u2019s most complex and lengthy differentiation processes, transforming spermatogonial stem cells into highly specialized sperm cells capable of fertilization. This elaborate differentiation program requires multiple transitions in mitochondrial morphology and extensive degradation of mitochondria, making it an attractive model system for investigating mitochondrial dynamics and mitophagy in vivo. Indeed, the field of mitochondrial dynamics has a long history with spermatogenesis. The first mitochondrial dynamics gene, Fuzzy onions (Fzo), was discovered in 1997 to mediate mitochondrial fusion during Drosophila spermatogenesis. However, the role of mitochondrial dynamics during mammalian spermatogenesis remained unknown for nearly two decades after discovery of Fzo. To address this gap in knowledge, we investigate mitochondrial dynamics and mitophagy during mammalian spermatogenesis. We uncover essential roles for mitochondrial fusion (Chapter 2), mitochondrial fission (Chapter 3), and mitophagy (Chapter 4) during spermatogenesis and show that each of these mitochondrial quality control mechanisms regulates a distinct stage of germ cell development. Our analyses reveal requirements for mitochondrial fusion, fission, and mitophagy that correspond to the mitochondrial and metabolic needs of the developing germ cells.
\r\n\r\nWe also investigate the role of mitochondrial fusion and fission in regulating subcellular mitochondrial domains upon fusion of a skeletal muscle stem cell with a myofiber (Chapter 5). Thus, the work presented in this thesis characterizes the in vivo role of mitochondrial dynamics in two systems: male germline development and skeletal muscle regeneration. However, we focus on the role of mitochondrial dynamics and mitophagy during male germline development.
" }, { "name": "Wang, Ruohan", "degree": "PhD", "year": "2021", "title": "Identification of New OPA1 Cleavage Site Reveals that Short Isoforms Regulate Mitochondrial Fusion", "advisor": "Chan, David C.", "url": "https://resolver.caltech.edu/CaltechTHESIS:11182020-224422123", "creators": [ { "name": { "family": "Wang", "given": "Ruohan" }, "id": "Wang-Ruohan", "orcid": "0000-0001-8361-4005", "display_name": "Wang, Ruohan" } ], "advisors": [ { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "advisor", "display_name": "Chan, David C." } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Voorhees", "given": "Rebecca M." }, "id": "Voorhees-R-M", "orcid": "0000-0003-1640-2293", "role": "member", "display_name": "Voorhees, Rebecca M." }, { "name": { "family": "Aravin", "given": "Alexei A." }, "id": "Aravin-A-A", "orcid": "0000-0002-6956-8257", "role": "member", "display_name": "Aravin, Alexei A." }, { "name": { "family": "van der Bliek", "given": "Alexander M." }, "id": "van-der-Bliek-A-M", "orcid": "0000-0002-6211-5765", "role": "member", "display_name": "van der Bliek, Alexander M." }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "member", "display_name": "Chan, David C." } ], "option_major": [ "biology" ], "doi": "10.7907/8nk6-1502", "abstract": "OPA1 is a 120kDa large GTPase belonging to the dynamin superfamily. It is the only known mitochondrial inner membrane fusion protein, mediating fusion of the mitochondrial inner membranes following outer membrane fusion. Additionally, OPA1 also regulates cristae morphology and maintains respiratory chain function.
\r\n\r\nOPA1 has two forms\u2014inner-membrane-anchored long forms (l-OPA1) and cleaved inter-membrane-space only short forms (s-OPA1). L-OPA1 are proteolytically processed by two mitochondrial proteases\u2014OMA1 and YME1L, acting at cleavage sites S1 and S2 respectively, to produce s-OPA1.
\r\n\r\nIn both mice and human, half of the mRNA splice forms of Opa1 are constitutively processed post translation to yield exclusively s-OPA1. However, the specific function of s-OPA1 in mitochondrial fusion has been debated\u2014under basal conditions, s-OPA1 are needed to maintain optimal fusion activity, but in certain stress conditions, s-OPA1 is dispensable for fusion. By constructing cells in which the Opa1 locus no longer produces transcripts with S2 cleavage sites using CRISPR-Cas9, we generated a simplified system to identify the novel YME1L-dependent site S3 that mediates constitutive and complete cleavage of OPA1. We found that S3 site locates within the C-terminal leucine string of Opa1 exon4b, slightly upstream of the well-established S1. We show that mitochondrial morphology is highly sensitive to the ratio of l-OPA1 to s-OPA1, indicating that s-OPA1 fine tunes mitochondrial fusion.
" }, { "name": "Wong, Wan-Rong", "degree": "PhD", "year": "2021", "title": "C. elegans Models of ASD-Associated Missense Variants", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03112021-005902753", "creators": [ { "name": { "family": "Wong", "given": "Wan-Rong" }, "id": "Wong-Wan-Rong", "orcid": "0000-0002-9757-8145", "display_name": "Wong, Wan-Rong" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "orcid": "0000-0002-7371-4675", "role": "chair", "display_name": "Prober, David A." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Hong", "given": "Elizabeth J." }, "id": "Hong-Elizabeth-J", "role": "member", "display_name": "Hong, Elizabeth J." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "neurobio" ], "doi": "10.7907/y9gj-y034", "abstract": "The evolving next-generation sequencing technology accelerates the identification of disease-associated genetic variants. However, interpretation of these variants remains challenging, especially variants with subtle effects such as missense variants. Missense variants account for a large proportion of genetic variants in human diseases, including autism spectrum disorder (ASD). The causal relationship of most missense variants in the pathogenesis of ASD has not yet been demonstrated, and an experimental method systematically prioritizing missense alleles can gain crucial insight into the molecular basis for disease pathology. Therefore, I developed an in vivo multi-cellular system using Caenorhabditis elegans to systematically evaluate the functional consequences of disease-associated missense variants. I identified highly conserved human ASD-associated missense variants in their C. elegans orthologs, used a CRISPR/Cas9-mediated homology-directed knock-in strategy to generate missense mutants, and analyzed their impact on behaviors and development via several broad-spectrum assays. Overall, I generated 60 ASD-associated missense variants and characterized these missense mutant strains using a fecundity assay, an automated locomotor tracking system, and a chemotaxis assay. I found that 19% of the human disease-associated alleles have conserved loci in their C. elegans orthologs. Among the genes I tested, 64-70% of the missense variants predicted to perturb protein function showed detectable phenotypic changes in morphology, locomotion, or fecundity. Our results also revealed that missense mutants in different gene networks displayed distinct phenotypic profiles. Moreover, I focused on studying the genetic properties of missense variants on two ASD risk genes. I discovered the developmental defects in the ALDH1A3 C174Y missense mutation involved in the retinoic acid signaling pathway. I also identified a conserved missense residue lin-45(K565N), orthologous to human BRAF(K499N), which displayed a hypersensitive non-dominant phenotype in the diacetyl chemotaxis assay that was capable of being inhibited by RNAi. The finding suggests a potential gain-of-function allele in BRAF, especially in the sensory function. To sum up, I established a working pipeline to systematically identify and generate evolutionarily conserved ASD-associated missense mutants in C. elegans. This approach will help assess the impact of a single missense mutation in the whole organism and prioritize consequential missense variants for further intensive analysis in vertebrate models and human cells.
" }, { "name": "Bogatyrev, Said R.", "degree": "PhD", "year": "2020", "title": "Development of Analytical Tools and Animal Models for Studies of Small-Intestine Dysbiosis", "advisor": "Ismagilov, Rustem F.", "url": "http://resolver.caltech.edu/CaltechTHESIS:10012019-095132591", "creators": [ { "name": { "family": "Bogatyrev", "given": "Said R." }, "id": "Bogatyrev-Said-R", "orcid": "0000-0003-0486-9451", "display_name": "Bogatyrev, Said R." } ], "advisors": [ { "name": { "family": "Ismagilov", "given": "Rustem F." }, "id": "Ismagilov-R-F", "orcid": "0000-0002-3680-4399", "role": "advisor", "display_name": "Ismagilov, Rustem F." } ], "committee": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "chair", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Ismagilov", "given": "Rustem F." }, "id": "Ismagilov-R-F", "orcid": "0000-0002-3680-4399", "role": "member", "display_name": "Ismagilov, Rustem F." }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "orcid": "0000-0003-2713-1513", "role": "member", "display_name": "Mazmanian, Sarkis K." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "bioeng" ], "doi": "10.7907/VJDZ-7B52", "abstract": "Our appreciation of the role of human-associated microbial communities in the context of human health and disease has grown dramatically in the past two decades, with modern research tools enabling deeper insights into the mechanisms of host-microbial interactions. The elusive notion of dysbiosis, a state of microbial imbalance related to a disease, has achieved widespread distribution across popular, scientific, and medical literature (on September 16, 2019 PubMed search yielded 6,064 records of scientific and medical publications containing this keyword). The conventional wisdom further narrows down the definition and understanding of dysbiosis towards a compositional \"imbalance\" of the microbiota (a community of all microorganisms inhabiting human body). There exists an additional and frequently overlooked aspect of microbial imbalance in the context of the human gastrointestinal system, something that we can define as a \"spatial imbalance\": a state of the microbial community in the host gastrointestinal system where even a \"healthy\" and \"balanced\" microbiota may be associated with or causative of a disease by being present in sections of the gastrointestinal tract where it is not \"supposed\" to be, with the most prominent example being small intestinal bacterial overgrowth (SIBO). This thesis describes the progress in the development of analytical tools (quantitative microbiome profiling described in Chapter I) and refinement of animal mouse models (non-coprophagic mouse model described in Chapter II) for exploring the normal function of small-intestine microbiota in health and for dissecting the mechanisms of emergence and the persistence of the small-intestine dysbiosis (SIBO) in the future.
" }, { "name": "Chure, Griffin Daniel", "degree": "PhD", "year": "2020", "title": "The Molecular Biophysics of Evolutionary and Physiological Adaptation", "advisor": "Phillips, Robert B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06022020-102020436", "creators": [ { "name": { "family": "Chure", "given": "Griffin Daniel" }, "id": "Chure-Griffin-Daniel", "orcid": "0000-0002-2216-2057", "display_name": "Chure, Griffin Daniel" } ], "advisors": [ { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "advisor", "display_name": "Phillips, Robert B." } ], "committee": [ { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "orcid": "0000-0002-7033-0844", "role": "chair", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." } ], "option_major": [ "biochem" ], "doi": "10.7907/q8h6-xr92", "abstract": "Central to any definition of Life is the ability to sense changes in one\u2019s environment and respond in kind. Adaptive phenomena can be found across the biological scales ranging from the nanosecond-scale conformational changes of proteins, to temporary rewiring of metabolic networks, to the 3.5 billion years of evolution that produced the enormous biodiversity we see today. This thesis presents a body of work which attempts to examine the overlap between these three scales of adaptation through the quantitative lens of statistical physics. Namely, we examine how molecular, physiological, and evolutionary adaptation governs a feature common to all life \u2013 the regulation of gene expression.
\r\n\r\nWe begin by examining the phenomenon of molecular adaptation in the context of allostery, specifically in the context of allosteric transcriptional repressors. Using simple tools of quasi-equilibrium thermodynamics, we derive and experimentally dissect a quantitative model of how such a repressor adapts to different concentrations of an extracellular inducer molecule, modulating the repressors activity and thereby gene expression. While the model is relatively simple, it is remarkably powerful in its ability to draw concrete, quantitative predictions about not only the level of gene expression at a given concentration of inducer, but details of how the repressor responds to changes in the inducer concentration. With a few lines of simple mathematics, we are able to use this model to derive a state variable of the simple repression motif which we term the free energy of the regulatory architecture. This permits us to collapse nearly 500 distinct measurements of the level of gene expression onto a master curve defined by this free energy.
\r\n\r\nWe leverage this feature of the model and use data collapse as a method to identify the effects of mutation, a strong evolutionary force responsible for much of the genetic diversity in bacteria. In Chapter 3, we examine how mutations within the allosteric repressor itself can be mapped to changes in the free energy. The precise value of these free energy shifts and their dependence on the inducer concentration reveal different classes of mutations with one class affecting only the DNA-repressor interaction and another class governing the allosteric nature of the repressor. We test these pen-and-paper predictions experimentally and illustrate that given sufficient knowledge of how single mutants behave, the complete phenotypic response of pairwise double mutants can be predicted with quantitative accuracy.
\r\n\r\nWith this framework in hand, we turn to exploring how changes in the physiological state of the cell influence the molecular biophysics of the regulatory architecture. We hypothesize that changes in the source of carbon in the growth medium or changes in the growth temperature can be accounted for by the existing model without any additional parameters. We experimentally show that the parameter values determined in one physiological state are inherited when the available carbon source is verified, but changes in the growth temperature require some additional considerations. Chapter 4 as a whole reveals that, while there remains work to be done both theoretically and experimentally when it comes to temperature variation, thermodynamic models can remain powerful tools to draw predictions of gene expression in different physiological contexts.
\r\n\r\nFinally, in Chapter 5, we explore physiological adaptation and cellular decision making where it counts \u2013 in the survival of cells to environmental insults. We turn our focus beyond transcriptional regulation and consider the relationship between osmotic shocks, the abundance of mechanosensitive channels, and cellular survival with single cell resolution. Using a combination of quantitative microscopy and tricks of statistical inference, we infer how the probability of a cell surviving an osmotic shock scales as a function of the cell\u2019s number of mechanosensitive channels.
" }, { "name": "Farhadi, Arash", "degree": "PhD", "year": "2020", "title": "Acoustic Reporter Genes for Noninvasive Imaging of Cellular Function", "advisor": "Shapiro, Mikhail G.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04222020-163929825", "creators": [ { "name": { "family": "Farhadi", "given": "Arash" }, "id": "Farhadi-Arash", "orcid": "0000-0001-9137-8559", "display_name": "Farhadi, Arash" } ], "advisors": [ { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "advisor", "display_name": "Shapiro, Mikhail G." } ], "committee": [ { "name": { "family": "Tirrell", "given": "David A." }, "id": "Tirrell-D-A", "orcid": "0000-0003-3175-4596", "role": "chair", "display_name": "Tirrell, David A." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Wang", "given": "Lihong" }, "id": "Wang-Lihong", "orcid": "0000-0001-9783-4383", "role": "member", "display_name": "Wang, Lihong" }, { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "member", "display_name": "Shapiro, Mikhail G." } ], "option_major": [ "bioeng" ], "doi": "10.7907/zght-4j47", "abstract": "The study of cellular function within the context of intact living organisms is a grand challenge in biological research. Addressing this challenge requires imaging tools that can visualize cells inside the body. If successful, this would greatly increase our ability to study a battery of processes from brain development to tumorigenesis, to monitoring cell-based therapeutics. To date, most common methods for imaging cellular processes such as gene expression have relied on optical reporters, such as fluorescent or luminescent proteins, which provide high molecular precision for studies in petri dishes and transparent organisms, but have limited performance in large animals due to the poor penetration of light in biological tissue. Conversely, magnetic resonance imaging (MRI) and ultrasound can image tissues at depth with high spatial and temporal resolution, but they lack molecular reporters analogous to the green fluorescent protein (GFP). As a result, they have made limited impact on biological research. To address this, we focus on developing biomolecular reporters for MRI and ultrasound \u2014 based on a unique class of air-filled protein nanostructures called gas vesicles \u2014 using them to image the location and function of cells deep inside the body.
\r\n\r\nThis thesis begins with a brief review of genetically encoded materials for noninvasive imaging, highlighting key advances over the past two decades and providing context for the work below. We discuss the development of increasingly sophisticated tools starting from early efforts to engineer single molecule reporters to recent work on multi-component genetic machinery (including gas vesicles) with multi-modality capabilities. In Chapter 2, we present a platform for engineering the surface of gas vesicles to modulate their acoustic, surface charge, and molecular- targeting properties as injectable acoustic biomolecules. In Chapter 3, we present the recombinant expression of gas vesicles as injectable contrast agents in common lab strain bacteria to facilitate the genetic engineering of the entire gas vesicle gene cluster and to assist this technology\u2019s adoption by other (non-specialist) research groups. This work characterized the ultrasound and hyperpolarized 129Xenon-MRI contrast of gas vesicles as nanoscale contrast agents.
\r\n\r\nIn a parallel effort, we developed a hybrid gene cluster that when introduced to microbes enables the imaging of their gene expression using ultrasound. These bacterial acoustic reporter genes were used to image the location of probiotic cells inside the gastrointestinal tract of mice. However, the ability for these genes to be expressed in mammalian cells had not been demonstrated and presented a major challenge in synthetic biology. In Chapter 4, we addressed this by introducing the first mammalian acoustic reporter genes \u2014 a genetic program whose introduction to mammalian cells resulted in the expression of gas vesicles that can be visualized by ultrasound. These mammalian acoustic reporter genes will enable previously impossible approaches to monitoring the location, viability and function of mammalian cells in vivo.
\r\n\r\nIn Chapter 5, we explore a new paradigm in MRI by taking advantage of the acousto-magnetic property of gas vesicles. Here, we present background-free MRI to address a longstanding challenge in untangling the signal of exogenous contrast agents from the endogenous MRI contrast produced by biological tissues. Chapter 6 explores the optical properties of gas vesicles as genetically encodable phase contrast agents in digital holographic imaging. Chapter 7 is a brief discussion of the potential future directions for this work.
\r\n\r\nThe data presented in this thesis lays the ground for exciting new research on developing noninvasive biomolecular tools that will enable the discovery of novel biological processes.
" }, { "name": "Gugel, Zhannetta V.", "degree": "PhD", "year": "2020", "title": "Effects of Sensory Experience on Early Stages of Olfactory Processing in the Fruit Fly", "advisor": "Hong, Elizabeth J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01062020-004548466", "creators": [ { "name": { "family": "Gugel", "given": "Zhannetta V." }, "id": "Gugel-Zhannetta-V", "orcid": "0000-0003-1082-3281", "display_name": "Gugel, Zhannetta V." } ], "advisors": [ { "name": { "family": "Hong", "given": "Elizabeth J." }, "id": "Hong-Elizabeth-J", "role": "advisor", "display_name": "Hong, Elizabeth J." } ], "committee": [ { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "orcid": "0000-0002-8587-9936", "role": "chair", "display_name": "Dickinson, Michael H." }, { "name": { "family": "Hong", "given": "Elizabeth J." }, "id": "Hong-Elizabeth-J", "role": "member", "display_name": "Hong, Elizabeth J." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "orcid": "0000-0003-2136-6506", "role": "member", "display_name": "Meister, Markus" } ], "option_major": [ "neurobio" ], "doi": "10.7907/e39b-w024", "abstract": "Plasticity is widely studied across different sensory systems and behavioral paradigms, but the underlying mechanisms are varied and incompletely understood. Previous work in the fruit fly Drosophila melanogaster reported changes in odor preference and walking behavior after chronic odor exposure during early adulthood. Here, we investigated the hypothesis that changes in behavior reflect changes in how odors are encoded in the first two layers of the fly olfactory circuit. We chronically exposed flies to naturalistic odor stimuli that selectively and robustly activate a single olfactory receptor neuron (ORN) class. We then performed targeted intracellular recordings from genetically identified second-order olfactory projection neurons (PNs) that either receive direct input from the activated ORN class, or receive indirect activity (via local lateral circuitry), during chronic odor exposure. In addition, we used existing reagents to create a novel optical method to characterize ORN-PN synaptic strength. We find that the fly antennal lobe is resistant to plasticity, with a few exceptions. Of the odors we tested, we find that rearing in trans-2-hexenal, a leaf aldehyde that selectively activates ab4a ORNs, weakly enhanced odor responses in some PNs. The effects of rearing on PNs were not explained by ORN odor responses or changes in ORN-PN synaptic strength. We find evidence that lateral excitation may increase across glomeruli following rearing, suggesting that some odors may alter PN responses globally. We discuss possible reasons for differences between our observations and prior work on olfactory plasticity in this circuit, which has been conducted primarily in the context of exposures to much higher, non-naturalistic concentrations of odor. Our results point to the stability of insect sensory circuits in the face of large perturbations in the sensory environment.
\r\n\r\n\u2028\u2028During our optical stimulation experiments, we find that driving Chrimson expression may abolish odor responses in some ORNs. We include sample data highlighting this observation in a population of pb1a olfactory neurons. Lastly, we include antennal local field potential recordings in response to a variety of odor concentrations to help guide future experiments seeking isointense odor panels.
" }, { "name": "Hanewich-Hollatz, Mikhail Henning", "degree": "PhD", "year": "2020", "title": "Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function via Dynamic RNA Nanotechnology", "advisor": "Pierce, Niles A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:11042019-135312842", "creators": [ { "name": { "family": "Hanewich-Hollatz", "given": "Mikhail Henning" }, "id": "Hanewich-Hollatz-Mikhail-Henning", "orcid": "0000-0002-5369-3846", "display_name": "Hanewich-Hollatz, Mikhail Henning" } ], "advisors": [ { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "advisor", "display_name": "Pierce, Niles A." } ], "committee": [ { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "orcid": "0000-0002-5899-7523", "role": "chair", "display_name": "Winfree, Erik" }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "member", "display_name": "Shapiro, Mikhail G." }, { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "member", "display_name": "Pierce, Niles A." } ], "option_major": [ "bioeng" ], "doi": "10.7907/NS2B-DJ96", "abstract": "A guide RNA (gRNA) directs the function of a CRISPR protein effector to a target gene of choice, providing a versatile programmable platform for engineering diverse modes of synthetic regulation (edit, silence, induce, bind). However, the fact that gRNAs are constitutively active places limitations on the ability to confine gRNA activity to a desired location and time. To achieve programmable control over the scope of gRNA activity, here we apply principles from dynamic RNA nanotechnology to engineer conditional guide RNAs (cgRNAs) whose activity is dependent on the presence or absence of an RNA trigger. These cgRNAs are programmable at two levels, with the trigger-binding sequence controlling the scope of the effector activity and the target-binding sequence determining the subject of the effector activity. There are two possible logical directions for single-input cgRNAs: constitutively active cgRNAs that are conditionally inactivated by an RNA trigger (ON\u2192OFF logic) and constitutively inactive cgRNAs that are conditionally activated by an RNA trigger (OFF\u2192ON logic). Using an in vitro assay for cgRNA activity with synthetic trigger, in vitro transcribed cgRNA, and recombinant dCas9, we observe a conditional (ON\u2192OFF logic) response for a set of four allosteric constitutively active cgRNAs with a median \u22486% crosstalk between noncognate cgRNA/trigger pairs. Motivated by the observed lack of conditional response of this mechanism when ported to E. coli, we describe a systematic study of unstructured sequence inserts into the standard gRNA structure and report the conditional response of a set of 34 candidate cgRNAs in living cells. Molecular mechanisms for both ON\u2192OFF and OFF\u2192ON cgRNAs are demonstrated in E. coli. For each mechanism, automated sequence design is performed using the reaction pathway designer within NUPACK to produce an orthogonal library of cgRNAs that respond to different RNA triggers. In E. coli expressing cgRNAs, triggers, and silencing dCas9 as the protein effector, we observe a median conditional response of \u224815-fold for a library of three orthogonal ON\u2192OFF \"splinted switch\" cgRNA/trigger pairs, and \u22483-fold for a library of three orthogonal OFF\u2192ON \"toehold switch\" cgRNA/trigger pairs; the median crosstalk within each library is <2% and \u224820% for the two mechanisms, respectively. By providing programmable control over both the scope and target of protein effector function, cgRNA regulators offer a promising platform for conditional gene regulation and synthetic biology.
" }, { "name": "Hesse, Janis Karan", "degree": "PhD", "year": "2020", "title": "Neural Construction of Conscious Perception", "advisor": "Tsao, Doris Y.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05302020-143859367", "creators": [ { "name": { "family": "Hesse", "given": "Janis Karan" }, "id": "Hesse-Janis-Karan", "orcid": "0000-0003-0405-8632", "display_name": "Hesse, Janis Karan" } ], "advisors": [ { "name": { "family": "Tsao", "given": "Doris Y." }, "id": "Tsao-D-Y", "orcid": "0000-0003-1083-1919", "role": "advisor", "display_name": "Tsao, Doris Y." } ], "committee": [ { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "orcid": "0000-0003-2136-6506", "role": "chair", "display_name": "Meister, Markus" }, { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "member", "display_name": "Adolphs, Ralph" }, { "name": { "family": "Rutishauser", "given": "Ueli" }, "id": "Rutishauser-U", "orcid": "0000-0002-9207-7069", "role": "member", "display_name": "Rutishauser, Ueli" }, { "name": { "family": "Tsao", "given": "Doris Y." }, "id": "Tsao-D-Y", "orcid": "0000-0003-1083-1919", "role": "member", "display_name": "Tsao, Doris Y." } ], "option_major": [ "cns" ], "doi": "10.7907/07r8-0845", "abstract": "Out of a myriad of sensory stimulations, our brain constructs a unified, self-consistent reality that we consciously experience. Little is known about how or where in the brain\u2019s processing stream of physical input a conscious percept emerges into awareness. A remarkable property of conscious perception is that even though external input is often ambiguous, the perceptual interpretation of the world that our brain generates is consistent across multiple layers of representation, e.g., figure-ground segmentation and object identity. We thus set out to study how the interaction between different nodes in the brain generates and propagates new conscious percepts. Since the code of object identity is already well-understood, in particular for faces as reviewed in this thesis, we decided to get a handle on segmentation signals first. It turned out that consistent segmentation signals are hard to find, however, we found functionally defined modules in the brain that contained consistent cells from which figure-ground signals can be decoded. We next investigated whether face cells in object recognition areas actually encode the conscious percept of a face or are just passive filters of visual input. To distill conscious perception from other cognitive processes, such as decision making, introspection, and reporting of the percept, which often accompany new conscious percepts, we developed a no-report binocular rivalry paradigm that relies on an active fixation task rather than report, and therefore eliminates these confounding factors. We found that face patches in inferotemporal cortex indeed encode the conscious percept of a face. Using novel high-yield electrodes, we were able to decode what the animal was consciously perceiving at a given time. Preliminary and future experiments of population recordings from multiple nodes of the cortical hierarchy simultaneously promise to go beyond correlates of consciousness and reveal the mechanisms of how and where conscious percepts are constructed.
" }, { "name": "Johnson, Robert Francis", "degree": "PhD", "year": "2020", "title": "Formal Design and Analysis for DNA Implementations of Chemical Reaction Networks", "advisor": "Winfree, Erik; Qian, Lulu", "url": "https://resolver.caltech.edu/CaltechTHESIS:04292020-052418975", "creators": [ { "name": { "family": "Johnson", "given": "Robert Francis" }, "id": "Johnson-Robert-Francis", "orcid": "0000-0002-5340-8347", "display_name": "Johnson, Robert Francis" } ], "advisors": [ { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "orcid": "0000-0002-5899-7523", "role": "co-advisor", "display_name": "Winfree, Erik" }, { "name": { "family": "Qian", "given": "Lulu" }, "id": "Qian-Lulu", "orcid": "0000-0003-4115-2409", "role": "co-advisor", "display_name": "Qian, Lulu" } ], "committee": [ { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "chair", "display_name": "Murray, Richard M." }, { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "member", "display_name": "Pierce, Niles A." }, { "name": { "family": "Bruck", "given": "Jehoshua" }, "id": "Bruck-J", "orcid": "0000-0001-8474-0812", "role": "member", "display_name": "Bruck, Jehoshua" }, { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "orcid": "0000-0002-5899-7523", "role": "member", "display_name": "Winfree, Erik" }, { "name": { "family": "Qian", "given": "Lulu" }, "id": "Qian-Lulu", "orcid": "0000-0003-4115-2409", "role": "member", "display_name": "Qian, Lulu" } ], "option_major": [ "bioeng" ], "doi": "10.7907/a74v-kv80", "abstract": "In molecular programming, the Chemical Reaction Network model is often used to describe systems of interacting molecules. This model can describe either real systems, allowing us to analyze and determine their computational function; or describe hypothetical systems, with known computational function but perhaps no known physical example. One significant breakthrough in the field is that any Chemical Reaction Network can be approximated by a system using DNA Strand Displacement mechanisms. This allows the Chemical Reaction Network model to be treated like a programming language, where programs can be written in the abstract and then compiled into physical molecules. Given a programming language and a proof-of-concept compiler, one would want to take the compiler from the proof-of-concept stage into a more reliable, more systematic, and better understood process. This thesis is made up of my contributions to that effort.
\r\n\r\nFirst, given a programming language and a compiler, it would be useful to formally verify that the compiler is correct. My collaborators, Qing Dong and Erik Winfree, and I defined a Chemical Reaction Network-specific form of bisimulation equivalence, which can compare two such networks and verify that one is (or is not) a correct implementation of the other. For example, the compiler-produced DNA circuit can be verified as an implementation of its abstract program, although this is not the only possible use. After defining this concept of equivalence, we show that it can be checked by algorithm; although various parts of the problem are NP-complete or PSPACE-complete, we give algorithms that meet these lower bounds. We also prove a number of interesting properties of Chemical Reaction Network bisimulation equivalence, including transitivity and modularity properties which are particularly useful for stepwise checking of large systems. We further extend this bisimulation method to linear Polymer Reaction Networks, a strictly more powerful abstraction which has been occasionally used in molecular programming. Again we prove complexity hardness results, which in this case are as expected uncomputable in the general case; however, many practical systems can still be verified, and we give one such example. Finally, we use bisimulation to identify a class of single-locus networks that are practical to implement. Thus we show a method of verification which can simplify use of the above-mentioned compiler by proving general statements of correctness about its results.
\r\n\r\nSecond, given a programming language and a concept of compiling it, it would be useful to optimize the result of the compilation. One particular area of optimization is the number of DNA strands per prepared complex; some experiments suggest that systems with no more than 2 strands per complex are more robust. Lulu Qian and I developed some proposed DNA Strand Displacement schemes for general Chemical Reaction Network implementations with no more than 2 strands per complex, and a number of other desirable properties. Meanwhile, having been shown to be useful for many reasons, the mechanisms of DNA Strand Displacement have recently been formalized, abstracted, and analyzed. I show that this formalization, combined with the bisimulation methods above, can prove various statements about the limits of DNA Strand Displacement systems. For example, a set of desirable conditions including the 2-strand limit cannot be achieved by any general Chemical Reaction Network implementation scheme. I also observe that two of the new schemes we discovered, each meeting all but one condition of the impossible set, were found in the process of coming up with this proof. I thus argue that through formalization of DNA Strand Displacement we can have a more systematic method of finding and designing molecular programs, and of knowing when the programs we want do not exist.
" }, { "name": "Jue, Erik Bradley", "degree": "PhD", "year": "2020", "title": "Improved Tools for Point-of-Care Nucleic Acid Amplification Testing", "advisor": "Ismagilov, Rustem F.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05292020-131840076", "creators": [ { "name": { "family": "Jue", "given": "Erik Bradley" }, "id": "Jue-Erik-Bradley", "orcid": "0000-0001-7585-3794", "display_name": "Jue, Erik Bradley" } ], "advisors": [ { "name": { "family": "Ismagilov", "given": "Rustem F." }, "id": "Ismagilov-R-F", "orcid": "0000-0002-3680-4399", "role": "advisor", "display_name": "Ismagilov, Rustem F." } ], "committee": [ { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "chair", "display_name": "Murray, Richard M." }, { "name": { "family": "Ismagilov", "given": "Rustem F." }, "id": "Ismagilov-R-F", "orcid": "0000-0002-3680-4399", "role": "member", "display_name": "Ismagilov, Rustem F." }, { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "member", "display_name": "Shapiro, Mikhail G." }, { "name": { "family": "Yang", "given": "Changhuei" }, "id": "Yang-Changhuei", "orcid": "0000-0001-8791-0354", "role": "member", "display_name": "Yang, Changhuei" } ], "option_major": [ "bioeng" ], "doi": "10.7907/d6mf-5081", "abstract": "There is a critical need for improved diagnostic tools to detect infectious diseases, especially in low-resource regions. A sample-to-answer point-of-care nucleic acid amplification test (NAAT) would be incredibly valuable for many different applications (e.g. COVID-19, Chlamydia/Gonorrhoeae, Influenza, Ebola, Zika/Chikungunya/Dengue, etc.). However, sample preparation (purification of pure nucleic acids) is a challenging bottleneck. In Chapter 2, commercial NA extraction methods were studied and improved. In Chapter 3, commercial stocks of SARS-CoV-2 RNA used in FDA emergency-use authorizations were found to be inaccurate and were independently quantified using reverse transcription digital PCR. In Chapter 4, a 3D printed meter-mix device was developed for initial processing prior to the sample preparation device. In Chapter 5, a 3D printed sample-to-device interface was prototyped to facilitate loading multi-volume SlipChip devices with purified template mixed with LAMP reactants. In Chapters 6-7, advancements were made for image processing of commercial chips to study digital LAMP reactions. In Chapter 8, additional tools were developed towards sample-to-answer point-of-care NAAT including a sample preparation module, amplification module, cell-phone readout, and automated base station." }, { "name": "Kim, Dong-Wook", "degree": "PhD", "year": "2020", "title": "Multimodal Analysis of Cell Types in a Hypothalamic Node Controlling Social Behavior in Mice", "advisor": "Anderson, David J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:12162019-183140887", "creators": [ { "name": { "family": "Kim", "given": "Dong-Wook" }, "id": "Kim-Dong-Wook", "orcid": "0000-0002-5497-5853", "display_name": "Kim, Dong-Wook" } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "advisor", "display_name": "Anderson, David J." } ], "committee": [ { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "chair", "display_name": "Pachter, Lior S." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Oka", "given": "Yuki" }, "id": "Oka-Yuki", "orcid": "0000-0003-2686-0677", "role": "member", "display_name": "Oka, Yuki" }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" } ], "option_major": [ "cns" ], "doi": "10.7907/RGVK-9962", "abstract": "The advent and recent advances of single-cell RNA sequencing (scRNA-seq) have yielded transformative insights into our understanding of cellular diversity in the central nervous system (CNS) with unprecedented detail. However, due to current experimental and computational limitations on defining transcriptomic cell types (T-types) and the multiple phenotypic features of cell types in the CNS, an integrative and multimodal approach should be required for the comprehensive classification of cell types.
\r\n\r\nTo this end, performing multimodal analysis of scRNA-seq in hypothalamus would be very beneficial in that hypothalamus, controlling homeostatic and innate survival behaviors which known to be highly conserved across a wide range of species and encoded in hard-wired brain circuits, is likely to display the more straightforward relationship between transcriptomic identity, axonal projections, and behavioral activation, respectively. In my dissertation, I have been focused on the cell type characterizations of a hypothalamic node controlling innate social behavior in mice, the ventrolateral subdivision of the ventromedial hypothalamus (VMHvl). VMHvl only contains ~4,000 neurons per hemisphere in mice but due to its behavioral, anatomical, and molecular heterogeneity, which T-types in VMHvl are related to connectivity and behavioral function is largely unknown.
\r\n\r\nIn Chapter II, I described my main thesis work to perform scRNA-seq in VMHvl using two independent platforms: SMART-seq2 (~4,500 neurons sequenced) and 10x (~78,000 neurons sequenced). Specifically, 17 joint VMHvl T-types including several sexually dimorphic clusters were identified by canonical correlation analysis (CCA) in Seurat, and the majority of them were validated by multiplexed single-molecule FISH (seqFISH). Correspondence between transcriptomic identity, and axonal projections or behavioral activation, respectively, was also investigated. Immediate early gene analysis identified T-types exhibiting preferential responses to intruder males versus females but only rare examples of behavior-specific activation. Unexpectedly, many VMHvl T-types comprise a mixed population of neurons with different projection target preferences. Overall our analysis revealed that, surprisingly, few VMHvl T-types exhibit a clear correspondence with behavior-specific activation and connectivity.
\r\n\r\nIn Chapter III, I will discuss about future directions for a deeper and better understanding of VMHvl cell types. Briefly, my previous data from whole-cell patch clamp recording in VMHvl slices suggested that there were at least 4 distinct electrophysiological cell types (E-types). Additionally, two distinct neuromodulatory effects on VMHvl were observed (persistently activated by vasopressin/oxytocin vs. silenced by nitric oxide) by monitoring populational activities using two-photon Ca2+ imaging in slices. Based on the results from the first part and combined with advanced molecular techniques (e.g. Patch-seq and CRISPR-Cas9), we can further dissect out the cellular diversity in VMHvl and their functional implications.
" }, { "name": "Lee, Sangjun", "degree": "PhD", "year": "2020", "title": "The Neural Basis of Sodium Appetite", "advisor": "Oka, Yuki", "url": "https://resolver.caltech.edu/CaltechTHESIS:05282020-000951837", "creators": [ { "name": { "family": "Lee", "given": "Sangjun" }, "id": "Lee-Sangjun", "orcid": "0000-0002-0846-8252", "display_name": "Lee, Sangjun" } ], "advisors": [ { "name": { "family": "Oka", "given": "Yuki" }, "id": "Oka-Yuki", "orcid": "0000-0003-2686-0677", "role": "advisor", "display_name": "Oka, Yuki" } ], "committee": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "chair", "display_name": "Lester, Henry A." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Lois", "given": "Carlos" }, "id": "Lois-Carlos", "orcid": "0000-0002-7305-2317", "role": "member", "display_name": "Lois, Carlos" }, { "name": { "family": "Oka", "given": "Yuki" }, "id": "Oka-Yuki", "orcid": "0000-0003-2686-0677", "role": "member", "display_name": "Oka, Yuki" } ], "option_major": [ "neurobio" ], "doi": "10.7907/bcxa-s404", "abstract": "Fluid homeostasis, which maintains a stable internal environment, is critical for survival. Body fluid is tightly monitored and regulated through its main components, water and salt. Here, I focus on the aspect of sodium regulation when sodium is the main cation in the extracellular fluid and is also required for primary metabolism. The depletion of sodium induces the retention of sodium but also a central mechanism to obtain sodium from the external sources. This need for sodium specifically drives animals towards sodium consumption, called sodium appetite. Even though sodium appetite is specific for only sodium ion, sodium appetite observed as an innate behavior across the animal kingdom.
\r\n\r\nSodium appetite is strictly regulated by both peripheral sensory signals and central appetite signals. Due to the development of genetic tools, I was able to investigate the neural basis of sodium appetite from searching sodium appetite dedicated neurons. Here, I identify two genetically defined neural circuits in mice that control sodium intake. The activation of these neurons drives robust sodium intake in sated animals. Particularly, prodynorphin expressing neurons in the pre-locus coeruleus shown specific consumption to sodium compounds, including rock salt. In terms of loss-of-function, inhibition of these neurons selectively reduced sodium consumption. It was further shown that these neurons receive sodium depleted signals by aldosterone-sensitive neurons.
\r\n\r\nPreviously, it was suggested that taste signals have a central role in sodium satiation. I demonstrate that the oral detection of sodium rapidly suppresses sodium appetite neurons. The blockage of the sodium taste or gastric infusion of sodium abolished the sodium suppression in the sodium appetite neurons. Consistently, gastric infusion of sodium did not cause sodium satiation. Moreover, retrograde-viral methods showed that specific inhibitory neurons partially mediate sensory modulation in the bed nucleus of the stria terminalis.
\r\n\r\nTogether, I identified a specific neural population as a functional unit for sodium appetite. By knowing the dedicated circuits for sodium appetite, I demonstrated chemosensory and physiological signals regulate the neural circuits. The genetically defined neural population can be handle as an entry point of further investigation of the neural basis of sodium appetite.
" }, { "name": "Neumann, Adam Patrick", "degree": "PhD", "year": "2020", "title": "Towards Single Molecule Imaging Using Nanoelectromechanical Systems", "advisor": "Roukes, Michael Lee", "url": "https://resolver.caltech.edu/CaltechTHESIS:05182020-141933604", "creators": [ { "name": { "family": "Neumann", "given": "Adam Patrick" }, "id": "Neumann-Adam-Patrick", "orcid": "0000-0002-2961-7640", "display_name": "Neumann, Adam Patrick" } ], "advisors": [ { "name": { "family": "Roukes", "given": "Michael Lee" }, "id": "Roukes-M-L", "orcid": "0000-0002-2916-6026", "role": "advisor", "display_name": "Roukes, Michael Lee" } ], "committee": [ { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "chair", "display_name": "Phillips, Robert B." }, { "name": { "family": "Roukes", "given": "Michael Lee" }, "id": "Roukes-M-L", "orcid": "0000-0002-2916-6026", "role": "member", "display_name": "Roukes, Michael Lee" }, { "name": { "family": "Beauchamp", "given": "Jesse L." }, "id": "Beauchamp-J-L", "orcid": "0000-0001-8839-4822", "role": "member", "display_name": "Beauchamp, Jesse L." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Sader", "given": "John E." }, "id": "Sader-J-E", "orcid": "0000-0002-7096-0627", "role": "member", "display_name": "Sader, John E." } ], "option_major": [ "bioeng" ], "doi": "10.7907/n4ap-7h91", "abstract": "We incorporate nanoelectromechanical systems (NEMS) into a state-of-the-art commercial mass spectrometer (Q Exactive Plus with Orbitrap detection). This unique hybrid instrument is capable of ionizing molecules up to 4.5 MDa in their intact native state, isolating molecules of interest according to their mass-to-charge ratio, performing high resolution mass spectrometry (MS), and delivering those molecules to the NEMS. We use NEMS optimized for detecting the inertial mass of adsorbed species directly, which contrasts with indirect measurements of the mass-to-charge ratio performed with typical instruments. This unique form of mass spectrometry, NEMS-MS, with its single-molecule sensitivity, has promising applications to the fields of proteomics and native mass spectrometry, including deep proteomic profiling, single-cell proteomics, mass spectrometry-based imaging, or identifying viruses in their in vivo state.
\r\n\r\nWe analyze intact E. coli GroEL chaperonin, a noncovalent 801 kDa complex consisting of 14 identical subunits. GroEL was sent to NEMS operated with the first two vibrational modes monitored in real time. Molecules physisorbing to the NEMS cause an abrupt shift in its resonance frequencies. The change in resonance frequencies is used to calculate the mass of each molecule. A mass spectrum is compiled with a main peak of 846 kDa, close to the expected value, and a secondary peak resolved near twice the mass of GroEL.
\r\nMeasurements are then performed operating the first three modes simultaneously. Using a technique called inertial imaging, frequency shifts are used to calculate the first three mass moments: mass, position, and variance (size). This is used to distinguish between adsorbates arriving in a single, point-like distribution or a more extended distribution, thus demonstrating a rudimentary form of molecular imaging.
\r\n\r\nTwo new theories are presented for analyzing frequency-shift data. The first approach offers a more streamlined approach for calculating the mass moments. This approach is used to improve the mass spectrum of the GroEL calculated using three-mode data, producing a main peak almost fully resolved at 805 kDa. An entirely different approach is presented that allows for obtaining the mass density distribution of an adsorbed molecule (i.e., imaging) with a higher number of modes.
" }, { "name": "Quinodoz, Sofia Agustina", "degree": "PhD", "year": "2020", "title": "Higher-Order RNA and DNA Hubs Shape Genome Organization in the Nucleus", "advisor": "Guttman, Mitchell", "url": "http://resolver.caltech.edu/CaltechTHESIS:03132019-143409731", "creators": [ { "name": { "family": "Quinodoz", "given": "Sofia Agustina" }, "id": "Quinodoz-Sofia-Agustina", "orcid": "0000-0003-1862-5204", "display_name": "Quinodoz, Sofia Agustina" } ], "advisors": [ { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "advisor", "display_name": "Guttman, Mitchell" } ], "committee": [ { "name": { "family": "Aravin", "given": "Alexei A." }, "id": "Aravin-A-A", "orcid": "0000-0002-6956-8257", "role": "chair", "display_name": "Aravin, Alexei A." }, { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "member", "display_name": "Guttman, Mitchell" }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Plath", "given": "Kathrin" }, "id": "Plath-K", "role": "member", "display_name": "Plath, Kathrin" } ], "option_major": [ "biology" ], "doi": "10.7907/k33x-9k36", "abstract": "Although the entire genome is present within the nucleus of every cell, distinct genes need to be accessed and expressed in different cellular conditions. Accordingly, the nucleus of each cell is a highly organized arrangement of DNA, RNA, and protein that is dynamically assembled and regulated in different cellular states. These dynamic nuclear structures are largely arranged around functionally related roles and often occur across multiple chromosomes. These include large nuclear bodies (i.e., nucleolus, nuclear speckle), smaller nuclear bodies (i.e., Cajal bodies and histone locus bodies), and gene-gene interactions (i.e., transcription compartments and loops). Yet, what molecular components are involved in establishing this dynamic organization have been largely unknown due to a lack of methods to measure the RNA and DNA components of nuclear bodies and their spatial arrangements in the nucleus. Here, we present Split-Pool Recognition of Interactions by Tag Extension (SPRITE), which enables genome-wide detection of higher-order interactions within the nucleus. In the second chapter, we introduce SPRITE and recapitulate known structures identified by proximity ligation and identify additional interactions occurring across larger distances, including two hubs of inter-chromosomal interactions that are arranged around the nucleolus and nuclear speckles. We show that a substantial fraction of the genome exhibits preferential organization relative to these nuclear bodies. Our results generate a global model whereby nuclear bodies act as inter-chromosomal hubs that shape the overall packaging of DNA in the nucleus. In the third chapter, we provide a detailed experimental protocol for performing SPRITE and an automated computational pipeline for analyzing SPRITE data. Finally, in the fourth chapter, we present a dramatically improved implementation of the SPRITE method that enables comprehensive mapping of all classes of RNA in the nucleus, from abundant RNAs encoded from DNA repeats to low abundance RNAs such as nascent pre-mRNAs and lncRNAs. We find that RNAs localize broadly across the nucleus, with individual RNAs localizing within discrete territories ranging from nuclear bodies to individual topologically associated domains. We uncover that nascent mRNAs interact in structures corresponding to nascent mRNA chromosome territories and compartments. Together, these results uncover a central and widespread role for non-coding RNA in demarcating 3D nuclear structures within the nucleus.
" }, { "name": "Ravindra Kumar, Sripriya", "degree": "PhD", "year": "2020", "title": "Engineering Vectors for Non-Invasive Gene Delivery to the Central Nervous System using Multiplexed-CREATE", "advisor": "Gradinaru, Viviana", "url": "https://resolver.caltech.edu/CaltechTHESIS:05312020-230436411", "creators": [ { "name": { "family": "Ravindra Kumar", "given": "Sripriya" }, "id": "Ravindra-Kumar-Sripriya", "orcid": "0000-0001-6033-7631", "display_name": "Ravindra Kumar, Sripriya" } ], "advisors": [ { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "advisor", "display_name": "Gradinaru, Viviana" } ], "committee": [ { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "chair", "display_name": "Guttman, Mitchell" }, { "name": { "family": "Lois", "given": "Carlos" }, "id": "Lois-Carlos", "orcid": "0000-0002-7305-2317", "role": "member", "display_name": "Lois, Carlos" }, { "name": { "family": "Voorhees", "given": "Rebecca M." }, "id": "Voorhees-R-M", "orcid": "0000-0003-1640-2293", "role": "member", "display_name": "Voorhees, Rebecca M." }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "member", "display_name": "Gradinaru, Viviana" } ], "option_major": [ "biology" ], "doi": "10.7907/sgez-zn27", "abstract": "Viruses are widely modified and used as gene delivery vectors for various applications in science and therapeutics. To this end, my thesis focuses on modifying the recombinant adeno-associated viral (rAAV) vectors that are identified as a safer choice for cargo delivery compared to other known viral vectors. They are widely used in the scientific communities, have seen promising outcomes in gene therapy clinical trials, and as of today have three products approved to use in humans. However, the natural repertoire of rAAVs have broad tropism when delivered systemically, and there is room for further improvement on the efficiency and specificity, especially for gene delivery in the central nervous system (CNS). The prior work done in Dr. Gradinaru lab addresses the issue by using a directed evolution approach called CREATE, Cre recombination-based AAV targeted evolution, to identify AAV-PHP.B and AAV-PHP.eB capsids, which broadly transduce the CNS (Deverman et al, 2016; Chan et al, 2017). CREATE selects for functional lox-flipped viral DNA that crosses the blood-brain barrier (BBB) and successfully transduces a specific nerve cell-type expressing Cre, thereby applying a strong selection pressure. However, the method is limited by its ability to identify a handful of enriched variants, and may also be prone to false positives resulting from experimental biases. The effort to fully understand the selection landscape, and to select for capsids that are not just efficient towards a cell-type but also specific towards it, led to the development of Multiplexed-CREATE (M-CREATE). M-CREATE allows parallel positive selections across different cell-types of interest, enables post-hoc negative selections across off-targets using a next-generation sequencing (NGS) based capsid recovery, and retains the principles of Cre-dependent functional recovery from CREATE. The method has a synthetic library generation approach to minimize biases within selection rounds, a variant replicate feature to identify the signal versus noise within a biological system, and an analysis pipeline to group families of enriched variants based on amino acid motifs, all of which together increases the confidence in the outcome and the throughput from a single experiment. Selections across brain endothelial cells, neurons, and astrocytes yielded several AAV-PHP.B-like variants that broadly transduce the CNS, AAV-PHP.V variants that can efficiently transduce the vascular cells forming the BBB, a AAV-PHP.N variant that transduces neurons with greater specificity, and AAV-PHP.C variants that cross the BBB without murine strain specificity across tested strains. The AAV-PHP.C variants have different amino acid motifs compared to the AAV-PHP.Bs that have been previously shown to have limited CNS transduction across some mouse strains due to its interaction with the strain specific host cell surface receptor, ly6a, a homolog of which is not found in humans. (Hordeaux et al, 2018, Hordeaux et al, 2019; Huang et al, 2019; Batista et al, 2019) Therefore AAV-PHP.Cs offer some hope towards translation across other species. In summary, the M-CREATE methodology turns out to be a high-confidence, robust selection platform to yield several novel viral capsids for use in neuroscience and potential gene therapy related applications.
" }, { "name": "Reichermeier, Kurt Michael", "degree": "PhD", "year": "2020", "title": "Quantitative Characterization of Composition and Regulation of Cullin-RING Ubiquitin Ligases", "advisor": "Deshaies, Raymond Joseph; Zinn, Kai George", "url": "https://resolver.caltech.edu/CaltechTHESIS:12122019-091017133", "creators": [ { "name": { "family": "Reichermeier", "given": "Kurt Michael" }, "id": "Reichermeier-Kurt-Michael", "orcid": "0000-0003-0613-690", "display_name": "Reichermeier, Kurt Michael" } ], "advisors": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "orcid": "0000-0002-3671-9354", "role": "advisor", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "advisor", "display_name": "Zinn, Kai George" } ], "committee": [ { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "chair", "display_name": "Chan, David C." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "orcid": "0000-0002-3671-9354", "role": "member", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Shan", "given": "Shu-ou" }, "id": "Shan-Shu-ou", "orcid": "0000-0002-6526-1733", "role": "member", "display_name": "Shan, Shu-ou" }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "member", "display_name": "Zinn, Kai George" } ], "option_major": [ "biology" ], "doi": "10.7907/0WX7-2Q56", "abstract": "Induced proteolysis of pathogenic proteins via degrader molecules, such as Proteolysis Targeting Chimeras (PROTACs), is emerging as a promising therapeutic strategy. In particular, induced proximity of Cullin-RING ubiquitin Ligases (CRLs) with various neo-substrates has proven successful in mediating proteasomal degradation of previously undruggable proteins. Hijacking enzymes to carry out biochemical reactions on neo-substrates stands in stark contrast to conventional pharmacological approaches and exposes degrader molecules to unusually complex pharmacodynamics. While the first PROTACs entered the clinic in 2019, much about the organization and regulation of the frequently co-opted CRLs remains elusive. In particular, the COP9 Signalosome (CSN) is essential to regulate CRL activity and assembly through cleaving Nedd8 from cullin scaffolds, yet it remains unknown how CSN becomes activated. We combine structural and kinetic analyses to identify mechanisms that contribute to CSN activation and Nedd8 deconjugation, detailing the kinetic picture of the deneddylation-disassembly cycle that promotes rapid remodeling of the cellular CRL network. Furthermore, we establish Protein Interaction Kinetics and Estimation of Stoichiometries (PIKES) analysis, a systematic proteomic profiling platform that integrates cellular engineering, affinity purification, chemical stabilization and quantitative mass spectrometry to investigate the dynamics of interchangeable multiprotein complexes. Using PIKES, we show that ligase assemblies of Cullin4 with individual substrate receptors differ in abundance by up to 200-fold and that Cand1 acts as an exchange factor to remodel the CRL4 ligase pool. Integrating quantitative data and model simulations of CRL-mediated substrate turnover, we show that high substrate receptor levels can enhance the potency of degraders. " }, { "name": "Saunders, Scott Harrison", "degree": "PhD", "year": "2020", "title": "Mechanisms of Phenazine-Mediated Extracellular Electron Transfer by Pseudomonas aeruginosa", "advisor": "Newman, Dianne K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04022020-212557295", "creators": [ { "name": { "family": "Saunders", "given": "Scott Harrison" }, "id": "Saunders-Scott-Harrison", "orcid": "0000-0003-4224-9106", "display_name": "Saunders, Scott Harrison" } ], "advisors": [ { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "advisor", "display_name": "Newman, Dianne K." } ], "committee": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "chair", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Barton", "given": "Jacqueline K." }, "id": "Barton-J-K", "orcid": "0000-0001-9883-1600", "role": "member", "display_name": "Barton, Jacqueline K." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." } ], "option_major": [ "microbio" ], "doi": "10.7907/P4Z5-5445", "abstract": "Extracellular electron transfer (EET), the process whereby cells access electron acceptors or donors that reside many cell lengths away, enables metabolic activity by microorganisms, particularly under oxidant-limited conditions that occur in multicellular bacterial biofilms. Although different mechanisms underpin this process in individual organisms, a potentially widespread strategy involves extracellular electron shuttles, redox-active metabolites that are secreted and recycled by diverse bacteria. Here, I first review general aspects of the electron shuttling strategy, such as the chemical diversity and potential distribution of electron shuttle producers and users, and the costs associated with electron shuttle biosynthesis. Then I address the long-standing question: how do these electron shuttles catalyze electron transfer within biofilms without being lost to the environment? I show that phenazine electron shuttles mediate efficient EET through interactions with extracellular DNA (eDNA) in Pseudomonas aeruginosa biofilms, which are important in nature and disease. Retention of pyocyanin (PYO) and phenazine carboxamide in the biofilm matrix is facilitated by binding to eDNA. In vitro, different phenazines can exchange electrons in the presence or absence of DNA and phenazines can participate directly in redox reactions through DNA; the biofilm eDNA can also support rapid electron transfer between redox-active intercalators. Electrochemical measurements of biofilms indicate that retained PYO supports an efficient redox cycle with rapid EET and slow loss from the biofilm. Together, these results establish that eDNA facilitates phenazine metabolic processes in P. aeruginosa biofilms, suggesting a model for how extracellular electron shuttles achieve retention and efficient EET in biofilms.
" }, { "name": "Thompson, John Warren Lenzi", "degree": "PhD", "year": "2020", "title": "Chemical Tools for Studying O-GlcNAc Glycosylation at the Systems Level", "advisor": "Hsieh-Wilson, Linda C.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06112020-001257003", "creators": [ { "name": { "family": "Thompson", "given": "John Warren Lenzi" }, "id": "Thompson-John-Warren-Lenzi", "orcid": "0000-0003-0061-4996", "display_name": "Thompson, John Warren Lenzi" } ], "advisors": [ { "name": { "family": "Hsieh-Wilson", "given": "Linda C." }, "id": "Hsieh-Wilson-L-C", "orcid": "0000-0001-5661-1714", "role": "advisor", "display_name": "Hsieh-Wilson, Linda C." } ], "committee": [ { "name": { "family": "Hoelz", "given": "Andre" }, "id": "Hoelz-A", "orcid": "0000-0003-0923-3284", "role": "chair", "display_name": "Hoelz, Andre" }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "member", "display_name": "Chan, David C." }, { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "member", "display_name": "Pachter, Lior S." }, { "name": { "family": "Hsieh-Wilson", "given": "Linda C." }, "id": "Hsieh-Wilson-L-C", "orcid": "0000-0001-5661-1714", "role": "member", "display_name": "Hsieh-Wilson, Linda C." } ], "option_major": [ "chemistry" ], "doi": "10.7907/gx3z-k069", "abstract": "The addition of O-linked \u03b2-N-acetylglucosamine (O-GlcNAc) to intracellular serine and threonine residues is a ubiquitous post-translational modification (PTM) found in all higher eukaryotes. Like other PTMs, it is finely regulated in response to stimuli and dysregulated in multiple diseases. However, unlike other PTMs, methods to detect and profile the dynamics of O-GlcNAc glycosylation are still in their infancy. Herein, we discuss the background, development, and application of new chemical tools that have allowed for some of the first systems-level investigations of O-GlcNAcylation in different cells, organ systems, and disease states. We also significantly advance established techniques for the detection and monitoring of O-GlcNAc on proteins of interest. Using these new techniques, we first uncover a novel O-GlcNAcylation site on Cdk5 and show that this site can dynamically regulate Cdk5 activity in the context of neurodegenerative disease. Next, we apply novel chemical, mass spectrometric, and computational tools to, for the first time, uncover cellular networks engaged by O-GlcNAcylation in vivo. Finally, we undertake the systematic optimization of mass spectrometry based O-GlcNAcomics and use these new insights to significantly advance our understanding of O-GlcNAcylation dynamics in metabolic diseases of the liver. Overall, the techniques developed and data generated herein are closing the methodological and intellectual gaps between the study of O-GlcNAc glycosylation and that of other PTMs.
" }, { "name": "Yoo, Bryan B.", "degree": "PhD", "year": "2020", "title": "Host-Microbe Interactions Impacting and Mediated by Nervous Systems", "advisor": "Mazmanian, Sarkis K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04282020-145500248", "creators": [ { "name": { "family": "Yoo", "given": "Bryan B." }, "id": "Yoo-Bryan-B", "orcid": "0000-0003-1450-2696", "display_name": "Yoo, Bryan B." } ], "advisors": [ { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "advisor", "display_name": "Mazmanian, Sarkis K." } ], "committee": [ { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "member", "display_name": "Mazmanian, Sarkis K." }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "role": "chair", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." } ], "option_major": [ "biology" ], "doi": "10.7907/1zv5-ve82", "abstract": "Animals and microbes coevolved, and thus it is not surprising that the trillions of microorganisms that harmoniously inhabit the mammalian gastrointestinal tract (GIT), collectively termed the gut microbiome, continue to be implicated in healthy and disease states. However, less is known about the mechanisms by which these states are maintained, and how deviations from homeostasis (i.e., dysbiosis) occurr. This thesis explores the relationship between host-microbe interactions and the central and peripheral nervous systems. Specifically, the first chapter of this thesis explores how the microbiome differs is patients with multiple sclerosis and how these differences alter diseases outcomes in a mouse model of the disease. Next, we introduce the enteric nervous system (ENS), the intrinsic nervous system of the GI tract which is supposed as a major conduit of the bidirectional communication between the gut and the brain. Lastly, by adopting biotechnologies in gene delivery and genetically encoded tools for neuroscience, we introduce a molecular toolkit to characterize the ENS in a robust and efficient manner and modulate the ENS to uncover novel mechanisms by which innervation of the GI mediates host-microbe interactions." }, { "name": "Zhang, Hanwen", "degree": "Senior Thesis", "year": "2020", "title": "Role of the Ventral CA1 to Primary Auditory Cortex Projection in Associative Learning", "advisor": "Siapas, Athanassios G.; Cassenaer, Stijn", "url": "https://resolver.caltech.edu/CaltechTHESIS:06112020-153709447", "creators": [ { "name": { "family": "Zhang", "given": "Hanwen" }, "id": "Zhang-Hanwen", "orcid": "0000-0001-7609-2122", "display_name": "Zhang, Hanwen" } ], "advisors": [ { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "orcid": "0000-0001-8837-678X", "role": "advisor", "display_name": "Siapas, Athanassios G." }, { "name": { "family": "Cassenaer", "given": "Stijn" }, "id": "Cassenaer-S", "role": "co-advisor", "display_name": "Cassenaer, Stijn" } ], "committee": [ { "name": { "family": "None", "given": "None" }, "display_name": "None, None" } ], "option_major": [ "cns" ], "abstract": "As we understand more about individual brain regions, more studies begin to focus on how different regions communicate with each other. Having long been established as the center for learning and memory, the hippocampus has connections to most of neocortex as well as many subcortical regions. Some recent findings revealed that the hippocampus not only receive input from primary sensory cortices but also provide feedback projections to these areas. Do these projections play a role in sensory associative learning? Our project focused on the role of hippocampus to primary auditory cortex (A1) projections in auditory associative learning. Optogenetics was used to study the function of a direct projection from the ventral CA1 region of the hippocampus to primary auditory cortex (A1) in an auditory go/no-go task using head-fixed mice. Preliminary results show that activation of this projection does not affect task acquisition or generalization. Our next step is to investigate the effect of inhibiting the CA1 to A1 projections on auditory associative learning." }, { "name": "Zocchi, Dhruv Sergio", "degree": "PhD", "year": "2020", "title": "Processing at Primary Chemosensory Neurons", "advisor": "Hong, Elizabeth J.; Oka, Yuki", "url": "https://resolver.caltech.edu/CaltechTHESIS:05032020-101047008", "creators": [ { "name": { "family": "Zocchi", "given": "Dhruv Sergio" }, "id": "Zocchi-Dhruv-Sergio", "display_name": "Zocchi, Dhruv Sergio" } ], "advisors": [ { "name": { "family": "Hong", "given": "Elizabeth J." }, "id": "Hong-Elizabeth-J", "role": "advisor", "display_name": "Hong, Elizabeth J." }, { "name": { "family": "Oka", "given": "Yuki" }, "id": "Oka-Yuki", "orcid": "0000-0003-2686-0677", "role": "advisor", "display_name": "Oka, Yuki" } ], "committee": [ { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "orcid": "0000-0003-2136-6506", "role": "chair", "display_name": "Meister, Markus" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "co-chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "orcid": "0000-0002-8587-9936", "role": "member", "display_name": "Dickinson, Michael H." }, { "name": { "family": "Hong", "given": "Elizabeth J." }, "id": "Hong-Elizabeth-J", "role": "member", "display_name": "Hong, Elizabeth J." }, { "name": { "family": "Oka", "given": "Yuki" }, "id": "Oka-Yuki", "orcid": "0000-0003-2686-0677", "role": "member", "display_name": "Oka, Yuki" } ], "option_major": [ "neurobio" ], "doi": "10.7907/yshd-3195", "abstract": "Chemosensory perception involves the detection of chemical compounds. In animals, there are 2 chemical senses: taste, and olfaction. The two are related in that they utilize ligand-gated receptors, expressed in primary sensory neurons, to detect chemical stimuli from the surrounding environment. However, the processing of these inputs is quite different in the two systems, leading to divergent roles for olfaction and taste in sensory perception. This dissertation highlights some of these differences, by looking at processing of ethologically relevant stimuli at the very peripheral receptor neurons. The work is divided into 2 parts: water sensing by the mammalian taste system, and CO\u2082 sensing by the Drosophila olfactory system.
\r\n\r\nIn Chapter 1, I talk about water sensing in the mammalian taste system. Initiation of drinking behavior relies on peripheral water detection. It is likely that this detection is mediated, at least in part, by the taste system. Here, I have shown that acid-sensing taste receptor cells (TRCs) that were previously suggested as the sensors for sour taste, also respond to water. This response is mediated by a bicarbonate-dependent molecular mechanism, likely involving the Carbonic Anhydrase enzyme family. Furthermore, optogenetic stimulation of the acid-sensing TRCs in thirsty animals induces robust licking responses towards the light source, even in the absence of water. Conversely, thirsty animals lacking functional acid-sensing TRCs show compromised discrimination between water and non-aqueous fluids. Taken together, this work reveals the cellular mechanism of water detection by the mammalian taste system.
\r\n\r\nIn chapter 2, I talk about CO\u2082 sensing in the fruit fly. The Drosophila olfactory system responds to most odors with the activation of a large subset of its olfactory receptors (ORs). This broad activation is a consequence of the ORs having affinity to multiple chemical compounds. In contrast, a small number of odors, like CO\u2082, elicit responses in only single ORs. These ORs are, in contrast to most ORs, very narrowly tuned, generally responding only to that one odor. It has been assumed up until now that the specificity of these unique ORs is inherited by the olfactory receptor neurons (ORNs) they are expressed in, and even in the projection neurons (PNs), that the ORNs synapse onto. I show here that CO\u2082, though it activates only a single OR, the GR63a/GR21a hetero-dimer complex, actually activates multiple ORN axon terminals. This activation is due to lateral excitatory connections between axon terminals of the GR63a/GR21a expressing ORNs, and at least 4 other ORN types. Focusing on one of these ORNs, Ab1B, I show the lateral connections bypass the ORN cell bodies, only driving responses at the axon terminals. Consequently, Ab1B ORN axon terminals receive 2 sources of excitatory input, a feed-forward excitation from its endogenous OR, and a lateral excitation from GR63a/GR21a. This effectively divides the ORN into 2 compartments, distinct in their odor tuning. Finally, I show that lateral excitation is a general feature of the ORN circuit by silencing the feed-forward input of another ORN class, Ab1A. The Ab1A cell body is completely silent, but the axon terminals retain odor responses from lateral excitatory inputs. Thus, there is a lateral flow of odor information between multiple ORNs of the Drosophila olfactory system.
" }, { "name": "Angeles-Albores, David", "degree": "PhD", "year": "2019", "title": "A Theory of Genetic Analysis Using Transcriptomic Phenotypes", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10232018-150005837", "creators": [ { "name": { "family": "Angeles-Albores", "given": "David" }, "id": "Angeles-Albores-David", "orcid": "0000-0001-5497-8264", "display_name": "Angeles-Albores, David" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "chair", "display_name": "Newman, Dianne K." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biochem" ], "doi": "10.7907/JRNS-NS05", "abstract": "This thesis deals with the conceptual and computational framework required to use transcriptomes as effective phenotypes for genetic analysis. I demonstrate that there are powerful theoretical reasons why Batesonian epistasis should feature prominently in transcriptional phenotypes. I also show how to compute and interpret the aggregate statistics for transcriptome-wide epistasis and transcriptome-wide dominance using whole-organism transcriptomic profiles of C. elegans mutants. Finally, I developed the WormBase Enrichment Suite for enrichment analysis of genomic data.
\r\n\r\nRNA-seq as a tool has enormous potential because it relies on protocols that are fast, simple and increasingly cheap. In spite of their potential, transcriptomes have seen their use largely limited to single-factor experiments. Even when many transcriptomes are collected, the main analytic approach is to apply clustering algorithms that correlate responses but do not have any power to identify causal mechanisms.
\r\n\r\nI demonstrate that if a complete genetic experimental design is used (in the form of a full two-factor matrix), transcriptomes can establish genetic interactions between a pair of genes without the need for clustering algorithms. Surprisingly, when we performed epistasis analyses of hypoxia pathway mutants in C. elegans we did not simply observe a generalized epistatic interaction between the mutants. In fact, the transcriptomes recapitulated the same Batesonian epistatic relationship that had been observed using classical phenotypes. In other words, we observed that the transcriptomic phenotype of one gene can be masked by the transcriptomic phenotype of a second gene, such that a double mutant of these two genes has exactly the same phenotype as a single mutant of the epistatic gene. Motivated by this observation, we developed methods to recognize and interpret Batesonian epistasis at the transcriptomic level. This method relies on the calculation of a single aggregate coefficient that we named the transcriptome-wide epistasis coefficient.
\r\n\r\nThe observation that Batesonian epistasis could be reproduced on a transcriptomic level was surprising. To explain how transcriptome-wide epistasis can arise, I studied a simplified model of transcriptional regulation using statistical mechanics. These studies demonstrate that epistatic analysis is equivalent to a perturbative analysis of the partition function of a promoter. Moreover, these studies revealed that a sufficient condition for Batesonian epistasis to occur is if the two genes encode variables that are transformed and multiplied together to form an effective single compound variable. Finally, these studies clearly demonstrate the connection between statistical (or generalized) epistasis and Batesonian epistasis and establish a physical basis for genetic logic.
\r\n\r\nGenetic analyses of gene functional units can also be carried out using allelic series in tandem with complementation (also known as dominance) tests. I developed a statistical coefficient known as transcriptome-wide dominance to enable analyses of allelic series using expression profiles. A crucial aspect of allelic series is the ability to enumerate the independent phenotypes associated with an arbitrary set of alleles. I developed the concept of phenotypic classes as a transcriptomic analogue of classical phenotypes for this purpose. Briefly, a phenotypic class is a set of transcripts that are differentially expressed in a specific set of genotypes. Thus, an allelic series consisting of two mutant alleles (and a wild-type) can at most result in 7 phenotypic classes. However, some of these phenotypic classes may be artifactual as a result of the significant false positive and false negative rates that are associated with RNA-seq. I developed a simple algorithm that tries to identify phenotypic classes that are artifactual, though often these classes may also be identified through a critical evaluation of their biological implications. I applied these concepts to a small allelic series of the dpy-22 gene, which encodes a Mediator subunit in C. elegans, and identified 3\u20134 functional units along with their sequence requirements.
\r\n\r\nFinally, I developed the WormBase Enrichment Suite by implementing a hypergeometric test on the tissue, gene and phenotype ontology for C. elegans. The importance of this tool derives mainly from its integration to WormBase, the repository of all C. elegans knowledge, which means that the databases that are tested will undergo continuous improvement and curation, and thus will yield the most accurate results.
" }, { "name": "Augustine, Vineet", "degree": "PhD", "year": "2019", "title": "Neural Architecture Underlying Thirst Regulation", "advisor": "Oka, Yuki", "url": "https://resolver.caltech.edu/CaltechThesis:06042019-143921851", "creators": [ { "name": { "family": "Augustine", "given": "Vineet" }, "id": "Augustine-Vineet", "orcid": "0000-0003-4431-1663", "display_name": "Augustine, Vineet" } ], "advisors": [ { "name": { "family": "Oka", "given": "Yuki" }, "id": "Oka-Yuki", "orcid": "0000-0003-2686-0677", "role": "advisor", "display_name": "Oka, Yuki" } ], "committee": [ { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "orcid": "0000-0001-8837-678X", "role": "chair", "display_name": "Siapas, Athanassios G." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "member", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Oka", "given": "Yuki" }, "id": "Oka-Yuki", "orcid": "0000-0003-2686-0677", "role": "member", "display_name": "Oka, Yuki" } ], "option_major": [ "cns" ], "doi": "10.7907/1H4D-7146", "abstract": "An important aspect of thirst is its quick quenching. When thirsty, you drink a glass of water for a few seconds; the water travels from the mouth to the stomach and you are satiated. The water has not yet been absorbed into the blood, so the brain needs to have mechanisms to signal stopping of drinking. It cannot simply depend on the body, as the body takes a good 15 - 30 minutes to even start absorption.
\r\n\r\nIn this dissertation, I describe dynamic thirst circuits that integrate the homeostatic-instinctive requirement for fluids, the consequent drinking behavior, and reward processing to maintain internal water balance.
\r\n\r\nIn Chapter 1, I show how neural populations in the lamina terminalis, a forebrain structure, form a hierarchical circuit architecture to regulate thirst. Among them, excitatory neurons in the median preoptic nucleus (MnPO) are essential for the integration of signals from the thirst-driving neurons of the subfornical organ (SFO). Thirst-driving neurons in the SFO receive temporarily distinct preabsorptive inhibition by drinking action and gastrointestinal osmolality sensing. A distinct inhibitory circuit, involving MnPO GABAergic neurons that express glucagon-like peptide 1 receptor (GLP1R), is activated immediately upon drinking and monosynaptically inhibits SFO thirst neurons. These responses are induced by the ingestion of fluids but not solids, and are time-locked to the onset and offset of drinking. Furthermore, loss-of-function manipulations of these neurons lead to a polydipsic, overdrinking phenotype. These neurons therefore facilitate rapid satiety of thirst by monitoring real-time fluid ingestion.
\r\n\r\nIn Chapters 2 and 3, I talk about how thirst triggers a strong motivational state that drives animals toward drinking behavior. The consequent fluid intake provides both satiation and pleasure of drinking to animals. However, how these two factors are processed and represented by the brain remains poorly understood. Here I will use in vivo optical recording, genetics, and intragastric infusion approaches to dissect thirst satiation circuits and their contribution to reward signals. Thirst-driving neurons in the subfornical organ (SFO) receive multiple temporally-distinct satiation signals prior to the homeostatic recovery: oropharyngeal stimuli induced by drinking action and gastrointestinal sensing of osmolality changes. In chapter 1, I have shown that drinking action is represented by inhibitory neurons in the median preoptic nucleus (MnPO). Here, I demonstrate that gut osmolality signals are mediated by specific GABAergic neurons in the SFO. These neurons were selectively activated by hypo-osmotic stimuli in the gut independent of drinking action. Optogenetic gain- and loss-of-function of this inhibitory population suppressed and increased water intake in thirsty animals, respectively. These results indicate that oropharyngeal- and gastrointestinal-driven satiation signals are transmitted to thirst neurons through different neural pathways. Furthermore, I investigated the contribution of thirst satiation signals to the reward circuit using a genetically-encoded ultrafast dopamine (DA) sensor. Interestingly, oral ingestion but not gut osmolality changes triggered robust DA release. Importantly, chemogenetic activation of thirst-quenching neurons did not induce DA release in water-deprived animals. Together, this dissected genetically-defined thirst satiation circuits, the activity of which are functionally separable from reward-related brain activity. Taken together, these finding provide answers to some longstanding questions in the neural control of fluid intake, and appetite in general.
\r\n" }, { "name": "Basta, David Wagdi", "degree": "PhD", "year": "2019", "title": "Genetic Determinants of Growth Arrest Survival in the Bacterial Pathogen Pseudomonas aeruginosa and the Role of Proteases", "advisor": "Newman, Dianne K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05282019-090132145", "creators": [ { "name": { "family": "Basta", "given": "David Wagdi" }, "id": "Basta-David-Wagdi", "orcid": "0000-0003-4176-6566", "display_name": "Basta, David Wagdi" } ], "advisors": [ { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "advisor", "display_name": "Newman, Dianne K." } ], "committee": [ { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "orcid": "0000-0003-2713-1513", "role": "chair", "display_name": "Mazmanian, Sarkis K." }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "member", "display_name": "Chan, David C." }, { "name": { "family": "Varshavsky", "given": "Alexander J." }, "id": "Varshavsky-A-J", "orcid": "0000-0002-4011-258X", "role": "member", "display_name": "Varshavsky, Alexander J." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." } ], "option_major": [ "microbio" ], "doi": "10.7907/K6X1-GS91", "abstract": "Growth arrest is the dominant mode of microbial existence on the planet, yet the molecular mechanisms that underpin survival during growth arrest remain far less studied than other growth states. A better understanding of these mechanisms would provide valuable insight into the activity of microbial communities in both biogeochemical and clinical contexts, including the treatment of chronic infections. This thesis investigates the genetic requirements for survival of the bacterium Pseudomonas aeruginosa, a metabolically versatile opportunistic pathogen that thrives in diverse environments in which growth arrest is often caused by energy limitation. After reviewing our current knowledge of the strategies used by growth-arrested bacteria to adjust metabolism, regulate transcription and translation, and maintain the chromosome, I perform a functional genomic screen to identify genes that promote fitness of P. aeruginosa during growth arrest caused by carbon or oxygen starvation. I find that P. aeruginosa can survive for days to weeks in these energy-starved conditions by maintaining a reduced steady-state level of ATP, and that many functional classes of genes are required for fitness. Intriguingly, a majority of genetic fitness determinants differ between carbon and oxygen starvation, despite the common endpoint of reduced ATP levels in these two conditions. Among the few genes generally required for fitness are the stress response sigma factor encoded by rpoS and the heat shock protease encoded by ftsH. Using independently-generated deletion strains, I show that mutants in distinct functional categories exhibit temporal fitness dynamics during oxygen starvation: regulatory genes generally manifest a phenotype early during growth arrest, whereas genes involved in cell wall metabolism are required later. Building on these findings, I investigate the functional role of FtsH during growth arrest more deeply and find a surprising negative genetic interaction between ftsH and rpoS, with mutations in rpoS alleviating the fitness defects of \u0394ftsH during growth arrest. I also find that FtsH functions coordinately with the other conserved heat shock proteases to maintain cellular integrity and delay aging of P. aeruginosa during growth arrest. Finally, I investigate the role of FtsH and the other heat shock proteases in a novel N-terminal protein degradation pathway and find that the molecular details of this pathway likely differ between E. coli and P. aeruginosa. Together, these findings uncover essential molecular processes that promote fitness of an important bacterial pathogen during growth and survival.
" }, { "name": "Cho, Jounhong Ryan", "degree": "PhD", "year": "2019", "title": "Optical Imaging of Dopamine Dynamics and Decoding its Role in Arousal and Salience", "advisor": "Gradinaru, Viviana", "url": "https://resolver.caltech.edu/CaltechTHESIS:05302019-104701860", "creators": [ { "name": { "family": "Cho", "given": "Jounhong Ryan" }, "id": "Cho-Jounhong-Ryan", "orcid": "0000-0001-9542-716X", "display_name": "Cho, Jounhong Ryan" } ], "advisors": [ { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "advisor", "display_name": "Gradinaru, Viviana" } ], "committee": [ { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "orcid": "0000-0001-8837-678X", "role": "chair", "display_name": "Siapas, Athanassios G." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Oka", "given": "Yuki" }, "id": "Oka-Yuki", "orcid": "0000-0003-2686-0677", "role": "member", "display_name": "Oka, Yuki" }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "member", "display_name": "Gradinaru, Viviana" } ], "option_major": [ "cns" ], "doi": "10.7907/TNGA-5X08", "abstract": "Dopamine (DA) is a key neuromodulator in the brain that can exert a profound impact on brain physiology and cognitive functions. There is consensus that DA plays critical roles in reward prediction error, reinforcement learning, and motor control, and that dysregulation of DA signaling is common in many neuropsychiatric diseases, such as Parkinson\u2019s disease, drug addiction, and depression. Although the tools to study the functional roles of DA have considerably expanded with novel genetic tools and optical imaging methods, we are still limited in our ability to record or visualize DA release in vivo with long-term stability and high spatiotemporal resolution. This is an unmet need in DA research, as DA release at the post-synaptic sites can be decoupled from DA cell body firing due to local circuit interaction and influence from other afferent activities. In parallel, there is growing evidence that DA is functionally heterogeneous beyond its classically described roles for reward and movement, based on its anatomical location, projection target, electrophysiological properties, and response patterns to stimuli with motivational valence. Pharmacological and genetic studies have provided indirect evidence that DA can promote strong behavioral arousal and signal salience, but the precise neural substrates for these functions remain largely unknown. Towards this end, my thesis work has been focused on 1) developing and characterizing optical tools to visualize DA release in vivo and 2) utilizing such optical and genetic tools to study the overlooked, sparse DA populations in the dorsal midbrain, demonstrating that they are functionally unique DA cells for broadcasting arousal and salience signals to the forebrain targets.
\r\n\r\nAs neuromodulatory systems exert profound influences on brain function, understanding how these systems modify the operating mode of target circuits requires spatiotemporally precise measurement of neuromodulator release. Towards this goal, in Chapter II, my colleagues and I developed dLight1, an intensity-based genetically encoded DA indicator, to enable optical recording of DA dynamics with high spatiotemporal resolution in behaving mice. We demonstrated the utility of dLight1 by imaging DA dynamics simultaneously with pharmacological manipulation, electrophysiological or optogenetic stimulation, and calcium imaging of local neuronal activity. dLight1 enabled chronic tracking of learning-induced changes in millisecond DA transients in mouse striatum. Further, we used dLight1 to image spatially distinct, functionally heterogeneous DA transients relevant to learning and motor control in mouse cortex. We also validated our sensor design platform for developing norepinephrine, serotonin, melatonin, and opioid neuropeptide indicators. Together, this tool provides a unique opportunity to optically monitor DA release dynamics in vivo with long-term stability and unprecedented spatiotemporal resolution.
\r\n\r\nIn Chapter III, I have characterized the functional roles of sparse DA populations in the dorsal raphe nucleus (DRN) and discovered that these neurons play key roles in promoting behavioral arousal. I first demonstrated that DRNDA neurons are activated by diverse forms of motivationally salient stimuli, irrespective of valence. Simultaneous fiber photometry and polysomnographic recordings showed that DRNDA neuronal activity is correlated with distinct sleep-wake states, showing highest activities during wakefulness over sleep states. Optogenetic activation of DRNDA neurons was sufficient to cause immediate sleep-to-wake transitions and promote longer wakefulness upon sustained stimulation. On contrary, DRNDA inhibition via chemogenetics reduced wakefulness and promoted non-rapid eye movement sleep, even in the presence of ethologically relevant salient stimuli. Taken together, this pinpoints DRNDA neurons as the critical contributor of arousal-promoting DA system in the brain.
\r\n\r\nIn Chapter IV, I further characterized the encoding dynamics of DRNDA neurons during classical conditioning tasks where mice learned the association between neutral cues and outcomes with positive or negative outcomes. DRNDA neurons developed phasic, positive responses to cues predicting both positive and negative unconditioned stimuli across learning, suggesting that these populations track motivational salience rather than valence. In addition, DRNDA neurons encoded unsigned prediction error, demonstrating higher neuronal activity to unexpected reward or punishment over fully expected outcomes. Collectively with Chapter III, these results expand on the existing literature on functionally heterogeneous roles of DA in the brain and propose that DRNDA neurons play critical roles in signaling arousal and motivational salience to the forebrain regions to coordinate appropriate behavior, depending on the nature of environmental stimuli.
" }, { "name": "Flytzanis, Nicholas C.", "degree": "PhD", "year": "2019", "title": "From Single-Cell to Whole-Body: Developing a Molecular Neuroscience Toolkit", "advisor": "Gradinaru, Viviana", "url": "https://resolver.caltech.edu/CaltechTHESIS:10122018-165154970", "creators": [ { "name": { "family": "Flytzanis", "given": "Nicholas C." }, "id": "Flytzanis-Nicholas-C", "orcid": "0000-0002-7921-9392", "display_name": "Flytzanis, Nicholas C." } ], "advisors": [ { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "advisor", "display_name": "Gradinaru, Viviana" } ], "committee": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "chair", "display_name": "Anderson, David J." }, { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "member", "display_name": "Arnold, Frances Hamilton" }, { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "member", "display_name": "Shapiro, Mikhail G." }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "member", "display_name": "Gradinaru, Viviana" } ], "option_major": [ "biology" ], "doi": "10.7907/S28C-DJ17", "abstract": "Throughout my Ph.D. I have worked on technology development, at first to answer basic scientific questions and eventually for therapeutic applications. This technology development applied to a variety of fields, from neuroscience to development to gene therapy, and acted upon biological systems in a wide range of scale, from the single-cell monitoring to organism-wide gene-transfer. My graduate research began with the engineering of microbial rhodopsin spectral properties and fluorescence. By making use of their ability to absorb light and emit fluorescence in a voltage-dependent manner, I aimed to interrogate neuronal activity during behavior at the single-cell level. That line of research ended with publication of the voltage-sensor Archer, which I used to track activity of a single cell in vivo in awake, behaving worms. I then shifted from tracking activity at the single cell level, to visualizing entire organisms, by developing clearing techniques that enable a high-resolution, three-dimensional analysis of a diverse range of tissues. I began by optimizing tissue-clearing parameters for various tissue types and a wide variety of experimental needs. I then took that knowledge and applied it to visualizing and tracking the developing neural crest in cleared, whole-mount chicken embryos, discovering some unexpected derivates. Finally, I became interested not only in visualizing entire organisms, but in developing technologies to facilitate gene transfer throughout the body. The rapidly growing field of gene therapy is in constant need of new tools that target specific tissues, avoiding off-target effects. The end of my Ph.D. has been spent engineering viruses that can be delivered body-wide, but target only specific areas of therapeutic interest, like the brain and lungs.
" }, { "name": "Fowler, Trevor Michael", "degree": "PhD", "year": "2019", "title": "Silicon Neural Probes for Stimulation of Neurons and the Excitation and Detection of Proteins in the Brain", "advisor": "Roukes, Michael Lee", "url": "https://resolver.caltech.edu/CaltechTHESIS:09172018-140652131", "creators": [ { "name": { "family": "Fowler", "given": "Trevor Michael" }, "id": "Fowler-Trevor-Michael", "display_name": "Fowler, Trevor Michael" } ], "advisors": [ { "name": { "family": "Roukes", "given": "Michael Lee" }, "id": "Roukes-M-L", "orcid": "0000-0002-2916-6026", "role": "advisor", "display_name": "Roukes, Michael Lee" } ], "committee": [ { "name": { "family": "Faraon", "given": "Andrei" }, "id": "Faraon-A", "orcid": "0000-0002-8141-391X", "role": "chair", "display_name": "Faraon, Andrei" }, { "name": { "family": "Roukes", "given": "Michael Lee" }, "id": "Roukes-M-L", "orcid": "0000-0002-2916-6026", "role": "member", "display_name": "Roukes, Michael Lee" }, { "name": { "family": "Yang", "given": "Changhuei" }, "id": "Yang-Changhuei", "orcid": "0000-0001-8791-0354", "role": "member", "display_name": "Yang, Changhuei" }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Moreaux", "given": "Laurent C." }, "id": "Moreaux-Laurent-C", "orcid": "0000-0003-1276-5062", "role": "member", "display_name": "Moreaux, Laurent C." } ], "option_major": [ "bioeng" ], "doi": "10.7907/2kvw-ad56", "abstract": "This thesis describes the development of a number of novel microfabricated neural probes for a variety of specific neuroscience applications. These devices rely on single mode waveguides and grating couplers constructed from silicon nitride thin films, which allows the use of planar lightwave circuits to create advanced device geometries and functions. These probes utilize array waveguide gratings to select an individual emitter from a large array of emitters using the wavelength of incoming light, allowing for spatial multiplexing of optical stimulation. These devices were tested in the laboratory and in living tissue to verify their efficacy. This technology was then modified to create steerable beam forming for stimulation of neurons using optical phase arrays. This technology was also tested for use in fluoresence lifetime imaging microscopy and the first application of pulsed light through the photonic circuits. Finally, this technology was again modified to create laminar illumination patterns for light sheet fluorescence microscopy applications. These devices were further improved by adding embedded microfluidics to the probes. The process of creating embedded microfluidic channels by the dig and seal method is described in detail, including modifications to the procedure that were added to address potential pitfalls in the fabrication process. Next, two projects which combine microfluidics with the optical devices described in the previous chapter are detailed. One project involves combining the use of optical emitters with microfluidic injections containing caged neurotransmitters to stimulate neurons is described. The other project involves microfluidic sampling of the extracellular space for neuropeptides which are detected using ring resonator biosensors. The sensitivity of these biosensors was analyzed in detail, determining both the physical limit of detection and the effect of biological noise due to non-specific binding on the sensors." }, { "name": "Frankiw, Luke Steven", "degree": "PhD", "year": "2019", "title": "mRNA Splicing-Mediated Gene Expression Regulation in Innate Immunity", "advisor": "Baltimore, David L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06052019-122355847", "creators": [ { "name": { "family": "Frankiw", "given": "Luke Steven" }, "id": "Frankiw-Luke-Steven", "display_name": "Frankiw, Luke Steven" } ], "advisors": [ { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "advisor", "display_name": "Baltimore, David L." } ], "committee": [ { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "role": "chair", "display_name": "Guttman, Mitchell" }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "member", "display_name": "Baltimore, David L." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "member", "display_name": "Mazmanian, Sarkis K." } ], "option_major": [ "molbiochem" ], "doi": "10.7907/NBQG-BS67", "abstract": "At the heart of an inflammatory response lies a tightly regulated gene expression program. Perturbations to this finely tuned response can result in unchecked or inappropriately scaled inflammation, shifting the balance from protective to destructive immunity. A variety of post-transcriptional mechanisms play a role in the fine-tuning of an inflammatory gene expression program. One such mechanism involves unproductive RNA splicing, whereby alternative splicing can frameshift the transcript or introduce a premature termination codon (PTC). These effects render the transcript nonfunctional and/or subject it to nonsense-mediated decay.
\r\n\r\nWe observed such an event in Irf7, the master regulator of the type I interferon response. We found a single intron was consistently retained at a level much greater than other introns in the Irf7 transcript. In an effort to understand trans-acting factors that regulate this retention, we used RNA-antisense purification followed by mass spectrometry (RAP-MS) to identify the factor BUD13 as a highly enriched protein on Irf7 transcripts. Deficiency in BUD13 was associated with increased retention, decreased mature Irf7 transcript and protein levels, and consequently a dampened type I interferon response, which compromised the ability of BUD13-deficient macrophages to withstand vesicular stomatitis virus (VSV) infection.
\r\n\r\nBeyond this intron retention event in Irf7, we identified a variety of other unproductive splicing events in a number of important genes involved with the innate immune response. This unproductive splicing was not restricted to intron retention events. For example, we identified a frequently used alternative splice site in the crucial murine antiviral response gene, oligoadenylate synthetase 1g (Oas1g) that led to both a frameshift and incorporation of a PTC. Genome editing was used to remove the alternative splice site in a macrophage cell line, which led to both increased Oas1g expression and improved viral clearance. We hypothesize these events exist as a means of mitigation for what might otherwise be an inappropriately scaled response. In doing so, they represent a previously underappreciated layer of gene expression regulation in innate immunity.
" }, { "name": "Frick, Christopher Lee", "degree": "PhD", "year": "2019", "title": "How Single Cells Sense Smad3 Signal", "advisor": "Goentoro, Lea A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05222019-160829503", "creators": [ { "name": { "family": "Frick", "given": "Christopher Lee" }, "id": "Frick-Christopher-Lee", "orcid": "0000-0001-6823-5920", "display_name": "Frick, Christopher Lee" } ], "advisors": [ { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "orcid": "0000-0002-3904-0195", "role": "advisor", "display_name": "Goentoro, Lea A." } ], "committee": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "chair", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." }, { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "orcid": "0000-0002-3904-0195", "role": "member", "display_name": "Goentoro, Lea A." } ], "option_major": [ "biochem" ], "doi": "10.7907/WNM4-4R58", "abstract": "Animal cells possess the remarkable ability to send, receive, and respond to molecular signals. Accurate processing of these signals is essential for the development and maintenance of complex cell fates and organization. The regulation of cell behavior in response to signal is mediated by signal transduction pathways, which are highly conserved protein-protein interaction networks. Recent work has shown that the activation of biomolecular networks is highly sensitive to natural cellular variation in protein levels, making it unclear how these pathways accurately and reliably transmit signals in single cells. In this thesis, I address this question in the Transforming Growth Factor-\u03b2 (Tgf-\u03b2) pathway, a major intercellular signaling pathway in animal cells. First, we asked whether extracellular signal is accurately transduced into pathway activation in single cells. Examining pathway dynamics in live reporter cells, we found evidence for fold-change detection. Although the level of nuclear Smad3 varied across cells, the fold change in the level of nuclear Smad3 was a more precise outcome of ligand stimulation. Indeed, by measuring Smad3 dynamics and gene expression in the same cells, we confirm that the fold-change in Smad3 carries signal in the pathway. These findings suggest that cells encode Tgf-\u03b2 signal in a precise Smad3 fold-change as a strategy for coping with cellular noise. Second, we brought two significant advancements, which enabled us to ask how tightly signaling dynamics dictates target gene expression. By imaging endogenous dynamics of both signaling and gene expression in clonal cells, and correlating the full dynamics with a non-manifold learning approach, we show that knowing the full dynamics of Smad3 is necessary but not sufficient to predict the full dynamics of target gene expression. Indeed, we find evidence for the role of mTOR, MEK5, and cell cycle as cell-specific variables that influence how a cell responds to Smad3. This demonstrates the extent to which, even across clonal cells, response to signal considerably varies, as each cell computes decisions based on its own internal state." }, { "name": "He, Peng", "degree": "PhD", "year": "2019", "title": "The Changing Mouse Embryo Transcriptome at Whole Tissue and Single-Cell Resolution", "advisor": "Wold, Barbara J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02192019-004236200", "creators": [ { "name": { "family": "He", "given": "Peng" }, "id": "He-Peng", "orcid": "0000-0002-2457-3554", "display_name": "He, Peng" } ], "advisors": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "advisor", "display_name": "Wold, Barbara J." } ], "committee": [ { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "chair", "display_name": "Pachter, Lior S." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Fejes Toth", "given": "Katalin" }, "id": "Fejes-Toth-K", "orcid": "0000-0001-6558-2636", "role": "member", "display_name": "Fejes Toth, Katalin" }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "member", "display_name": "Guttman, Mitchell" }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." } ], "option_major": [ "biology" ], "doi": "10.7907/35S4-HG18", "abstract": "Mammalian histogenesis is a sophisticated process of coordinated changes of cellular composition governed by selective gene expression. This thesis focuses on the systematic application of modern RNA-seq methods to histogenesis processes in developing mouse embryos. Most of the work presented here is conducted as part of the ENCODE (ENCyclopedia Of DNA Elements) Project. Chapter 1 introduces the current advances of transcriptome studies on tissue development. Chapter 2 discusses a large-scale study on the whole-tissue transcriptome of 12 embryonic tissues at up to 8 time points and 5 additional perinatal tissues. Coherent themes of biological function and underlying regulatory mechanisms are revealed from the large-scale analysis. Chapter 3 presents a high-resolution single-cell RNAseq study focused on the developing forelimb of the mouse embryo. This approach enables the assignment of differential genes to corresponding lineages and provides an even more accurate picture of RNA level patterns and regulatory modes. Finally, whole-tissue and single-cell methods are compared, contrasted, and integrated in Chapter 4 to extrapolate from the main discoveries of this thesis.
" }, { "name": "Jafari, Matiar", "degree": "PhD", "year": "2019", "title": "Neural Correlates of Sensorimotor Control in Human Cortex: State Estimates and Reference Frames", "advisor": "Andersen, Richard A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05302019-163325527", "creators": [ { "name": { "family": "Jafari", "given": "Matiar" }, "id": "Jafari-Matiar", "orcid": "0000-0002-2224-4896", "display_name": "Jafari, Matiar" } ], "advisors": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "role": "advisor", "display_name": "Andersen, Richard A." } ], "committee": [ { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "chair", "display_name": "Chan, David C." }, { "name": { "family": "Aflalo", "given": "Tyson" }, "id": "Aflalo-Tyson", "role": "member", "display_name": "Aflalo, Tyson" }, { "name": { "family": "Rutishauser", "given": "Ueli" }, "id": "Rutishauser-U", "role": "member", "display_name": "Rutishauser, Ueli" }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "role": "member", "display_name": "Prober, David A." }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "role": "member", "display_name": "Andersen, Richard A." } ], "option_major": [ "biology" ], "doi": "10.7907/TQ35-KH33", "abstract": "Interacting with our environment involves multiple sensory-motor circuits throughout the human brain. How do these circuits transform sensory inputs into discernable motor actions? Our understanding of this question is critical to behavioral neuroscience and implementation of brain-machine interfaces (BMIs). In this thesis, we present experiments that explore the contributions of human cerebral cortex (parietal, premotor, and primary somatosensory cortices) to sensory-motor transformations. First, we provide evidence in support of primary somatosensory cortex (S1) encoding cognitive motor signals. Next, we describe a series of experiments that explore contributions of posterior parietal cortex (PPC) to the internal state estimate. Neural correlates for the state estimate are found in PPC; furthermore, it is found to be encoded with respect to gaze position. Finally, we investigate reference frame encoding in regions throughout human cortex (AIP, SMG, PMv, and S1) during an imagined reaching task. We find the greatest heterogeneity among brain regions during movement planning, which collapses to a largely single reference frame representation (hand-centered) during execution of the imagined reach. However, this result is dependent upon brain region. These findings yield new perspectives and evidence on the organization of sensory-motor transformations and the location the human brain\u2019s internal estimate of the body\u2019s state.
" }, { "name": "Khazaei, Tahmineh", "degree": "PhD", "year": "2019", "title": "Metabolic Bi-Stability and Hysteresis in a Model Microbiome Community", "advisor": "Ismagilov, Rustem F.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05312019-135625690", "creators": [ { "name": { "family": "Khazaei", "given": "Tahmineh" }, "id": "Khazaei-Tahmineh", "orcid": "0000-0002-4743-2383", "display_name": "Khazaei, Tahmineh" } ], "advisors": [ { "name": { "family": "Ismagilov", "given": "Rustem F." }, "id": "Ismagilov-R-F", "orcid": "0000-0002-3680-4399", "role": "advisor", "display_name": "Ismagilov, Rustem F." } ], "committee": [ { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "chair", "display_name": "Murray, Richard M." }, { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "orcid": "0000-0002-7033-0844", "role": "member", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Doyle", "given": "John C." }, "id": "Doyle-J-C", "orcid": "0000-0002-1828-2486", "role": "member", "display_name": "Doyle, John C." }, { "name": { "family": "Henry", "given": "Christopher S." }, "id": "Henry-Christopher-S", "role": "member", "display_name": "Henry, Christopher S." }, { "name": { "family": "Ismagilov", "given": "Rustem F." }, "id": "Ismagilov-R-F", "orcid": "0000-0002-3680-4399", "role": "member", "display_name": "Ismagilov, Rustem F." } ], "option_major": [ "bioeng" ], "doi": "10.7907/Z588-5H60", "abstract": "Changes in the species composition of the human microbiome are associated with a broad range of diseases, but elucidating causal mechanisms has been challenging. Some microbiome disease states persist in seemingly unfavorable conditions, e.g., the proliferation of aerobe\u2013anaerobe communities in oxygen-exposed environments in wounds or small intestinal bacterial overgrowth. In Chapter I, using two microbes relevant to the human microbiome, we combine genome-scale mathematical modeling, bioreactor experiments, transcriptomics, and control theory to show that multi-stability and hysteresis (MSH) is a mechanism that can describe shifts to a resilient aerobe\u2013anaerobe community. We examine the impact of changing oxygen and nutrient regimes and identify factors, including changes in metabolism and gene expression, that lead to MSH. Where MSH explains microbiome shifts, it can profoundly improve our conceptual understanding of these paradoxically persistent disease states, and thereby facilitate effective interventions.
\r\n\r\nChapter II details a method for rapidly detecting the susceptibility and resistance of Neisseria gonorrhoeae to the antibiotic ciprofloxacin. Antimicrobial-resistant Neisseria gonorrhoeae is an urgent public-health threat, with continued worldwide incidents of infection and rising resistance to antimicrobials. Traditional culture-based methods for antibiotic susceptibility testing are unacceptably slow (1\u20132 days), resulting in the use of broad-spectrum antibiotics and the further development and spread of resistance. Critically needed is a rapid antibiotic susceptibility test (AST) that can guide treatment at the point-of-care. In our approach, we explore the use of RNA signatures, which are among the first cellular responses to drug exposure, as an indicator of antibiotic susceptibility. Using RNA sequencing, we identified antibiotic-responsive transcripts. Significant shifts (>4-fold change) in transcript levels occurred within 5 minutes of antibiotic exposure. We designed assays for responsive transcripts with the highest abundances and fold changes, and validated gene expression using digital PCR. Using the top two markers (porB and rpmB), we correctly determined the antibiotic susceptibility and resistance of 49 clinical isolates after 10-min exposure to ciprofloxacin. RNA signatures are therefore promising as an approach on which to build rapid AST devices for N. gonorrhoeae at the point-of-care, which is critical for disease management, surveillance, and antibiotic stewardship efforts.
" }, { "name": "Kim, Kibeom", "degree": "PhD", "year": "2019", "title": "Memory and Decoding in Signaling Transduction Pathways", "advisor": "Goentoro, Lea A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06092019-220453694", "creators": [ { "name": { "family": "Kim", "given": "Kibeom" }, "id": "Kim-Kibeom", "orcid": "0000-0002-0764-4875", "display_name": "Kim, Kibeom" } ], "advisors": [ { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "orcid": "0000-0002-3904-0195", "role": "advisor", "display_name": "Goentoro, Lea A." } ], "committee": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "chair", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "orcid": "0000-0002-3904-0195", "role": "member", "display_name": "Goentoro, Lea A." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "member", "display_name": "Zinn, Kai George" } ], "option_major": [ "sysbiol" ], "doi": "10.7907/BJDQ-JX45", "abstract": "Intercellular communication allows cells to broadcast and receive necessary information for decision making, and is essential for development, growth, and maintenance of a community of cells in a multicellular organism. Signaling pathways are highly conserved systems of communication between cells, each composed of a distinct network of protein interactions that detect extracellular signal and transduce the signal information for cellular response. A signaling pathway typically encodes information from signaling events into dynamics of second messengers, intracellular molecules in the signaling pathway that activate in response to signal and initiate cellular response. Therefore, understanding how information is encoded in second messenger dynamics, and how transcriptional machinery decode and generate output response is an important aspect in investigating how signaling information is transduced inside a cell. In the first chapter, we investigate the timescales of memory in endogenous \u03b2-catenin and Smad3, second messengers in the Wnt and Tgf-\u03b2 pathways, through single cell timelapse microscopy. The findings demonstrate that both second messengers have short memory and high cell-to-cell variability, and that their memory is tunable through modulating cellular contexts. In the second chapter, we investigate decoding of information from \u03b2-catenin in the Wnt pathway. We identify a novel 11-bp DNA element that recruit \u03b2-catenin for transcriptional suppression. This negative regulatory element is shown to act in conjunction with the canonical Wnt responsive element to form an incoherent feedforward loop (IFFL). Through mathematical simulations, we present how the IFFL circuit can generate complex output functions in decoding \u03b2-catenin dynamics, which include those that confer robustness against perturbations in signaling response such as band-pass filtering and fold change detection.
\r\n" }, { "name": "Lakshmanan, Anupama", "degree": "PhD", "year": "2019", "title": "Engineering Acoustic Protein Nanostructures for Non-Invasive Molecular Imaging using Ultrasound", "advisor": "Shapiro, Mikhail G.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06062019-165907194", "creators": [ { "name": { "family": "Lakshmanan", "given": "Anupama" }, "id": "Lakshmanan-Anupama", "orcid": "0000-0002-6702-837X", "display_name": "Lakshmanan, Anupama" } ], "advisors": [ { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "advisor", "display_name": "Shapiro, Mikhail G." } ], "committee": [ { "name": { "family": "Tirrell", "given": "David A." }, "id": "Tirrell-D-A", "orcid": "0000-0003-3175-4596", "role": "chair", "display_name": "Tirrell, David A." }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "member", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Yang", "given": "Changhuei" }, "id": "Yang-Changhuei", "orcid": "0000-0001-8791-0354", "role": "member", "display_name": "Yang, Changhuei" }, { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "member", "display_name": "Shapiro, Mikhail G." } ], "option_major": [ "bioeng" ], "doi": "10.7907/ASX5-KB62", "abstract": "Visualizing biomolecular and cellular processes in real time within deep tissues is fundamental to our understanding of the normal and pathological activity underlying health and disease. Ultrasound provides the ability to non-invasively image deep inside biological tissues with high spatial and temporal resolution. However, this technology has limited capacity to monitor molecular and cellular processes, due to the lack of appropriate intra-cellular and endogenously producible nanoscale contrast agents, which can directly couple sound waves to the activity or concentration of physiologically relevant molecules. This problem could in principle be solved by developing genetically encodable ultrasound sensors \u2013 biomolecules that can get illuminated in ultrasound imaging in response to specific cellular or molecular activity. This thesis describes the engineering and characterization of acoustic protein nanostructures called 'gas vesicles', or 'GVs', to accomplish this task.
\r\n\r\nGVs are protein-shelled gas-filled nanostructures produced by buoyant microbes, and were recently shown to be capable of scattering sound waves to produce ultrasound contrast. Owing to this property, they were initially conceptualized as a new class of ultrasound contrast agents. However, little was known about their tunability to enable molecular ultrasound imaging for a wide range of applications. In this thesis, we leveraged the genetic encodability of GVs to modify them at the level of their DNA sequence and constituent proteins, and thereby tune their mechanical, acoustic, surface and targeting properties. We accomplished this by establishing a facile and modular molecular engineering platform, to produce GVs that provide enhanced nonlinear signals for sensitive and specific detection in deep tissues, target specific cell types such as cancer and immune cells, and also provide distinct acoustic collapse spectra for multiplexed imaging. We then extended this platform to build GV-based biosensors that modulate their nonlinear ultrasound signals in response to changes in the activity or concentration of specific molecules in their environment. Specifically, we engineered acoustic sensors for three different types of enzymes and for calcium \u2013 whose activity or flux underlie a wide range of important cellular processes. Furthermore, we succeeded in transferring the genetic code of gas vesicles from their species of origin into a variety of other microbes that do not naturally produce them, in order to unlock their potential as ultrasound reporter genes. Our results establish GVs as reliable acoustic biomolecules, and thereby extend the capabilities of ultrasound for molecular and cellular imaging in a manner analogous to green fluorescent protein (GFP) and its derivatives in optical microscopy. When combined with the advantages of ultrasound for non-invasive imaging, this work facilitates novel technology to significantly enhance our understanding of molecular and cellular processes in basic biology, as well as enable improved diagnosis, monitoring and treatment of diseases.
" }, { "name": "Lee, James Siho", "degree": "PhD", "year": "2019", "title": "The Genomics of Stress-Induced Life Cycle Decisions in Nematodes", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:12082018-135910865", "creators": [ { "name": { "family": "Lee", "given": "James Siho" }, "id": "Lee-James-Siho", "orcid": "0000-0002-4959-7237", "display_name": "Lee, James Siho" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "chair", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Parker", "given": "Joseph" }, "id": "Parker-J", "orcid": "0000-0001-9598-2454", "role": "member", "display_name": "Parker, Joseph" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/WZ8Y-ZM04", "abstract": "Organisms including bacteria, insects, and mammals make decisions to alter aspects of their development based on signals from the environment. The roundworm Caenorhabditis elegans can escape environmental collapse by halting reproductive growth and entering the stress-resistant dauer larval stage. Dauer larvae are spore-like and have specialized behaviors for finding and stowing onto carrier animals for dispersal. The decision to enter dauer is an anticipatory decision that is based on the inputs of food, pheromone, and temperature.
\r\n\r\nHere, I show that touch is an overlooked input into the dauer entry decision. Using quantitative dauer entry assays on CRISPR knock-ins and existing mutants in mechanosensation, I demonstrate that gentle, harsh, and piezo touch promote dauer entry. By measuring pheromone sensation and signal tranmission in mechnanosensation-defective mutants, I show that mechanosensation likely inputs into the decision in parallel with pheromone. Further confirmation that touch promotes dauer entry is provided using direct mechanical stimulation of C. elegans, and I provide a plausible role for touch in sensing dauer-promoting weather and crowding conditions.
\r\n\r\nUsing RNA-seq, I also show that 8,042 genes are differentially expressed between dauer and reproductive development. Within this dataset, we observed the striking up-regulation of 64 neuropeptide genes (encoding 215 peptides) during dauer. By comparison, the entire human genome contains 97 neuropeptide genes (encoding 270 peptides). In particular, we observed coordinated up-regulation of the FMRFamide-like neuropeptides (FLPs). Using sbt-1 mutants to knock down neuropeptide processing, we demonstrate that peptidergic signaling promotes the dauer entry decision, promotes vigorous waving during the dauer-specific nictation behavior (carrier animal-hitchhiking), and is necessary for switching from repulsion to CO2 (a carrier animal cue) in non-dauers to CO2 attraction in dauers. By testing individual neuropeptides using CRISPR knockouts and existing strains, we show that 7 FLPs promote dauer entry while 4 FLPs inhibit. I therefore propose plausible roles for these FLPs in acting downstream of and/or modulating the sensation of food, pheromone, temperature, and touch inputs. We also demonstrate that FLP-10/FLP-17, which are expressed in the CO2-sensing BAG neuron, promote CO2 chemotaxis and nictation in dauers. These findings reveal that neuropeptides can alter decision-making and behavior during C. elegans dauer entry. Through a meta-analysis, we discovered similar up-regulation of FLPs in the dauer-like infective juveniles of diverse parasitic nematodes, suggesting that this may be an ancient mechanism for expanding the behavioral repertoire of nematodes.
\r\n\r\nFurther utilizing our RNA-seq dataset, I identified several markers for conveniently tracking and manipulating the dauer entry decision. These include col-183 (which tracks dauer fate in the hypodermis), ets-10 (neurons and intestine), nhr-246 (intestine and muscle), and led-1 (reproductive fate in hypodermis). Using condition shift experiments, we demonstrate that the dauer markers label animals during dauer-commitment. We show that these markers can be used to manipulate the entry decision by driving the reproduction-promoting gene daf-9/Cytochrome P450 under the control of the dauer-commitment markers. We further demonstrate that the markers can be used to track tissue coordination and its breakdown in partial dauer mutants, and propose strategies for using the markers to identify the intercellular signals that coordinate the dauer entry decision.
\r\n\r\nI have discovered that the C. elegans dauer entry decision is more complex than previously realized, I have shown that C. elegans dauers obtain new behaviors through FLP signaling, and I have engineered tools for conveniently tracking and manipulating the dauer entry decision. My findings may illuminate how animals make robust decisions in uncertain environments, and have implications for how densely information and behaviors can be packed into a nervous system.
" }, { "name": "Lewis, Russell DeRieux", "degree": "PhD", "year": "2019", "title": "Evolution and Characterization of Carbene Transferases for Cyclopropanation and Carbon\u2013Silicon Bond Formation", "advisor": "Arnold, Frances Hamilton", "url": "https://resolver.caltech.edu/CaltechTHESIS:06052019-172451535", "creators": [ { "name": { "family": "Lewis", "given": "Russell DeRieux" }, "id": "Lewis-Russell-DeRieux", "orcid": "0000-0002-5776-7347", "display_name": "Lewis, Russell DeRieux" } ], "advisors": [ { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "advisor", "display_name": "Arnold, Frances Hamilton" } ], "committee": [ { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "chair", "display_name": "Shapiro, Mikhail G." }, { "name": { "family": "Stoltz", "given": "Brian M." }, "id": "Stoltz-B-M", "orcid": "0000-0001-9837-1528", "role": "member", "display_name": "Stoltz, Brian M." }, { "name": { "family": "Dougherty", "given": "Dennis A." }, "id": "Dougherty-D-A", "orcid": "0000-0003-1464-2461", "role": "member", "display_name": "Dougherty, Dennis A." }, { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "member", "display_name": "Arnold, Frances Hamilton" } ], "option_major": [ "bioeng" ], "doi": "10.7907/RMEX-Q134", "abstract": "Heme proteins have recently been demonstrated to catalyze cyclopropanation reactions via a putative carbene transfer mechanism. Carbene transfer reactions are not known to occur in natural biological systems, but are highly useful synthetic reactions. There is growing interest in developing new \"carbene transferases\" that bring new chemical reactions into the realm of biology, and growing interest in engineering these enzymes for use in organic synthesis. Additionally, the mechanistic details of iron porphyrin-catalyzed carbene transfer reactions are largely unknown, especially with regards to how the enzyme environment influences the outcome of a carbene transfer reaction. This thesis details both the engineering of carbene transferases with novel catalytic capabilities and investigations into how these enzymes catalyze carbene transfer reactions. Chapter 1 introduces heme protein-catalyzed carbene transfer reactions and describes the directed evolution of new enzymes that allow access to a range of useful cyclopropane products. Chapter 2 describes the evolution of an enzyme that performs carbene transfer to silicon\u2013hydrogen bonds, resulting in a highly efficient and selective carbon\u2013silicon bond-forming enzyme, the first of its kind. Chapter 3 focuses on the characterization of a key reactive intermediate, the iron-porphyrin carbene, in the active site of the evolved carbon\u2013silicon bond-forming enzyme. This study provides an explanation of the remarkable enantioselectivity of the enzyme and provides a foundation from which to investigate the enzyme reaction mechanism. The mechanism of carbon\u2013 silicon bond formation is elucidated in Chapter 4, and the is then used to explain how the enzyme achieves chemoselectivity, which in turn guides the evolution of enzyme variants with altered chemoselectivity. Finally, two off-cycle catalytic pathways that cause inactivation of the carbene transferase are characterized, and methods to prevent and/or circumvent inactivation are investigated (Chapter 5). Overall, the work presented here expands the repertoire of enzyme-catalyzed reactions and facilitates the continuing development of new carbene transferases by developing our mechanistic understanding of this novel class of enzymes.
" }, { "name": "Liu, Jonathan C.", "degree": "PhD", "year": "2019", "title": "Engineering and Application of cGAL, a GAL4 Bipartite Expression System for Caenorhabditis elegans", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06072019-163128348", "creators": [ { "name": { "family": "Liu", "given": "Jonathan C." }, "id": "Liu-Jonathan-C", "orcid": "0000-0002-5761-8704", "display_name": "Liu, Jonathan C." } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "chair", "display_name": "Anderson, David J." }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "role": "member", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/V1DD-9108", "abstract": "The core objectives of genetics are to dissect and understand the function of genes, the consequence of their perturbation on an organism, and how their collective action influences an organism\u2019s biology. For genetic model organisms, transgenesis is a tool that allows researchers to introduce synthetic genetic constructs to determine where a gene acts, when it is required, and infer its function. Caenorhabditis elegans is a powerful genetic model organism, with a variety of transgenesis methods available to researchers. Each has its own advantages in speed, efficiency, control of copy number, and control of integration site. However, all methods suffer from issues of reproducibility, reusability, and labor cost. Bipartite systems offer solutions to these issues- they separate the promoter element from the gene product producing strains in which one sex contains the promoter (\u2018driver\u2019 strain) and the other contains the gene (\u2018effector\u2019 strain). Crossing driver and effector strains reunites promoter and gene in the progeny, which are assayed and analyzed for gene function. This separation of drivers from effectors allows for a variety of benefits. Driver and effector strains can be combinatorially reused, meaning less time-consuming strain construction. Reusing strains allows for more reproducibility and consistency between experiments and between laboratories. Additionally, novel genes and promoters can be crossed to existing strains for novel transgenic patterns requiring minimal effort. Thus, bipartite systems greatly increase the rigor and pace of genetic analysis. This thesis details the engineering of cGAL, a GAL4-based bipartite system for C. elegans. It uses a novel GAL4 gene from Saccharomyces cerevisiae, a yeast whose optimal growth temperature is similar to that of C. elegans. This thesis also describes an intein-based split bipartite system that offers more refined spatiotemporal control, by allowing two promoters to dictate gene expression instead of one. This split method is used to analyze rhythmic feeding in C. elegans. Finally, engineering of cGAL using single copy methodology is detailed, with a discussion of future improvements to, and usage of, single copy cGAL. This development of a new bipartite system will greatly accelerate genetic analysis for the C. elegans, improve reproducibility for the field, and generate a valuable resource for the community." }, { "name": "Nunns, Harry James Rogan", "degree": "PhD", "year": "2019", "title": "Linearity in Cell Signaling Pathways", "advisor": "Goentoro, Lea A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01222019-133152032", "creators": [ { "name": { "family": "Nunns", "given": "Harry James Rogan" }, "id": "Nunns-Harry- James-Rogan", "orcid": "0000-0002-9669-0039", "display_name": "Nunns, Harry James Rogan" } ], "advisors": [ { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "orcid": "0000-0002-3904-0195", "role": "advisor", "display_name": "Goentoro, Lea A." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "orcid": "0000-0002-3904-0195", "role": "member", "display_name": "Goentoro, Lea A." } ], "option_major": [ "sysbiol" ], "doi": "10.7907/WP0J-E945", "abstract": "Accurate cellular communication is of paramount importance for the development, growth, and maintenance of multi-cellular organisms. Communication between cells is carried out by a highly conserved set of signaling pathways, whose dysregulation can lead to many diseases. The molecular details of these signaling pathways are now well-characterized, allowing researchers to investigate the emergent properties that arise from the complex signaling networks. These properties often arise from counter-intuitive or paradoxical mechanisms, meaning that systems-level analysis is necessary. Importantly, mathematical models have been constructed for many pathways that capture measured reaction rates and protein levels. These mathematical models successfully recapitulate dynamic responses of each pathway. Here, I investigated the input-output response of the Wnt, MAPK/ERK, and Tgf\u03b2 pathways using analytical and numerical treatment of mathematical models. Using this approach, I found that the distinct architectures of the three signaling pathways lead to a convergent behavior, linear input-output response. Specifically, mathematical analysis reveals that a futile cycle in the Wnt pathway, a kinase cascade coupled to feedback in the ERK pathway, and nucleocytoplasmic shuttling in the Tgf\u03b2 pathways all yield linear signal transmission. I then verified this finding experimentally in the Wnt and ERK pathways. For the Wnt pathway, direct measurements of the input-output response reveal that \u03b2-catenin is linear with respect to Wnt co-receptor LRP5/6 activity up until receptor saturation. For the ERK pathway, direct measurements indicate a linear relationship between phosphorylated ERK1/2 and the concentration of EGF ligand, up until saturation of ERK1/2. Finally, mathematical modeling reveals that linear response in the Wnt pathway, in conjunction with a recently identified cis-regulatory motif, is sufficient to explain gene expression buffering to perturbations. Therefore, this thesis demonstrates how linearity emerges across three dissimilar architectures, and introduces a novel benefit for linear signal transmission in biology.
" }, { "name": "Ramesh, Pradeep", "degree": "PhD", "year": "2019", "title": "Imaging and Control of Engineered Cells using Magnetic Fields", "advisor": "Shapiro, Mikhail G.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06052019-181520170", "creators": [ { "name": { "family": "Ramesh", "given": "Pradeep" }, "id": "Ramesh-Pradeep", "orcid": "0000-0001-6243-8145", "display_name": "Ramesh, Pradeep" } ], "advisors": [ { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "advisor", "display_name": "Shapiro, Mikhail G." } ], "committee": [ { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "orcid": "0000-0001-8723-8190", "role": "chair", "display_name": "Baltimore, David L." }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." }, { "name": { "family": "Orphan", "given": "Victoria J." }, "id": "Orphan-V-J", "orcid": "0000-0002-5374-6178", "role": "member", "display_name": "Orphan, Victoria J." }, { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "member", "display_name": "Shapiro, Mikhail G." } ], "option_major": [ "bioeng" ], "doi": "10.7907/KY00-7Y74", "abstract": "Making cells magnetic is a long-standing goal of synthetic biology, aiming to enable the separation of cells from complex biological samples and their non-invasive visualization in vivo using Magnetic Resonance Imaging (MRI). Previous efforts towards this goal, focused on engineering cells to biomineralize superparamagnetic or ferromagnetic iron oxides, have largely been unsuccessful due to the stringent required chemical conditions. In this thesis, we introduce an alternative approach to making cells magnetic, focusing on biochemically maximizing cellular paramagnetism. Here, we show that a novel genetic construct combining the functions of ferroxidation and iron-chelation enables engineered bacteria to accumulate iron in 'ultraparamagnetic' macromolecular complexes, which subsequently allows for these cells to be trapped using strong magnetic field gradients and imaged using MRI in vitro and in vivo. We characterize the properties of these cells and complexes using magnetometry, an array of spectroscopic techniques, biochemical assays, and computational modeling to elucidate the unique mechanisms and implications of this 'ultraparamagnetic' concept.
\r\n\r\nIn addition to making cells magnetic, remote control of cellular localization in deep tissue is another long-standing goal of synthetic biology. Such an ability to non-invasively direct cells to sites of interest will not only improve therapeutic outcomes by minimizing off-target activity, but more broadly enable new research on complex cellular communities, such as the gut microbiome, in living animals. Given their deep penetrance through tissues, magnetic fields are ideally suited for facilitating non-invasive targeting of cells; however, the rapid decay of magnetic flux density from its source currently limits the depths to which magnetic targeting can be employed to within 1-2 mm from the surface. Here, we demonstrate a new approach wherein the retention of orally-administered and synthetically magnetized cell-like-particles is selectively enhanced within the murine intestinal tract to depths of up to 13 mm from the surface. Our cellular localization assisted by magnetic particles (CLAMP) strategy can potentially be generalized to any cell (bacterial, mammalian) or drug-containing nanoparticle of interest, and can be combined with existing non-invasive imaging modalities thereby facilitating remote environmental sensing at sites of interest.
\r\n \r\nFinally, while magnetic fields in MRI scanners are widely used today to safely and non-invasively image anatomical structures in living animals, much of the image contrast in MRI is the result of microscale magnetic-field variations in tissues. However, the connection between these microscopic patterns and the appearance of macroscopic MR images has not been the subject of direct experimental studies due to a lack of methods to map microscopic fields in biological samples under ambient conditions. Here, we optically probed magnetic fields in mammalian cells and tissues with submicron resolution and nanotesla sensitivity using nitrogen-vacancy (NV) diamond magnetometry and combined these measurements with simulations of nuclear-spin precession to predict the corresponding MRI contrast. Additionally, we demonstrate the broad utility of this technology for imaging an in vitro model of cellular iron uptake, as well as imaging histological samples from a mouse model of hepatic iron overload. Taken together, our approach bridges a fundamental intellectual gap between a macroscopic MRI voxel and its microscopic constituents.
\r\n" }, { "name": "Schretter, Catherine Elizabeth", "degree": "PhD", "year": "2019", "title": "Microbial Modulation of Host Locomotion", "advisor": "Mazmanian, Sarkis K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08222018-105935103", "creators": [ { "name": { "family": "Schretter", "given": "Catherine Elizabeth" }, "id": "Schretter-Catherine-Elizabeth", "orcid": "0000-0002-3957-6838", "display_name": "Schretter, Catherine Elizabeth" } ], "advisors": [ { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "advisor", "display_name": "Mazmanian, Sarkis K." } ], "committee": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "chair", "display_name": "Lester, Henry A." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "role": "member", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "member", "display_name": "Mazmanian, Sarkis K." } ], "option_major": [ "biology" ], "doi": "10.7907/Z1DX-4J03", "abstract": "Coordinated locomotor behavior is critical for the survival and propagation of an individual and is modulated by internal and external sensory inputs. The microbiota regulates host metabolism, which is closely intertwined with motor behavior. However, little is known regarding influences by the gut microbiome on host locomotion, or the pathways involved. The work presented in this thesis examines microbial regulation of locomotor behavior from both bacterial and host perspectives. Removal of the microbiota results in hyperactivity in female D. melanogaster, which is reversible through colonization with specific bacteria or administration of bacterial-derived products, including xylose isomerase (Xi) from Lactobacillus brevis. We found that Xi modulates host speed via sugar metabolism and octopamine signaling in flies. Additionally, aspects of microbial regulation of host locomotion appear to be conserved in mice. This work suggests that microbial modulation of host physiology extends beyond local intestinal effects to locomotor behavior through alterations in energy-related pathways." }, { "name": "Shao, Zixuan", "degree": "PhD", "year": "2019", "title": "Biological Responses to Therapeutic Treatments of Human Vascular Diseases", "advisor": "Kornfield, Julia A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05302019-145256322", "creators": [ { "name": { "family": "Shao", "given": "Zixuan" }, "id": "Shao-Zixuan", "orcid": "0000-0002-4676-6023", "display_name": "Shao, Zixuan" } ], "advisors": [ { "name": { "family": "Kornfield", "given": "Julia A." }, "id": "Kornfield-J-A", "role": "advisor", "display_name": "Kornfield, Julia A." } ], "committee": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Tirrell", "given": "David A." }, "id": "Tirrell-D-A", "role": "chair", "display_name": "Tirrell, David A." }, { "name": { "family": "Kornfield", "given": "Julia A." }, "id": "Kornfield-J-A", "role": "member", "display_name": "Kornfield, Julia A." } ], "option_major": [ "bioeng" ], "doi": "10.7907/P2S4-AY40", "abstract": "Diseases of the retina affect hundreds of millions of patients worldwide, with limited treatment options available. ALG-1001 is an investigational drug that showed success in mitigating disease symptoms in animal models and improved patient vision in multiple clinical trials. To gain a better understanding of the drug\u2019s mechanism of action, RNA sequencing (RNA-seq) and shotgun proteomics were employed to study the drug-induced transcriptome change in retinal tissue and cell culture models. Chapter 2 focuses on application of this approach in an animal model of the disease that showed the drug can reversely modulate hypoxia-activated angiogenesis and inflammation gene expression changes. Chapter 3 discusses the study of drug-induced transcriptome response in two cell culture models relevant to pathophysiology of the retinal diseases. Chapter 4 explores retinal cell transcriptome after short and long-term exposure to disease-relevant hypoxia condition and after hypoxia recovery. Appendix A documents our shotgun proteomics protocol and includes results from the application of this method in the study of drug mechanism.
\r\n\r\nTypical RNA-seq studies use few biological replicates for differential expression analysis, mainly due to the high cost of generating sequencing data. As a result, not all comparisons have the proper statistical power, which result in false positives and false negatives that can lead the researcher to the wrong conclusion. Chapter 5 discusses a novel algorithm and software that help users perform quality control of their dataset to identify whether the appropriate sample size was used for differential gene discovery. The chapter covers demonstration of the software with four publicly available RNA-seq datasets to illustrate its utility.
\r\n\r\nBioresorbable vascular scaffolds (BVSs) are the application of biocompatible polymer in the treatment of coronary heart disease, one of the leading causes of death worldwide. BVSs are designed to replace metal stents, which stay permanently in the body after surgery and can lead to various complications, such as lethal thrombosis. In contrast, BVSs provide the necessary support and are resorbed by the body to leave behind a healthy artery after 2-3 years. Improving on the existing BVS material, chapter 6 explores a new polymer nanocomposite that increases the structure\u2019s radial strength in a thinner profile and provides radio-opacity to enhance surgery success.
" }, { "name": "Shih, Pei-Yin", "degree": "PhD", "year": "2019", "title": "The Ethology of Stress in Nematodes \r ", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01062019-215120039", "creators": [ { "name": { "family": "Shih", "given": "Pei-Yin" }, "id": "Shih-Pei-Yin", "orcid": "0000-0003-3082-9242", "display_name": "Shih, Pei-Yin" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "role": "chair", "display_name": "Prober, David A." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/1E7G-K373", "abstract": "Animals can respond to stress in two ways: one is through innate, reflexive behaviors and physiological responses. For example, bees sting invaders when they feel threatened, and heat shock proteins in our body ensure the proper folding of proteins under stressful conditions. The other strategy is through the more active and dynamic phenotypic plasticity responses, for example the transformation of spadefoot tadpoles into cannibals in crowded environments.
\r\n\r\nWhen Caenorhabditis elegans roundworms face harsh environmental conditions they can develop into the dauer larvae stage instead of reproductive adult. Dauers are long-lived, stress-resistant, and specialized for dispersal. Dauer biology has much to reveal about stress resistance, neural state, and tissue coordination.
\r\n\r\nUsing RNA-seq we compared dauers vs non-dauers and found 8,042 genes that are differentially expressed. By bioinformatically clustering these genes, we discovered the significant up-regulation of neuropeptide genes during dauer development. In particular, the FMRFamide neuropeptides are coordinatelly up-regulated as a family. Peptidergic signaling downstream of sbt-1 promotes dauer entry decision and nication coordination, and it is necessary for CO2 chemoattraction. We further identified that flp-10 and flp-17 together have the same effect as sbt-1 on nictation and CO2 attraction. Finally, we showed that the upregulation of flp might be a shared strategy in the host-seeking parasitic infective juvenile (IJ) stage.
\r\n\r\nFrom the RNA-seq data we also identified four good marker genes for labeling the dauer entry decision and driving gene expression, specifically during dauer commitment. By overexpressing daf-9 in the hypodermis during dauer-commitment, we can manipulate the decision and promote reproductive development. Combining the markers with partial dauer mutants allowed me to confirm their subtle phenotypes in tissue-coordination breakdown. Furthermore, this approach allowed me to uncover the novel neuronal partial dauer phenotype for daf-18 mutants.
\r\n\r\nIn work done outside of the lab, I investigated the innate stress response of extremophiles to Mono Lake. I isolated nine new nematode species that were diversely related in phylogeny, morphology, and feeding lifestyles. We were able to culture one of the species, Auanema tufa, in the laboratory, and demonstrated a high level of arsenic stress-resistance in the species. These data suggest that Mono Lake\u2014particularly its more buffered tide zone\u2014has been invaded independently and multiple times by nematodes. We also speculate that pre-adaptation to arsenic in the tide zones on Mono Lake could lead to the genomic evolution necessary to adapt to the high pH and salinity of inner Mono Lake.
\r\n\r\nAltogether, I have investigated innate and plastic stress responses in and outside of the lab through my work on dauer development and arsenic resistance in Mono Lake. This has allowed me to survey the strategies nematodes use to maximize the use of their simple body plans. In particular, dauers up-regulate 64 neuropeptide genes that encode for 215 peptides to massively rewire their neural state. This likely allows them to overcome the physical limitations of their un-compartmentalized nervous system, and I speculate that such a strategy would be useful in other organisms lacking compartmentalized brains, as well as in local regions of a brain that are low complexity.
" }, { "name": "Singhal, Vipul", "degree": "PhD", "year": "2019", "title": "Modeling, Computation, and Characterization to Accelerate the Development of Synthetic Gene Circuits in Cell-Free Extracts", "advisor": "Murray, Richard M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08262018-213846283", "creators": [ { "name": { "family": "Singhal", "given": "Vipul" }, "id": "Singhal-Vipul", "orcid": "0000-0003-1670-1824", "display_name": "Singhal, Vipul" } ], "advisors": [ { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "role": "advisor", "display_name": "Murray, Richard M." } ], "committee": [ { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "role": "chair", "display_name": "Winfree, Erik" }, { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "role": "member", "display_name": "Goentoro, Lea A." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "role": "member", "display_name": "Murray, Richard M." } ], "option_major": [ "cns" ], "doi": "10.7907/g31j-ch52", "abstract": "Synthetic biology may be defined as an attempt at using engineering principles to design and build novel biological functionalities. An important class of such functionalities involves the bottom up design of genetic networks (or 'circuits') to control cellular behavior. Performing design iterations on these circuits in vivo is often a time consuming process. One approach that has been developed to address these long design times is to use E. coli cell extracts as simplified circuit prototyping environments. The analogy with similar approaches in engineering, such as prototyping using wind tunnels and breadboards, may be extended by developing accompanying computer aided design tools. In this thesis, we discuss the development of computational and mathematical tools to accelerate circuit prototyping in the TX-TL cell free prototyping platform, and demonstrate some applications of these tools.
\r\n\r\nWe start by discussing the problem of reducing circuit behavior variability between different batches of TX-TL cell extracts. To this end, we demonstrate a model-based methodology for calibrating extract batches, and for using the calibrations to 'correct' the behavior of genetic circuits between batches. We also look at the interaction of this methodology with the phenomenon of parameter non-identifiability, which occurs when the parameter identification inverse problem has multiple solutions. In particular, we derive conditions under which parameter non-identifiability does not hinder our modeling objectives, and subsequently demonstrate the use of such non-identifiable models in performing data variability reduction.
\r\n\r\nNext, we describe txtlsim, a MATLAB Simbiology based toolbox for automatically generating models of genetic circuits in TX-TL, and for using these models for part characterization and circuit behavior prediction. Large genetic circuits can have non-negligible resource usage needs, leading to unintended interactions between circuit nodes arising due to the loading of cellular machinery, transcription factors or other regulatory elements. The usage of consumable resources like nucleotides and amino acids can also have non-trivial effects on complex genetic circuits. These types of effects are handled by the modeling framework of txtlsim in a natural way.
\r\n\r\nWe also highlight mcmc-simbio, a smaller toolbox within txtlsim for performing concurrent Bayesian parameter inference on Simbiology models. Concurrent inference here means that a common set of parameters can be identified using data from an ensemble of different circuits and experiments, with each experiment informing a subset of the parameters. The combination of the concurrence feature with the fact that Markov chain Monte Carlo based Bayesian inference methods allow for the direct visualization of parameter non-identifiability enables the design of ensembles of experiments that reduce such non-identifiability.
\r\n\r\nFinally, we end with a method for performing model order reduction on transcription and translation elongation models while maintaining the ability of these models to track resource consumption. We show that due to their network topology, our models cannot be brought into the two-timescale form of singular perturbation theory when written in species concentration coordinates. We identify a coordinate system in which singular perturbation theory may be applied to chemical reaction networks more naturally, and use this to achieve the desired model reduction.
" }, { "name": "Wang, Haoqing", "degree": "PhD", "year": "2019", "title": "Structure and Dynamics of HIV-1 Env Trimer", "advisor": "Bjorkman, Pamela J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02272019-142326751", "creators": [ { "name": { "family": "Wang", "given": "Haoqing" }, "id": "Wang-Haoqing", "orcid": "0000-0003-0277-3018", "display_name": "Wang, Haoqing" } ], "advisors": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "advisor", "display_name": "Bjorkman, Pamela J." } ], "committee": [ { "name": { "family": "Jensen", "given": "Grant J." }, "id": "Jensen-G-J", "orcid": "0000-0003-1556-4864", "role": "chair", "display_name": "Jensen, Grant J." }, { "name": { "family": "Clemons", "given": "William M." }, "id": "Clemons-W-M", "orcid": "0000-0002-0021-889X", "role": "member", "display_name": "Clemons, William M." }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." } ], "option_major": [ "biochem" ], "doi": "10.7907/65VD-VM42", "abstract": "The human immunodeficiency virus-1 (HIV-1) infects CD4+ helper T cells by fusing its lipid membrane to the host cell plasma membrane. HIV-1 Envelope (Env) glycoprotein, a trimer of gp120-gp41 heterodimers, is the fusion machinery that mediates viral and host cell membrane fusion, which is initiated by interaction between the host cell receptor CD4 and viral glycoprotein gp120. Receptor binding induces conformational changes in the Env trimer such that coreceptor CCR5/CXCR4 can bind to gp120 and trigger subsequent membrane fusion steps. Previous cryo-electron tomography (cryoET) and spectroscopy studies have shown that CD4 binding induces changes in Env trimer from the closed, prefusion conformation to an open, CD4-bound conformation. However, the CD4-bound Env trimer is dynamic and flexible such that structural biology study of CD4-bound Env trimer is difficult.
\r\n\r\nAs the sole glycoprotein located on HIV-1 viral surface, Env trimer is the only target for neutralizing antibodies. Since HIV-1 mutates rapidly, most neutralizing antibodies are strain-specific. However, a small percent of HIV-1 infected patients can develop broadly neutralizing antibodies (bNAbs) that neutralize a wide spectrum of viruses. In 2011, our collaborator isolated a bNAb called 8ANC195. To understand how 8ANC195 recognizes Env trimer and prevents infection, we used a combination of structural biology and biochemistry tools. Interestingly, we found that 8ANC195 can recognize Env trimer in both its prefusion and CD4-bound conformations by targeting an epitope at gp120-gp41 interface. Furthermore, 8ANC195 stabilizes the Env-CD4 complex, allowing us to investigate the conformational changes induced by CD4 in atomic resolution. By solving and comparing single-particle cryo-EM structures of Env trimer in complex with CD4 and/or antibodies, we found that: 1) CD4 binding induces coreceptor binding site exposure through a \u03b2-sheet rearrangements in each gp120 monomer. Several key residues allosterically regulate this coreceptor binding site exposure. 2) CD4 binding induces Env trimer opening together with gp41 conformational changes, which represents an intermediate between prefusion gp41 and postfusion gp41. 3) Env trimer opening is necessary but not sufficient for coreceptor binding site exposure, while CD4 binding induces both, some antibodies can open Env trimer without exposing the coreceptor binding site. These conclusions further illuminate how Env trimer mediates membrane fusion and inform potential strategies blocking viral entry.
\r\n" }, { "name": "Yi, Lynn Donglin", "degree": "PhD", "year": "2019", "title": "Statistical Methods for Gene Differential Expression Analysis of RNA-Sequencing", "advisor": "Pachter, Lior S.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10102018-143313907", "creators": [ { "name": { "family": "Yi", "given": "Lynn Donglin" }, "id": "Yi-Lynn-Donglin", "orcid": "0000-0003-4575-0158", "display_name": "Yi, Lynn Donglin" } ], "advisors": [ { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "advisor", "display_name": "Pachter, Lior S." } ], "committee": [ { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "chair", "display_name": "Chan, David C." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Pachter", "given": "Lior S." }, "id": "Pachter-L", "orcid": "0000-0002-9164-6231", "role": "member", "display_name": "Pachter, Lior S." }, { "name": { "family": "Chandrasekaran", "given": "Venkat" }, "id": "Chandrasekaran-V", "role": "member", "display_name": "Chandrasekaran, Venkat" } ], "option_major": [ "sysbiol" ], "doi": "10.7907/0YE6-2217", "abstract": "RNA-Sequencing (\"RNA-Seq\") is performed to measure gene expression, often to ask the question of what genes are differentially expressed across various biological conditions. Statistical methods have been used to model RNA-Seq quantifications in order to determine differential expression, and have traditionally be divided into gene-level methods and transcript-level methods. There has been little attempt to connect the statistical divide, although transcript expression and gene expression are biologically inextricably linked. In this thesis, we provide a case study of a comparative differential expression analysis, demonstrating that many differential expression events happen on the isoform-level, and that performing an analysis using only summarized gene quantifications would fail to capture these events. Furthermore, we develop statistical methods that unify the transcript-level and gene-level analysis. In bulk RNA-Seq, by using p-value aggregation methods, we are able to translate transcript-level results into gene-level results under a unified framework. For single cell RNA-Seq, we propose using multiple logistic regression, leveraging the high dimensionality of the data in order to determine if the transcript quantifications pertaining to a gene are able to constitute a linear discriminant for cell type. This method combines differential transcript expression analysis and differential gene expression analysis into a unified framework which we call \u201cgene differential expression.\u201d Lastly, we demonstrate that our methods could be used on transcript compatibility counts instead of transcript quantifications in order to bypass ambiguous read assignment and improve accuracy. We show that transcript compatibility counts obtained via transcriptome pseudoalignment are comparable in quantification accuracy to quantifications from genome alignment methods.
" }, { "name": "Abrams, Michael Jacob", "degree": "PhD", "year": "2018", "title": "Self-Repair and Sleep in Jellyfish", "advisor": "Goentoro, Lea A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06042018-104549400", "creators": [ { "name": { "family": "Abrams", "given": "Michael Jacob" }, "id": "Abrams-Michael-Jacob", "orcid": "0000-0003-1864-1706", "display_name": "Abrams, Michael Jacob" } ], "advisors": [ { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "orcid": "0000-0002-3904-0195", "role": "advisor", "display_name": "Goentoro, Lea A." } ], "committee": [ { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "chair", "display_name": "Hay, Bruce A." }, { "name": { "family": "Dabiri", "given": "John O." }, "id": "Dabiri-J-O", "orcid": "0000-0002-6722-9008", "role": "member", "display_name": "Dabiri, John O." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "orcid": "0000-0002-3904-0195", "role": "member", "display_name": "Goentoro, Lea A." } ], "option_major": [ "biology" ], "doi": "10.7907/69br-kb59", "abstract": "Studying the cnidarian jellyfish, we have pursued basic biological questions related to self-repair mechanisms and sleep behavior. Working in Aurelia we have discovered a novel strategy of self-repair; we determined that they can undergo body reorganization after amputations that culminates in the recovery of essential radial symmetry without rebuilding lost parts. Working with Cassiopea, we have, for the first time, identified a behavioral sleep-like state in an animal without a centralized nervous system, supporting the hypothesis that sleep is ancestral in animals.
" }, { "name": "Barnes, Stephanie Loos", "degree": "PhD", "year": "2018", "title": "Decoding the Regulatory Genome: Quantitative Analysis of Transcriptional Regulation in Escherichia coli", "advisor": "Phillips, Robert B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05292018-133205686", "creators": [ { "name": { "family": "Barnes", "given": "Stephanie Loos" }, "id": "Barnes-Stephanie-Loos", "orcid": "0000-0002-5237-603X", "display_name": "Barnes, Stephanie Loos" } ], "advisors": [ { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "advisor", "display_name": "Phillips, Robert B." } ], "committee": [ { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "chair", "display_name": "Newman, Dianne K." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." } ], "option_major": [ "bioeng" ], "doi": "10.7907/D13T-7868", "abstract": "Over the past decades DNA sequencing has become significantly cheaper and faster, which has enabled the accumulation of a huge amount of genomic data. However, much of this genomic data is illegible to us. For noncoding regions of the genome in particular, it is difficult to determine what role is played by specific DNA sequences. Here we focus on regions of DNA that play a role in transcriptional regulation. We develop models and techniques that allow us to discover new regulatory sequences and better understand how DNA sequence determines regulatory output.
\r\n\r\nWe start by considering how quantitative models serve as a powerful tool for testing our understanding of biological systems. We apply a statistical mechanical framework that incorporates the Monod-Wyman-Changeux model to analyze the effects of allostery in simple repression, using the lac operon as a test case. By fitting our model to experimental data, we are able to determine the values of the unknown parameter values in our model. We then show that we can use the model to accurately predict the induction responses of an array of simple repression constructs with a variety of repressor copy numbers and repressor binding energies.
\r\n\r\nNext, we consider how the DNA sequence of a promoter region can provide details about how the promoter is regulated. We begin by describing an approach for discovering regulatory architectures for promoters whose regulation has not previously been studied. We focus on six promoters from E. coli including three well-studied promoters (rel, mar, and lac) to serve as test cases. We use the massively parallel reporter assay Sort-Seq to identify transcription factor binding sites with base-pair resolution, determine the regulatory role of each binding site, and infer energy matrices for each binding site. Then, we use DNA affinity chromatography and mass spectrometry to identify each transcription factor.
\r\n\r\nWe conclude with an in vivo approach for analyzing the sequence-dependence of transcription factor binding energies. Again using Sort-Seq, we show that we can represent transcription factor binding sites using energy matrices in absolute energy units. We then show that these energy matrices can be used to accurately predict the binding energies of mutated binding sites. We provide several examples of how understanding the relationship between DNA sequence and transcription factor binding provides us with a foundation for addressing additional scientific topics, such as the co-evolution of transcription factors and their binding sites.
" }, { "name": "Bedbrook, Claire Nicole", "degree": "PhD", "year": "2018", "title": "Engineering Novel Rhodopsins for Neuroscience", "advisor": "Gradinaru, Viviana; Arnold, Frances Hamilton", "url": "https://resolver.caltech.edu/CaltechTHESIS:05302018-095950094", "creators": [ { "name": { "family": "Bedbrook", "given": "Claire Nicole" }, "id": "Bedbrook-Claire-Nicole", "orcid": "0000-0003-3973-598X", "display_name": "Bedbrook, Claire Nicole" } ], "advisors": [ { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "co-advisor", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "co-advisor", "display_name": "Arnold, Frances Hamilton" } ], "committee": [ { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "chair", "display_name": "Shapiro, Mikhail G." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "member", "display_name": "Arnold, Frances Hamilton" }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "member", "display_name": "Gradinaru, Viviana" } ], "option_major": [ "bioeng" ], "doi": "10.7907/7RA0-BC29", "abstract": "The overarching goal of my PhD research has been engineering proteins capable of controlling and reading out neural activity to advance neuroscience research. I engineered light-gated microbial rhodopsins, primarily focusing on the algal derived, light-gated channel, channelrhodopsin (ChR), which can be used to modulate neuronal activity with light. This work has required overcoming three major challenges. First, rhodopsins are trans-membrane proteins, which are inherently difficult to engineer because the sequence and structural determinants of membrane protein expression and plasma membrane localization are highly constrained and poorly understood (Chapter 3-5). Second, protein properties of interest for neuroscience applications are assayed using very low throughput patch-clamp electrophysiology preventing the use of high-throughput assays required for directed evolution experiments (Chapter 2, 5-6). And third, in vivo application of these improved tools require either retention or optimization of multiple protein properties in a single protein tool; for example, we must optimize expression and localization of these algal membrane proteins in mammalian cells while at the same time optimizing kinetic and functional properties (Chapter 5-6). These challenges restricted the field to low-throughput, conservative methods for discovery of improved ChRs, e.g., structure-guided mutagenesis and testing of natural ChR variants. I used an alternative approach: data-driven machine learning to model the fitness landscape of ChRs for different properties of interest and applying these models to select ChR sequences with optimal combinations of properties (Chapters 5-6). ChR variants identified from this work have unprecedented conductance properties and light sensitivity that could enable non-invasive activation of populations of cells throughout the nervous system. These ChRs have the potential to change how optogenetics experiments are done. This work is a convincing demonstration of the power of machine learning guided protein engineering for a class of proteins that present multiple engineering challenges. A component of the novel application of these new ChR tools relies on recent advances in gene delivery throughout the nervous system facilitated by engineered AAVs (Chapter 7). And finally, I developed a behavioral tracking system to monitor behavior and demonstrate sleep behavior in the jellyfish Cassiopea, the most primitive organism to have this behavior formally characterized (Chapter 8).
" }, { "name": "Belliveau, Nathan Maurice", "degree": "PhD", "year": "2018", "title": "Quantitative Dissection of the Allosteric and Sequence-Dependent Regulatory Genome in E. coli", "advisor": "Phillips, Robert B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01052018-221609680", "creators": [ { "name": { "family": "Belliveau", "given": "Nathan Maurice" }, "id": "Belliveau-Nathan-Maurice", "orcid": "0000-0002-1536-1963", "display_name": "Belliveau, Nathan Maurice" } ], "advisors": [ { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "advisor", "display_name": "Phillips, Robert B." } ], "committee": [ { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "chair", "display_name": "Phillips, Robert B." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Thomson", "given": "Matthew" }, "id": "Thomson-M-W", "orcid": "0000-0003-1021-1234", "role": "member", "display_name": "Thomson, Matthew" }, { "name": { "family": "Van Valen", "given": "David A." }, "id": "Van-Valen-D", "orcid": "0000-0001-7534-7621", "role": "member", "display_name": "Van Valen, David A." } ], "option_major": [ "bioeng" ], "doi": "10.7907/Z9DN438T", "abstract": "Transcriptional regulation of gene expression is one of the most ubiquitous processes in biology. But while the catalog of bacterial genomes continues to expand rapidly, we remain ignorant about how almost all of the genes in these genomes are regulated. One of the ways genes are regulated is through external signals. To that end, we begin by presenting a general theory of allosteric transcriptional regulation using a statistical formulation of the Monod-Wyman-Changeux model, which we rigorously test using the ubiquitous simple repression motif in Escherichia coli. We then move to consider the consequence of the regulatory sequences themselves on gene expression. Here we apply a massively parallel reporter assay, Sort-Seq, to build models that describe the sequence-dependent binding energies of transcription factors and RNA polymerase to DNA. By coupling such models to our thermodynamic models of regulation, we construct a genotype to phenotype mapping that predicts gene expression as a function of regulatory sequence. We first demonstrate this approach in the context of the allosteric simple repression motif, and then show how it can be applied broadly across a bacterial genome, in conjunction with mass spectrometry, to uncover how genes are regulated.
" }, { "name": "Chen, Chun-Kan", "degree": "PhD", "year": "2018", "title": "Revealing the Mechanism of Xist-mediated Silencing", "advisor": "Guttman, Mitchell", "url": "https://resolver.caltech.edu/CaltechTHESIS:11032017-102218314", "creators": [ { "name": { "family": "Chen", "given": "Chun-Kan" }, "id": "Chen-Chun-Kan", "orcid": "0000-0002-1194-9137", "display_name": "Chen, Chun-Kan" } ], "advisors": [ { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0002-1221-0967", "role": "advisor", "display_name": "Guttman, Mitchell" } ], "committee": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "chair", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "orcid": "0000-0003-4748-9352", "role": "member", "display_name": "Guttman, Mitchell" }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Aravin", "given": "Alexei A." }, "id": "Aravin-A-A", "orcid": "0000-0002-6956-8257", "role": "member", "display_name": "Aravin, Alexei A." } ], "option_major": [ "molbiochem" ], "doi": "10.7907/Z94J0C9J", "abstract": "Xist initiates XCI by spreading across the future inactive X-chromosome, excluding RNA polymerase II, recruiting the polycomb repressive complex and its associated repressive chromatin modifications, and repositioning active genes into a transcriptionally silenced nuclear compartment. While much is known about the events that occur during XCI, the mechanism by which Xist carries out these various roles remains unclear. Here we identify ten proteins that directly associate with Xist, and we further show that three of these proteins are required for Xist-mediated transcriptional silencing. One of these proteins, SHARP, which is known to interact with the SMRT co-repressor that activates HDAC3, is not only essential for silencing, but is also required for the exclusion of PolII from the inactive X. We show that both SMRT and HDAC3 are required for Xist-mediated silencing and RNA polymerase II exclusion. Another of these proteins, LBR, is required for repositioning actively transcribed genes into the Xist-silenced compartment. We further show that Xist, through its interaction with LBR, a protein that is anchored in the inner nuclear membrane, would effectively reposition Xist-coated DNA to the nuclear lamina, thereby changing the accessibility of other genes on the X-chromosome to enable Xist to spread to active genes across the entire chromosome to silence chromosome-wide transcription. Together, these results present an integrative picture of how Xist can scaffold multiple proteins to orchestrate the complex functions required for the establishment of the inactive X-chromosome.
" }, { "name": "Colas, Jaron Taylor", "degree": "PhD", "year": "2018", "title": "Value-Based Decision Making and Learning as Algorithms Computed by the Nervous System", "advisor": "O'Doherty, John P.", "url": "https://resolver.caltech.edu/CaltechThesis:11162017-001051515", "creators": [ { "name": { "family": "Colas", "given": "Jaron Taylor" }, "id": "Colas-Jaron-Taylor", "orcid": "0000-0003-1872-7614", "display_name": "Colas, Jaron Taylor" } ], "advisors": [ { "name": { "family": "O'Doherty", "given": "John P." }, "id": "O'Doherty-J-P", "role": "advisor", "display_name": "O'Doherty, John P." } ], "committee": [ { "name": { "family": "O'Doherty", "given": "John P." }, "id": "O'Doherty-J-P", "role": "chair", "display_name": "O'Doherty, John P." }, { "name": { "family": "Rangel", "given": "Antonio" }, "id": "Rangel-A", "role": "member", "display_name": "Rangel, Antonio" }, { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "role": "member", "display_name": "Adolphs, Ralph" }, { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "role": "member", "display_name": "Shimojo, Shinsuke" } ], "option_major": [ "cns" ], "doi": "10.7907/Z90R9MK8", "abstract": "How do we do what we do? Casting light on this essential question, the blossoming perspective of computational cognitive neuroscience gives rise to the present exposition of the nervous system and its phenomena of value-based decision making and learning. As justified herein by not only theory but also simulation against empirical data, human decision making and learning are framed mathematically in the explicit terms of two fundamental classes of algorithms--namely, sequential sampling and reinforcement learning. These counterparts are complementary in their coverage of the dynamics of unified neural, mental, and behavioral processes at different temporal scales. Novel variants of models based on such algorithms are introduced here to account for findings from experiments including measurements of both behavior and the brain in human participants.
\r\n\r\nIn principle, formal dynamical models of decision making hold the potential to represent fundamental computations underpinning value-based (i.e., preferential) decisions in addition to perceptual decisions. Sequential-sampling models such as the race model and the drift-diffusion model that are grounded in simplicity, analytical tractability, and optimality remain popular, but some of their more recent counterparts have instead been designed with an aim for more feasibility as architectures to be implemented by actual neural systems. In Chapter 2, connectionist models are proposed at an intermediate level of analysis that bridges mental phenomena and underlying neurophysiological mechanisms. Several such models drawing elements from the established race, drift-diffusion, feedforward-inhibition, divisive-normalization, and competing-accumulator models were tested with respect to fitting empirical data from human participants making choices between foods on the basis of hedonic value rather than a traditional perceptual attribute. Even when considering performance at emulating behavior alone, more neurally plausible models were set apart from more normative race or drift-diffusion models both quantitatively and qualitatively despite remaining parsimonious. To best capture the paradigm, a novel six-parameter computational model was formulated with features including hierarchical levels of competition via mutual inhibition as well as a static approximation of attentional modulation, which promotes \"winner-take-all\" processing. Moreover, a meta-analysis encompassing several related experiments validated the robustness of model-predicted trends in humans' value-based choices and concomitant reaction times. These findings have yet further implications for analysis of neurophysiological data in accordance with computational modeling, which is also discussed in this new light.
\r\n\r\nDecision making in any brain is imperfect and costly in terms of time and energy. Operating under such constraints, an organism could be in a position to improve performance if an opportunity arose to exploit informative patterns in the environment being searched. Such an improvement of performance could entail both faster and more accurate (i.e., reward-maximizing) decisions. Chapter 3 investigated the extent to which human participants could learn to take advantage of immediate patterns in the spatial arrangement of serially presented foods such that a region of space would consistently be associated with greater subjective value. Eye movements leading up to choices demonstrated rapidly induced biases in the selective allocation of visual fixation and attention that were accompanied by both faster and more accurate choices of desired goods as implicit learning occurred. However, for the control condition with its spatially balanced reward environment, these subjects exhibited preexisting lateralized biases for eye and hand movements (i.e., leftward and rightward, respectively) that could act in opposition not only to each other but also to the orienting biases elicited by the experimental manipulation, producing an asymmetry between the left and right hemifields with respect to performance. Potentially owing at least in part to learned cultural conventions (e.g., reading from left to right), the findings herein particularly revealed an intrinsic leftward bias underlying initial saccades in the midst of more immediate feedback-directed processes for which spatial biases can be learned flexibly to optimize oculomotor and manual control in value-based decision making. The present study thus replicates general findings of learned attentional biases in a novel context with inherently rewarding stimuli and goes on to further elucidate the interactions between endogenous and exogenous biases.
\r\n\r\nPrediction-error signals consistent with formal models of \"reinforcement learning\" (RL) have repeatedly been found within dopaminergic nuclei of the midbrain and dopaminoceptive areas of the striatum. However, the precise form of the RL algorithms implemented in the human brain is not yet well determined. For Chapter 4, we created a novel paradigm optimized to dissociate the subtypes of reward-prediction errors that function as the key computational signatures of two distinct classes of RL models--namely, \"actor/critic\" models and action-value-learning models (e.g., the Q-learning model). The state-value-prediction error (SVPE), which is independent of actions, is a hallmark of the actor/critic architecture, whereas the action-value-prediction error (AVPE) is the distinguishing feature of action-value-learning algorithms. To test for the presence of these prediction-error signals in the brain, we scanned human participants with a high-resolution functional magnetic-resonance imaging (fMRI) protocol optimized to enable measurement of neural activity in the dopaminergic midbrain as well as the striatal areas to which it projects. In keeping with the actor/critic model, the SVPE signal was detected in the substantia nigra. The SVPE was also clearly present in both the ventral striatum and the dorsal striatum. However, alongside these purely state-value-based computations we also found evidence for AVPE signals throughout the striatum. These high-resolution fMRI findings suggest that model-free aspects of reward learning in humans can be explained algorithmically with RL in terms of an actor/critic mechanism operating in parallel with a system for more direct action-value learning.
" }, { "name": "DeSalvo, Gilberto", "degree": "PhD", "year": "2018", "title": "Transcriptional Enhancer Activity of Biochemically Marked Genomic Elements", "advisor": "Wold, Barbara J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06112018-020802925", "creators": [ { "name": { "family": "DeSalvo", "given": "Gilberto" }, "id": "DeSalvo-Gilberto", "orcid": "0000-0002-8957-1699", "display_name": "DeSalvo, Gilberto" } ], "advisors": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "advisor", "display_name": "Wold, Barbara J." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Hardison", "given": "Ross C." }, "id": "Hardison-R-C", "role": "member", "display_name": "Hardison, Ross C." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." } ], "option_major": [ "biology" ], "doi": "10.7907/J73E-Y429", "abstract": "Functional genomics aspires to explain how a transcription factor (TF) and its measured biochemical occupancy relates to the enhancer activity of the underlying sequence elements. Tissue-specific TFs exhibit remarkable selectivity and reproducibility in the available genome-wide sequence motifs accessed. A consistent central conclusion is that, irrespective of the element selection criteria used, ~50% of candidate Enhancers score as transcriptionally active in both mouse and human cell types, while the remaining 50% of similarly biochemically marked regions are unable to activate transcription on their own. This finding is based on an integrated comparison of a group of functionally assayed elements containing TF-occupied elements, evolutionarily conserved elements, and TF agnostic elements with hallmark biochemical signatures of known enhancers. Quantitatively, the level of TF occupancy signal was the best predictor of the proportion of active enhancers detected, but overall (and contrary to expectation) it is a weak predictor of the magnitude of enhancer activity readout. In specific cell types, elements can display all of the hallmark signatures of enhancers, but can remain inactively poised prior to a stimulus that either activates them or releases a repressive factor. Against previous expectations these poised occupancy sites, once released, behave comparatively in magnitude of enhancer activity as their counterparts that are only directly accessed upon stimulation. Based on our findings, the vast majority of active enhancers in the genome, including some of the most individually powerful ones, are expected to display relatively modest biochemical signatures. Finally, the combined set of over a hundred genomic regions that lacked biochemical marks, even while containing the motifs known to be necessary to bind the relevant TFs, did not support significant enhancer function. We also found evidence that both enhancer orientation and combinations of relatively closely spaced candidate Enhancers, can yield additive functions, with possible fine tuning of the enhancer activity controlled by the type and the distance between individually accessed motifs. In special cases, these elements might cooperate to recruit stable complexes resulting in a synergistic transcriptional activation, suggesting that both local \"super-enhancers\" and recruited multi-element combinatorics are likely to play an important role in vivo. These findings provide an expectation for enhancer function in the comprehensive annotations provided by the new ENCODE encyclopedia and may help guide future efforts to define the mechanisms by which enhancer activity is achieved and conferred selectively to target genes. Surprisingly, elements that deeply sample the biochemical occupancy of complex loci, match a random population of selected elements remarkably well. Our findings also indicate that carefully designed and lower throughput approaches, rather than high numerical assays that focus on the outstanding features, will bring widely applicable answers to the remaining questions of how relative enhancers are tuned and how seemingly identical regions at a biochemical and motif level are selected for or against function." }, { "name": "Donaldson, Gregory Paul", "degree": "PhD", "year": "2018", "title": "Colonization of the Intestinal Surface by Indigenous Microbiota", "advisor": "Mazmanian, Sarkis K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05082018-122340793", "creators": [ { "name": { "family": "Donaldson", "given": "Gregory Paul" }, "id": "Donaldson-Gregory-Paul", "orcid": "0000-0002-8551-374X", "display_name": "Donaldson, Gregory Paul" } ], "advisors": [ { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "orcid": "0000-0003-2713-1513", "role": "advisor", "display_name": "Mazmanian, Sarkis K." } ], "committee": [ { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "chair", "display_name": "Newman, Dianne K." }, { "name": { "family": "Orphan", "given": "Victoria J." }, "id": "Orphan-V-J", "orcid": "0000-0002-5374-6178", "role": "member", "display_name": "Orphan, Victoria J." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "orcid": "0000-0002-3671-9354", "role": "member", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "orcid": "0000-0003-2713-1513", "role": "member", "display_name": "Mazmanian, Sarkis K." } ], "option_major": [ "microbio" ], "doi": "10.7907/6EZ0-3007", "abstract": "The mammalian gut evolved to foster the development and maintenance of a community of specific bacterial symbionts that persist for years. Bacteroides fragilis is one of a number of species that are able to colonize the mucus of the large intestine in mice and humans. This thesis explores the mechanisms and functions of mucosal colonization, most notably by using reductionist approaches with gnotobiotic mice. Harnessing genetics on both the host and microbial side allowed the dissection of a pathway by which immunoglobulin A enhances mucosal colonization by B. fragilis. Novel colonization assays were developed to explore the importance of mucosal colonization to bacterial fitness. Finally, an enrichment method for host-associated bacterial transcriptomics was used to define the behavior of this symbiont within the mucus layer.
" }, { "name": "Gethers, Matthew Leroy, III", "degree": "PhD", "year": "2018", "title": "Therapeutic Opportunities and Approaches to Sequence Control for Nucleic Acids", "advisor": "Goddard, William A., III; Weiss, Paul S.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06012018-190621868", "creators": [ { "name": { "family": "Gethers", "given": "Matthew Leroy, III" }, "id": "Gethers-Matthew-Leroy-III", "orcid": "0000-0001-7455-4709", "display_name": "Gethers, Matthew Leroy, III" } ], "advisors": [ { "name": { "family": "Goddard", "given": "William A., III" }, "id": "Goddard-W-A-III", "orcid": "0000-0003-0097-5716", "role": "co-advisor", "display_name": "Goddard, William A., III" }, { "name": { "family": "Weiss", "given": "Paul S." }, "id": "Weiss-P-S", "orcid": "0000-0001-5527-6248", "role": "co-advisor", "display_name": "Weiss, Paul S." } ], "committee": [ { "name": { "family": "Tirrell", "given": "David A." }, "id": "Tirrell-D-A", "orcid": "0000-0003-3175-4596", "role": "chair", "display_name": "Tirrell, David A." }, { "name": { "family": "Rothemund", "given": "Paul W. K." }, "id": "Rothemund-P-W-K", "orcid": "0000-0002-1653-3202", "role": "member", "display_name": "Rothemund, Paul W. K." }, { "name": { "family": "Goddard", "given": "William A., III" }, "id": "Goddard-W-A-III", "orcid": "0000-0003-0097-5716", "role": "member", "display_name": "Goddard, William A., III" }, { "name": { "family": "Weiss", "given": "Paul S." }, "id": "Weiss-P-S", "orcid": "0000-0001-5527-6248", "role": "member", "display_name": "Weiss, Paul S." } ], "option_major": [ "bioeng" ], "doi": "10.7907/WE1E-EZ49", "abstract": "RNA interference (RNAi) is a powerful mechanism to regulate gene expression. A key feature of RNAi is its sequence specificity: a short interfering RNA (siRNA) assembles into the RNA induced silencing complex (RISC) and then targets cellular transcripts complementary to the siRNA for degradation. RNAi has been adapted for therapeutic applications, but is challenged by the need to identify unique target transcripts for each disease that are both effective and result in few off-target effects. This challenge could be eased if siRNAs could be activated only and specifically in diseased cells. If this were the case, rather than targeting a new transcript for each new disease, the same cellular housekeeping genes could be reused. Targeting housekeeping genes would result in greater potency, both effectively treating the disease and requiring less drug for treatment, alleviating problems associated with toxicity and delivery. A new class of nucleic acid therapeutics called conditional siRNAs (Cond-siRNA) is designed to act in this environment-specific manner. The first part of this thesis uses molecular dynamics simulations to understand the structure of Cond-siRNA and to suggest improvements in future designs.
\r\n\r\nBioengineering like the work done in the development of Cond-siRNAs depends on the existence of tools that make work simple, fast, cheap, and reproducible. In the case of nucleic acids, de novo synthesis of custom constructs is a fundamental tool. While approaches to synthesis have improved immensely since their inception, increasing ambition demands increasingly powerful tools. As target constructs get longer, the synthesis can become intractably complicated, slowing the process, increasing costs, and making it less likely to be replicated by others. The source of complexity in nucleic acid synthesis is the inability to directly synthesize long fragments without errors. Finding a new means of sequence-controlled synthesis that results in fewer errors and perhaps allows for correction could address this challenge. The second part of this thesis looks at using graphene as a mask for patterning the deposition of molecules on a surface with an eye towards arranging and coupling reactants in a sequence-specific way.
" }, { "name": "Jensen, Elizabeth Hwang", "degree": "PhD", "year": "2018", "title": "Elucidating the Role of O-GlcNAc Glycosylation in Neurobiology and Neurodegeneration", "advisor": "Hsieh-Wilson, Linda C.", "url": "https://resolver.caltech.edu/CaltechTHESIS:12192017-155744407", "creators": [ { "name": { "family": "Jensen", "given": "Elizabeth Hwang" }, "id": "Jensen-Elizabeth-Hwang", "orcid": "0000-0002-6177-4304", "display_name": "Jensen, Elizabeth Hwang" } ], "advisors": [ { "name": { "family": "Hsieh-Wilson", "given": "Linda C." }, "id": "Hsieh-Wilson-L-C", "role": "advisor", "display_name": "Hsieh-Wilson, Linda C." } ], "committee": [ { "name": { "family": "Dervan", "given": "Peter B." }, "id": "Dervan-P-B", "role": "chair", "display_name": "Dervan, Peter B." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "role": "member", "display_name": "Prober, David A." }, { "name": { "family": "Hsieh-Wilson", "given": "Linda C." }, "id": "Hsieh-Wilson-L-C", "role": "member", "display_name": "Hsieh-Wilson, Linda C." } ], "option_major": [ "biochem" ], "doi": "10.7907/Z9JD4TZ9", "abstract": "O-GlcNAc glycosylation is a dynamic, inducible post-translational modification (PTM) essential for neuronal homeostasis and found on proteins associated with neurodegenerative diseases such as \u03b1-synuclein, amyloid precursor protein, and tau. Intracellularly, O-GlcNAc modification is cycled by two enzymes in mammalian cells: O-GlcNAc transferase (OGT) appends O-GlcNAc to serine or threonine residues and O-GlcNAcase (OGA) removes O-GlcNAc. OGT modifies over 1000 different proteins, but the lack of a well-defined consensus sequence or substrate structural constraints has hampered efforts to predict sites a priori. Furthermore, the identification of O-GlcNAc modification sites has been obstructed by the difficulty of enriching and detecting O-GlcNAc using traditional biochemical methods. Here, we established and employed biological and chemical tools to illuminate the role of O-GlcNAc in neuronal function.
\r\n\r\nIn Chapter 2, we sought to determine the role of O-GlcNAc in learning, memory, and neurodegeneration. Deletion of the OGT gene causes early postnatal lethality in mice, complicating efforts to study O-GlcNAc glycosylation in mature neuronal function and dysfunction. We demonstrated that the loss of OGT in the forebrain of adult mice (OGT cKO) leads to progressive neurodegeneration, including neuronal death, neuroinflammation, hyperphosphorylated tau, amyloidogenic A\u03b2-peptides, and memory deficits. In the hippocampus, we showed that OGT ablation lead to the upregulation of neuroinflammatory genes and the downregulation of cholesterol biosynthetic genes. Additionally, a gene network analysis (WGCNA), qPCR, and immunohistochemistry (IHC) revealed that loss of O-GlcNAc perturbed cell cycle progression in the hippocampal neurons. In the hippocampus, we identified increased neuroinflammatory gene transcription in OGT cKO mice and both tau neurofibrillary tangle (NFT)-forming and amyloid-forming Alzheimer\u2019s disease (AD) mouse models. However, only OGT cKO and NFT-forming mice displayed decreased synaptic gene expression, suggesting that NFT formation and OGT cKO compromise hippocampal synaptic transcription. These studies indicate that O-GlcNAcylation regulates pathways vital for the maintenance of neuronal health and suggest that dysfunctional O-GlcNAc signaling may be an important contributor to neurodegenerative diseases.
\r\n\r\nIn order to understand the critical O-GlcNAc-mediated neuronal functions that underlie OGT cKO dysfunction, we next developed and utilized novel biological and chemical tools in order to identify key OGT interactors and substrates in the brain in Chapter 3. Due to the lack of a well-defined OGT substrate sequence and structural constraints, OGT is believed to obtain its substrate specificity through its interactome where specific interactors target OGT to specific substrates. In order to identify these interactors, we used CRISPR/Cas9 to generate a novel mouse with a minimally tagged OGT in order to identify the endogenous OGT brain interactome using tandem affinity purification and MS methods. The preliminary OGT brain interactome consisted of previously identified OGT interactors and substrates as well as novel interactors. The identified OGT interactors were enriched for ribosomal and cytoskeletal proteins in addition to axonal, dendritic, and neuronal cell body proteins, implicating OGT as a pivotal mediator of neuronal structure and function.
\r\n\r\nIn addition to the OGT interactome, we sought to uncover OGT\u2019s substrates or the O-GlcNAcome. We developed an improved approach to quantitatively label and enrich O-GlcNAcylated proteins for site identification. Chemoenzymatic labeling followed by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) installed a new MS-compatible linker designed for facile purification and release of O-GlcNAcylated proteins for downstream MS analysis. We validated the approach by identifying several established O-GlcNAc sites on the proteins \u03b1-crystallin and OGT as well as discovering new, previously unreported sites on both proteins. Notably, these novel sites on OGT lie in key functional domains of OGT, underscoring how this site identification method can reveal important biological insights into protein activity and regulation.
\r\n\r\nFinally, in Chapters 4 and 5, we focus on the post-translational modification (PTM) code on a specific transcription factor (TF), CREB (cAMP response element binding protein). CREB regulates memory formation through its transcriptional control of neuronal metabolism, activity, differentiation, development, and survival. CREB phosphorylation at serine 133 has been previously shown to enhance CREB-mediated transcription while CREB glycosylation at serine 40 has been shown to decrease CREB-mediated transcription. However, the exact gene networks modulated by and potential interplay between CREB glycosylation and phosphorylation have not been explored. Through differential expression analysis with glycosylation-deficient (S40A) and phosphorylation-deficient (S133A) CREB mutants, we showed that CREB O-GlcNAcylation is important for neuronal activity and excitability while phosphorylation at serine 133 regulated the expression of genes involved in neuronal differentiation. Using WGCNA, we demonstrated that CREB O-GlcNAcylation at serine 40 and phosphorylation at serine 133 mediate mutually exclusive gene networks. The glycosylation-deficient mutant enhanced neuronal activity- and excitotoxicity-related gene networks while the phosphorylation-deficient mutant perturbed neuronal differentiation and amino and fatty acid metabolism-related gene networks. Our work sheds light on the regulation of CREB through PTMs to modulate neuronal function and delineate the roles of O-GlcNAcylation and phosphorylation in modulating neuronal excitability and neuronal development and metabolism respectively. Altogether, these studies demonstrate that O-GlcNAc modification is a critical mediator of neuronal homeostasis and neurodegeneration.
" }, { "name": "Lin, Ben Albert", "degree": "PhD", "year": "2018", "title": "Ultrasound Speckle Image Velocimetry: Studies on System Performance and Application to Cardiovascular Fluid Dynamics", "advisor": "Gharib, Morteza", "url": "https://resolver.caltech.edu/CaltechTHESIS:08152017-194113650", "creators": [ { "name": { "family": "Lin", "given": "Ben Albert" }, "id": "Lin-Ben-Albert", "display_name": "Lin, Ben Albert" } ], "advisors": [ { "name": { "family": "Gharib", "given": "Morteza" }, "id": "Gharib-M", "orcid": "0000-0003-0754-4193", "role": "advisor", "display_name": "Gharib, Morteza" } ], "committee": [ { "name": { "family": "Gharib", "given": "Morteza" }, "id": "Gharib-M", "orcid": "0000-0003-0754-4193", "role": "chair", "display_name": "Gharib, Morteza" }, { "name": { "family": "Yang", "given": "Changhuei" }, "id": "Yang-Changhuei", "orcid": "0000-0001-8791-0354", "role": "member", "display_name": "Yang, Changhuei" }, { "name": { "family": "Dabiri", "given": "John O." }, "id": "Dabiri-J-O", "orcid": "0000-0002-6722-9008", "role": "member", "display_name": "Dabiri, John O." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "bioeng" ], "doi": "10.7907/Z998856J", "abstract": "Knowledge of detailed blood flow characteristics can be extremely valuable in a variety of settings. Examples range from studying disease processes such as\r\natherosclerosis to aiding in the design of medical devices such as prosthetic cardiac valves. For in vivo and optically inaccessible in vitro flows, accurate measurements of velocity fields and shear stresses can be difficult to obtain. Doppler ultrasound and magnetic resonance imaging are the most commonly used techniques, but have important limitations. Recently, there has been increased interest in the application of particle image velocimetry principles towards tracking of ultrasound speckle patterns to determine multidimensional flow velocities with increased temporal resolution. We refer to our implementation as ultrasound speckle image velocimetry (USIV). In this research project, our first objective was to obtain a detailed characterization of the factors unique to ultrasound imaging that can influence the accuracy of velocity measurements. By conducting in vitro experiments with uniform speckle phantom translation as well as steady tube flow, we have shown that characteristics such as transducer focal depth and beam sweep speed as well as particle motion direction and velocity can all influence USIV results. Our second objective was to demonstrate the utility of USIV for analyzing in vivo blood flows. After administering ultrasound contrast agent to anesthetized\r\npigs, we were able to obtain detailed images of both left ventricular flow and\r\nabdominal aortic flow. Velocity profiles were measured during both left ventricular filling and ejection. Our most interesting finding was the presence in certain cases of highly asymmetric retrograde flow in the infrarenal aorta. The factors that lead to such flows may have relevance to the development of atherosclerosis and abdominal aneurysms. USIV is likely to be very useful for further studies both in vivo and with in vitro elastic aorta models." }, { "name": "Lin, Yong-Jun", "degree": "PhD", "year": "2018", "title": "Human Duration Perception Mechanisms in the Subsecond Range: Psychophysics and Electroencephalography Investigations", "advisor": "Shimojo, Shinsuke", "url": "https://resolver.caltech.edu/CaltechTHESIS:06012018-150815346", "creators": [ { "name": { "family": "Lin", "given": "Yong-Jun" }, "id": "Lin-Yong-Jun", "orcid": "0000-0003-1239-5217", "display_name": "Lin, Yong-Jun" } ], "advisors": [ { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "role": "advisor", "display_name": "Shimojo, Shinsuke" } ], "committee": [ { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "role": "chair", "display_name": "Meister, Markus" }, { "name": { "family": "O'Doherty", "given": "John P." }, "id": "O'Doherty-J-P", "role": "member", "display_name": "O'Doherty, John P." }, { "name": { "family": "Yun", "given": "Kyongsik" }, "id": "Yun-Kyongsik", "role": "member", "display_name": "Yun, Kyongsik" }, { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "role": "member", "display_name": "Shimojo, Shinsuke" } ], "option_major": [ "cns" ], "doi": "10.7907/KGG5-9375", "abstract": "In a world full of fleeting events, how do humans perceive time intervals as short as half a second? Unlike primary senses, there are no time receptors. Is sub-second time perception reconstructed from memory traces in the primary senses, or based on the output of a modality-independent internal clock? In analogy to bugs in computer programs or mutations in genetics studies, I studied two types of subjective time warp illusions in order to understand how time perception normally works. One illusion that I examined is called oddball chronostasis, which is a duration distortion effect that happens to an unusual item. The other illusion is called debut chronostasis, which is a time warp effect that occurs to the first item among other identical ones.
\r\n\r\nRegarding oddball chronostasis, we solved a theoretical dispute over its underlying mechanisms and dissociated three causes. The necessary component is top-down attention to the target item. The other two components are contingent factors. This suggests that a pure sensory modality-dependent view of time perception mechanisms is less likely.\r\nRegarding debut chronostasis, we discovered auditory debut chronostasis and found that its illusion strength is about the same as the visual case. At first glance, this seems to suggest that time perception is independent of the primary sensory modalities. However, when visual and auditory events were compared against each other (inter-modal comparison), debut chronostasis disappeared. Therefore, modality-dependent mechanisms of time perception do exist. Further, we found a special factor that could counteract debut chronostasis and thus re-interpreted the main cause of debut chronostasis as internal duration template uncertainty. By examining both intra- and inter-modal comparisons, this uncertainty effect turned out to be a modality-independent effect. Therefore, modality-independent mechanisms of time perception also exist.
\r\n\r\nIn conclusion, this dissertation work contributed to novel theoretical understanding of two types of time perception illusions. Unlike many simplified theories in the literature either holding a modality-dependent or independent view, our findings altogether indicate that time perception involves both intra- and supra-modal stages. Future experimental work could thus target on separating intra- and supra-modal time perception mechanisms.
" }, { "name": "Mahmoudabadi, Gita", "degree": "PhD", "year": "2018", "title": "Virology By The Numbers: A Quantitative Exploration of Viral Energetics, Genomics, and Ecology", "advisor": "Phillips, Robert B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04172018-174233725", "creators": [ { "name": { "family": "Mahmoudabadi", "given": "Gita" }, "id": "Mahmoudabadi-Gita", "orcid": "0000-0002-8812-7246", "display_name": "Mahmoudabadi, Gita" } ], "advisors": [ { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "advisor", "display_name": "Phillips, Robert B." } ], "committee": [ { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "chair", "display_name": "Phillips, Robert B." }, { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "orcid": "0000-0002-7033-0844", "role": "member", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "orcid": "0000-0002-3904-0195", "role": "member", "display_name": "Goentoro, Lea A." }, { "name": { "family": "Orphan", "given": "Victoria J." }, "id": "Orphan-V-J", "orcid": "0000-0002-5374-6178", "role": "member", "display_name": "Orphan, Victoria J." }, { "name": { "family": "Gelbart", "given": "William" }, "id": "Gelbart-W", "role": "member", "display_name": "Gelbart, William" } ], "option_major": [ "bioeng" ], "doi": "10.7907/Z9Q81B91", "abstract": "Over the past couple of decades, technological advancements in sequencing and imaging have unequivocally proven that the world of viruses is far bigger and more consequential than previously imagined. There are 1031 viruses estimated to inhabit our planet, outnumbering even bacteria. Despite their astronomical numbers and staggering sequence diversity, environmental viruses are poorly characterized. In this thesis we will demonstrate our three-pronged exploration of viruses through the lenses of energetics (Chapters 2 and 3), genomics (Chapter 4) and ecology (Chapter 5). We will first focus on one of the defining features of viruses, namely their reliance on their host for energy, and demonstrate the energetic cost of building a virus and mounting an infection. In our second study, we present one of the largest surveys of complete viral genomes, providing a comprehensive and quantitative snapshot of viral genomic trends for thousands of viruses. In our third study, we shift our focus towards ecological questions surrounding the large number of commensal phages inhabiting the human body. We discovered that phage community composition could serve as a fingerprint, or a \"phageprint\" \u2013 highly personal and stable over time. To our knowledge, this study is one of the largest studies of human phages and the first to demonstrate the feasibility of human identification based on phage sequences.
" }, { "name": "Nandagopal, Nagarajan", "degree": "PhD", "year": "2018", "title": "New Capabilities of the Notch Signaling Pathway", "advisor": "Elowitz, Michael B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:12282017-140713708", "creators": [ { "name": { "family": "Nandagopal", "given": "Nagarajan" }, "id": "Nandagopal-Nagarajan", "orcid": "0000-0002-0469-6549", "display_name": "Nandagopal, Nagarajan" } ], "advisors": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "advisor", "display_name": "Elowitz, Michael B." } ], "committee": [ { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "orcid": "0000-0002-3904-0195", "role": "chair", "display_name": "Goentoro, Lea A." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." } ], "option_major": [ "bioeng" ], "doi": "10.7907/Z98050TB", "abstract": "Animal cells use a conserved repertoire of signaling pathways to exchange information during and after development. The constituent molecules of these pathways and their individual interactions are now well-characterized. However, it is becoming clear that pathways often possess unexpected signal-processing capabilities, which are typically collective, systems-level, features. Recent work shows that these capabilities are best investigated using quantitative, single-cell, dynamic analyses of pathway behavior. Here, we used this approach to study Notch signaling pathway, which is widely utilized for juxtacrine signaling during the development and maintenance of most tissues. Our work reveals two new capabilities of this pathway. First, the receptor Notch1 is capable of discriminating between two similar ligands, Dll1 and Dll4, and can use this ability to enact ligand-specific developmental programs. To enable this, the pathway encodes ligand identity in the dynamics of Notch1 signaling, and later decodes it for controlling gene expression. We show that dynamic encoding by Dll1 and Dll4 results from different requirements for ligand-receptor clustering during activation. Second, the pathway is capable of cell-autonomous signaling (cis-activation). This mode of signaling is general to multiple ligand-receptor combinations, and possesses many attributes of intercellular signaling. We show that cis-activation occurs in natural stem-cell contexts, where it could be important for self-renewal and prevents premature differentiation. These new capabilities of this central signaling pathway have implications for understanding the role of Notch in development and homeostasis, diagnosing and treating its misregulation in disease, and controlling it for tissue engineering and regeneration.
" }, { "name": "Nath, Ravi David", "degree": "PhD", "year": "2018", "title": "The Evolutionary Construction of Sleep", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05272018-025533397", "creators": [ { "name": { "family": "Nath", "given": "Ravi David" }, "id": "Nath-Ravi-David", "orcid": "0000-0003-0905-2707", "display_name": "Nath, Ravi David" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "role": "chair", "display_name": "Prober, David A." }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "role": "member", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/JPDW-JW81", "abstract": "To understand the biological basis of sleep we need to understand its neuronal and genetic regulation. In this thesis, I explore how individual behaviors serve as building blocks to construct the sleep state where a block is defined as a set of measurable behaviors. These behavioral blocks are shaped by evolutionary forces. From one animal to the next, blocks may remain or change. If a block remains across all the sleep states in the metazoan lineage then it must have an important and conserved role in sleep regulation. For example, reduced locomotion is a behavior that is often observed during sleep. There are two possible explanations for the changing of a block: either the block was vestigial or the block was easily replaceable with another block that fulfills the same function. Consider sleep duration: some animals may require five hours of sleep, while others only require one hour. The changing of a block is one way that the sleep state could evolve. Blocks may also be added during the evolution of the sleep state, increasing the dimensions and number of tasks that are accomplished during sleep. Here, I discuss the origin of sleep, as well as its conserved neuronal and genetic regulation. I report the following: the discovery of sleep in jellyfish which are among the first animals to evolve neurons and the identification of novel sleep regulators in the roundworm Nematode Caenorhabiditis elegans (C. elegans). The sleep regulators discovered in C. elegans may have conserved functions in vertebrates. These studies show that some sleep behaviors and various sleep molecules change or remain homologous across metazoans. The studies are united by our simple block hypothesis of sleep construction.
" }, { "name": "Park, Jin", "degree": "PhD", "year": "2018", "title": "Circuits of Dynamically Interacting Sigma Factors in Single Cells", "advisor": "Elowitz, Michael B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:12312017-234413682", "creators": [ { "name": { "family": "Park", "given": "Jin" }, "id": "Park-Jin", "display_name": "Park, Jin" } ], "advisors": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "advisor", "display_name": "Elowitz, Michael B." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "role": "member", "display_name": "Goentoro, Lea A." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "member", "display_name": "Elowitz, Michael B." } ], "option_major": [ "bioeng" ], "doi": "10.7907/CT0B-Q853", "abstract": "How do cells integrate multiple, dynamic genetic circuits? I study this question in the context of the alternative sigma factors of B. subtilis.
\r\n\r\nThe first project proposes a novel mode of gene regulation called timesharing. The key idea is that a limited resource is shared dynamically in time. Here we show that the alternative sigma factors of B. subtilis use dynamic sharing to share a limited supply of core RNA Polymerase (RNAP). We show that 5 alternative sigma factors activate in pulses, and that these pulses operate in a competitive regime. Interestingly, we found that pairwise correlations between these sigma factors contained a mixture of positive and negative correlations, whereas one may naively expect all correlations to be negative. We show with a mathematical model that competitive pulsing can lead to non-intuitive sets of mixed correlations.
\r\n\r\nThe second project take a closer, quantitative look at sigma factor competition. Although competition between the housekeeping sigma and a single alternative sigma has been well studied, competition between alternative sigmas themselves has been relatively unexplored. To address this issue, we systematically investigated the pairwise competitive relationships between 7 alternative sigma factors in B. subtilis. The main experimental tool was a 7x7 'deletion' matrix of strains, where every matrix strain was deleted for one sigma, and reported on another sigma via a fluorescent reporter. The deletion matrix revealed that competition is highly asymmetric. Deletion of any given sigma factor increased \u03c3W activity, but did not affect other sigma factors. These results are recreated by a minimal mathematical model of sigma factor competition, where importantly \u03c3W is relatively high in abundance but weak in affinity for core RNAP. We used the model to predict how overexpressing sigma factors affect each other, and these predictions were matched by experiments.
\r\n\r\nThe third project reports a novel activator for alternative sigma factors. Alternative sigmas factors are activated by many forms of stress, such as nutrient limitation, temperature shifts, and molecular stresses like antibiotics. Here we show that surprisingly, cell lysis causes adjacent cells to specifically activate \u03c3X. This cell lysis-\u03c3X response is a general phenomenon, as it is observed under multiple experimental conditions. We show this relationship between cell death and \u03c3X is causal, since harvested cell extract activates \u03c3X. Finally, we hypothesize that cell death and \u03c3X play an important role in biofilm wrinkle formation.
" }, { "name": "Petersen, Philip Fai", "degree": "PhD", "year": "2018", "title": "Engineering Molecular Self-assembly and Reconfiguration in DNA Nanostructures", "advisor": "Qian, Lulu", "url": "https://resolver.caltech.edu/CaltechTHESIS:05312018-112853523", "creators": [ { "name": { "family": "Petersen", "given": "Philip Fai" }, "id": "Petersen-Philip-Fai", "orcid": "0000-0002-9912-389X", "display_name": "Petersen, Philip Fai" } ], "advisors": [ { "name": { "family": "Qian", "given": "Lulu" }, "id": "Qian-Lulu", "orcid": "0000-0003-4115-2409", "role": "advisor", "display_name": "Qian, Lulu" } ], "committee": [ { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "orcid": "0000-0002-5899-7523", "role": "chair", "display_name": "Winfree, Erik" }, { "name": { "family": "Rothemund", "given": "Paul W. K." }, "id": "Rothemund-P-W-K", "orcid": "0000-0002-1653-3202", "role": "member", "display_name": "Rothemund, Paul W. K." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Qian", "given": "Lulu" }, "id": "Qian-Lulu", "orcid": "0000-0003-4115-2409", "role": "member", "display_name": "Qian, Lulu" } ], "option_major": [ "biology" ], "doi": "10.7907/7FXP-8402", "abstract": "Smart electronics have developed ubiquitously to assist people in everything from navigation to health monitoring. The rise of complex electronics relied on rational design of platforms to build ever larger and more complex circuit networks and for frameworks to test those electronics. Biochemical circuits have also seen dramatic advancement in the last two decades within the field of DNA nanotechnology. As with electronics, DNA nanotechnology applied rational design to DNA molecules to build ever more complex biochemical networks that, beyond current electronics, also retain a significant measure of biological compatibility and plasticity akin to many networks of biological origin. Well situated for promising applications in diagnostics and therapeutics, advancing DNA nanotechnology devices will also rely upon larger platforms and testing frameworks.
\r\n\r\nIn roughly the last decade, researchers have been building upon the invention of DNA origami, a technique allowing the robust construction of biomolecular nano-structures capable of precise nanometer positioning of proteins, nanoparticles, and other molecules. DNA circuits have computed on the nanostructures; DNA robots have moved nanoparticles, made choices, and have even sorted cargo on the surface of a nanostructure. The complexity of circuits and devices continues to rise.
\r\n\r\nIn this thesis, we will discuss our contributions to the field of DNA nanotechnology by developing design rules and systematic approaches to controlling nanostructure complex assembly. These rules and approaches allow for the construction of molecular structures with a tunable diversity, large systems approaching the size of bacteria yet retaining nanometer precision, and biological plasticity inspired dynamic systems for arbitrary reconfiguration.
\r\n\r\nUsing a DNA origami tile tailored for array formation with a high continuous surface area, we create a framework inspired from molecular stochasticity for programming DNA array formation and gaining control over diversity of global properties through simple local rules. Three general forms of planar networks, random loops, mazes, and trees, were manipulated on the micron scale upon the self-assembled DNA arrays. We demonstrate control of several properties of the networks, such as branching rules, growth directions, the proximity between adjacent networks, and size distributions. The large diversity, in principle, allows for a wide, but tunable, testing environment for molecular circuits. By further applying these principles to subunits of finite assemblies, variable components may be mixed with fixed components potentially opening additional applications in high throughput device or drug screening.
\r\n\r\nNext we turned to expanding the platform size biochemical circuits may be built upon. While DNA origami allows nanometer precise placement, the size remains roughly below 0.05 um2. Toward making large arbitrarily complex structures with only a set of simple tiles, multi-stage self-assembly has been explored in theory and for small DNA tiles. None were successful experimentally with DNA origami. We developed a strategy for DNA origami: a simple rule set applied recursively in each stage of a hierarchical self-assembly process, and to significantly reduce costs, a constant set of unique DNA strands regardless of size. We also developed a software tool to automatically compile a designed surface pattern into experimental protocols. We experimentally demonstrated DNA origami arrays approaching the size of small bacteria, 0.5 um2, with several arbitrary patterns, each consisting of 8,704 specifically chosen pixel locations with nanometer precision, including a bacteria sized portrait of a bacteria. The large platform opens the door to more advanced molecular circuits for applications such as diagnostics.
\r\n\r\nFinally we demonstrated control over the dynamics of DNA origami reconfiguration in tile arrays. In an approach we call DNA tile displacement, we showed that a DNA origami array may have tiles arbitrarily replaced by another tile, including tiles of another shape or surface pattern. We also demonstrated control over the kinetics of tile displacement and performed several general purpose reconfigurations of DNA nanostructures. Examples include sequential reconfiguration, competitive reconfiguration, cooperative reconfiguration, and finally the scalability of multi-step reconfiguration as demonstrated through a fully playable nano-scale biomolecular tic-tac-toe game. The major ramifications are a plasticity more common to biology than to electronics\u2014molecular platforms with arbitrary patterning that can reconfigure an arbitrary part of the nanostructure in an arbitrary order based on environmental signals. In principle, such reconfiguration can allow advanced circuits with the capacity to adapt to environmental needs or heal damaged components.
\r\n" }, { "name": "Rogers, Alicia Kathryn", "degree": "PhD", "year": "2018", "title": "Mechanisms of Transcriptional Silencing by the Nuclear Piwi Protein in Drosophila Germ Cells", "advisor": "Fejes T\u00f3th, Katalin; Aravin, Alexei A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04092018-141017685", "creators": [ { "name": { "family": "Rogers", "given": "Alicia Kathryn" }, "id": "Rogers-Alicia-Kathryn", "orcid": "0000-0001-5525-6095", "display_name": "Rogers, Alicia Kathryn" } ], "advisors": [ { "name": { "family": "Fejes T\u00f3th", "given": "Katalin" }, "id": "Fejes-T\u00f3th-K", "orcid": "0000-0001-6558-2636", "role": "advisor", "display_name": "Fejes T\u00f3th, Katalin" }, { "name": { "family": "Aravin", "given": "Alexei A." }, "id": "Aravin-A-A", "orcid": "0000-0002-6956-8257", "role": "co-advisor", "display_name": "Aravin, Alexei A." } ], "committee": [ { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "chair", "display_name": "Hay, Bruce A." }, { "name": { "family": "Fejes T\u00f3th", "given": "Katalin" }, "id": "Fejes-T\u00f3th-K", "orcid": "0000-0001-6558-2636", "role": "member", "display_name": "Fejes T\u00f3th, Katalin" }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Aravin", "given": "Alexei A." }, "id": "Aravin-A-A", "orcid": "0000-0002-6956-8257", "role": "member", "display_name": "Aravin, Alexei A." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "molbiochem" ], "doi": "10.7907/Z9KK991X", "abstract": "An important characteristic for life is the ability to persist \u2013 to reproduce and defend oneself against different stresses. The ability of a species to persist from one generation to the next heavily depends on the integrity of the genetic material being passed down, and thus organisms have developed strategies to ensure the integrity of their genomes remain in tact. In Metazoan germlines, piwi proteins and their associated piwi-interacting RNAs (piRNAs) provide a RNA-interference (RNAi) based defense system against the expression of transposable elements (TEs). TE expression is detrimental to an organism\u2019s genome \u2013 resulting in disruption of genes, double-stranded DNA breaks, and germ cell death \u2013 ultimately leading to the sterility of the organism. In Drosophila melanogaster, the piRNA pathway is composed of two cytoplasmic piwi clade Argonuate proteins, Aubergine (Aub) and Argonaute3 (Ago3), and a single nuclear piwi clade Argonuate protein, Piwi. The piwi clade Argonaute proteins bind piRNAs to form effector complexes that repress TE sequences.
\r\n\r\nThe work presented in this thesis examines the role of the nuclear piwi clade Argonaute \u2013 Piwi \u2013 and the mechanisms by which Piwi accomplishes its functions. Chapter Two presents how Piwi/piRNA complexes identify genomic loci expressing TEs and direct the establishment of a repressive chromatin state to transcriptionally silence the loci. In Chapter Three, we explore the piRNA-induced transcriptional silencing (piRITS) pathway using a heterologous reporter based tethering system in vivo. We discuss how the recruitment of Piwi alone to a locus is not sufficient to induce repression, and establish a model for the connection bridging the Piwi/piRNA complex and effector silencing complex in the piRITS pathway. In Chapter Four, we employ our heterologous reporter based tethering system to explore the mechanism of piRNA precursor selection in the two cell types that make up Drosophila ovaries. We uncover a common mechanism of piRNA biogenesis in the two cell types and establish a unifying model of piRNA substrate selection. Finally, in Chapter Five, as essential step to understanding how Piwi achieves its nuclear function, we developed a heterologous two-hybrid system to identify factors that directly interact with Piwi. Overall, the work presented in this thesis provides a piece of the groundwork in understanding the mechanisms of transcriptional silencing of TEs in germ cells by Piwi. The work proposes that Piwi has dual functions in the nucleus. First, upon target recognition, Piwi recruits the piRITS complex to target loci to accomplish Piwi- mediated transcriptional silencing by deposition of H3K9me3. Then, Piwi recruits the RDC complex to specifically bind H3K9me3 at target loci to allow piRNA-production from the locus.
" }, { "name": "Schmidt, William Charles", "degree": "Senior Thesis", "year": "2018", "title": "What Makes a Narrative? Understanding the Portrayals of Hermenegild's Rebellion", "advisor": "Brown, Warren C.", "url": "https://resolver.caltech.edu/CaltechTHESIS:09252018-124342094", "creators": [ { "name": { "family": "Schmidt", "given": "William Charles" }, "id": "Schmidt-William-Charles", "orcid": "0000-0001-9780-9495", "display_name": "Schmidt, William Charles" } ], "advisors": [ { "name": { "family": "Brown", "given": "Warren C." }, "id": "Brown-Warren-C", "role": "advisor", "display_name": "Brown, Warren C." } ], "committee": [ { "name": { "family": "None", "given": "None" }, "display_name": "None, None" } ], "option_major": [ "bioeng", "history" ], "doi": "10.7907/R6G8-3B60", "abstract": "When one studies an event through the perspectives of multiple accounts, one\u2019s first instinct might be to reconcile the sources into a single cohesive narrative. One can try to assign a likelihood that various portions of each story are factually correct, and then reconstruct what happened based upon which parts seem the most trustworthy.However, in doing so, one cannot be certain of the results. Each of us has our own subjectivity and biases, as our experiences can implicitly shape the decisions we make about what is reliable or plausible and what is not. Moreover, when one tries to compile the \u201ctruth\u201d of an event from multiple sources, such \"truth\" comes at the cost of understanding what made the sources different in the first place. An account is not written in a vacuum, and the way that an author chooses to portray an event is determined by their own personal background, circumstances, and purposes for crafting their narratives. In condensing a host of different accounts into a single internally consistent version, one loses sight of how the authors themselves viewed the events. Keeping different accounts of the same event separate, and investigating each individually in its own context, may not provide a simple solution to the question of \u201cwhat happened?\u201d, but it will teach us more about the authors\u2019 motivations and what they understood to be important about an event. Such an understanding provides more substantial and reliable information than trying to reconcile the accounts would be able to provide." }, { "name": "Urban, Luke Stuart", "degree": "PhD", "year": "2018", "title": "An Electrophysiological Study Of Voluntary Movement and Spinal Cord Injury", "advisor": "Burdick, Joel Wakeman", "url": "https://resolver.caltech.edu/CaltechTHESIS:06012018-140912331", "creators": [ { "name": { "family": "Urban", "given": "Luke Stuart" }, "id": "Urban-Luke-Stuart", "display_name": "Urban, Luke Stuart" } ], "advisors": [ { "name": { "family": "Burdick", "given": "Joel Wakeman" }, "id": "Burdick-J-W", "role": "advisor", "display_name": "Burdick, Joel Wakeman" } ], "committee": [ { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "role": "chair", "display_name": "Perona, Pietro" }, { "name": { "family": "Abu-Mostafa", "given": "Yaser S." }, "id": "Abu-Mostafa-Y-S", "role": "member", "display_name": "Abu-Mostafa, Yaser S." }, { "name": { "family": "Edgerton", "given": "V. Reggie" }, "id": "Edgerton-V-R", "role": "member", "display_name": "Edgerton, V. Reggie" }, { "name": { "family": "Burdick", "given": "Joel Wakeman" }, "id": "Burdick-J-W", "role": "member", "display_name": "Burdick, Joel Wakeman" } ], "option_major": [ "cns" ], "doi": "10.7907/K6P2-ZH75", "abstract": "Voluntary movement is generated from the interaction between neurons in our brain and the neurons in our spinal cord that engage our muscles. A spinal cord injury destroys the connection between these two regions, but parts of their underlying neural circuits survive. A new class of treatment (the brain-machine interface) takes advantage of this fact by either a) recording neural activity from the brain and predicting the intended movement (neural prosthetics) or b) stimulating neural activity in the spinal cord to facilitate muscle activity (spinal stimulation). This thesis covers new research studying the brain-machine interface and its application for spinal injury.
\r\n\r\nFirst, the electrical properties of the microelectrode (the main tool of the brain-machine interface) are studied during deep brain recording and stimulation. This work shows that the insulation coating the electrode forms a capacitor with the surrounding neural tissue. This capacitance causes large spikes of voltage in the surrounding tissue during deep brain stimulation, which will cause electrical artifacts in neural recordings and may damage the surrounding neurons. This work also shows that a coaxially shielded electrode will block this effect.
\r\n\r\nSecond, the activity of neurons in the parietal cortex is studied during hand movements, which has applications for neural prosthetics. Prior work suggests that the parietal cortex encodes a state-estimator [1], which combines sensory feedback with the internal efference copy to predict the state of the hand. To test this idea, we used a visual lag to misalign sensory feedback from the efference copy. The expectation was that a state-estimator would unknowingly combine the delayed visual feedback with the current efference information, resulting in incorrect predictions of the hand. Our results show a drop in correlation between neural activity in the parietal cortex and hand movement during a visual lag, supporting the idea that the parietal cortex encodes a state-estimator. This correlation gradually recovers over time, showing that parietal cortex is adaptive to sensory delays.
\r\n\r\nThird, while the intention of spinal stimulation was to interact locally with neural circuits in the spinal cord, results from the clinic show that electrical stimulation of the lumbosacral enlargement enables paraplegic patients to regain voluntary movement of their legs [2]. This means that spinal stimulation facilitates communication across an injury site. To further study this effect, we developed a new behavioral task in the rodent. Rats were trained to kick their right hindlimb in response to an auditory cue. The animals then received a spinal injury that caused paraplegia. After injury, the animals recovered the behavior (they could kick in response to the cue), but only during spinal stimulation. Their recovered behavior was slower and more stereotyped than their pre-injury response. Administering quipazine to these rodents disrupted their ability to respond to the cue, suggesting that serotonin plays an important role in the recovered pathway. This work proves that the new behavioral task is a successful tool for studying the recovery of voluntary movement.
\r\n\r\nFuture work will combine cortical recordings with this behavioral task in the rodent to study plasticity in the nervous system and improve treatment of spinal cord injuries.
\r\n\r\n[1] Mulliken, Grant H., Sam Musallam, and Richard A. Andersen. \"Forward estimation of movement state in posterior parietal cortex.\" Proceedings of the National Academy of Sciences105.24 (2008): 8170-8177.
\r\n\r\n[2] Harkema, Susan, et al. \"Effect of epidural stimulation of the lumbosacral spinal cord on voluntary movement, standing, and assisted stepping after motor complete paraplegia: a case study.\" The Lancet 377.9781 (2011): 1938-1947.
" }, { "name": "Valencia, Jonathan Exiquio", "degree": "PhD", "year": "2018", "title": "The Developmental Organization of Regulatory States in the Sea Urchin Larva", "advisor": "Davidson, Eric H.; Peter, Isabelle S.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05292018-131620231", "creators": [ { "name": { "family": "Valencia", "given": "Jonathan Exiquio" }, "id": "Valencia-Jonathan-Exiquio", "orcid": "0000-0002-3498-8980", "display_name": "Valencia, Jonathan Exiquio" } ], "advisors": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "advisor", "display_name": "Davidson, Eric H." }, { "name": { "family": "Peter", "given": "Isabelle S." }, "id": "Peter-I-S", "role": "advisor", "display_name": "Peter, Isabelle S." } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "role": "member", "display_name": "Goentoro, Lea A." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Peter", "given": "Isabelle" }, "id": "Peter-I-S", "role": "member", "display_name": "Peter, Isabelle" } ], "option_major": [ "biodev" ], "doi": "10.7907/NVWB-AP91", "abstract": "Development is an inherently dynamic process where cell fate specification occurs continuously and in a progressive manner. Thus, a major focus in developmental biology is solving the gene regulatory networks (GRNs) that underlie specification of cell fates. GRNs specify new spatial domains of cells by controlling the expression of their changing regulatory states throughout development. Regulatory states are composed of combinations of expressed regulatory genes which encode transcription factors that form regulatory circuits which function to carry out the specific developmental tasks involved in cell fate specification.
\r\n\r\nTo investigate the differences of GRNs operating in the embryo and their change over development, we sought to identify and characterize the regulatory states present in multiple developmental stages of sea urchin embryogenesis. We performed a genome-wide survey and embryo-wide annotation of regulatory gene expression by whole mount in situ hybridization at five consecutive developmental time-points in order to determine regulatory states and their developmental trajectory. We determined at least 74 distinct regulatory states expressed in discrete developmental domains which coincide with larval morphological structures and show that their progenitor domains foreshadow the ensuing larval morphology. Among these domains, we identified bilateral ciliary photoreceptors in the larva which express a distinct regulatory state that include factors known in ciliary photoreceptor specification. We show that this photoreceptor regulatory state does not express the genes of the retinal determination network that specify eyes in both flies and vertebrates. In addition, we show that though the sizes of regulatory states are comparable over developmental time, no two regulatory states are equal, even those expressed in a given domain at previous or subsequent developmental time-points. Lastly, we found that similarities among regulatory states reflect a common developmental function but not necessarily a common developmental history. The results suggest that the combinations of transcription factors defining regulatory states are both spatially and temporally dynamic in their progressive specification of cell fates during development and that regulatory state expression is tightly associated with the developing morphology of the larva.
" }, { "name": "Zhang, Carey Yuzhe", "degree": "PhD", "year": "2018", "title": "Partially Mixed Selectivity and Parietal Cortex", "advisor": "Andersen, Richard A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05212018-145923990", "creators": [ { "name": { "family": "Zhang", "given": "Carey Yuzhe" }, "id": "Zhang-Carey-Yuzhe", "orcid": "0000-0001-9867-4510", "display_name": "Zhang, Carey Yuzhe" } ], "advisors": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "role": "advisor", "display_name": "Andersen, Richard A." } ], "committee": [ { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "role": "chair", "display_name": "Murray, Richard M." }, { "name": { "family": "Burdick", "given": "Joel Wakeman" }, "id": "Burdick-J-W", "role": "member", "display_name": "Burdick, Joel Wakeman" }, { "name": { "family": "Rutishauser", "given": "Ueli" }, "id": "Rutishauser-U", "role": "member", "display_name": "Rutishauser, Ueli" }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "role": "member", "display_name": "Andersen, Richard A." } ], "option_major": [ "bioeng" ], "doi": "10.7907/R1RS-RJ59", "abstract": "Brain-machine interfaces (BMIs) decode intention signals and other variables from the brain in order to control a computer, tablet, or prosthetic limb. In order to improve the technology, a better understanding of the representational mechanisms within the brain is necessary. Here we study how the anterior intraparietal area (AIP) of human posterior parietal cortex is able to represent many variables within a small patch of cortex. We record single unit activity using a 4 x 4 mm microelectrode array implanted in AIP of a human tetraplegic volunteer. Testing movements of different cognitive strategies, body parts, and body sides, we find that the neural population represents information in a high-dimensional way, termed \"mixed selectivity\", with individual units coding for idiosyncratic combinations of variables. Furthermore, we find that the variables are not randomly mixed but exhibited \"partially mixed selectivity\" with certain variables more randomly mixed than others. Representations were \"functionally segregated\", with representations of the hand and shoulder largely orthogonal despite the high degree of anatomical overlap; representations of body side and strategy were organized by body part. We also examine how the representations changed between BMI training and online BMI control. We find that the structure of the movement representations was preserved, with the different representations found during calibration maintained during online control. Finally, we study the sensory mirror system, a system that processes observed sensations similarly to experienced sensations. We once again find partially mixed selectivity and functional segregation by body parts, showing that this method of encoding information exists not just in the action intention domain but also in the sensory domain. Our results propose partially mixed selectivity as a general mechanism for encoding high dimensional in formation in a small neural population, while also advancing the possibility of limited electrode-array BMIs decoding movements of a large extent of the body.
" }, { "name": "de Oliveira Penna Tavares, Gabriela", "degree": "PhD", "year": "2018", "title": "Computation and Comparison of Value Signals in Simple Perceptual and Economic Choices", "advisor": "Rangel, Antonio", "url": "https://resolver.caltech.edu/CaltechTHESIS:12272017-080109402", "creators": [ { "name": { "family": "de Oliveira Penna Tavares", "given": "Gabriela" }, "id": "de-Oliveira-Penna-Tavares-Gabriela", "orcid": "0000-0002-8624-1758", "display_name": "de Oliveira Penna Tavares, Gabriela" } ], "advisors": [ { "name": { "family": "Rangel", "given": "Antonio" }, "id": "Rangel-A", "role": "advisor", "display_name": "Rangel, Antonio" } ], "committee": [ { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "role": "chair", "display_name": "Perona, Pietro" }, { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "role": "member", "display_name": "Adolphs, Ralph" }, { "name": { "family": "O'Doherty", "given": "John P." }, "id": "O'Doherty-J-P", "role": "member", "display_name": "O'Doherty, John P." }, { "name": { "family": "Rangel", "given": "Antonio" }, "id": "Rangel-A", "role": "member", "display_name": "Rangel, Antonio" } ], "option_major": [ "cns" ], "doi": "10.7907/Z9P55KP1", "abstract": "How do we choose between different foods from a restaurant menu, or between a vacation overseas and more money in our savings account? Certain mechanisms in our brains allow us to make these and many other kinds of decisions effectively and efficiently. In this dissertation, I describe three projects which aim to advance our understanding of the systems and algorithms involved in the process of human decision making.
\r\n\r\nChapter 2 investigates the application of the attentional Drift-Diffusion Model to a perceptual decision making task. Perceptual decisions requiring the comparison of spatially distributed stimuli that are fixated sequentially might be influenced by fluctuations in visual attention. We used two psychophysical tasks with human subjects to investigate the extent to which visual attention influences simple percep- tual choices, and to test the extent to which the attentional Drift-Diffusion Model provides a good computational description of how attention affects the underlying decision processes. We found that this model provides a reasonable quantitative description of the relationship between fluctuations in visual attention, choices, and response times. We also found evidence for the sizable attentional choice biases predicted by the model, and that exogenous manipulations of attention induce choice biases consistent with these predictions.
\r\n\r\nChapter 3 compares two methods for fitting the parameters of the Drift-Diffusion Model using experimental data. A large number of studies have proposed that sequential integrator models of decision making, such as the Drift-Diffusion Model and its variants, provide a simple computational description of the algorithms used to make a large number of simple decisions. This is based on the fact that this class of models has been able to produce reasonably accurate descriptions of how choices, response times, and fixations are related to each other and to exogenous trial parameters, in a wide range of tasks. A difficult step in those studies is the estimation of a small number of free parameters to find the ones that explain the observed data best. The estimation method used in most studies is computationally very expensive since it approximates the likelihood of the observed data by simulating the model thousands of times and then counting the frequency with which the outcomes match the observed data. This problem is exacerbated with more complex models, such as the attentional Drift-Diffusion Model, or models with collapsing bounds, which contain a larger number of free parameters. We propose an alternative method for estimating the free parameters which relies on computing only the probability of the actual observed data, bypassing the need for the additional simulations. We present the results of simulation tests which show that our approach provides two key advantages over the alternative widely used method: a smaller number of experimental trials is needed in order to obtain comparable estimation accuracy, and the execution time of the estimation algorithms is substantially reduced.
\r\n\r\nFinally, Chapter 4 studies simple economic choices involving two distinct classes of valuation systems: an experiential system, which assigns value based on the history of previous reward experiences with similar options, and a descriptive system, which computes values using information about the options and environment available at the time of decision. Although these two systems often assign similar relative desirability to the different options, they do not always do so. When conflict arises with the experiential system favoring one option and the descriptive system favoring another, the brain needs to resolve the conflict to select a single option. We present the results of a psychometric study designed to characterize the basic interactions of these two valuation systems, with and without conflict.
" }, { "name": "Chan, Ken Yee", "degree": "PhD", "year": "2017", "title": "Engineered Viral Vectors and Developed Tissue Clearing Methods for Single-cell Phenotyping in Whole Organs", "advisor": "Gradinaru, Viviana", "url": "https://resolver.caltech.edu/CaltechTHESIS:05302017-222932176", "creators": [ { "name": { "family": "Chan", "given": "Ken Yee" }, "id": "Chan-Ken-Yee", "orcid": "0000-0002-8853-5186", "display_name": "Chan, Ken Yee" } ], "advisors": [ { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "role": "advisor", "display_name": "Gradinaru, Viviana" } ], "committee": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "chair", "display_name": "Lester, Henry A." }, { "name": { "family": "Cai", "given": "Long" }, "id": "Cai-Long", "role": "member", "display_name": "Cai, Long" }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "role": "member", "display_name": "Gradinaru, Viviana" } ], "option_major": [ "biology" ], "doi": "10.7907/Z9NC5Z7H", "abstract": "A central question in biology is how different cell types interact with each other and their native environment to form complex functional systems and networks. Although our ability to investigate this question has considerably expanded from the development of genetically encoded tools, some limitations still persist. For instance, we are limited in our ability to visualize the native three dimensional environments of whole organs. Additionally, it is challenging to efficiently deliver transgenes into difficult-to-target areas through direct-injections, such as the cardiac ganglia, or broadly distributed networks, such as the myenteric nervous system, which limits our ability to extensively study these areas. Therefore, tools and methods that overcome these limitations are needed. Towards this end, my thesis work has been focused on developing tools for single-cell resolution phenotyping in whole organs. I have been developing tissue clearing technologies to render whole organs transparent for optical interrogation and characterizing viral capsids and engineering viral vectors for noninvasive widespread gene delivery to the central and peripheral nervous system.
\r\n\r\nTissue clearing techniques for three dimensional optical interrogation were invented over a century ago. However, these earlier methods used harsh organic chemicals and failed to retain the tissue\u2019s native fluorescence or epitopes. These earlier methods eventually became incompatible to the hundreds of newly generated transgenic mouse lines that allowed for cell type-specific expression of fluorescent transgenes or to fluorescent labeling techniques, such as immunohistochemistry (IHC). The first part of my dissertation is aimed at addressing these limitations by further developing and standardizing a tissue clearing method that utilizes the vasculature to perfuse clearing reagents. This technique, called perfusion assisted agent release in situ (PARS) enables (i) whole organ clearing of soft tissue, (ii) preservation of native fluorescence, and (iii) preservation of epitopes compatible with IHC.
\r\n\r\nAlthough PARS allows us to optically investigate whole soft tissue organs, it is unsuitable for clearing bone tissue. The clearing of bone is important as it may provide optical access to delicate environments, such as the lymphatic vessels lining the dural sinuses beneath the skull that would otherwise be damaged through traditional methods. However, clearing bone tissue is challenging since it is composed of both soft (bone marrow) and hard (mineral) tissue. To overcome this challenge, I developed a clearing method that rendered intact bone tissue transparent by using EDTA to decalcify bones and by constructing a convective flow chamber to efficiently clear bones. This method, called Bone CLARITY, is able to preserve native fluorescence and epitopes. In order to demonstrate the utility of Bone CLARITY, I collaborated with colleagues to quantitatively access a rare and non-uniformly distributed population of osteoprogenitor cells in their native three dimensional environment. Bone CLARITY in conjunction with light-sheet microscope enabled the early detection of an increase to this osteoprogenitor population after administration of a novel anabolic drug, which may have been undetected with traditional techniques.
\r\n\r\nTowards my second goal, I have been working on characterizing adeno-associated viruses (AAVs) for non-invasive widespread gene delivery across the central or peripheral nervous system. Through systemic delivery, these novel AAVs are able to efficiently deliver transgenes to (i) difficult-to-target areas, such as the dorsal root ganglia; (ii) cellular populations that are widely distributed across the mouse body, such as neurons in the myenteric nervous system, and (iii) through highly selective barriers, such as the blood-brain barrier. These viruses enable rapid expression of transgenes for perturbing and monitoring cellular circuits, or for potentially treating neurological diseases. In addition, I worked on engineering or validating several different gene regulatory elements to achieve cell type restricted expression in transgenic and non-transgenic animals with AAVs. These viral vectors may prove useful in rapidly testing newly developed genetic tools. Finally, I developed and characterized two different two-component viral vector systems to control the density of labeling when systemically delivering genes with our novel engineered viruses. I utilized this two-component system to perform single-cell morphology studies in the CNS and PNS. Collectively, these capsids and vectors expand the AAV toolbox and enable efficient and versatile gene delivery into the CNS and PNS of transgenic and non-transgenic animals.
\r\n" }, { "name": "Fisher-Aylor, Katherine Irene", "degree": "PhD", "year": "2017", "title": "Chromatin Topology and Transcription in Myogenesis", "advisor": "Wold, Barbara J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:09302016-110627143", "creators": [ { "name": { "family": "Fisher-Aylor", "given": "Katherine Irene" }, "id": "Fisher-Aylor-Katherine-Irene", "orcid": "0000-0003-3371-2947", "display_name": "Fisher-Aylor, Katherine Irene" } ], "advisors": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "advisor", "display_name": "Wold, Barbara J." } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "chair", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "member", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." } ], "option_major": [ "sysbiol" ], "doi": "10.7907/Z9K07290", "abstract": "High-throughput sequencing and the resulting development of biochemical \"-Seq\" experiments such as ChIP-Seq, DNase-Seq, and Methyl-Seq over the past decade has given rise to a wealth of predicted enhancers and other cis-regulatory regions (CRMs). These new assays provide a new opportunity to compare the number, location, and possible nature of CRMs that are predicted by various new biochemical techniques to instances of known CRMs, which until recently have primarily been located\u2014for reasons of technological limitations\u2014at a few tens of highly expressed, mostly developmentally-specific genes and the several kilobases (kb) upstream of their promoters. For example, an early surprise in the first ChIP-Seq experiments was that the number of predicted tissue-specific transcription factor-occupied sites outnumbered the number of tissue-specific genes by at least a factor of 10, and that many of these occupied sites were nowhere near developmentally relevant genes. In this thesis, I use the ChIA-PET technique, which preserves factor-containing physical interactions between loci in the genome that are far from each other (10kb-2Mb), where the factors used in this thesis are RNA Polymerase II (pol2) to capture active genes, and separately the developmental transcription factor Myogenin to additionally capture CRMs not at promoters. Overall, I report that (1) the closer together two occupied regions are, the more likely they are to be connected, and (2) that a gene\u2019s activity level is highly correlated with its likelihood of being physically engaged with a distant occupied locus. These lead to the discoveries that occupied regions tend to engage with the active genes nearest to them regardless of the developmental profile of the genes, that many genes engage with multiple individual loci, and that many occupied regions interact with multiple genes, including genes that are not at all related in terms of their expression patterns. Individual elements that have multiple connections likely represent sequential rather than simultaneous interactions, and developmental genes may require more engaged enhancers than genes that are expressed in all cell types. Most excitingly, it is possible that many genes with unchanging expression patterns, including so-called \"housekeeping genes,\" use CRMs; very few such genes have ever been assayed with respect to gene regulation, and they are the vast majority of genes in the genome." }, { "name": "Hulse, Bradley Kline", "degree": "PhD", "year": "2017", "title": "Membrane Potential Dynamics of Hippocampal Neurons During Ripples in Awake Mice", "advisor": "Siapas, Athanassios G.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01052017-162955208", "creators": [ { "name": { "family": "Hulse", "given": "Bradley Kline" }, "id": "Hulse-Bradley-Kline", "orcid": "0000-0002-7117-7036", "display_name": "Hulse, Bradley Kline" } ], "advisors": [ { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "role": "advisor", "display_name": "Siapas, Athanassios G." } ], "committee": [ { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "role": "chair", "display_name": "Dickinson, Michael H." }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "role": "member", "display_name": "Prober, David A." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "role": "member", "display_name": "Siapas, Athanassios G." } ], "option_major": [ "neurobio" ], "doi": "10.7907/Z95Q4T3S", "abstract": "During periods of slow wave sleep and quiet wakefulness, the hippocampal formation generates spontaneous population bursts that are organized as a high-frequency \"ripple\" oscillation. The neurons that participate in these bursts often replay previously experienced activity patterns encoded during alert behavior, and interfering with ripple generation produces deficits in learning and memory tasks. For these reasons, ripples play a prominent role in theories of memory consolidation and retrieval. While spiking during ripples has been extensively studied, our understanding of the subthreshold behavior of hippocampal neurons during these events remains incomplete. Here, we combine in vivo whole-cell recordings with multisite extracellular and behavioral measurements to study the membrane potential dynamics of hippocampal neurons during ripples in awake mice. We find that the subthreshold depolarization of CA1 pyramidal neurons is uncorrelated with net excitatory input, clarifying the circuit mechanism keeping most neurons silent during ripples. On a finer time scale, the phase delay between intracellular and extracellular ripple oscillations varies systematically with the membrane potential, which is inconsistent with models of intracellular ripple generation involving perisomatic inhibition alone. In addition, we find that membrane potential statistics (mean, variability, distance to threshold) of CA1 pyramidal neurons and dentate granule cells are systematically modulated across brain states, that rapid variations in pupil diameter are reflected in subthreshold fluctuations, and that many neurons begin depolarizing about one second before ripple onset. These results provide evidence that coordinated shifts in the subthreshold dynamics of individual neurons may contribute to the emergence of state-dependent hippocampal activity patterns. Finally, we present evidence that area CA3 provides the major excitatory input to dentate granule cells during ripples and that there are coordinated interactions between hippocampal ripples and population events in the dentate gyrus, both of which inform network-level models of ripple generation." }, { "name": "Li, Hanqing", "degree": "PhD", "year": "2017", "title": "Development of a High-Throughput Protein Interaction Assay and its Applications", "advisor": "Zinn, Kai George", "url": "https://resolver.caltech.edu/CaltechTHESIS:05292017-183651116", "creators": [ { "name": { "family": "Li", "given": "Hanqing" }, "id": "Li-Hanqing", "display_name": "Li, Hanqing" } ], "advisors": [ { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "advisor", "display_name": "Zinn, Kai George" } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "member", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" } ], "option_major": [ "biology" ], "doi": "10.7907/Z9Q23X9R", "abstract": "Protein-protein interactions (PPIs) form the backbone for a vast array of biological processes in an organism, ranging from signal transduction to gene regulation to intercellular signaling. Therefore, mapping out protein interactomes has been a crucial and prolific area of scientific research. In recent years, much progress has been made in generating high throughput protein interaction data in a variety of organisms, including S. cerevisiae, C. elegans, and D. melanogaster, as well as in human cell culture. The strength of protein interactions varies widely, from almost irreversible assembly of complexes to highly transient interactions. Because of their diversity and complexity, a wide variety of methods have been used to study protein interactions. This includes such commonly-used assays like yeast two-hybrid, to mass spectrometry, ELISA, affinity pulldowns, etc.
\r\n\r\nDespite the plethora of assays and data sets generated for PPIs, interactions involving cell surface and secreted proteins (CSSPs) remains underrepresented in the results. This is due to the fact that CSSP interactions tend to have lower KDs (around the \u03bcM range), and are generally highly transient and difficult to perform using standard assays such as yeast two-hybrid. To circumvent these problems, we designed a high-throughput PPI assay with high sensitivity. To validate the effectiveness of the assay, we utilized it to probe for interactions among two families of Drosophila CSS proteins, the Beats and the Sides, to see if we could recapitulate known interactions and uncover new ones. We were able to recapitulate almost all of the known interactions, as well as discover three novel ones. Additionally, we also studied the expression patterns of members of the Beat and Side families in Drosophila embryos and larvae, as well as analyzed the effects of mutations of Side-VI and Beat-Vs in embryos.
\r\n" }, { "name": "Liu, Raymond", "degree": "PhD", "year": "2017", "title": "Mechanisms of Drp1 Recruitment to Mitochondria", "advisor": "Chan, David C.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05312017-190514797", "creators": [ { "name": { "family": "Liu", "given": "Raymond" }, "id": "Liu-Raymond", "display_name": "Liu, Raymond" } ], "advisors": [ { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "advisor", "display_name": "Chan, David C." } ], "committee": [ { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "role": "chair", "display_name": "Campbell, Judith L." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "member", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "role": "member", "display_name": "Dunphy, William G." }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "member", "display_name": "Chan, David C." } ], "option_major": [ "biology" ], "doi": "10.7907/Z99Z92ZF", "abstract": "Dynamin-related protein 1 (Drp1) is a GTPase of the dynamin superfamily that catalyzes mitochondrial fission in the cell. Cytosolic Drp1 is recruited to mitochondria by receptors anchored to the outer mitochondrial membrane. Once there, Drp1 assembles into a complex around the mitochondrial circumference to drive division via a GTP-dependent constriction process. The four known receptors of Drp1 are Fis1, Mff, MiD51, and MiD49, but stable interactions between Drp1 and these proteins have not been established. In addition, though mounting evidence suggests these receptors have non-redundant roles in their interaction with Drp1, mechanistic details explaining these distinctions are lacking. Here we address these questions, and show that the Insert B domain of Drp1 inhibits its interaction with Mff. Removal of this domain stabilizes a complex of Drp1 and Mff in vitro. In addition, we show that Drp1 oligomerization is a requirement for Mff recruitment, but not for MiD51 or MiD49-mediated recruitment. Together the results suggest a model in which Drp1 recruitment to mitochondria is regulated by the oligomeric state of Drp1, such that Mff, MiD51, and MiD49 recruit different subpopulations of Drp1 from the cytosol.
\r\n\r\nWith this model as a framework, we analyze the effect that a Drp1 R403C mutant, identified in several human patients presenting with encephalopathy and refractory epilepsy, has on mitochondrial morphology in cultured cells. We find that the loss of Drp1 oligomerization in these mutants impedes its ability to be recruited by Mff, leading to abnormal elongation of the mitochondrial population.
\r\n" }, { "name": "Sandler, Jeremy Edward", "degree": "PhD", "year": "2017", "title": "Genome Activation and Regulation of Signaling in the Rapidly Dividing Drosophila Embryo", "advisor": "Stathopoulos, Angelike", "url": "https://resolver.caltech.edu/CaltechTHESIS:05112017-102839369", "creators": [ { "name": { "family": "Sandler", "given": "Jeremy Edward" }, "id": "Sandler-Jeremy-Edward", "orcid": "0000-0001-8340-7583", "display_name": "Sandler, Jeremy Edward" } ], "advisors": [ { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "advisor", "display_name": "Stathopoulos, Angelike" } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "member", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." } ], "option_major": [ "biology" ], "doi": "10.7907/Z9K935KD", "abstract": "Embryonic development of the fruit fly Drosophila melanogaster is unique among model organisms and animals in general, as rapid and syncytial nuclear divisions characterize the early stages before cell membranes form. These nuclear divisions occur every eight to fifteen minutes, culminating with a 45-minute cell cycle where cell membranes form and the 6000 nuclei become 6000 cells before the embryo undergoes gastrulation. At the beginning of development, maternally deposited transcripts define the major axes of the embryo and control all processes that occur. As the syncytial nuclear cycles slow and nuclei migrate to the periphery of the embryo, maternal transcripts are degraded and the zygotic genome is first activated. The rapid pace of nuclear divisions concurrent with the activation of the zygotic genome presents unique challenges to the developing embryo, as the constraints imposed by mitosis limit the ability to transcribe new genes. This switch of control, the Maternal to Zygotic Transition, has been the subject of studies at the molecular and genetic level for almost 30 years. Here, we use new tools and approaches to study the developing embryo at a time scale not previously achieved. We show how the gene regulatory network along the dorsal-ventral axis, including entire signaling pathways, is activated using time point intervals of 10 minutes. Using mutants, we show the contribution of individual genes in the process of development and the resulting changes in expression levels for the entire network. Finally, we examine the transcription of long genes during the rapid syncytial nuclear cycles, when time constraints limit the ability to transcribe the entire gene. We show how an RNA binding protein regulates the truncation of the transcripts into short isoforms with novel coding sequences, and how these short gene products code for functional proteins that regulate the spatiotemporal activation of key signaling pathways in the embryo.
" }, { "name": "Shah, Sheel Mukesh", "degree": "PhD", "year": "2017", "title": "Highly Multiplexed Single Cell In Situ RNA Detection", "advisor": "Cai, Long", "url": "https://resolver.caltech.edu/CaltechTHESIS:12152016-144548062", "creators": [ { "name": { "family": "Shah", "given": "Sheel Mukesh" }, "id": "Shah-Sheel-Mukesh", "orcid": "0000-0002-6321-4669", "display_name": "Shah, Sheel Mukesh" } ], "advisors": [ { "name": { "family": "Cai", "given": "Long" }, "id": "Cai-Long", "role": "advisor", "display_name": "Cai, Long" } ], "committee": [ { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "role": "member", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "chair", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Cai", "given": "Long" }, "id": "Cai-Long", "role": "member", "display_name": "Cai, Long" } ], "option_major": [ "molbiochem" ], "doi": "10.7907/Z9X63JXH", "abstract": "Identifying the genetic basis of cellular function and identity has become a central question in understanding the functioning of complex biological systems in recent years. Single cell sequencing techniques have provided a great deal of insight into the transcriptional profiles of various cell types. However, single cell RNAseq studies require cells to be removed from their native environments resulting in the loss of spatial relationships between cells and suffer from low detection efficiency. Moving forward, a central question in further understanding large biological systems consisting of many disparate cell types will be how do these cells interact with each other to form functional tissues. To accomplish this goal, a method that keeps the tissue architecture intact is required. Single molecule fluorescence in situ hybridization (smFISH) is one such technique, but suffers from a lack of multiplex measurement capability as only a very few genes can be measured in any given sample and has low signal to noise ratio. Here I present a method that overcomes the low signal to noise ratio by using an amplification technique known as single molecule hybridization chain reaction (smHCR). smHCR coupled with the existing sequential FISH (seqFISH) method, which overcomes the inherent multiplexing limit of smFISH, provides a powerful tool to measure the copy numbers of 100\u2019s of genes in single cell in situ.
\r\n\r\nThe mouse brain contains 100,000,000 cells arranged into distinct anatomical structures. While cell types have been previously characterized by morphology and electrophysiology, single cell RNA sequencing has recently identified many cell types based on gene expression profiles. On the other hand, the Allen Brain Atlas (ABA) provides a systematic gene expression database using in situ hybridization (ISH) of the entire mouse brain, but lacks the ability to correlate the expression of different genes in the same cell. Using the smHCR-seqFISH technique to measure the expression profiles of up to 249 genes in single cells in coronal brain sections, we have identified distinct cell clusters based on the expression profiles of 15000 cells and observed spatial patterning of cells in the hippocampus. In the dentate gyrus, we resolved lamina-layered patterns of cell clusters with a clear separation between the granule cell layer and the sub-granular zone. In CA1 and CA3, the data revealed distinct subregions, each with unique combinations of cell clusters. Particularly, we observed that the dorso-lateral CA1 is almost completely cellular homogeneous with increasing cellular heterogeneity on the dorsal to ventral axis. Together, these results demonstrate the power of highly multiplex in situ analysis to the brain, with further application to a wide range of biological systems.
" }, { "name": "Thubagere Jagadeesh, Anupama", "degree": "PhD", "year": "2017", "title": "Programming Complex Behavior in DNA-based Molecular Circuits and Robots", "advisor": "Qian, Lulu; Murray, Richard M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06082017-194534497", "creators": [ { "name": { "family": "Thubagere Jagadeesh", "given": "Anupama" }, "id": "Thubagere-Jagadeesh-Anu", "display_name": "Thubagere Jagadeesh, Anupama" } ], "advisors": [ { "name": { "family": "Qian", "given": "Lulu" }, "id": "Qian-Lulu", "role": "advisor", "display_name": "Qian, Lulu" }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "role": "co-advisor", "display_name": "Murray, Richard M." } ], "committee": [ { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "role": "chair", "display_name": "Murray, Richard M." }, { "name": { "family": "Rothemund", "given": "Paul W. K." }, "id": "Rothemund-P-W-K", "role": "member", "display_name": "Rothemund, Paul W. K." }, { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "role": "member", "display_name": "Shapiro, Mikhail G." }, { "name": { "family": "Qian", "given": "Lulu" }, "id": "Qian-Lulu", "role": "member", "display_name": "Qian, Lulu" } ], "option_major": [ "bioeng" ], "doi": "10.7907/Z9WD3XMS", "abstract": "Integrated electronic circuits, like those found in cellphones and computers, are ubiquitous in our information-driven society. The success of electronics has, in part, been due its modular architecture that enables individual components to be independently improved while the overall device functionality remains unchanged. Over the last two decades the emerging field of dynamic DNA nanotechnology has been trying to apply the underlying philosophy of electronics to biochemical circuits. DNA nanotechnology employs rationally designed DNA molecules as building blocks of biochemical circuits that can, in principle, enable powerful applications like diagnostics and therapeutics.
\r\n\r\nResearchers in the field of DNA nanotechnology have developed simple elements to construct biomolecular systems with desired functions. They have also developed molecular compilers for defining design principles. The cost of DNA synthesis has decreased by over three orders of magnitude in the past decade. This has lead to a non-trivial number of small scale circuits, like DNA-based logic gates and chemical oscillators, being implemented. However, the scalability of this approach has yet to be clearly demonstrated. n this thesis, we will discuss our main contributions to facilitating the advancement of DNA nanotechnology by developing systematic approaches for constructing modular DNA building blocks. These modules can be used to construct biochemical circuits and molecular robotic systems. The performance of the modules can be individually tuned and integrated into large-scale systems.
\r\n\r\nUsing automated circuit-design software and cheap unpurified DNA, we demonstrated the design and construction of a complex synthetic biochemical circuit consisting of 78 distinct DNA species. The circuit is capable of computing the transition rules of a cell updating its state based on its neighboring cells, defined in a classic computational model called cellular automata. Using a bottom-up approach, we first characterized the component necessary for basic Boolean logic computation. We then systematically integrated more circuit elements and eventually constructed the full circuit. By developing a systematic procedure for building DNA-based circuits using unpurified components, we significantly simplified the experimental procedure. By using unpurified DNA components, we reduced the cost and technical barrier for circuit construction, thus making the design and synthesis of complex DNA circuits accessible to even novice researchers.
\r\n\r\nNext we demonstrated a cargo sorting DNA nano-robot, using a simple algorithm and modular building blocks. The DNA robot has a leg and two foot domains for exploring a two-dimensional DNA origami surface, and an arm and hand domain for picking up randomly located cargos and dropping them off at their designated locations. It is completely autonomous and is programmed to perform a random walk without requiring an external energy source. Further, we demonstrated sorting multiple copies of two distinct cargo species on the same origami. Additionally, by compartmentalizing each sorting task on a single origami, we showed that two distinct sorting tasks can be implemented on different origami simultaneously in the same test tube. The recognition of a cargo is embedded in its destination, therefore it is possible to scale up the system simply by having multiple types of cargos. The same robot design can be used for performing multiple instances of distinct tasks in parallel. The different modules can be integrated to perform diverse functions, including applications in time-release targeted therapeutics.
" }, { "name": "Acharya, Aneesh", "degree": "PhD", "year": "2016", "title": "Multiplexed Analysis of Diverse RNA Classes via Hybridization Chain Reaction", "advisor": "Pierce, Niles A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06032016-143422651", "creators": [ { "name": { "family": "Acharya", "given": "Aneesh" }, "id": "Acharya-Aneesh", "orcid": "0000-0002-4402-7147", "display_name": "Acharya, Aneesh" } ], "advisors": [ { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "role": "advisor", "display_name": "Pierce, Niles A." } ], "committee": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "chair", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "role": "member", "display_name": "Pierce, Niles A." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "member", "display_name": "Mazmanian, Sarkis K." } ], "option_major": [ "bioeng" ], "doi": "10.7907/Z95M63N0", "abstract": "Gene circuits are complex biological networks composed of numerous regulatory elements, including transcription factors, mRNAs, and microRNAs. Fluorescent in situ hybridization (FISH) is a powerful method for spatially mapping expression levels of RNA elements within an intact organism, but traditional methods exhibit at least one of the following drawbacks: low signal-to-background, arduous and/or destructive multiplexing, and non-quantitative signal. These issues are all overcome using in situ amplification based on the mechanism of hybridization chain reaction (HCR). With this approach, nucleic acid probes complementary to RNA targets trigger the self-assembly of fluorophore-labeled nucleic acid hairpins into tethered fluorescent amplification polymers. In situ HCR enables straightforward multiplexing, high signal-to-background, and quantitative signal. Here, we address three key scenarios in which HCR enables novel applications for in situ hybridization. First, we address the challenge of sorting cell subpopulations based on mRNA abundance using flow cytometry to enable high-throughput measurement of the signal intensity from individual cells. High signal is required to overcome the background autofluorescence integrated over the volume of each cell. Quantitative HCR signal amplification enables multi-dimensional sorting of mammalian cell lines based on expression levels of multiple target mRNAs. Second, we address the challenge of mapping multiple microRNA and mRNA targets simultaneously. Traditional methods enable mapping of single microRNA targets in isolation and use costly LNA probes with proprietary compositions that differ for each target. Here we develop in situ HCR for multiplexed mapping not only of microRNAs, but of microRNAs and mRNAs together, using non-proprietary 2'OMe-RNA probes for miRNA targets and DNA probes for mRNA targets. Third, to enable studies of gut flora, we address the challenge of mapping spatial relationships between different bacterial species within the intact mouse colon. In situ HCR enables multiplexed discrimination of multiple closely-related Bacteroides species with rRNAs that differ by only a few nucleotides. In summary, this thesis presents in situ HCR as a tool for multiplexed analysis of diverse RNA classes and expands the range of gene circuit regulatory elements that can be spatially and quantitatively mapped." }, { "name": "Chen, Shijia", "degree": "PhD", "year": "2016", "title": "Light Dependent Regulation of Sleep/Wake States by Prokineticin 2 in Larval Zebrafish", "advisor": "Prober, David A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08082015-121950812", "creators": [ { "name": { "family": "Chen", "given": "Shijia" }, "id": "Chen-Shijia", "display_name": "Chen, Shijia" } ], "advisors": [ { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "role": "advisor", "display_name": "Prober, David A." } ], "committee": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "chair", "display_name": "Anderson, David J." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "role": "member", "display_name": "Prober, David A." } ], "option_major": [ "biology" ], "doi": "10.7907/Z99P2ZNR", "abstract": "Sleep is an evolutionarily conserved behavior and essential to survival. The classic two process model of sleep regulation proposes that sleep results from the interaction between circadian and homeostatic processes, but the details remain elusive. Most sleep research is performed using nocturnal rodents, and diurnal vertebrates are under-represented. It is unclear whether circadian regulatory mechanisms of sleep in nocturnal animals can be directly translated into diurnal animals. In this thesis, I first briefly describe sleep behavior and the two process model of sleep regulation, focusing on the circadian process, and then discuss the advantages of using larval zebrafish as a model to study sleep behavior in diurnal vertebrates. In Chapter 2, I characterize the role of Prokineticin 2, a proposed circadian output factor in nocturnal animals, in sleep/wake regulation in larval zebrafish. I show that, similar to nocturnal rodents, Prok2 is both necessary for daytime sleep/wake behavior and sufficient to modulate sleep/wake states in a light dependent manner. However, unlike nocturnal rodents and similar to humans, Prok2 is not required for maintaining circadian rhythmicity in larval zebrafish after removing external light cue. This result demonstrates the potential functional difference of circadian output factors in different chronotypes, and establishes larval zebrafish as an alternative model for studying circadian regulation of sleep and possibly other behaviors in humans. In Chapter 3, I describe the adaptation and development of TRP channels to manipulate neuronal activity in larval zebrafish, in an effort to expand the existing repertoire of genetic tools for studying behavior in zebrafish. I show that three TRP channels, TRPV1, TRPM8 and TRPA1, can inducibly activate specific populations of neurons in larval zebrafish by using their appropriate agonists. At high agonist concentrations, TRPV1, can rapidly induce cell ablation. Adaptation of TRP channels for use in larval zebrafish expands the variety of behavioral experiments and combinatorial manipulation of neuronal activity that can be performed in zebrafish. In summary, this work deepens our understanding of sleep regulation, establishes larval zebrafish as an appropriate model for studying circadian regulation of sleep in diurnal vertebrates, and presents novel genetic tools for studying behavior in larval zebrafish." }, { "name": "Cui, Miao", "degree": "PhD", "year": "2016", "title": "Refining Sea Urchin Developmental Gene Regulatory Network Models by Incorporating Wnt Signaling and Information Processed at the hox11/13b Locus", "advisor": "Davidson, Eric H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05232016-193319268", "creators": [ { "name": { "family": "Cui", "given": "Miao" }, "id": "Cui-Miao", "orcid": "0000-0003-3150-3930", "display_name": "Cui, Miao" } ], "advisors": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "advisor", "display_name": "Davidson, Eric H." } ], "committee": [ { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "chair", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Peter", "given": "Isabelle S." }, "id": "Peter-I-S", "role": "member", "display_name": "Peter, Isabelle S." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." } ], "option_major": [ "biology" ], "doi": "10.7907/Z9251G6D", "abstract": "The availability of advanced GRN models for sea urchin development presents a unique opportunity to address the function of signaling interactions in cell fate specification at a system-wide level. Here we take a global approach to investigate the regulatory functions of the Wnt signaling system during pre-gastrular development. We examine the embryonic specification processes in order to determine in which embryonic lineages and at what time specific Wnt signals are required. We show a functional divergence among individual Wnt ligands despite their similar and partially overlapped spatial expression. By studying TF activators, we show that expression of wnt genes is tightly controlled and is correlated with their respective functions. In particular Wnt1 and Wnt16, which regulate endodermal specification, are activated by the endoderm regulator Hox11/13b. Motivated by these results and in an effort to further enhance our understanding of endodermal specification, we conducted a cis-regulatory analysis of the hox11/13b gene across a range of developmental stages up to 60 hours post-fertilization. We identify Ets, Eve, and Tcf as direct regulators of hox11/13b, and we show that their combinatorial control drives the endoderm-specific and dynamic early expression of hox11/13b. Furthermore, we show that its late expression in the hindgut is controlled by an inter-modular AND logic gate, in which two separate regulatory modules are both required but neither alone is sufficient. \r\n" }, { "name": "Erkenbrack, Eric Matthew", "degree": "PhD", "year": "2016", "title": "Evolution of Developmental Gene Regulatory Networks in Echinoids", "advisor": "Davidson, Eric H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04262016-110444326", "creators": [ { "name": { "family": "Erkenbrack", "given": "Eric Matthew" }, "id": "Erkenbrack-Eric-Matthew", "orcid": "0000-0001-9375-3279", "display_name": "Erkenbrack, Eric Matthew" } ], "advisors": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "advisor", "display_name": "Davidson, Eric H." } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." }, { "name": { "family": "Peter", "given": "Isabelle S." }, "id": "Peter-I-S", "orcid": "0000-0003-3685-3147", "role": "member", "display_name": "Peter, Isabelle S." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." } ], "option_major": [ "biology" ], "doi": "10.7907/Z95X26WZ", "abstract": "Developmental gene regulatory networks (dGRNs) are assemblages of regulatory genes that direct embryonic development of animal body plans and their morpho-logical structures. dGRNs exhibit recursively-wired circuitry that is encoded in the genome and executed during development. Alteration to the regulatory architecture of dGRNs causes variation in developmental programs both during the development of an individual organism and during the evolution of an individual lineage. The ex-planatory power of these networks is best exempli\ufb01ed by the global dGRN directing early development of the euechinoid sea urchin Strongylocentrotus purpuratus. This network consists of numerous regulatory genes engaging in hundreds of genomic regulatory transactions that collectively direct the delineation of early embryonic domains and the speci\ufb01cation of cell lineages. Research on closely-related euechi-noid sea urchins, e.g. Lytechinus variegatus and Paracentrotus lividus, has revealed marked conservation of dGRN architecture in echinoid development, suggesting little appreciable alteration has occurred since their divergence in evolution at least 90 million years ago (mya).
\r\n\r\nWe sought to test whether this observation extends to all sea urchins (echinoids) and undertook a systematic analysis of over 50 regulatory genes in the cidaroid sea urchin Eucidaris tribuloides, surveing their regulatory activity and function in a sea urchin that diverged from euechinoid sea urchins at least 268 mya. Our results revealed extensive alterations have occurred to all levels of echinoid dGRN archi-tecture since the cidaroid-euechinoid divergence. Alterations to mesodermal sub-circuits were particularly striking, including functional di\u02d9erences in speci\ufb01cation of non-skeletogenic mesenchyme (NSM), skeletogenic mesenchyme (SM), and en-domesodermal segregation. Speci\ufb01cation of endomesodermal embryonic domains revealed that, while their underlying network circuitry had clearly diverged, regu-latory states established in pregastrular embryos of these two groups are strikingly similar. Analyses of E. tribuloides speci\ufb01cation leading to the estab-lishment of dorsal-ventral (aboral-oral) larval polarity indicated that regulation of regulatory genes expressed in mesodermal embryonic domains had incurred signi\ufb01cantly more alterations than those expressed in endodermal and ectodermal domains. Taken together, this study highlights the ability of dGRN architecture to buffer extensive alterations in the evolution and early development of echinoids and adds further support to the notion that alterations can occur at all levels of dGRN architecture and all stages of embryonic development.
" }, { "name": "Galimidi, Rachel P.", "degree": "PhD", "year": "2016", "title": "Combating HIV with Novel Antibody Architectures ", "advisor": "Bjorkman, Pamela J.; Baltimore, David L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06082016-141626444", "creators": [ { "name": { "family": "Galimidi", "given": "Rachel P." }, "id": "Galimidi-Rachel-P", "display_name": "Galimidi, Rachel P." } ], "advisors": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "advisor", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "orcid": "0000-0001-8723-8190", "role": "co-advisor", "display_name": "Baltimore, David L." } ], "committee": [ { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "orcid": "0000-0003-2713-1513", "role": "chair", "display_name": "Mazmanian, Sarkis K." }, { "name": { "family": "Clemons", "given": "William M." }, "id": "Clemons-W-M", "orcid": "0000-0002-0021-889X", "role": "member", "display_name": "Clemons, William M." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Zack", "given": "Jerome" }, "id": "Zack-J-A", "role": "member", "display_name": "Zack, Jerome" }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "orcid": "0000-0001-8723-8190", "role": "member", "display_name": "Baltimore, David L." } ], "option_major": [ "immun" ], "doi": "10.7907/Z9QC01FR", "abstract": "More than 30 years has passed since the discovery of Human Immunodeficiency Virus (HIV) yet it remains one of the most important current threats to global public health. HIV is a T-lymphotrophic retrovirus that is the causative agent of Acquired Immune Deficiency Syndrome, and despite decades of research, there remains no cure. Vaccines are most effective when they are able to induce broadly neutralizing antibodies at concentrations capable of blocking viral infection. Notwithstanding all of the effort, a successful vaccine that is capable of inducing complete protection from the immune system has yet to be found. In this thesis, the first chapter provides a history of the discovery of HIV, the origins of the virus, description of the HIV genome, focusing primarily on the envelope glycoprotein, a trimeric spike on the surface of the HIV virion necessary for viral fusion and the sole epitope for broadly neutralizing antibodies. Lastly, the first chapter reviews an overview of the antiviral immune response specifically the role of humoral immune branch and broadly neutralizing antibodies, as well as their limitations in protection against HIV. Antibodies developed during HIV-1 infection lose efficacy as the viral spike mutates. In addition to structural features of HIV\u2019s envelope spike that facilitate antibody evasion, we proposed that the low-density and limited lateral mobility of HIV spikes impedes bivalent binding by antibodies. The resulting predominantly monovalent binding minimizes avidity and thereby high affinity binding and potent neutralization, thus expanding the range of HIV mutations permitting antibody evasion. The work described in subsequent chapters attempts to overcome HIV\u2019s evasion strategy of low spike density through the design of novel antibody architectures.
\r\n\t\r\nWe postulated that anti-HIV-1 spike antibodies primarily bind monovalently because HIV\u2019s low spike density impedes bivalent binding through inter-spike crosslinking, and the spike trimer structure prohibits bivalent binding through intra-spike crosslinking. Monovalent binding reduces avidity and neutralization potency, thus expanding the range of mutations permitting antibody evasion. To test this idea, we engineered antibody-based molecules capable of bivalent binding through intra-spike crosslinking. We used DNA as a \u201cmolecular ruler\u201d to measure intra-epitope distances on virion-bound spikes and to construct intra-spike crosslinking molecules. Optimal bivalent reagents exhibited up to 2.5 orders of magnitude of increased potency (>100-fold average increases across a virus panel) and identified conformational states of virion-bound spikes. The demonstration that intra-spike crosslinking lowers the concentration of antibodies required for neutralization supports the hypothesis that low spike densities facilitate antibody evasion and the use of molecules capable of intra-spike crosslinking for therapy or passive protection. These results shed light on dynamic spike conformations and are relevant to therapeutic interventions.
\r\n" }, { "name": "Hsiao, Victoria", "degree": "PhD", "year": "2016", "title": "Synthetic Circuits for Feedback and Detection in Bacteria", "advisor": "Murray, Richard M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05082016-170628018", "creators": [ { "name": { "family": "Hsiao", "given": "Victoria" }, "id": "Hsiao-Victoria", "orcid": "0000-0001-9297-1522", "display_name": "Hsiao, Victoria" } ], "advisors": [ { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "role": "advisor", "display_name": "Murray, Richard M." } ], "committee": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "chair", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "role": "member", "display_name": "Pierce, Niles A." }, { "name": { "family": "Rothemund", "given": "Paul W. K." }, "id": "Rothemund-P-W-K", "role": "member", "display_name": "Rothemund, Paul W. K." } ], "option_major": [ "bioeng" ], "doi": "10.7907/Z9WD3XJW", "abstract": "Synthetic biology, by co-opting molecular machinery from existing organisms, can be used as a tool for building new genetic systems from scratch, for understanding natural networks through perturbation, or for hybrid circuits that piggy-back on existing cellular infrastructure. Although the toolbox for genetic circuits has greatly expanded in recent years, it is still difficult to separate the circuit function from its specific molecular implementation. In this thesis, we discuss the function-driven design of two synthetic circuit modules, and use mathematical models to understand the fundamental limits of circuit topology versus operating regimes as determined by the specific molecular implementation. First, we describe a protein concentration tracker circuit that sets the concentration of an output protein relative to the concentration of a reference protein. The functionality of this circuit relies on a single negative feedback loop that is implemented via small programmable protein scaffold domains. We build a mass-action model to understand the relevant timescales of the tracking behavior and how the input/output ratios and circuit gain might be tuned with circuit components. Second, we design an event detector circuit with permanent genetic memory that can record order and timing between two chemical events. This circuit was implemented using bacteriophage integrases that recombine specific segments of DNA in response to chemical inputs. We simulate expected population-level outcomes using a stochastic Markov-chain model, and investigate how inferences on past events can be made from differences between single-cell and population-level responses. Additionally, we present some preliminary investigations on spatial patterning using the event detector circuit as well as the design of stationary phase promoters for growth-phase dependent activation. These results advance our understanding of synthetic gene circuits, and contribute towards the use of circuit modules as building blocks for larger and more complex synthetic networks." }, { "name": "Kim, Jocelyn Tammy", "degree": "PhD", "year": "2016", "title": "The Innate Immune System in Dendritic Cell-Targeted Lentiviral Vector Immunization and Cell-to-Cell Transmission of HIV-1", "advisor": "Baltimore, David L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06072016-121745268", "creators": [ { "name": { "family": "Kim", "given": "Jocelyn Tammy" }, "id": "Kim-Jocelyn-Tammy", "display_name": "Kim, Jocelyn Tammy" } ], "advisors": [ { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "advisor", "display_name": "Baltimore, David L." } ], "committee": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "chair", "display_name": "Mazmanian, Sarkis K." }, { "name": { "family": "Yang", "given": "Otto" }, "id": "Yang-Otto", "role": "member", "display_name": "Yang, Otto" }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "member", "display_name": "Baltimore, David L." } ], "option_major": [ "biology" ], "doi": "10.7907/Z93N21CS", "abstract": "Dendritic cells (DCs) are the sentinels of the immune system, and thus specialized in transporting foreign antigen to T cells and initiating activation of innate and adaptive immune responses. In this work, we first explore how DCs sense viral pathogens and stimulate antigen-specific T cell responses. In particular, we find the DC-targeting HIV-1 derived lentiviral vector (LV) is a potent T cell vaccine in vivo. However, the exact mechanism behind such efficient immunization is not clear. Interestingly, we find that DC activation is triggered by cellular DNA packaged in LVs and at least partially dependent on the STING protein. Innate immune activation is independent of MyD88, TRIF and IPS-1, ruling out an involvement of Toll-like receptors or RIG-I-like receptor signaling. Further, we find that antigenic protein delivered in viral particles via pseudotransduction is sufficient to stimulate an antigen-specific immune response. Delivery of the viral genome encoding the antigen increases the magnitude of this response in vivo, but is irrelevant in vitro. Thus, pseudotransduction, genomic transduction, and STING-mediated activation thus collaborate to make the DC-targeted LV a uniquely powerful immunogen. In addition, we explore how DCs mediate HIV-1 infection of T cells via cell-to-cell infection. In particular, we assess how DC-to-T cell transmission of HIV-1 allows for a concentrated amount of virus to be directed to an uninfected T cell. We report that DCs amplify the efficiency of T cell infection, resulting in anti-retroviral drug insensitivity compared to T cell infection in the absence of DCs. The DC-mediated amplification and drug-insensitivity of T cell infection are both entirely dependent on physical cellular interactions. Further, we find that the input of a virus is important to the drug insensitivity of DC-to-T cell infection, but not DC-free T cell infection. Thus, we have studied two separate roles of DCs: initiating immune responses to LVs and mediating transmission of infectious HIV-1. The study of these roles is important to discovering novel immune adjuvants and identifying targeted therapeutics to inhibit viral dissemination. " }, { "name": "Leighton, Daniel H. W.", "degree": "PhD", "year": "2016", "title": "Mating at Advanced Age: How Old Nematodes Modulate Pheromone Production to Attract Young Males", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06062016-184616124", "creators": [ { "name": { "family": "Leighton", "given": "Daniel H. W." }, "id": "Leighton-Daniel-H-W", "orcid": "0000-0002-1379-0078", "display_name": "Leighton, Daniel H. W." } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "role": "chair", "display_name": "Prober, David A." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/Z9RB72KK", "abstract": "Nematodes have been studied for centuries in their roles as pathogens of humans, crops, and livestock. In more recent times, the free-living nematode Caenorhabditis elegans and its close relatives have been heavily studied as genetic and developmental model organisms. Despite the extent of research into nematode biology and lifestyle, relatively little is known about communication between nematodes. In the last decade, there has been a burst of research into identifying the pheromones secreted by nematodes, as well as determining their effect on other nematodes in the population.
\r\n\r\nThe bulk of pheromone research has focused on the chemical identification of olfactory signals, and studying the behavioral and physiological responses of worms exposed to these signals. We report the discovery of a new C. elegans mating pheromone, and an attempt to dissect the pathway that regulates its production. Instead of studying what a worm \u201chears\u201d when this signal is received, we hope to understand what the worm that produces the signal is trying to \u201csay\u201d.
\r\n\r\nWe also review the existing literature on nematode mating pheromones, highlighting the most stunning recent discoveries, and point out several questionable claims frequently made by authors in the field.
\r\n" }, { "name": "Rios, Gustavo", "degree": "PhD", "year": "2016", "title": "Nanofabricated Neural Probe System for Dense 3-D Recordings of Brain Activity", "advisor": "Siapas, Athanassios G.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05172016-133516099", "creators": [ { "name": { "family": "Rios", "given": "Gustavo" }, "id": "Rios-Gustavo", "orcid": "0000-0003-1411-4933", "display_name": "Rios, Gustavo" } ], "advisors": [ { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "role": "advisor", "display_name": "Siapas, Athanassios G." } ], "committee": [ { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "role": "chair", "display_name": "Dickinson, Michael H." }, { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "role": "member", "display_name": "Siapas, Athanassios G." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Lubenov", "given": "Evgueniy V." }, "id": "Lubenov-E-V", "role": "member", "display_name": "Lubenov, Evgueniy V." } ], "option_major": [ "bioeng" ], "doi": "10.7907/Z9BG2M0B", "abstract": "Computations in brain circuits involve the coordinated activation of large populations of neurons distributed across brain areas. However, monitoring neuronal activity in the brain of intact animals with high temporal and spatial resolution has remained a technological challenge. Here we address this challenge by developing dense, three-dimensional (3-D) electrode array system for electrophysiology. The front-end of the system is composed of nanofabricated neural probes with ultrathin shanks that are engineered to minimize tissue damage. The probes are connected via flexible cables to custom PCBs that multiplex the electrophysiological signals. This system architecture decouples the front-end both mechanically and thermally from the PCB which carries all active electronics for signal conditioning and multiplexing. This system was validated in vivo with hippocampal recordings from head-fixed mice. The culmination of these efforts was a 3-D array with 1024 sites packed within 0.6 mm3 of tissue that yielded the densest electrophysiological recordings to date." }, { "name": "Rojansky, Rebecca Bloom", "degree": "PhD", "year": "2016", "title": "A Core Mitophagic Machinery Promotes Selective Degradation of Paternal Mitochondria in Mouse Embryos and MEF Cells", "advisor": "Chan, David C.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04292016-193643737", "creators": [ { "name": { "family": "Rojansky", "given": "Rebecca Bloom" }, "id": "Rojansky-Rebecca-Bloom", "orcid": "0000-0002-3735-8159", "display_name": "Rojansky, Rebecca Bloom" } ], "advisors": [ { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "advisor", "display_name": "Chan, David C." } ], "committee": [ { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "chair", "display_name": "Newman, Dianne K." }, { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "orcid": "0000-0001-6597-2036", "role": "member", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Shan", "given": "Shu-ou" }, "id": "Shan-Shu-ou", "orcid": "0000-0002-6526-1733", "role": "member", "display_name": "Shan, Shu-ou" }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "member", "display_name": "Chan, David C." } ], "option_major": [ "biology" ], "doi": "10.7907/Z96Q1V75", "abstract": "The maternal mode of mitochondrial inheritance is conserved across mammalian species; however, little is known about how mitochondria from the sperm are eliminated from early mammalian embryos. Mitophagy, the regulated degradation of mitochondria in the lysosome, has been proposed as a possible mechanism. Mitophagy is an important means by which the cell responds to changes in mitochondrial fitness, and has been observed under a number of physiological and non-physiological circumstances, including, but not limited to, hypoxia, mitochondrial depolarization, mitochondrial fission, and erythrocyte differentiation.
\r\n\r\nHere we examine the core component of mitophagy proteins involved in three physiological states: respiration-induced mitophagy in cultured mouse fibroblasts, mitophagy of dysfunctional mitochondria in the absence of mitochondrial fusion, and degradation of paternal mitochondria in pre-implantation mouse embryos. We find that a common pathway is used for elimination of mitochondria, involving mitochondrial depolarization, and the E3 ubiquitin ligases PARKIN and MUL1. We find that PARKIN and MUL1 play partially redundant roles in elimination of paternal mitochondria that is also dependent on PINK1 kinase, the fission factor, FIS1, and the autophagy receptor, p62. We find that p62 is specifically recruited to defective mitochondria in fusion deficient cells by a mechanism independent of ubiquitin binding. Our results elucidate the molecular mechanism of strict maternal transmission of mitochondria and uncover a collaboration between MUL1 and PARKIN in mitophagy.
\r\n" }, { "name": "Shai, Adam S.", "degree": "PhD", "year": "2016", "title": "The Physiology and Computation of Pyramidal Neurons", "advisor": "Meister, Markus", "url": "https://resolver.caltech.edu/CaltechTHESIS:01042016-124746578", "creators": [ { "name": { "family": "Shai", "given": "Adam S." }, "id": "Shai-Adam-S", "display_name": "Shai, Adam S." } ], "advisors": [ { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "orcid": "0000-0003-2136-6506", "role": "advisor", "display_name": "Meister, Markus" } ], "committee": [ { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "orcid": "0000-0003-2136-6506", "role": "chair", "display_name": "Meister, Markus" }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "member", "display_name": "Koch, Christof" }, { "name": { "family": "Anastassiou", "given": "Costas" }, "id": "Anastassiou-C", "role": "member", "display_name": "Anastassiou, Costas" }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "orcid": "0000-0001-5868-348X", "role": "member", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Tsao", "given": "Doris Y." }, "id": "Tsao-D-Y", "orcid": "0000-0003-1083-1919", "role": "member", "display_name": "Tsao, Doris Y." } ], "option_major": [ "neurobio" ], "doi": "10.7907/Z92R3PMW", "abstract": "A variety of neural signals have been measured as correlates to consciousness. In particular, late current sinks in layer 1, distributed activity across the cortex, and feedback processing have all been implicated. What are the physiological underpinnings of these signals? What computational role do they play in the brain? Why do they correlate to consciousness? This thesis begins to answer these questions by focusing on the pyramidal neuron. As the primary communicator of long-range feedforward and feedback signals in the cortex, the pyramidal neuron is set up to play an important role in establishing distributed representations. Additionally, the dendritic extent, reaching layer 1, is well situated to receive feedback inputs and contribute to current sinks in the upper layers. An investigation of pyramidal neuron physiology is therefore necessary to understand how the brain creates, and potentially uses, the neural correlates of consciousness. An important part of this thesis will be in establishing the computational role that dendritic physiology plays. In order to do this, a combined experimental and modeling approach is used.
\r\n\r\nThis thesis beings with single-cell experiments in layer 5 and layer 2/3 pyramidal neurons. In both cases, dendritic nonlinearities are characterized and found to be integral regulators of neural output. Particular attention is paid to calcium spikes and NMDA spikes, which both exist in the apical dendrites, considerable distances from the spike initiation zone. These experiments are then used to create detailed multicompartmental models. These models are used to test hypothesis regarding spatial distribution of membrane channels, to quantify the effects of certain experimental manipulations, and to establish the computational properties of the single cell. We find that the pyramidal neuron physiology can carry out a coincidence detection mechanism. Further abstraction of these models reveals potential mechanisms for spike time control, frequency modulation, and tuning. Finally, a set of experiments are carried out to establish the effect of long-range feedback inputs onto the pyramidal neuron. A final discussion then explores a potential way in which the physiology of pyramidal neurons can establish distributed representations, and contribute to consciousness.
" }, { "name": "Sun, Zachary Zhipeng", "degree": "PhD", "year": "2016", "title": "An in vitro Biomolecular Breadboard for Prototyping Synthetic Biological Circuits", "advisor": "Murray, Richard M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10012015-221355676", "creators": [ { "name": { "family": "Sun", "given": "Zachary Zhipeng" }, "id": "Sun-Zachary-Zhipeng", "orcid": "0000-0002-9425-2924", "display_name": "Sun, Zachary Zhipeng" } ], "advisors": [ { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "role": "advisor", "display_name": "Murray, Richard M." } ], "committee": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "chair", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "member", "display_name": "Baltimore, David L." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Noireaux", "given": "Vincent" }, "id": "Noireaux-V", "role": "member", "display_name": "Noireaux, Vincent" } ], "option_major": [ "biology" ], "doi": "10.7907/Z9TB14TW", "abstract": "Biomolecular circuit engineering is critical for implementing complex functions in vivo, and is a baseline method in the synthetic biology space. However, current methods for conducting biomolecular circuit engineering are time-consuming and tedious. A complete design-build-test cycle typically takes weeks' to months' time due to the lack of an intermediary between design ex vivo and testing in vivo. In this work, we explore the development and application of a \"biomolecular breadboard\" composed of an in-vitro transcription-translation (TX-TL) lysate to rapidly speed up the engineering design-build-test cycle. We first developed protocols for creating and using lysates for conducting biological circuit design. By doing so we simplified the existing technology to an affordable ($0.03/uL) and easy to use three-tube reagent system. We then developed tools to accelerate circuit design by allowing for linear DNA use in lieu of plasmid DNA, and by utilizing principles of modular assembly. This allowed the design-build-test cycle to be reduced to under a business day. We then characterized protein degradation dynamics in the breadboard to aid to implementing complex circuits. Finally, we demonstrated that the breadboard could be applied to engineer complex synthetic circuits in vitro and in vivo. Specifically, we utilized our understanding of linear DNA prototyping, modular assembly, and protein degradation dynamics to characterize the repressilator oscillator and to prototype novel three- and five-node negative feedback oscillators both in vitro and in vivo. We therefore believe the biomolecular breadboard has wide application for acting as an intermediary for biological circuit engineering." }, { "name": "Tobin, Cory James", "degree": "PhD", "year": "2016", "title": "Morphogenesis of the Arabidopsis Shoot Apical Meristem", "advisor": "Meyerowitz, Elliot M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05162016-155222105", "creators": [ { "name": { "family": "Tobin", "given": "Cory James" }, "id": "Tobin-Cory-James", "display_name": "Tobin, Cory James" } ], "advisors": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "advisor", "display_name": "Meyerowitz, Elliot M." } ], "committee": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "chair", "display_name": "Wold, Barbara J." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "member", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/Z9NK3C0N", "abstract": "Phyllotaxis patterns in plants, or the arrangement of leaves and flowers radially around the shoot, have fascinated both biologists and mathematicians for centuries. The current model of this process involves the lateral transport of the hormone auxin through the first layer of cells in the shoot apical meristem via the auxin efflux carrier protein PIN1. Locations around the meristem with high auxin concentration are sites of organ formation and differentiation. Many of the molecular players in this process are well known and characterized. Computer models composed of all these components are able to produce many of the observed phyllotaxis patterns. To understand which parts of this model have a large effect on the phenotype I automated parameter testing and tried many different parameter combinations. Results of this showed that cell size and meristem size should have the largest effect on phyllotaxis. This lead to three questions: (1) How is cell geometry regulated? (2) Does cell size affect auxin distribution? (3) Does meristem size affect phyllotaxis? To answer the first question I tracked cell divisions in live meristems and quantified the geometry of the cells and the division planes using advanced image processing techniques. The results show that cell shape is maintained by minimizing the length of the new wall and by minimizing the difference in area of the daughter cells. To answer the second question I observed auxin patterning in the meristem, shoot, leaves, and roots of Arabidopsis mutants with larger and smaller cell sizes. In the meristem and shoot, cell size plays an important role in determining the distribution of auxin. Observations of auxin in the root and leaves are less definitive. To answer the third question I measured meristem sizes and phyllotaxis patterns in mutants with altered meristem sizes. These results show that there is no correlation between meristem size and average divergence angle. But in an extreme case, making the meristem very small does lead to a switch on observed phyllotaxis in accordance with the model." }, { "name": "Uy, Benjamin Robert", "degree": "PhD", "year": "2016", "title": "Insights into Neural Crest Evolution", "advisor": "Bronner, Marianne E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03092016-182739355", "creators": [ { "name": { "family": "Uy", "given": "Benjamin Robert" }, "id": "Uy-Benjamin-Robert", "display_name": "Uy, Benjamin Robert" } ], "advisors": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "advisor", "display_name": "Bronner, Marianne E." } ], "committee": [ { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "chair", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Moore", "given": "Jonathan" }, "id": "Moore-J", "role": "member", "display_name": "Moore, Jonathan" }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." } ], "option_major": [ "biology" ], "doi": "10.7907/Z99K486C", "abstract": "Neural crest cells are unique to vertebrates and essential to the development and evolution of the craniofacial skeleton. Using a combination of DiI cell lineage tracing, transcriptomics, and analysis of key transcription factors of the Sox Family, I examined neural crest development in the sea lamprey, Petromyzon marinus, as the most basal extant vertebrate from which it is possible to get embryos. The results have uncovered distinct cranial and trunk neural crest subpopulations along the anterior-posterior axis of the lamprey embryo, with a clear separation between the two. However, no evidence of the presence of an intermediate vagal neural crest population was uncovered. Comparing cranial neural crest genes between lamprey and chick, either by examining individual candidate genes or whole genome transcriptome analysis, reveals significant changes in the cranial neural crest gene regulatory network of lamprey compared with chick. In particular, the lamprey cranial neural crest is \"missing\" several gnathostome cranial crest genes. We speculate that these may underlie the evolutionary divergence of craniofacial development between jawed and jawless vertebrates. Despite the absence of vagal neural crest, DiI-labeling shows that trunk neural crest-derived cells, likely homologous to mammalian Schwann cell precursors, contribute to the lamprey enteric nervous system, potentially representing the most primitive form of neural crest cells contribution to the ENS. Finally, I characterized key members of the Sox Family (Sox B-F) due to their importance in neural crest specification in other species. In comparative studies of the SoxC genes (Sox4, Sox11, and Sox12) in both lamprey and Xenopus, I found similar expression patterns and a novel key role in early neural crest specification, suggesting a conserved role of the SoxC genes amongst vertebrates. Taken together, this work represents important progress in characterizing the early evolution of the neural crest in vertebrates and its role in the transition from jawless to jawed vertebrates." }, { "name": "Wadas, Brandon Christopher", "degree": "PhD", "year": "2016", "title": "Biochemical and Genetic Studies of the N-End Rule Pathway in Yeast and Mammals ", "advisor": "Varshavsky, Alexander J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06022016-162251299", "creators": [ { "name": { "family": "Wadas", "given": "Brandon Christopher" }, "id": "Wadas-Brandon-Christopher", "display_name": "Wadas, Brandon Christopher" } ], "advisors": [ { "name": { "family": "Varshavsky", "given": "Alexander J." }, "id": "Varshavsky-A-J", "role": "advisor", "display_name": "Varshavsky, Alexander J." } ], "committee": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "chair", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "role": "member", "display_name": "Dunphy, William G." }, { "name": { "family": "Shan", "given": "Shu-ou" }, "id": "Shan-Shu-ou", "role": "member", "display_name": "Shan, Shu-ou" }, { "name": { "family": "Varshavsky", "given": "Alexander J." }, "id": "Varshavsky-A-J", "role": "member", "display_name": "Varshavsky, Alexander J." } ], "option_major": [ "biology" ], "doi": "10.7907/Z91V5BZQ", "abstract": "Regulation of the in vivo half-lives of intracellular proteins is an important cellular process. Many intracellular proteins are short-lived, owing to their regulated and processive degradation by the Ubiquitin (Ub)-Proteasome System (UPS). In eukaryotes, the N-end rule pathway is one specific pathway within the UPS. The N-end rule pathway relates the identity of the N-terminal residue of a protein, or a protein fragment, to its in vivo half-life. Substrates of the N-end rule pathway are recognized by the presence of degradation signals, termed N-degrons. Recognition components of the N-end rule pathway are E3 ubiquitin ligases that are capable of binding to N-degrons. The N-end rule pathway consists of two distinct branches: the Arg/N-end rule pathway and the Ac/N-end rule pathway.
\r\n\r\nIn the present studies, we demonstrate a complementary targeting of the rat serotonin N-acetyltransferase (AANAT), an important mediator of circadian physiology, by both branches of the N-end rule pathway. The co-targeting results from incomplete N-terminal (Nt-) acetylation of a Met-\u0424 motif at the N-terminus of AANAT in vivo. In the same study, we demonstrate that human AANAT is substantially longer-lived than its rat counterpart, owing to differences in their N-terminal sequences. This molecular genetic investigation of the degradation of a physiological N-end rule substrate followed an analogous earlier study, in which we reported that a clinically-relevant (blood pressure-increasing) Q2L mutant of human RGS2 (termed ML-RGS2), a regulator of G proteins, could likewise be co-targeted by both branches of the N-end rule pathway. Together, AANAT and RGS2 are the first identified and characterized physiological substrates of the Ac/N-end rule pathway in mammals.
\r\n\r\nWe also report on the development and use of in vitro N-terminal arginylation (Nt-arginylation) assays using CelluSpots peptide arrays, in conjunction with pulse-chase assays in rabbit reticulocyte extract, for the systematic investigation of the effects of N-terminus-proximal sequence context on the Nt-arginylation activity of the Ate1 arginyltransferase, a component of the Arg/N-end rule pathway. These experiments help to define the sequence requirements for efficient Nt-arginylation by Ate1. Finally, we demonstrate that Rec8, a subunit of the cohesin protein complex during meiosis, is a natural short-lived substrate of the mammalian Arg/N-end rule pathway.
\r\n" }, { "name": "Ahmed, Alysia Ashley", "degree": "PhD", "year": "2015", "title": "Structural Characterization of Pro-inflammatory and Anti-inflammatory Immunoglobulin G Fc Proteins", "advisor": "Bjorkman, Pamela J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05292015-064617250", "creators": [ { "name": { "family": "Ahmed", "given": "Alysia Ashley" }, "id": "Ahmed-Alysia-Ashley", "display_name": "Ahmed, Alysia Ashley" } ], "advisors": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "advisor", "display_name": "Bjorkman, Pamela J." } ], "committee": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "orcid": "0000-0002-3671-9354", "role": "chair", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "orcid": "0000-0001-8291-5551", "role": "member", "display_name": "Campbell, Judith L." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." } ], "option_major": [ "molbiochem" ], "doi": "10.7907/Z9GT5K33", "abstract": "Immunoglobulin G (IgG) is central in mediating host defense due to its ability to target and eliminate invading pathogens. The fragment antigen binding (Fab) regions are responsible for antigen recognition; however the effector responses are encoded on the Fc region of IgG. IgG Fc displays considerable glycan heterogeneity, accounting for its complex effector functions of inflammation, modulation and immune suppression. Intravenous immunoglobulin G (IVIG) is pooled serum IgG from multiple donors and is used to treat individuals with autoimmune and inflammatory disorders such as rheumatoid arthritis and Kawasaki\u2019s disease, respectively. It contains all the subtypes of IgG (IgG1-4) and over 120 glycovariants due to variation of an Asparagine 297-linked glycan on the Fc. The species identified as the activating component of IVIG is sialylated IgG Fc. Comparisons of wild type Fc and sialylated Fc X-ray crystal structures suggests that sialylation causes an increase in conformational flexibility, which may be important for its anti-inflammatory properties.
\r\n\r\nAlthough glycan modifications can promote the anti-inflammatory properties of the Fc, there are amino acid substitutions that cause Fcs to initiate an enhanced immune response. Mutations in the Fc can cause up to a 100-fold increase in binding affinity to activating Fc gamma receptors located on immune cells, and have been shown to enhance antibody dependent cell-mediated cytotoxicity. This is important in developing therapeutic antibodies against cancer and infectious diseases. Structural studies of mutant Fcs in complex with activating receptors gave insight into new protein-protein interactions that lead to an enhanced binding affinity.
\r\n\r\nTogether these studies show how dynamic and diverse the Fc region is and how both protein and carbohydrate modifications can alter structure, leading to IgG Fc\u2019s switch from a pro-inflammatory to an anti-inflammatory protein.
" }, { "name": "Bagert, John David", "degree": "PhD", "year": "2015", "title": "Quantitative, Time-Resolved Proteomic Analysis Using Bio-Orthogonal Non-Canonical Amino Acid Tagging", "advisor": "Tirrell, David A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05272015-123731363", "creators": [ { "name": { "family": "Bagert", "given": "John David" }, "id": "Bagert-John-David", "orcid": "0000-0001-7768-2853", "display_name": "Bagert, John David" } ], "advisors": [ { "name": { "family": "Tirrell", "given": "David A." }, "id": "Tirrell-D-A", "orcid": "0000-0003-3175-4596", "role": "advisor", "display_name": "Tirrell, David A." } ], "committee": [ { "name": { "family": "Tirrell", "given": "David A." }, "id": "Tirrell-D-A", "orcid": "0000-0003-3175-4596", "role": "chair", "display_name": "Tirrell, David A." }, { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "member", "display_name": "Arnold, Frances Hamilton" }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." }, { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "orcid": "0000-0002-7033-0844", "role": "member", "display_name": "Leadbetter, Jared R." } ], "option_major": [ "bioeng" ], "doi": "10.7907/Z9QC01D9", "abstract": "Bio-orthogonal non-canonical amino acid tagging (BONCAT) is an analytical method that allows the selective analysis of the subset of newly synthesized cellular proteins produced in response to a biological stimulus. In BONCAT, cells are treated with the non-canonical amino acid L-azidohomoalanine (Aha), which is utilized in protein synthesis in place of methionine by wild-type translational machinery. Nascent, Aha-labeled proteins are selectively ligated to affinity tags for enrichment and subsequently identified via mass spectrometry. The work presented in this thesis exhibits advancements in and applications of the BONCAT technology that establishes it as an effective tool for analyzing proteome dynamics with time-resolved precision.
\r\n\r\nChapter 1 introduces the BONCAT method and serves as an outline for the thesis as a whole. I discuss motivations behind the methodological advancements in Chapter 2 and the biological applications in Chapters 2 and 3.
\r\n\r\nChapter 2 presents methodological developments that make BONCAT a proteomic tool capable of, in addition to identifying newly synthesized proteins, accurately quantifying rates of protein synthesis. I demonstrate that this quantitative BONCAT approach can measure proteome-wide patterns of protein synthesis at time scales inaccessible to alternative techniques.
\r\n\r\nIn Chapter 3, I use BONCAT to study the biological function of the small RNA regulator CyaR in Escherichia coli. I correctly identify previously known CyaR targets, and validate several new CyaR targets, expanding the functional roles of the sRNA regulator.
\r\n\r\nIn Chapter 4, I use BONCAT to measure the proteomic profile of the quorum sensing bacterium Vibrio harveyi during the time-dependent transition from individual- to group-behaviors. My analysis reveals new quorum-sensing-regulated proteins with diverse functions, including transcription factors, chemotaxis proteins, transport proteins, and proteins involved in iron homeostasis.
\r\n\r\nOverall, this work describes how to use BONCAT to perform quantitative, time-resolved proteomic analysis and demonstrates that these measurements can be used to study a broad range of biological processes.
\r\n" }, { "name": "Buhler, Vivian Huang", "degree": "Senior Thesis", "year": "2015", "title": "Mary and Magdalene Reintegrated Through Sisterhood in a Male-Dominated Goblin Market", "advisor": "Gilmartin, Kevin M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04182018-090000178", "creators": [ { "name": { "family": "Buhler", "given": "Vivian Huang" }, "id": "Buhler-Vivian-Huang", "display_name": "Buhler, Vivian Huang" } ], "advisors": [ { "name": { "family": "Gilmartin", "given": "Kevin M." }, "id": "Gilmartin-K-M", "role": "advisor", "display_name": "Gilmartin, Kevin M." } ], "committee": [ { "name": { "family": "None", "given": "None" }, "display_name": "None, None" } ], "option_major": [ "bioeng", "english" ], "doi": "10.7907/Z9959FST", "abstract": "In her most celebrated and controversial poem, Goblin Market, Christina Rossetti presents an allegory of her own spiritual journey toward redemption as a woman, a sinner, and a reform worker. Through the stories of three young maidens\u2019 misadventures among the goblin men\u2019s \u201chaunted glen,\u201d Rossetti reveals the unjust Victorian binaries of male versus female, as well as virgin versus prostitute, and proposes an alternative definition of virtue attained through redemption that is accessible to all regardless of gender or social background. Using Jeanie, Lizzie, and Laura as figures for fallen woman, penitent, and sister, Rossetti further recasts the parallel feminine archetypes Eve, Mary, and Mary Magdalene as biblical paradigms of humanity united against the evils of the age, all equally destined for eternal salvation even if patriarchal standards should bar any such woman from an earthly life of honor." }, { "name": "Cornejo de los Santos, Emmanuel Lorenzo", "degree": "PhD", "year": "2015", "title": "Expanding the Toolkit for Synthetic Biology: Frameworks for Native-like Non-natural Gene Circuits", "advisor": "Murray, Richard M.; Mayo, Stephen L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05182015-163708506", "creators": [ { "name": { "family": "Cornejo de los Santos", "given": "Emmanuel Lorenzo" }, "id": "Cornejo-de-los-Santos-Emmanuel-Lorenzo-Cornejo", "display_name": "Cornejo de los Santos, Emmanuel Lorenzo" } ], "advisors": [ { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "advisor", "display_name": "Murray, Richard M." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "co-advisor", "display_name": "Mayo, Stephen L." } ], "committee": [ { "name": { "family": "Tirrell", "given": "David A." }, "id": "Tirrell-D-A", "orcid": "0000-0003-3175-4596", "role": "chair", "display_name": "Tirrell, David A." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." } ], "option_major": [ "bioeng" ], "doi": "10.7907/Z9M61H64", "abstract": "Synthetic biology combines biological parts from different sources in order to engineer non-native, functional systems. While there is a lot of potential for synthetic biology to revolutionize processes, such as the production of pharmaceuticals, engineering synthetic systems has been challenging. It is oftentimes necessary to explore a large design space to balance the levels of interacting components in the circuit. There are also times where it is desirable to incorporate enzymes that have non-biological functions into a synthetic circuit. Tuning the levels of different components, however, is often restricted to a fixed operating point, and this makes synthetic systems sensitive to changes in the environment. Natural systems are able to respond dynamically to a changing environment by obtaining information relevant to the function of the circuit. This work addresses these problems by establishing frameworks and mechanisms that allow synthetic circuits to communicate with the environment, maintain fixed ratios between components, and potentially add new parts that are outside the realm of current biological function. These frameworks provide a way for synthetic circuits to behave more like natural circuits by enabling a dynamic response, and provide a systematic and rational way to search design space to an experimentally tractable size where likely solutions exist. We hope that the contributions described below will aid in allowing synthetic biology to realize its potential." }, { "name": "Gandhi, Avni Vasant", "degree": "PhD", "year": "2015", "title": "The Regulation of Sleep and Circadian Rhythms: The Role of Melatonin and Adenosine in Zebrafish", "advisor": "Prober, David A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05292015-184053369", "creators": [ { "name": { "family": "Gandhi", "given": "Avni Vasant" }, "id": "Gandhi-Avni-Vasant", "display_name": "Gandhi, Avni Vasant" } ], "advisors": [ { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "role": "advisor", "display_name": "Prober, David A." } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Gradinaru", "given": "Viviana" }, "id": "Gradinaru-V", "role": "member", "display_name": "Gradinaru, Viviana" }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "role": "member", "display_name": "Prober, David A." } ], "option_major": [ "biology" ], "doi": "10.7907/Z99Z92TN", "abstract": "Sleep is a highly conserved behavioral state whose regulation is still unclear. In this thesis I initially briefly introduce the known sleep circuitry and regulation in vertebrates, and why zebrafish is seen as a good model to study sleep-regulation. I describe the existing two-process model of sleep regulation, which posits that the two processes C (circadian) and S (homeostatic) control timing of sleep-wake behavior. I then study the role melatonin plays in the circadian regulation of sleep using zebrafish. Firstly, we find that the absence of melatonin results in a reduction of sleep at night, establishing that endogenous melatonin is required for sleep at night. Secondly, melatonin mutants show a reduction in sleep in animals with no functional behavioral rhythms suggesting that melatonin does not require intact circadian rhythms for its effect on sleep. Thirdly, melatonin mutants do not exhibit any changes in circadian rhythms, suggesting that the circadian clock does not require melatonin for its function. Fourthly, we find that in the absence of melatonin, there is no rhythmic expression of sleep, suggesting that melatonin is the output molecule of process C. Lastly, we describe a connection between adenosine signaling (output molecules of process S), and melatonin. Following this we proceed to study the role adenosine signaling plays in sleep-wake behavior. We find that firstly, adenosine receptor A1 and A2 are involved in sleep- wake behavior in zebrafish, based on agonist/antagonist behavioral results. Secondly, we find that several brain regions such as PACAP cells in the rostral midbrain, GABAergic cells in the forebrain and hindbrain, Dopamine and serotonin cells in the caudal hypothalamus and sox2 cells lining the hindbrain ventricle are activated in response to the A1 antagonist and VMAT positive cells are activated in response to the A2A agonist, suggesting these areas are involved in adenosine signaling in zebrafish. Thirdly, we find that knocking out the zebrafish adenosine receptors has no effect on sleep architecture. Lastly, we find that while the A1 agonist phenotype requires the zfAdora1a receptor, the antagonist and the A2A agonist behavioral phenotypes are not mediated by the zfAdora1a, zfAdora1b and zfAdoraA2Aa, zfAdora2Ab receptors respectively." }, { "name": "Gharib, Alma Mariam", "degree": "PhD", "year": "2015", "title": "Visual Behavior and Preference Decision-Making in Response to Faces in High-Functioning Autism", "advisor": "Shimojo, Shinsuke; Adolphs, Ralph", "url": "https://resolver.caltech.edu/CaltechTHESIS:06052015-203007731", "creators": [ { "name": { "family": "Gharib", "given": "Alma Mariam" }, "id": "Gharib-Alma-Mariam", "orcid": "0000-0002-7588-407X", "display_name": "Gharib, Alma Mariam" } ], "advisors": [ { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "role": "advisor", "display_name": "Shimojo, Shinsuke" }, { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "role": "co-advisor", "display_name": "Adolphs, Ralph" } ], "committee": [ { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "role": "chair", "display_name": "Adolphs, Ralph" }, { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "role": "member", "display_name": "Shimojo, Shinsuke" }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "role": "member", "display_name": "Perona, Pietro" } ], "option_major": [ "neurobio" ], "doi": "10.7907/Z9DN4307", "abstract": "How do we come to the decision that we like a face? This thesis investigates this important aspect of social processing and communication by examining preference decisions for faces and the role that visual behavior plays in the process. I present a series of studies designed to investigate face preference formation and gaze patterns using eye-tracking and self-reported preference ratings. I tested healthy control subjects and two clinical populations known to have deficits in social processing: people with autism and patients with amygdala lesions. In studies one and two, I explore whether known social cognition deficits in people with autism and amygdala lesions also impair subjective decision-making regarding the attractiveness of faces. In study three, I investigate the flexibility of rule-based visual strategies used by these populations during face perception. Additionally, I present a custom algorithm developed to process raw eyetracking data, which was used to analyze all eyetracking data in this thesis.
\r\nPeople with autism and patients with amygdala lesions are known to have general deficits in social processing, including difficulty orienting toward and evaluating faces. Nevertheless, I find that their behavior is markedly similar in many areas where we would expect them to have abnormalities or deficiencies. Their preference decisions when judging facial attractiveness were highly correlated with those made by controls, and both groups showed the same biases for familiar faces over novel faces. In addition, people with autism exhibit the same visual sampling behavior linking preference and attentional orienting, but reach their decisions faster than controls and also appear insensitive to the difficulty of the choice. Finally, gaze to the eye region appears normal in the absence of an explicit decision-making task, but only when analyzed in a similar manner as previous studies. However, when face sub-regions were analyzed in greater detail, people with autism demonstrate abnormalities in face gaze patterns, failing to emphasize the most information-rich regions of the face. Furthermore, people with autism demonstrate impairments in their ability to update those gaze patterns to accommodate different viewing restrictions. Taken together, these findings support the idea that the normal formation of face preferences can be preserved in the presence of general social processing impairments. Patterns in the eyetracking and behavioral data indicate that this is made possible, in part, by compensatory atypical processing and visual strategies.
\r\n" }, { "name": "Ghosh, Srimoyee", "degree": "PhD", "year": "2015", "title": "Establishing the C. elegans Uterine Seam Cell (utse) as a Novel Model for Studying Cell Behavior", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05202015-102200658", "creators": [ { "name": { "family": "Ghosh", "given": "Srimoyee" }, "id": "Ghosh-Srimoyee", "orcid": "0000-0002-7820-6741", "display_name": "Ghosh, Srimoyee" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "member", "display_name": "Chan, David C." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/Z92F7KCX", "abstract": "The molecular inputs necessary for cell behavior are vital to our understanding of development and disease. Proper cell behavior is necessary for processes ranging from creating one\u2019s face (neural crest migration) to spreading cancer from one tissue to another (invasive metastatic cancers). Identifying the genes and tissues involved in cell behavior not only increases our understanding of biology but also has the potential to create targeted therapies in diseases hallmarked by aberrant cell behavior.
\r\n\r\nA well-characterized model system is key to determining the molecular and spatial inputs necessary for cell behavior. In this work I present the C. elegans uterine seam cell (utse) as an ideal model for studying cell outgrowth and shape change. The utse is an H-shaped cell within the hermaphrodite uterus that functions in attaching the uterus to the body wall. Over L4 larval stage, the utse grows bidirectionally along the anterior-posterior axis, changing from an ellipsoidal shape to an elongated H-shape. Spatially, the utse requires the presence of the uterine toroid cells, sex muscles, and the anchor cell nucleus in order to properly grow outward. Several gene families are involved in utse development, including Trio, Nav, Rab GTPases, Arp2/3, as well as 54 other genes found from a candidate RNAi screen. The utse can be used as a model system for studying metastatic cancer. Meprin proteases are involved in promoting invasiveness of metastatic cancers and the meprin-likw genes nas-21, nas-22, and toh-1 act similarly within the utse. Studying nas-21 activity has also led to the discovery of novel upstream inhibitors and activators as well as targets of nas-21, some of which have been characterized to affect meprin activity. This illustrates that the utse can be used as an in vivo model for learning more about meprins, as well as various other proteins involved in metastasis.
\r\n" }, { "name": "Goldberg, Mark David", "degree": "PhD", "year": "2015", "title": "Development of Microfluidic Devices with the Use of Nanotechnology to Aid in the Analysis of Biological Systems Including Membrane Protein Separation, Single Cell Analysis, and Genetic Markers", "advisor": "Scherer, Axel", "url": "https://resolver.caltech.edu/CaltechTHESIS:06022015-150554177", "creators": [ { "name": { "family": "Goldberg", "given": "Mark David" }, "id": "Goldberg-Mark-David", "display_name": "Goldberg, Mark David" } ], "advisors": [ { "name": { "family": "Scherer", "given": "Axel" }, "id": "Scherer-A", "role": "advisor", "display_name": "Scherer, Axel" } ], "committee": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "chair", "display_name": "Davidson, Eric H." }, { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "role": "member", "display_name": "Campbell, Judith L." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Kartalov", "given": "Emil P." }, "id": "Kartalov-E-P", "role": "member", "display_name": "Kartalov, Emil P." }, { "name": { "family": "Scherer", "given": "Axel" }, "id": "Scherer-A", "role": "member", "display_name": "Scherer, Axel" } ], "option_major": [ "biology" ], "doi": "10.7907/Z9P848V6", "abstract": "Computation technology has dramatically changed the world around us; you can hardly find an area where cell phones have not saturated the market, yet there is a significant lack of breakthroughs in the development to integrate the computer with biological environments. This is largely the result of the incompatibility of the materials used in both environments; biological environments and experiments tend to need aqueous environments. To help aid in these development chemists, engineers, physicists and biologists have begun to develop microfluidics to help bridge this divide. Unfortunately, the microfluidic devices required large external support equipment to run the device. This thesis presents a series of several microfluidic methods that can help integrate engineering and biology by exploiting nanotechnology to help push the field of microfluidics back to its intended purpose, small integrated biological and electrical devices. I demonstrate this goal by developing different methods and devices to (1) separate membrane bound proteins with the use of microfluidics, (2) use optical technology to make fiber optic cables into protein sensors, (3) generate new fluidic devices using semiconductor material to manipulate single cells, and (4) develop a new genetic microfluidic based diagnostic assay that works with current PCR methodology to provide faster and cheaper results. All of these methods and systems can be used as components to build a self-contained biomedical device. " }, { "name": "Ho, Margaret Ching Wai (MC)", "degree": "PhD", "year": "2015", "title": "Discovery of Active Cis-Regulatory Elements and Transcription Factor Footprints in Nematodes Using Functional Genomics Approaches", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05282015-161443045", "creators": [ { "name": { "family": "Ho", "given": "Margaret Ching Wai (MC)" }, "id": "Ho-Margaret-Ching-Wai", "orcid": "0000-0002-2007-2226", "display_name": "Ho, Margaret Ching Wai (MC)" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Aravin", "given": "Alexei A." }, "id": "Aravin-A-A", "orcid": "0000-0002-6956-8257", "role": "chair", "display_name": "Aravin, Alexei A." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/Z9G73BVF", "abstract": "High throughput DNA sequencing has emerged as a versatile and inexpensive readout of functional activity in biological organisms. In this study I describe the implementation of DNaseI hypersensitivity assays using deep sequencing (DNase-seq) to systematically identify Caenorhabditis elegans cis-regulatory modules (CRMs) in embryonic and L1 arrest larval life stages in an unbiased and de novo manner. We validated our data by comparison to many known enhancers of lin-39/ceh-13 Hox complex and of hlh-1, myo-2, myo-3, lin-26, and other important developmental genes and are also able to predict novel cis-regulatory modules. We predict novel regulatory motifs from our DNase-seq data and predict potential regulatory functions using gene ontology and anatomy enrichment analysis. In addition, our data are high-resolution enough to identify binding sites of transcription factors in the genome. Our data provide support for many distal CRMs in C. elegans and for a significant portion of genes possessing multiple CRMs. DNase-seq data can also be used to refine prediction of tissue-specific genes such as those regulated by C. elegans pan-neuronal N1 and intestinal ELT-2 DNA motifs. Overall, we identify 24,128 putative CRMS containing over 55,000 footprints. In L1 arrest, we identify 15,841 putative CRMs in the L1 arrest larvae containing 32,000 TF footprints. From comparison of these datasets, we identify an additional 1,854 noncoding DHS that appear to be specific to the L1 arrest larvae condition. These genes include downstream targets of signaling pathways known to be regulated during L1 arrest such as insulin-like signaling via DAF-16/FOXO and Forkhead box transcription factor PHA-4/FOXA that impacts starvation survival in the L1 arrest condition. Having established the first proof-of-principle DNase-seq in nematodes using C. elegans, I am applying DNase-seq to a distantly related entomopathogenic nematode, Steinernema carpocapsae, with a recently sequenced genome and transcriptome. Finally, I am using a massively parallel reporter assay to test the functional activity of the CRMs we have discovered from DNase-seq using two reporter designs based on MPRA and STARR-seq and by performing DNA and RNA sequencing on transgenic C. elegans.
" }, { "name": "Kreamer, Naomi N.", "degree": "PhD", "year": "2015", "title": "Ferrous Iron Sensing and Responding in Pseudomonas aeruginosa", "advisor": "Newman, Dianne K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03092015-135659710", "creators": [ { "name": { "family": "Kreamer", "given": "Naomi N." }, "id": "Kreamer-Naomi-N", "display_name": "Kreamer, Naomi N." } ], "advisors": [ { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "advisor", "display_name": "Newman, Dianne K." } ], "committee": [ { "name": { "family": "Gray", "given": "Harry B." }, "id": "Gray-H-B", "orcid": "0000-0002-7937-7876", "role": "chair", "display_name": "Gray, Harry B." }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Shan", "given": "Shu-ou" }, "id": "Shan-Shu-ou", "orcid": "0000-0002-6526-1733", "role": "member", "display_name": "Shan, Shu-ou" }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." } ], "option_major": [ "biochem" ], "doi": "10.7907/Z9DN4324", "abstract": "Controlling iron distribution is important for all organisms, and is key in bacterial pathogenesis. It has long been understood that cystic fibrosis (CF) patient sputum contains elevated iron concentrations. However, anaerobic bacteria have been isolated from CF sputum and hypoxic zones in sputum have been measured. Because ferrous iron [Fe(II)] is stable in reducing, acidic conditions, it could exist in the CF lung. I show that a two-component system, BqsRS, specifically responds to Fe(II) in the CF pathogen, Pseudomonas aeruginosa. Concurrently, a clinical study found that Fe(II) is present in CF sputum at all stages of lung function decline. Fe(II), not Fe(III) correlates with patients in the most severe disease state. Furthermore, transcripts of the newly identified BqsRS were detected in sputum. Two component systems are the main method bacteria interact with their extracellular environment. A typical two-component system contains a sensor histidine kinase, which upon activation phosphorylates a response regulator that then acts as a transcription factor to elicit a cellular response to stimuli. To explore the mechanism of BqsRS, I describe the Fe(II)-sensing RExxE motif in the sensor BqsS and determine the consensus DNA sequence BqsR binds. With the BqsR binding sequence, I identify novel regulon members through bioinformatic and molecular biology techniques. From the predicted function of new BqsR regulon members, I find that Fe(II) elicits a response that globally protects the cells against cationic stressors, including clinically relevant antibiotics. Subsequently, I use BqsR as a case study to determine if promoter outputs can accurately be predicted based only on a deep understanding of a transcriptional activator\u2019s operator or if a broader regulatory context is required for accurate predictions at all genomic loci. This work highlights the importance of Fe(II) as a (micro)environmental factor, even in conditions typically thought of as aerobic. Since the presence of Fe(II) can alter P. aeruginosa\u2019s antibiotic susceptibility, combining the current strategy of targeting Fe(III) with a new approach targeting Fe(II) may help eradicate infections in the CF lung in the future." }, { "name": "Lim, Rod S.", "degree": "PhD", "year": "2015", "title": "How Resources Control Aggression in Drosophila", "advisor": "Anderson, David J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:11222014-220142839", "creators": [ { "name": { "family": "Lim", "given": "Rod S." }, "id": "Lim-Rod-S", "display_name": "Lim, Rod S." } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "advisor", "display_name": "Anderson, David J." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "co-chair", "display_name": "Zinn, Kai George" }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "role": "member", "display_name": "Perona, Pietro" } ], "option_major": [ "sysbiol" ], "doi": "10.7907/Z9R78C4T", "abstract": "How animals use sensory information to weigh the risks vs. benefits of behavioral decisions remains poorly understood. Inter-male aggression is triggered when animals perceive both the presence of an appetitive resource, such as food or females, and of competing conspecific males. How such signals are detected and integrated to control the decision to fight is not clear. Here we use the vinegar fly, Drosophila melanogaster, to investigate the manner in which food and females promotes aggression.
\r\n\r\nIn the first chapter, we explore how food controls aggression. As in many other species, food promotes aggression in flies, but it is not clear whether food increases aggression per se, or whether aggression is a secondary consequence of increased social interactions caused by aggregation of flies on food. Furthermore, nothing is known about how animals evaluate the quality and quantity of food in the context of competition. We show that food promotes aggression independently of any effect to increase the frequency of contact between males. Food increases aggression but not courtship between males, suggesting that the effect of food on aggression is specific. Next, we show that flies tune the level of aggression according to absolute amount of food rather than other parameters, such as area or concentration of food. Sucrose, a sugar molecule present in many fruits, is sufficient to promote aggression, and detection of sugar via gustatory receptor neurons is necessary for food-promoted aggression. Furthermore, we show that while food is necessary for aggression, too much food decreases aggression. Finally, we show that flies exhibit strategies consistent with a territorial strategy. These data suggest that flies use sweet-sensing gustatory information to guide their decision to fight over a limited quantity of a food resource.
\r\n\r\nFollowing up on the findings of the first chapter, we asked how the presence of a conspecific female resource promotes male-male aggression. In the absence of food, group-housed male flies, who normally do not fight even in the presence of food, fight in the presence of females. Unlike food, the presence of females strongly influences proximity between flies. Nevertheless, as group-housed flies do not fight even when they are in small chambers, it is unlikely that the presence of female indirectly increases aggression by first increasing proximity. Unlike food, the presence of females also leads to large increases in locomotion and in male-female courtship behaviors, suggesting that females may influence aggression as well as general arousal. Female cuticular hydrocarbons are required for this effect, as females that do not produce CH pheromones are unable to promote male-male aggression. In particular, 7,11-HD\u2013\u2013a female-specific cuticular hydrocarbon pheromone critical for male-female courtship\u2013\u2013is sufficient to mediate this effect when it is perfumed onto pheromone-deficient females or males. Recent studies showed that ppk23+ GRNs label two population of GRNs, one of which detects male cuticular hydrocarbons and another labeled by ppk23 and ppk25, which detects female cuticular hydrocarbons. I show that in particular, both of these GRNs control aggression, presumably via detection of female or male pheromones. To further investigate the ways in which these two classes of GRNs control aggression, I developed new genetic tools to independently test the male- and female-sensing GRNs. I show that ppk25-LexA and ppk25-GAL80 faithfully recapitulate the expression pattern of ppk25-GAL4 and label a subset of ppk23+ GRNs. These tools can be used in future studies to dissect the respective functions of male-sensing and female-sensing GRNs in male social behaviors.
\r\n\r\nFinally, in the last chapter, I discuss quantitative approaches to describe how varying quantities of food and females could control the level of aggression. Flies show an inverse-U shaped aggressive response to varying quantities of food and a flat aggressive response to varying quantities of females. I show how two simple game theoretic models, \u201cprisoner\u2019s dilemma\u201d and \u201ccoordination game\u201d could be used to describe the level of aggression we observe. These results suggest that flies may use strategic decision-making, using simple comparisons of costs and benefits.
\r\n\r\nIn conclusion, male-male aggression in Drosophila is controlled by simple gustatory cues from food and females, which are detected by gustatory receptor neurons. Different quantities of resource cues lead to different levels of aggression, and flies show putative territorial behavior, suggesting that fly aggression is a highly strategic adaptive behavior. How these resource cues are integrated with male pheromone cues and give rise to this complex behavior is an interesting subject, which should keep researchers busy in the coming years.
" }, { "name": "Liu, Justin", "degree": "PhD", "year": "2015", "title": "Development and Function of Sleep Regulatory Circuits in Zebrafish", "advisor": "Prober, David A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06012015-124819012", "creators": [ { "name": { "family": "Liu", "given": "Justin" }, "id": "Liu-Justin", "display_name": "Liu, Justin" } ], "advisors": [ { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "role": "advisor", "display_name": "Prober, David A." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "member", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "role": "member", "display_name": "Prober, David A." } ], "option_major": [ "neurobio" ], "doi": "10.7907/Z9WM1BDC", "abstract": "Sleep is widely accepted as an essential behavior for optimum mental and physical health, yet the genetic and neural circuits that govern sleep remain poorly understood. In this thesis, I briefly introduce the behavioral criteria that define sleep, currently known sleep regulatory mechanisms, and the distinct advantages of the zebrafish, Danio rerio, as a simple animal model for studying sleep. I then investigate two factors previously implicated in sleep behavior: epidermal growth factor receptor and hypocretin. First, I show that epidermal growth factor receptor signaling is both necessary and sufficient for normal sleep behavior in zebrafish, just as it is in invertebrates. This demonstrates that sleep regulatory mechanisms can be conserved over large evolutionary distances, and is the first genetic study showing that the epidermal growth factor receptor signaling is necessary for normal sleep behavior in a vertebrate. Second, I capitalize upon the rapid external development of zebrafish embryos to screen for developmental factors that specify hypocretin neurons, which are known to promote arousal and consolidate sleep/wake bouts. I identify the LIM homeobox 9 transcription factor as necessary for hypocretin neuronal development in zebrafish and sufficient to specify additional hypocretin neurons in both zebrafish and mice. This is the first time any factor has been shown to induce hypocretin neurons in vivo and may be an important step towards curing narcolepsy, a debilitating sleep disorder caused by the selective loss of hypocretin neurons. These studies deepen our understanding of how sleep is regulated at a genetic and cellular level and underscore the potential for zebrafish to make future contributions to sleep research." }, { "name": "Malladi, Chaitanya Lakshmidhar", "degree": "Other", "year": "2015", "title": "Shakespeare\u2019s Crafting of the Ideal King in Henry V", "advisor": "Unknown, Unknown", "url": "https://resolver.caltech.edu/CaltechTHESIS:04062016-111338363", "creators": [ { "name": { "family": "Malladi", "given": "Chaitanya Lakshmidhar" }, "id": "Malladi-Chaitanya-Lakshmidhar", "display_name": "Malladi, Chaitanya Lakshmidhar" } ], "advisors": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioeng" ], "doi": "10.7907/2B07-N545", "abstract": "[Introduction] In Shakespeare\u2019s history Henry V, the playwright depicts the reign of King Harry\u2014\r\nspecifically with respect to his claim to the throne of France and his subsequent invasion. In the\r\nprequel to this play, Henry IV, Harry is shown as an impetuous young boy who engages in\r\nmischief around town with his immature friends. However, by the end of the first part of Henry\r\nIV, Harry matures and becomes a brave, strong warrior on the battlefield as well as a courageous,\r\nhonorable man. He is ultimately portrayed as the rightful heir to the throne. At the beginning of\r\nHenry V, the audience sees a mature king who has been in charge of the kingdom for some time\r\nalready. While Shakespeare bases his plays on historical events as documented in works like\r\nRaphael Holinshed\u2019s Chronicles of England, Scotland, and Ireland, the playwright uses his\r\nliterary liberty to alter the chronological order of events and even sometimes the actions that\r\ncertain characters take or do not take. Shakespeare\u2019s dramatization of several historical elements\r\nof Holinshed serves to aggrandize the glory and maturity of Henry V as a just, idealized king;\r\nwith his decisions to change what is written in the history books, Shakespeare highlights the king\u2019s military aptitude, his modesty in dealing with his soldiers and citizens, and his political\r\nsavviness." }, { "name": "Mehta, Arnav", "degree": "PhD", "year": "2015", "title": "MicroRNA-132 is a Physiological Regulator of Hematopoietic Stem Cell Function and B-cell Development", "advisor": "Baltimore, David L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03252015-144135628", "creators": [ { "name": { "family": "Mehta", "given": "Arnav" }, "id": "Mehta-Arnav", "display_name": "Mehta, Arnav" } ], "advisors": [ { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "advisor", "display_name": "Baltimore, David L." } ], "committee": [ { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "chair", "display_name": "Mazmanian, Sarkis K." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "role": "member", "display_name": "Guttman, Mitchell" }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "member", "display_name": "Baltimore, David L." } ], "option_major": [ "immun" ], "doi": "10.7907/Z9XS5SBD", "abstract": "MicroRNAs are a class of small non-coding RNAs that negatively regulate gene expression. Several microRNAs have been implicated in altering hematopoietic cell fate decisions. Importantly, deregulation of many microRNAs can lead to deleterious consequences in the hematopoietic system, including the onset of cancer, autoimmunity, or a failure to respond effectively to infection. As such, microRNAs fine-tune the balance between normal hematopoietic output and pathologic consequences. In this work, we explore the role of two microRNAs, miR-132 and miR-125b, in regulating hematopoietic stem cell (HSC) function and B cell development. In particular, we uncover the role of miR-132 in maintaining the appropriate balance between self-renewal, differentiation, and survival in aging HSCs by buffering the expression of a critical transcription factor, FOXO3. By maintain this balance, miR-132 may play a critical role in preventing aging-associated hematopoietic conditions such as autoimmune disease and cancer. We also find that miR-132 plays a critical role in B cell development by targeting a key transcription factor, Sox4, that is responsible for the differentiation of pro-B cells into pre-B cells. We find that miR-132 regulates B cell apoptosis, and by delivering miR-132 to mice that are predisposed to developing B cell cancers, we can inhibit the formation of these cancers and improve the survival of these mice. In addition to miR-132, we uncovered the role of another critical microRNA, miR-125b, that potentiates hematopoietic stem cell function. We found that enforced expression of miR-125b causes an aggressive myeloid leukemia by downregulation of its target Lin28a. Importantly, miR-125b also plays a critical role in inhibiting the formation of pro-B cells. Thus, we have discovered two microRNAs with important roles in regulating normal hematopoiesis, and whose dregulation can lead to deleterious consequences such as cancer in the aging hematopoietic system. Both miR-132 and miR-125b may therefore be targeted for therapeutics to inhibit age-related immune diseases associated with the loss of HSC function and cancer progression." }, { "name": "Miles, Timothy Francis", "degree": "PhD", "year": "2015", "title": "Binding Site Structure and Stoichiometry in Serotonin Type 3 Receptors", "advisor": "Dougherty, Dennis A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:07142014-132743727", "creators": [ { "name": { "family": "Miles", "given": "Timothy Francis" }, "id": "Miles-Timothy-Francis", "display_name": "Miles, Timothy Francis" } ], "advisors": [ { "name": { "family": "Dougherty", "given": "Dennis A." }, "id": "Dougherty-D-A", "role": "advisor", "display_name": "Dougherty, Dennis A." } ], "committee": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "chair", "display_name": "Lester, Henry A." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "member", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Dougherty", "given": "Dennis A." }, "id": "Dougherty-D-A", "role": "member", "display_name": "Dougherty, Dennis A." } ], "option_major": [ "molbiochem" ], "doi": "10.7907/Z9T151MC ", "abstract": "This dissertation primarily describes studies of serotonin type 3 (5-HT3) receptors of the Cys-loop super-family of ligand gated ion channels. The first chapter provides a general introduction to these important proteins and the methods used to interrogate their structure and function. The second chapter details the delineation of a structural unit of the ligand binding site of homomeric 5-HT3A receptors on which the ligands serotonin (5-HT) and m-chlorophenyl biguanide (mCPBG) are reliant for effective receptor activation. Unnatural amino acid mutagenesis results show that the details of each ligand\u2019s interaction with this organizing feature of the binding site differ, providing insights into general principles of receptor activation.
\r\n\r\nThe third chapter describes a study in which florescent protein fusions of the A and B subunits of the heteromeric 5-HT3AB receptor are employed to determine the subunit stoichiometry and order within functional receptors. Strong evidence is found for an A3B2 stoichiometry with A-A-B-A-B order. The fourth chapter investigates the potential for ligand binding across heteromeric binding sites in the 5-HT3AB receptor. Unlike serotonin, mCPBG is found to bind the receptor at heteromeric binding sites. Further mCPBG is capable of allosterically modulating the response of serotonin on the 5-HT3AB receptor from these heteromeric sites.
\r\n\r\nFinally, the fifth chapter describes progress towards the application of unnatural amino acid mutagenesis to an important new class of proteins, transcription factors. Experiments optimizing novel methods for the detection of function are described, using RAR\u03b1 of the nuclear receptor family of transcription factors.
" }, { "name": "Mosadeghi, Ruzbeh", "degree": "PhD", "year": "2015", "title": "Mechanistic Dissection of the Cop9 Signalosome\u2019s Deneddylation Activity on Cullin-RING Ligases", "advisor": "Deshaies, Raymond Joseph", "url": "https://resolver.caltech.edu/CaltechTHESIS:05312015-231120184", "creators": [ { "name": { "family": "Mosadeghi", "given": "Ruzbeh" }, "id": "Mosadeghi-Ruzbeh", "display_name": "Mosadeghi, Ruzbeh" } ], "advisors": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "advisor", "display_name": "Deshaies, Raymond Joseph" } ], "committee": [ { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "chair", "display_name": "Chan, David C." }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "member", "display_name": "Mazmanian, Sarkis K." }, { "name": { "family": "Shan", "given": "Shu-ou" }, "id": "Shan-Shu-ou", "role": "member", "display_name": "Shan, Shu-ou" }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "member", "display_name": "Deshaies, Raymond Joseph" } ], "option_major": [ "biology" ], "doi": "10.7907/Z9P26W3G", "abstract": "We set out to understand the precise mechanisms that regulate the activation and deactivation of Cullin-RING Ligases (CRLs). While a great deal of work has already gone into identifying the players involved in these pathways and the cellular consequences associated with the loss of each, the biochemical mechanisms regulating these steps have remained elusive. In this work we sought to gain a better understanding of the mechanisms behind these steps by teasing apart specific their biochemical reactions. By measuring the individual microscopic rate constants of the reactions we have shed light on both the proper sequence of events in the regulation of CRLs as well as how they are in fact controlled.
\r\n\r\nPrior to this work, it was believed that CSN deactivated CRLs by binding them and enzymatically removing the activating post-translation modification Nedd8. It was believed that CSN could not bind to CRLs while they were active due to the steric hindrance by the CRL substrates, and that they would remain bound to deneddylated CRLs as a sequestering agent until a new substrate could displace it. We now have some insight that substrates themselves cannot inhibit CSN very well, but that the active ubiquitination by an E2 enzyme precludes CSN binding and activity. When the substrate for a CRL becomes depleted, CSN then binds to the CRL in a low affinity, low activity conformation. This triggers a conformational change that pulls the autoinhibitory Ins-1 loop away from the active site in the catalytic subunit Csn5, resulting in a large increase in affinity and cleavage of the isopeptide bond between CRLs and Nedd8. Upon dissociation of Nedd8, CSN rapidly returns to the low affinity state and dissociates from the CRL, allowing it reenter its activation cycle.
\r\n" }, { "name": "Parker, Rell Lin", "degree": "PhD", "year": "2015", "title": "Lynx1 Modulation of Nicotinic Acetylcholine Receptors", "advisor": "Lester, Henry A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06292014-232713483", "creators": [ { "name": { "family": "Parker", "given": "Rell Lin" }, "id": "Parker-Rell-Lin", "display_name": "Parker, Rell Lin" } ], "advisors": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "advisor", "display_name": "Lester, Henry A." } ], "committee": [ { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "chair", "display_name": "Chan, David C." }, { "name": { "family": "Meister", "given": "Markus" }, "id": "Meister-M", "role": "member", "display_name": "Meister, Markus" }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "role": "member", "display_name": "Prober, David A." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "member", "display_name": "Lester, Henry A." } ], "option_major": [ "neurobio" ], "doi": "10.7907/Z9HM56DT", "abstract": "Nicotinic receptors are the target of nicotine in the brain. They are pentameric ion channels. The pentamer structure allows many combinations of receptors to be formed. These various subtypes exhibit specific properties determined by their subunit composition. Each brain region contains a fixed complement of nicotinic receptor subunits. The midbrain region is of particular interest because the dopaminergic neurons of the midbrain express several subtypes of nicotinic receptors, and these dopaminergic neurons are important for the rewarding effects of nicotine. The \u03b16 nicotinic receptor subunit has garnered intense interest because it is present in dopaminergic neurons but very few other brain regions. With its specific and limited presence in the brain, targeting this subtype of nicotinic receptor may prove advantageous as a method for smoking cessation. However, we do not fully understand the trafficking and membrane localization of this receptor or its effects on dopamine release in the striatum. We hypothesized that lynx1, a known modulator of other nicotinic receptor subtypes, is important for the proper function of \u03b16 nicotinic receptors. lynx1 has been found to act upon several classes of nicotinic receptors, such as \u03b14\u03b22 and \u03b17, the two most common subtypes in the brain. To determine whether lynx1 affects \u03b16 containing nicotinic receptors we used biochemistry, patch clamp electrophysiology, fast scan cyclic voltammetry, and mouse behavior. We found that lynx1 has effects on \u03b16 containing nicotinic receptors, but the effects were subtle. This thesis will detail the observed effects of lynx1 on \u03b16 nicotinic receptors." }, { "name": "Ricci, Jessica Nicole", "degree": "PhD", "year": "2015", "title": "Constraining the Interpretation of 2-Methylhopanoids through Genetic and Phylogenetic Methods", "advisor": "Newman, Dianne K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05282015-153534197", "creators": [ { "name": { "family": "Ricci", "given": "Jessica Nicole" }, "id": "Ricci-Jessica-Nicole", "display_name": "Ricci, Jessica Nicole" } ], "advisors": [ { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "advisor", "display_name": "Newman, Dianne K." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "orcid": "0000-0003-2713-1513", "role": "member", "display_name": "Mazmanian, Sarkis K." }, { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "orcid": "0000-0002-7033-0844", "role": "member", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Sessions", "given": "Alex L." }, "id": "Sessions-A-L", "orcid": "0000-0001-6120-2763", "role": "member", "display_name": "Sessions, Alex L." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." } ], "option_major": [ "biology" ], "doi": "10.7907/Z9MC8X0S", "abstract": "Hopanoids are a class of sterol-like lipids produced by select bacteria. Their preservation in the rock record for billions of years as fossilized hopanes lends them geological significance. Much of the structural diversity present in this class of molecules, which likely underpins important biological functions, is lost during fossilization. Yet, one type of modification that persists during preservation is methylation at C-2. The resulting 2-methylhopanoids are prominent molecular fossils and have an intriguing pattern over time, exhibiting increases in abundance associated with Ocean Anoxic Events during the Phanerozoic. This thesis uses diverse methods to address what the presence of 2-methylhopanes tells us about the microbial life and environmental conditions of their ancient depositional settings. Through an environmental survey of hpnP, the gene encoding the C-2 hopanoid methylase, we found that many different taxa are capable of producing 2-methylhopanoids in more diverse modern environments than expected. This study also revealed that hpnP is significantly overrepresented in organisms that are plant symbionts, in environments associated with plants, and with metabolisms that support plant-microbe interactions; collectively, these correlations provide a clue about the biological importance of 2-methylhopanoids. Phylogenetic reconstruction of the evolutionary history of hpnP revealed that 2-methylhopanoid production arose in the Alphaproteobacteria, indicating that the origin of these molecules is younger than originally thought. Additionally, we took genetic approach to understand the role of 2-methylhopanoids in Cyanobacteria using the filamentous symbiotic Nostoc punctiforme. We found that hopanoids likely aid in rigidifying the cell membrane but do not appear to provide resistance to osmotic or outer membrane stressors, as has been shown in other organisms. The work presented in this thesis supports previous findings that 2-methylhopanoids are not biomarkers for oxygenic photosynthesis and provides new insights by defining their distribution in modern environments, identifying their evolutionary origin, and investigating their role in Cyanobacteria. These efforts in modern settings aid the formation of a robust interpretation of 2-methylhopanes in the rock record. " }, { "name": "Singer, Zakary Sean", "degree": "PhD", "year": "2015", "title": "Metastability and Dynamics of Stem Cells: From Direct Observations to Inference at the Single Cell Level", "advisor": "Elowitz, Michael B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05262015-152206802", "creators": [ { "name": { "family": "Singer", "given": "Zakary Sean" }, "id": "Singer-Zakary-Sean", "display_name": "Singer, Zakary Sean" } ], "advisors": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "advisor", "display_name": "Elowitz, Michael B." } ], "committee": [ { "name": { "family": "Cai", "given": "Long" }, "id": "Cai-Long", "role": "chair", "display_name": "Cai, Long" }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "role": "member", "display_name": "Guttman, Mitchell" }, { "name": { "family": "Plath", "given": "Kathrin" }, "id": "Plath-K", "role": "member", "display_name": "Plath, Kathrin" } ], "option_major": [ "cns" ], "doi": "10.7907/Z9ST7MS1", "abstract": "Organismal development, homeostasis, and pathology are rooted in inherently probabilistic events. From gene expression to cellular differentiation, rates and likelihoods shape the form and function of biology. Processes ranging from growth to cancer homeostasis to reprogramming of stem cells all require transitions between distinct phenotypic states, and these occur at defined rates. Therefore, measuring the fidelity and dynamics with which such transitions occur is central to understanding natural biological phenomena and is critical for therapeutic interventions.
\r\n\r\nWhile these processes may produce robust population-level behaviors, decisions are made by individual cells. In certain circumstances, these minuscule computing units effectively roll dice to determine their fate. And while the 'omics' era has provided vast amounts of data on what these populations are doing en masse, the behaviors of the underlying units of these processes get washed out in averages.
\r\n\r\nTherefore, in order to understand the behavior of a sample of cells, it is critical to reveal how its underlying components, or mixture of cells in distinct states, each contribute to the overall phenotype. As such, we must first define what states exist in the population, determine what controls the stability of these states, and measure in high dimensionality the dynamics with which these cells transition between states.
\r\n\r\nTo address a specific example of this general problem, we investigate the heterogeneity and dynamics of mouse embryonic stem cells (mESCs). While a number of reports have identified particular genes in ES cells that switch between 'high' and 'low' metastable expression states in culture, it remains unclear how levels of many of these regulators combine to form states in transcriptional space. Using a method called single molecule mRNA fluorescent in situ hybridization (smFISH), we quantitatively measure and fit distributions of core pluripotency regulators in single cells, identifying a wide range of variabilities between genes, but each explained by a simple model of bursty transcription. From this data, we also observed that strongly bimodal genes appear to be co-expressed, effectively limiting the occupancy of transcriptional space to two primary states across genes studied here. However, these states also appear punctuated by the conditional expression of the most highly variable genes, potentially defining smaller substates of pluripotency.
\r\n\r\nHaving defined the transcriptional states, we next asked what might control their stability or persistence. Surprisingly, we found that DNA methylation, a mark normally associated with irreversible developmental progression, was itself differentially regulated between these two primary states. Furthermore, both acute or chronic inhibition of DNA methyltransferase activity led to reduced heterogeneity among the population, suggesting that metastability can be modulated by this strong epigenetic mark.
\r\n\r\nFinally, because understanding the dynamics of state transitions is fundamental to a variety of biological problems, we sought to develop a high-throughput method for the identification of cellular trajectories without the need for cell-line engineering. We achieved this by combining cell-lineage information gathered from time-lapse microscopy with endpoint smFISH for measurements of final expression states. Applying a simple mathematical framework to these lineage-tree associated expression states enables the inference of dynamic transitions. We apply our novel approach in order to infer temporal sequences of events, quantitative switching rates, and network topology among a set of ESC states.
\r\n\r\nTaken together, we identify distinct expression states in ES cells, gain fundamental insight into how a strong epigenetic modifier enforces the stability of these states, and develop and apply a new method for the identification of cellular trajectories using scalable in situ readouts of cellular state.
" }, { "name": "Szablowski, Jerzy Olgierd", "degree": "PhD", "year": "2015", "title": "Biological Activity of Pyrrole-Imidazole Polyamides in vivo", "advisor": "Dervan, Peter B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05212015-030950854", "creators": [ { "name": { "family": "Szablowski", "given": "Jerzy Olgierd" }, "id": "Szablowski-Jerzy-Olgierd", "display_name": "Szablowski, Jerzy Olgierd" } ], "advisors": [ { "name": { "family": "Dervan", "given": "Peter B." }, "id": "Dervan-P-B", "orcid": "0000-0001-8852-7306", "role": "advisor", "display_name": "Dervan, Peter B." } ], "committee": [ { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "chair", "display_name": "Arnold, Frances Hamilton" }, { "name": { "family": "Dervan", "given": "Peter B." }, "id": "Dervan-P-B", "orcid": "0000-0001-8852-7306", "role": "member", "display_name": "Dervan, Peter B." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." } ], "option_major": [ "bioeng" ], "doi": "10.7907/Z9Q23X6D", "abstract": "This thesis focuses on biological activity of pyrrole-imidazole polyamides in vivo. The work presented includes experiments underlining sequence selectivity of these compounds in living cells and potential methods to improve it. A large fraction of this thesis is devoted to activity of Py-Im in murine models of cancer. We investigated the pharmacokinetics and biodistribution of two compounds \u2013 targeted to 5'-WGGWCW-3' and 5'-WTWCGW-3' sequences \u2013 and characterized their activity by measuring their effects on tumor growth, gene expression in vivo and in tissue culture, and their effects on physiology of tumors. The initial theoretical studies suggested that a large fraction of genomic sites are bound by Py-Im polyamides non-specifically and experimental data shows that the programmed binding sequence is not a sole determinant of the patterns of gene regulation. Despite the likely presence of non-specific effects of Py-Im polyamides in living cells, in vivo administration of Py-Im polyamides resulted in tolerable host toxicity and anti-tumor activity. Py-Im polyamide targeted to Estrogen Receptor Response Element showed downregulation of ER-driven gene expression in tumor cells, while the compound targeted to hypoxia response element reduced vascularization of tumors and their growth rate, induced apoptosis of cells in hypoxic areas and reduced expression of proangiogenic and prometastatic factors. Further studies, showed that polyamides distributed to many of the tested tissues and their FITC-conjugates showed nuclear uptake. The gene expression effects were also present in murine tissues, such as liver and kidneys, indicating a potential for use for Py-Im polyamides in non-cancerous diseases." }, { "name": "Trisnadi, Nathanie Alna", "degree": "PhD", "year": "2015", "title": "Regulation of Gastrulation Through Dynamic Patterning in the Drosophila Embryo", "advisor": "Stathopoulos, Angelike", "url": "https://resolver.caltech.edu/CaltechTHESIS:09082014-171122678", "creators": [ { "name": { "family": "Trisnadi", "given": "Nathanie Alna" }, "id": "Trisnadi-Nathanie-Alna", "display_name": "Trisnadi, Nathanie Alna" } ], "advisors": [ { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "advisor", "display_name": "Stathopoulos, Angelike" } ], "committee": [ { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "chair", "display_name": "Zinn, Kai George" }, { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "member", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "member", "display_name": "Chan, David C." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biology" ], "doi": "10.7907/Z9CJ8BGK", "abstract": "Gene patterning delineates an embryo into precise domains of differential gene expression. However, throughout gastrulation, these patterns are spatiotemporally dynamic due to the changing environment inherent in development and the contribution of multiple inputs. We investigated how the spatiotemporal dynamics of gene expressions influence two processes in the early Drosophila embryo: the establishment of the dorsal-ventral axis and the subsequent mesoderm migration. We found that genes are able to integrate many forms of regulation over space and time in order to refine their expression boundaries and guide gastrulation. Live imaging of the Dorsal transcription factor morphogen gradient revealed spatiotemporal dynamics that never reached steady state. Computational simulations correlated these changes with shifts in the boundaries of downstream target genes. For early mesoderm development, we conducted a screen to ectopically express proteins in specific domains to identify factors involved in migration. We showed that modulation of fibroblast growth factor (FGF) signaling switches between two proteoglycans to transition cells from migration to differentiation. In addition, multiple contributions regulate the complementary expression of cadherins, which is required to provide the proper balance of cell-cell interactions during mesoderm migration. We conclude that the changing environment of the embryo is an important factor during gastrulation and give examples of its impact in defining gene expression domains, supporting specificity of signaling pathways, and regulating adhesion during collective movements." }, { "name": "Trivedi, Vikas", "degree": "PhD", "year": "2015", "title": "From Molecules to Organs : Microscopy and Multi-Scale Nature of Development", "advisor": "Fraser, Scott E.; Pierce, Niles A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05292015-080950114", "creators": [ { "name": { "family": "Trivedi", "given": "Vikas" }, "id": "Trivedi-Vikas", "orcid": "0000-0003-0953-0553", "display_name": "Trivedi, Vikas" } ], "advisors": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "co-advisor", "display_name": "Fraser, Scott E." }, { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "co-advisor", "display_name": "Pierce, Niles A." } ], "committee": [ { "name": { "family": "Ravichandran", "given": "Guruswami" }, "id": "Ravichandran-G", "orcid": "0000-0002-2912-0001", "role": "chair", "display_name": "Ravichandran, Guruswami" }, { "name": { "family": "Gharib", "given": "Morteza" }, "id": "Gharib-M", "orcid": "0000-0003-0754-4193", "role": "member", "display_name": "Gharib, Morteza" }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "member", "display_name": "Pierce, Niles A." } ], "option_major": [ "bioeng" ], "doi": "10.7907/Z9SX6B59", "abstract": "Morphogenesis is a phenomenon of intricate balance and dynamic interplay between processes occurring at a wide range of scales (spatial, temporal and energetic). During development, a variety of physical mechanisms are employed by tissues to simultaneously pattern, move, and differentiate based on information exchange between constituent cells, perhaps more than at any other time during an organism's life. To fully understand such events, a combined theoretical and experimental framework is required to assist in deciphering the correlations at both structural and functional levels at scales that include the intracellular and tissue levels as well as organs and organ systems. Microscopy, especially diffraction-limited light microscopy, has emerged as a central tool to capture the spatio-temporal context of life processes. Imaging has the unique advantage of watching biological events as they unfold over time at single-cell resolution in the intact animal. In this work I present a range of problems in morphogenesis, each unique in its requirements for novel quantitative imaging both in terms of the technique and analysis. Understanding the molecular basis for a developmental process involves investigating how genes and their products- mRNA and proteins-function in the context of a cell. Structural information holds the key to insights into mechanisms and imaging fixed specimens paves the first step towards deciphering gene function. The work presented in this thesis starts with the demonstration that the fluorescent signal from the challenging environment of whole-mount imaging, obtained by in situ hybridization chain reaction (HCR), scales linearly with the number of copies of target mRNA to provide quantitative sub-cellular mapping of mRNA expression within intact vertebrate embryos. The work then progresses to address aspects of imaging live embryonic development in a number of species. While processes such as avian cartilage growth require high spatial resolution and lower time resolution, dynamic events during zebrafish somitogenesis require higher time resolution to capture the protein localization as the somites mature. The requirements on imaging are even more stringent in case of the embryonic zebrafish heart that beats with a frequency of ~ 2-2.5 Hz, thereby requiring very fast imaging techniques based on two-photon light sheet microscope to capture its dynamics. In each of the hitherto-mentioned cases, ranging from the level of molecules to organs, an imaging framework is developed, both in terms of technique and analysis to allow quantitative assessment of the process in vivo. Overall the work presented in this thesis combines new quantitative tools with novel microscopy for the precise understanding of processes in embryonic development. " }, { "name": "Wall, Teagan Rose", "degree": "PhD", "year": "2015", "title": "Effects of TI-299423 on Neuronal Nicotinic Acetylcholine Receptors", "advisor": "Lester, Henry A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03262015-100311493", "creators": [ { "name": { "family": "Wall", "given": "Teagan Rose" }, "id": "Wall-Teagan-Rose", "display_name": "Wall, Teagan Rose" } ], "advisors": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "advisor", "display_name": "Lester, Henry A." } ], "committee": [ { "name": { "family": "Camerer", "given": "Colin F." }, "id": "Camerer-C-F", "role": "chair", "display_name": "Camerer, Colin F." }, { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "role": "member", "display_name": "Adolphs, Ralph" }, { "name": { "family": "Dougherty", "given": "Dennis A." }, "id": "Dougherty-D-A", "role": "member", "display_name": "Dougherty, Dennis A." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "member", "display_name": "Lester, Henry A." } ], "option_major": [ "cns" ], "doi": "10.7907/Z9JD4TQ5", "abstract": "Nicotinic acetylcholine receptors (nAChRs) are pentameric, ligand-gated, cation channels found throughout the central and peripheral nervous system, whose endogenous ligand is acetylcholine, but which can also be acted upon by nicotine. The subunit compositions of nAChR determine their physiological and pharmacological properties, with different subunits expressed in different combinations or areas throughout the brain. The behavioral and physiological effects of nicotine are elicited by its agonistic and desensitizing actions selectively on neuronal nAChRs. The midbrain is of particular interest due to its population of nAChRs expressed on dopaminergic neurons, which are important for reward and reinforcement, and possibly contribute to nicotine dependence. The \u03b16-subunit is found on dopaminergic neurons but very few other regions of the brain, making it an interesting drug target. We assayed a novel nicotinic agonist, called TI-299423 or TC299, for its possible selectivity for \u03b16-containing nAChRs. Our goal was to isolate the role of \u03b16-containing nAChRs in nicotine reward and reinforcement, and provide insight into the search for more effective smoking cessation compounds. This was done using a variety of in vitro and behavioral assays, aimed dually at understanding TI-299423\u2019s exact mechanism of action and its downstream effects. Additionally, we looked at the effects of another compound, menthol, on nicotine reward. Understanding how reward is generated in the cholinergic system and how that is modulated by other compounds contributes to a better understand of our complex neural circuitry and provides insight for the future development of therapeutics." }, { "name": "Wannier, Timothy Milton", "degree": "PhD", "year": "2015", "title": "Computationally Guided Monomerization of Red Fluorescent Proteins of the Class Anthozoa", "advisor": "Mayo, Stephen L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01272015-110638252", "creators": [ { "name": { "family": "Wannier", "given": "Timothy Milton" }, "id": "Wannier-Timothy-Milton", "display_name": "Wannier, Timothy Milton" } ], "advisors": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "advisor", "display_name": "Mayo, Stephen L." } ], "committee": [ { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "chair", "display_name": "Pierce, Niles A." }, { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "member", "display_name": "Arnold, Frances Hamilton" }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." } ], "option_major": [ "molbiochem" ], "doi": "10.7907/Z9571908", "abstract": "Red fluorescent proteins (RFPs) have attracted significant engineering focus because of the promise of near infrared fluorescent proteins, whose light penetrates biological tissue, and which would allow imaging inside of vertebrate animals. The RFP landscape, which numbers ~200 members, is mostly populated by engineered variants of four native RFPs, leaving the vast majority of native RFP biodiversity untouched. This is largely due to the fact that native RFPs are obligate tetramers, limiting their usefulness as fusion proteins. Monomerization has imposed critical costs on these evolved tetramers, however, as it has invariably led to loss of brightness, and often to many other adverse effects on the fluorescent properties of the derived monomeric variants. Here we have attempted to understand why monomerization has taken such a large toll on Anthozoa class RFPs, and to outline a clear strategy for their monomerization. We begin with a structural study of the far-red fluorescence of AQ143, one of the furthest red emitting RFPs. We then try to separate the problem of stable and bright fluorescence from the design of a soluble monomeric \u03b2-barrel surface by engineering a hybrid protein (DsRmCh) with an oligomeric parent that had been previously monomerized, DsRed, and a pre-stabilized monomeric core from mCherry. This allows us to use computational design to successfully design a stable, soluble, fluorescent monomer. Next we took HcRed, which is a previously unmonomerized RFP that has far-red fluorescence (\u03bbemission = 633 nm) and attempted to monomerize it making use of lessons learned from DsRmCh. We engineered two monomeric proteins by pre-stabilizing HcRed\u2019s core, then monomerizing in stages, making use of computational design and directed evolution techniques such as error-prone mutagenesis and DNA shuffling. We call these proteins mGinger0.1 (\u03bbem = 637 nm / \u03a6 = 0.02) and mGinger0.2 (\u03bbem = 631 nm \u03a6 = 0.04). They are the furthest red first generation monomeric RFPs ever developed, are significantly thermostabilized, and add diversity to a small field of far-red monomeric FPs. We anticipate that the techniques we describe will be facilitate future RFP monomerization, and that further core optimization of the mGingers may allow significant improvements in brightness." }, { "name": "Webster, Alexandre", "degree": "PhD", "year": "2015", "title": "Mechanisms of Transposable Element Repression by Piwi Proteins in the piRNA Pathway of Drosophila Germ Cells", "advisor": "Aravin, Alexei A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05112015-230250866", "creators": [ { "name": { "family": "Webster", "given": "Alexandre" }, "id": "Webster-Alexandre", "orcid": "0000-0002-1416-5872", "display_name": "Webster, Alexandre" } ], "advisors": [ { "name": { "family": "Aravin", "given": "Alexei A." }, "id": "Aravin-A-A", "orcid": "0000-0002-6956-8257", "role": "advisor", "display_name": "Aravin, Alexei A." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Shan", "given": "Shu-ou" }, "id": "Shan-Shu-ou", "orcid": "0000-0002-6526-1733", "role": "member", "display_name": "Shan, Shu-ou" }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "orcid": "0000-0001-8723-8190", "role": "member", "display_name": "Baltimore, David L." }, { "name": { "family": "Aravin", "given": "Alexei A." }, "id": "Aravin-A-A", "orcid": "0000-0002-6956-8257", "role": "member", "display_name": "Aravin, Alexei A." } ], "option_major": [ "molbiochem" ], "doi": "10.7907/Z9WQ01RS", "abstract": "The ability to reproduce is a defining characteristic of all living organisms. During reproduction, the integrity of genetic material transferred from one generation to the next is of utmost importance. Organisms have diverse strategies to ensure the fidelity of genomic information inherited between generations of individuals. In sexually reproducing animals, the piRNA pathway is an RNA-interference (RNAi) mechanism that protects the genomes of germ cells from the replication of \u2018selfish\u2019 genetic sequences called transposable elements (TE). When left unabated, the replication of TE sequences can cause gene disruption, double-stranded DNA breaks, and germ cell death that results in sterility of the organism. In Drosophila, the piRNA pathway is divided into a cytoplasmic and nuclear branch that involves the functions of three Piwi-clade Argonaute proteins\u2014Piwi, Aubergine (Aub) and Argonaute-3 (Ago3)\u2014which bind piwi-interacting RNA (piRNA) to form the effector complexes that represses deleterious TE sequences.
\r\n\r\nThe work presented in this thesis examines the function and regulation of Piwi proteins in Drosophila germ cells. Chapter 1 presents an introduction to piRNA biogenesis and to the essential roles occupied by each Piwi protein in the repression of TE. We discuss the architecture and function of germ granules as the cellular compartments where much of the piRNA pathway operates. In Chapter 2, we present how Piwi in the nucleus co-transcriptionally targets genomic loci expressing TE sequences to direct the deposition of repressive chromatin marks. Chapter 3 examines the cytoplasmic function of the piRNA pathway, where we find that the protein Krimper coordinates Aub and Ago3 in the piRNA ping-pong pathway to adaptively target and destroy TE transcripts. Chapter 4 explores how interactions of Piwis with associated proteins are modulated by arginine methylation modifications. Lastly, in Chapter 5 I present evidence that the cytoplasmic branch of the piRNA pathway can potentially \u2018cross-talk\u2019 with the nuclear branch to transfer sequence information to better target and co-transcriptionally silence the genomic loci coding active TE sequences. Overall, the work presented in this thesis constitutes a part of the first steps in understanding the molecular mechanisms that protect germ cells from invasion by TE sequences.
" }, { "name": "Wu, Yunji", "degree": "PhD", "year": "2015", "title": "Structural Characterizations of the Dimeric Anti-HIV Antibody 2G12 and the HIV-2 Envelope Glycoprotein", "advisor": "Bjorkman, Pamela J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05212015-194407438", "creators": [ { "name": { "family": "Wu", "given": "Yunji" }, "id": "Wu-Yunji", "display_name": "Wu, Yunji" } ], "advisors": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "advisor", "display_name": "Bjorkman, Pamela J." } ], "committee": [ { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "chair", "display_name": "Chan, David C." }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "orcid": "0000-0001-8723-8190", "role": "member", "display_name": "Baltimore, David L." }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." } ], "option_major": [ "molbiochem" ], "doi": "10.7907/Z98050K6", "abstract": "More than thirty years after the discovery that Human Immunodeficiency Virus (HIV) was the causative agent of Acquired Immunodeficiency Syndrome (AIDS), the disease remains pandemic as long as no effective universal vaccine is found. Over 34 million individuals in the world are infected with the virus, and the vast majority of them have no access to the antiretroviral therapies that have largely reduced HIV to a chronic disease in the developed world. The first chapter of this thesis introduces the history of the virus. The key to the infectious mechanism of the virus lies in its envelope glycoprotein (Env), a trimeric spike on the viral surface that utilizes host T cell receptors for entry. Though HIV-1 Env is immunogenic, most infected patients do not mount an effective neutralizing antibody response against it. Broadly-neutralizing anti-Env antibodies (bNAbs) present in the serum of a minority of infected individuals are usually sufficient to prevent the progression to full blown AIDS. Thus, the molecular details of these bNAbs as well as the antibody-antigen interface are of prime interest for structural studies, as insight gained would contribute to the design of a more effective immunogen and potential vaccine candidate. The second chapter of this thesis describes the low-resolution crystal structure of one such antibody, 2G12 dimer, which targets a high mannose epitope on the surface of Env. Patients infected with HIV-2, a related virus with ~35% sequence identity in the Env region, can generally mount a robust antibody response sufficient for viral control for reasons still unknown. The final two chapters of this thesis focus on the first reported structural studies of HIV-2 Env, the molecular details of which may inform HIV-1 therapy and immunogen design. " }, { "name": "Yong, John", "degree": "PhD", "year": "2015", "title": "Dynamics and Heterogeneity of Gene Expression and Epigenetic Regulation at the Single-Cell Level", "advisor": "Elowitz, Michael B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04292015-153123844", "creators": [ { "name": { "family": "Yong", "given": "John" }, "id": "Yong-John", "orcid": "0000-0002-4914-2259", "display_name": "Yong, John" } ], "advisors": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "advisor", "display_name": "Elowitz, Michael B." } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "chair", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "role": "member", "display_name": "Guttman, Mitchell" }, { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "role": "member", "display_name": "Siapas, Athanassios G." }, { "name": { "family": "Plath", "given": "Kathrin" }, "id": "Plath-K", "role": "member", "display_name": "Plath, Kathrin" } ], "option_major": [ "biology" ], "doi": "10.7907/Z9ZC80VX", "abstract": "The ability of cells to establish and remember their gene expression states is a cornerstone of multicellular life. This thesis explores how gene expression states are regulated dynamically, and how these regulations differ in individual cells even under the same conditions. These properties underlie cellular state decisions and often determine the balance between different cell types in a multicellular system, but are typically inaccessible to conventional techniques that rely on static snapshots and population averaging. We address these issues in two separate contexts, one natural and one synthetic, using time-lapse imaging and other single-cell techniques.
\r\n\t\t\r\nIn the first context, we use embryonic stem cells (ES), which were shown to exist in a mixed population of at least two cellular states with distinct differentiation propensities, as a model to study natural dynamics of cellular states. These cells display rare, stochastic, and spontaneous transitions between the two states, as well as more frequent fluctuations in gene expression levels within each state. Our system enables us to further investigate how these dynamics are modulated under a cell signaling environment that enhances pluripotency, and the role DNA methylation plays in maintaining these states.
\r\n\t\t\t\r\nIn the second context, we investigate how chromatin regulators (CRs), part of a complex system that enables cells to modulate gene expression and epigenetic memory, operate dynamically in individual cells. We build a synthetic platform to measure the isolated effect of recruitment and de-recruitment of four individual CRs. In contrast to conventional transcription factor control, all CRs tested regulate gene expression in all-or-none events, controlling the probability of stochastic transitions between fully active and silent states rather than the strength of gene expression. The qualitative and quantitative responses of a cell population are determined by the set of event rates associated with each CR, as well as the duration of CR recruitment. These results provide a framework for understanding and engineering chromatin-based cellular states and their dynamics.\r\n
" }, { "name": "Abelin, Anna Cecilia Therese", "degree": "PhD", "year": "2014", "title": "A Ratiometric-Based Measure of Gene Co-Expression", "advisor": "Wold, Barbara J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05072014-160838588", "creators": [ { "name": { "family": "Abelin", "given": "Anna Cecilia Therese" }, "id": "Abelin-Anna-Cecilia-Therese", "display_name": "Abelin, Anna Cecilia Therese" } ], "advisors": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "advisor", "display_name": "Wold, Barbara J." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Guttman", "given": "Mitchell" }, "id": "Guttman-M", "role": "member", "display_name": "Guttman, Mitchell" } ], "option_major": [ "biology" ], "doi": "10.7907/Z9JH3J5B", "abstract": "Current measures of global gene expression analyses, such as correlation and mutual information-based approaches, largely depend on the degree of association between mRNA levels and to a lesser extent on variability. I develop and implement a new approach, called the Ratiometric method, which is based on the coefficient of variation of the expression ratio of two genes, relying more on variation than previous methods. The advantage of such modus operandi is the ability to detect possible gene pair interactions regardless of the degree of expression dispersion across the sample group. Gene pairs with low expression dispersion, i.e., their absolute expressions remain constant across the sample group, are systematically missed by correlation and mutual information analyses. The superiority of the Ratiometric method in finding these gene pair interactions is demonstrated in a data set of RNA-seq B-cell samples from the 1000 Genomes Project Consortium. The Ratiometric method renders a more comprehensive recovery of KEGG pathways and GO-terms. " }, { "name": "Basalova Buchman, Anna", "degree": "PhD", "year": "2014", "title": "Engineered Underdominance as a Method of Insect Population Replacement and Reproductive Isolation", "advisor": "Hay, Bruce A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05262014-104457839", "creators": [ { "name": { "family": "Basalova Buchman", "given": "Anna" }, "id": "Basalova-Buchman-Anna", "display_name": "Basalova Buchman, Anna" } ], "advisors": [ { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "advisor", "display_name": "Hay, Bruce A." } ], "committee": [ { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "chair", "display_name": "Zinn, Kai George" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Aravin", "given": "Alexei A." }, "id": "Aravin-A-A", "orcid": "0000-0002-6956-8257", "role": "member", "display_name": "Aravin, Alexei A." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "member", "display_name": "Hay, Bruce A." } ], "option_major": [ "biology" ], "doi": "10.7907/3YMP-DB79", "abstract": "Insect vector-borne diseases, such as malaria and dengue fever (both spread by mosquito vectors), continue to significantly impact health worldwide, despite the efforts put forth to eradicate them. Suppression strategies utilizing genetically modified disease-refractory insects have surfaced as an attractive means of disease control, and progress has been made on engineering disease-resistant insect vectors. However, laboratory-engineered disease refractory genes would probably not spread in the wild, and would most likely need to be linked to a gene drive system in order to proliferate in native insect populations. Underdominant systems like translocations and engineered underdominance have been proposed as potential mechanisms for spreading disease refractory genes. Not only do these threshold-dependent systems have certain advantages over other potential gene drive mechanisms, such as localization of gene drive and removability, extreme engineered underdominance can also be used to bring about reproductive isolation, which may be of interest in controlling the spread of GMO crops. Proof-of-principle establishment of such drive mechanisms in a well-understood and studied insect, such as Drosophila melanogaster, is essential before more applied systems can be developed for the less characterized vector species of interest, such as mosquitoes. This work details the development of several distinct types of engineered underdominance and of translocations in Drosophila, including ones capable of bringing about reproductive isolation and population replacement, as a proof of concept study that can inform efforts to construct such systems in insect disease vectors." }, { "name": "Chitsaz, Mohsen", "degree": "PhD", "year": "2014", "title": "Protein Structure Refinement Algorithms", "advisor": "Mayo, Stephen L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06072014-074257676", "creators": [ { "name": { "family": "Chitsaz", "given": "Mohsen" }, "id": "Chitsaz-Mohsen", "orcid": "0000-0001-6016-7687", "display_name": "Chitsaz, Mohsen" } ], "advisors": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "advisor", "display_name": "Mayo, Stephen L." } ], "committee": [ { "name": { "family": "Goddard", "given": "William A., III" }, "id": "Goddard-W-A-III", "orcid": "0000-0003-0097-5716", "role": "chair", "display_name": "Goddard, William A., III" }, { "name": { "family": "Bjorkman", "given": "Pamela Jane" }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "co-chair", "display_name": "Bjorkman, Pamela Jane" }, { "name": { "family": "Umans", "given": "Christopher M." }, "id": "Umans-C-M", "orcid": "0000-0002-6390-9401", "role": "member", "display_name": "Umans, Christopher M." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." } ], "option_major": [ "biochem" ], "doi": "10.7907/7731-QM74", "abstract": "Protein structure prediction has remained a major challenge in structural biology for more than half a century. Accelerated and cost efficient sequencing technologies have allowed researchers to sequence new organisms and discover new protein sequences. Novel protein structure prediction technologies will allow researchers to study the structure of proteins and to determine their roles in the underlying biology processes and develop novel therapeutics.
\r\n\r\nDifficulty of the problem stems from two folds: (a) describing the energy landscape that corresponds to the protein structure, commonly referred to as force field problem; and (b) sampling of the energy landscape, trying to find the lowest energy configuration that is hypothesized to be the native state of the structure in solution. The two problems are interweaved and they have to be solved simultaneously. This thesis is composed of three major contributions. In the first chapter we describe a novel high-resolution protein structure refinement algorithm called GRID. In the second chapter we present REMCGRID, an algorithm for generation of low energy decoy sets. In the third chapter, we present a machine learning approach to ranking decoys by incorporating coarse-grain features of protein structures.
\r\n" }, { "name": "Chiu, Cindy Nicole", "degree": "PhD", "year": "2014", "title": "A Perfect Day for Zebrafish: Neuromodulation of Sleep in a Diurnal Vertebrate", "advisor": "Prober, David A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06102014-154051538", "creators": [ { "name": { "family": "Chiu", "given": "Cindy Nicole" }, "id": "Chiu-Cindy-Nicole", "display_name": "Chiu, Cindy Nicole" } ], "advisors": [ { "name": { "family": "Prober", "given": "David A." }, "role": "advisor", "display_name": "Prober, David A." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "role": "member", "display_name": "Prober, David A." }, { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "role": "member", "display_name": "Siapas, Athanassios G." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "neurobio" ], "doi": "10.7907/Z9FN1452", "abstract": "Every day, we shift among various states of sleep and arousal to meet the many demands of our bodies and environment. A central puzzle in neurobiology is how the brain controls these behavioral states, which are essential to an animal's well-being and survival. Mammalian models have predominated sleep and arousal research, although in the past decade, invertebrate models have made significant contributions to our understanding of the genetic underpinnings of behavioral states. More recently, the zebrafish (Danio rerio), a diurnal vertebrate, has emerged as a promising model system for sleep and arousal research.
\r\n\r\nIn this thesis, I describe two studies on sleep/arousal pathways that I conducted using zebrafish, and I discuss how the findings can be combined in future projects to advance our understanding of vertebrate sleep/arousal pathways. In the first study, I discovered a neuropeptide that regulates zebrafish sleep and arousal as a result of a large-scale effort to identify molecules that regulate behavioral states. Taking advantage of facile zebrafish genetics, I constructed mutants for the three known receptors of this peptide and identified the one receptor that exclusively mediates the observed behavioral effects. I further show that the peptide exerts its behavioral effects independently of signaling at a key module of a neuroendocrine signaling pathway. This finding contradicts the hypothesis put forth in mammalian systems that the peptide acts through the classical neuroendocrine pathway; our data further generate new testable hypotheses for determining the central nervous system or alternative neuroendocrine pathways involved.
\r\n\r\nSecond, I will present the development of a chemigenetic method to non-invasively manipulate neurons in the behaving zebrafish. I validated this technique by expressing and inducing the chemigenetic tool in a restricted population of sleep-regulating neurons in the zebrafish. As predicted by established models of this vertebrate sleep regulator, chemigenetic activation of these neurons induced hyperactivity, whereas chemigenetic ablation of these neurons induced increased sleep behavior. Given that light is a potent modulator of behavior in zebrafish, our proof-of-principle data provide a springboard for future studies of sleep/arousal and other light-dependent behaviors to interrogate genetically-defined populations of neurons independently of optogenetic tools.
" }, { "name": "Chow, Elly Suk Hen", "degree": "PhD", "year": "2014", "title": "The C. elegans ALA Neuron: Its Transcriptions and Roles in Inducing Sleep", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06052014-104149263", "creators": [ { "name": { "family": "Chow", "given": "Elly Suk Hen" }, "id": "Chow-Elly-Suk-Hen", "display_name": "Chow, Elly Suk Hen" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "role": "chair", "display_name": "Prober, David A." }, { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "member", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/BFB7-Y645", "abstract": "A long-standing yet to be accomplished task in understanding behavior is to dissect the function of each gene involved in the development and function of a neuron. The C. elegans ALA neuron was chosen in this study for its known function in sleep, an ancient but less understood animal behavior. Single-cell transcriptome profiling identified 8,133 protein-coding genes in the ALA neuron, of which 57 are neuropeptide-coding genes. The most enriched genes are also neuropeptides. In combination with gain-of-function and loss-of-function assays, here I showed that the ALA-enriched FMRFamide neuropeptides, FLP-7, FLP-13, and FLP-24, are sufficient and necessary for inducing C. elegans sleep. These neuropeptides act as neuromodulators through GPCRs, NPR-7, and NPR-22. Further investigation in zebrafish indicates that FMRFamide neuropeptides are sleep-promoting molecules in animals. To correlate the behavioral outputs with genomic context, I constructed a gene regulatory network of the relevant genes controlling C. elegans sleep behavior through EGFR signaling in the ALA neuron. First, I identified an ALA cell-specific motif to conduct a genome-wide search for possible ALA-expressed genes. I then filtered out non ALA-expressed genes by comparing the motif-search genes with ALA transcriptomes from single-cell profiling. In corroborating with ChIP-seq data from modENCODE, I sorted out direct interaction of ALA-expressed transcription factors and differentiation genes in the EGFR sleep regulation pathway. This approach provides a network reference for the molecular regulation of C. elegans sleep behavior, and serves as an entry point for the understanding of functional genomics in animal behaviors. \r\n" }, { "name": "Hamdouche, Samy", "degree": "PhD", "year": "2014", "title": "Engineered Antibody and Monobody Domains with T Cell Receptor-Like Selectivity for Tumor Associated Peptide-MHC Antigens", "advisor": "Mayo, Stephen L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06062014-175608943", "creators": [ { "name": { "family": "Hamdouche", "given": "Samy" }, "id": "Hamdouche-Samy", "display_name": "Hamdouche, Samy" } ], "advisors": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "advisor", "display_name": "Mayo, Stephen L." } ], "committee": [ { "name": { "family": "Miller", "given": "Thomas F." }, "id": "Miller-T-F", "orcid": "0000-0002-1882-5380", "role": "chair", "display_name": "Miller, Thomas F." }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "orcid": "0000-0001-8723-8190", "role": "member", "display_name": "Baltimore, David L." }, { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "member", "display_name": "Pierce, Niles A." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." } ], "option_major": [ "biochem" ], "doi": "10.7907/Z9WH2N02", "abstract": "Monoclonal antibody (mAb)-based therapeutics have established themselves as meaningful components of the treatment paradigm for a variety of tumors. However, since the approval of rituximab in 1997 as the first mAb-based therapy for cancer, there has been a paucity of novel, validated cancer targets for therapeutic intervention by mAbs. In effect, numerous challenges lie in the discovery of suitable extracellular or transmembrane antigens that permit the differentiation of tumor from healthy tissue. The adaptive immune system, though, mediates recognition of foreign antigens derived from the intracellular proteome by T cell receptor (TCR) binding to peptide-loaded major histocompatibility complex (pMHC) molecules. Because cancer is associated with large-scale alterations in the genome, there are a vast number of novel epitopes presented to the adaptive immune system. Although natural TCRs have exquisite functionality in distinguishing these foreign epitopes, and several tumor-reactive TCRs have, in fact, been characterized, the molecules themselves are poorly developable as therapeutic candidates. Thus, in order to enable TCR-like binding of a broader class of protein agents, this study explores the transfer of TCR binding domains to other mAb-based scaffolds, including the fibronectin-derived Fn3 and the IgG-derived 4D5 scaffolds. By using a combination of rational design and directed evolution to guide binding domain transfer, evidence for TCR-like binding was demonstrated for several engineered molecules. In addition to conferring binding functionality, the grafted TCR domains had a deleterious effect on the biophysical properties of these inherently robust protein scaffolds. Thus, this work provides novel insight into the objective of developing mAb-based agents with TCR-like binding specificity for pMHC antigens, informing future efforts to target the abundance of intracellular tumor epitopes." }, { "name": "Inagaki, Hidehiko K.", "degree": "PhD", "year": "2014", "title": "Neuronal Mechanism of State Control in Drosophila melanogaster", "advisor": "Anderson, David J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02112014-133121063", "creators": [ { "name": { "family": "Inagaki", "given": "Hidehiko K." }, "id": "Inagaki-Hidehiko-K", "display_name": "Inagaki, Hidehiko K." } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "advisor", "display_name": "Anderson, David J." } ], "committee": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "chair", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "role": "member", "display_name": "Prober, David A." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." } ], "option_major": [ "neurobio" ], "doi": "10.7907/MPZX-TN59", "abstract": "The changes in internal states, such as fear, hunger and sleep affect behavioral responses in animals. In most of the cases, these state-dependent influences are \u201cpleiotropic\u201d: one state affects multiple sensory modalities and behaviors; \u201cscalable\u201d: the strengths and choices of such modulations differ depending on the imminence of demands; and \u201cpersistent\u201d: once the state is switched on the effects last even after the internal demands are off. These prominent features of state-control enable animals to adjust their behavioral responses depending on their internal demands. Here, we studied the neuronal mechanisms of state-controls by investigating energy-deprived state (hunger state) and social-deprived state of fruit flies, Drosophila melanogaster, as prototypic models. To approach these questions, we developed two novel methods: a genetically based method to map sites of neuromodulation in the brain and optogenetic tools in Drosophila.
\r\n\r\nThese methods, and genetic perturbations, reveal that the effect of hunger to alter behavioral sensitivity to gustatory cues is mediate by two distinct neuromodulatory pathways. The neuropeptide F (NPF) \u2013 dopamine (DA) pathway increases sugar sensitivity under mild starvation, while the adipokinetic hormone (AKH)- short neuropeptide F (sNPF) pathway decreases bitter sensitivity under severe starvation. These two pathways are recruited under different levels of energy demands without any cross interaction. Effects of both of the pathways are mediated by modulation of the gustatory sensory neurons, which reinforce the concept that sensory neurons constitute an important locus for state-dependent control of behaviors. Our data suggests that multiple independent neuromodulatory pathways are underlying pleiotropic and scalable effects of the hunger state.
\r\n\r\nIn addition, using optogenetic tool, we show that the neural control of male courtship song can be separated into probabilistic/biasing, and deterministic/command-like components. The former, but not the latter, neurons are subject to functional modulation by social experience, supporting the idea that they constitute a locus of state-dependent influence. Interestingly, moreover, brief activation of the former, but not the latter, neurons trigger persistent behavioral response for more than 10 min. Altogether, these findings and new tools described in this dissertation offer new entry points for future researchers to understand the neuronal mechanism of state control.
\r\n" }, { "name": "Jiang, Siduo (Stone)", "degree": "Senior Thesis", "year": "2014", "title": "Structure-Guided Design and Biophysical Characterization of Novel Anti-HIV Reagents", "advisor": "Bjorkman, Pamela J.; Galimidi, Rachel P.; West, Anthony P.; Sievers, Stuart A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06172014-114402762", "creators": [ { "name": { "family": "Jiang", "given": "Siduo (Stone)" }, "id": "Jiang-Siduo-Stone", "orcid": "0000-0002-2143-4030", "display_name": "Jiang, Siduo (Stone)" } ], "advisors": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "advisor", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Galimidi", "given": "Rachel P." }, "id": "Galimidi-R-P", "role": "co-advisor", "display_name": "Galimidi, Rachel P." }, { "name": { "family": "West", "given": "Anthony P." }, "id": "West-A-P", "role": "co-advisor", "display_name": "West, Anthony P." }, { "name": { "family": "Sievers", "given": "Stuart A." }, "id": "Sievers-S-A", "role": "co-advisor", "display_name": "Sievers, Stuart A." } ], "committee": [ { "name": { "family": "None", "given": "None" }, "display_name": "None, None" } ], "option_major": [ "biology", "chemistry" ], "doi": "10.7907/SP19-9R34", "abstract": "Despite over 30 years of effort, an HIV-1 vaccine that elicits protective antibodies still does not exist. Recent clinical studies have identified that during natural infection about 20% of the population is capable of mounting a potent and protective antibody response. Closer inspection of these individuals reveal that a subset of these antibodies, recently termed potent VRC01-like (PVL), derive exclusively from a single human germline heavy chain gene. Induced clonal expansion of the B cell encoding this gene is the first step through which PVL antibodies may be elicited. Unfortunately, naturally occurring HIV gp120s fail to bind to this germline, and as a result cannot be used as the initial prime for a vaccine regimen. We have determined the crystal structure of an important germline antibody that is a promising target for vaccine design efforts, and have set out to engineer a more likely candidate using computationally-guided rational design.
\r\n\r\nIn addition to prevention efforts on the side of vaccine design, recently characterized broadly neutralizing anti-HIV antibodies have excellent potential for use in gene therapy and passive immunotherapy. The separation distance between functional Fabs on an antibody is important due to the sparse distribution of envelop spikes on HIV compared to other viruses. We set out to build and characterize novel antibody architectures by incorporating structured linkers into the hinge region of an anti-HIV antibody b12. The goal was to observe whether these linkers increased the arm-span of the IgG dimer. When incorporated, flexible Gly4Ser repeats did not result in detectable extensions of the IgG antigen binding domains, by contrast to linkers including more rigid domains such as \u03b22-microglobulin, Zn-\u03b12-glycoprotein, and tetratricopeptide repeats (TPRs). This study adds an additional set of linkers with varying lengths and rigidities to the available linker repertoire, which may be useful for the modification and construction of antibodies and other fusion proteins.
\r\n" }, { "name": "Khosravi, Arya", "degree": "PhD", "year": "2014", "title": "Gut Microbiota Promote Hematopoiesis to Control Bacterial Infection", "advisor": "Mazmanian, Sarkis K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04242014-100521885", "creators": [ { "name": { "family": "Khosravi", "given": "Arya" }, "id": "Khosravi-Arya", "display_name": "Khosravi, Arya" } ], "advisors": [ { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "advisor", "display_name": "Mazmanian, Sarkis K." } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "chair", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "member", "display_name": "Baltimore, David L." }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "member", "display_name": "Mazmanian, Sarkis K." } ], "option_major": [ "biology" ], "doi": "10.7907/XM07-4X53", "abstract": "The commensal microbiota impacts specific immune cell populations and their functions at peripheral sites, such as gut mucosal tissues. However, it remains unknown whether gut microbiota control immunity through regulation of hematopoiesis at primary immune sites. We reveal that germ-free mice display reduced proportions and differentiation potential of specific myeloid cell progenitors of both yolk sac and bone marrow origin. Homeostatic innate immune defects may lead to impaired early responses to pathogens. Indeed, following systemic infection with Listeria monocytogenes, germ-free and oral antibiotic-treated mice display increased pathogen burden and acute death. Recolonization of germ-free mice with a complex microbiota restores defects in myelopoiesis and resistance to Listeria. These findings reveal that gut bacteria direct innate immune cell development via promoting hematopoiesis, contributing to our appreciation of the deep evolutionary connection between mammals and their microbiota. " }, { "name": "Kirilusha, Anthony George", "degree": "PhD", "year": "2014", "title": "Transcription Factor Occupancy in Differentiating Skeletal Muscle", "advisor": "Wold, Barbara J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06112014-114426786", "creators": [ { "name": { "family": "Kirilusha", "given": "Anthony George" }, "id": "Kirilusha-Anthony-George", "display_name": "Kirilusha, Anthony George" } ], "advisors": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "advisor", "display_name": "Wold, Barbara J." } ], "committee": [ { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "chair", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Mjolsness", "given": "Eric D." }, "id": "Mjolsness-E-D", "role": "member", "display_name": "Mjolsness, Eric D." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." } ], "option_major": [ "biology" ], "doi": "10.7907/4Q24-CJ96", "abstract": "With recent advances in high-throughput sequencing, mapping of genome-wide transcription factor occupancy has become feasible. To advance the understanding of skeletal muscle differentiation specifically and transcriptional regulation in general, I determined the genome-wide occupancy map for myogenin in differentiating C2C12 myocyte cells. I then analyzed the myogenin map for underlying sequence content and the association between occupied elements and expression trajectories of adjacent genes. Having determined that myogenin primarily associates with expressed genes, I performed a similar analysis on occupancy maps of other transcription factors active during skeletal muscle differentiation, including an extensive analysis of co-occupancy. This analysis provided strong motif evidence for protein-protein interactions as the primary driving force in the formation of Myogenin / Mef2 and MyoD / AP-1 complexes at jointly-occupied sites. Finally, factor occupancy analysis was extended to include bHLH transcription factors in tissues other than skeletal muscle. The cross-tissue analysis led to the emergence of a motif structure used by bHLH TFs to encode either tissue-specific or \"general\" (public) access in a variety of lineages." }, { "name": "Kym, Eugene Yongshik (Gene)", "degree": "PhD", "year": "2014", "title": "Engineered Discoidin Domain from Factor VIII Binds \u03b1v\u03b23 Integrin with Antibody-like Affinity", "advisor": "Mayo, Stephen L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05302014-171245991", "creators": [ { "name": { "family": "Kym", "given": "Eugene Yongshik (Gene)" }, "id": "Kym-Eugene-Yongshik-Gene", "display_name": "Kym, Eugene Yongshik (Gene)" } ], "advisors": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "advisor", "display_name": "Mayo, Stephen L." } ], "committee": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "orcid": "0000-0002-3671-9354", "role": "chair", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "member", "display_name": "Chan, David C." }, { "name": { "family": "Tirrell", "given": "David A." }, "id": "Tirrell-D-A", "orcid": "0000-0003-3175-4596", "role": "member", "display_name": "Tirrell, David A." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." } ], "option_major": [ "biology" ], "doi": "10.7907/PTZH-PB29", "abstract": "Alternative scaffolds are non-antibody proteins that can be engineered to bind new targets. They have found useful niches in the therapeutic space due to their smaller size and the ease with which they can be engineered to be bispecific. We sought a new scaffold that could be used for therapeutic ends and chose the C2 discoidin domain of factor VIII, which is well studied and of human origin. Using yeast surface display, we engineered the C2 domain to bind to \u03b1v\u03b23 integrin with a 16 nM affinity while retaining its thermal stability and monomeric nature. We obtained a crystal structure of the engineered domain at 2.1 A\u030a resolution. We have christened this discoidin domain alternative scaffold the \u201cdiscobody.\u201d" }, { "name": "Lakhanpal, Amit", "degree": "PhD", "year": "2014", "title": "Experimental and Theoretical Studies of Notch Signaling-Mediated Spatial Pattern", "advisor": "Elowitz, Michael B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10312013-102814054", "creators": [ { "name": { "family": "Lakhanpal", "given": "Amit" }, "id": "Lakhanpal-Amit", "display_name": "Lakhanpal, Amit" } ], "advisors": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "advisor", "display_name": "Elowitz, Michael B." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." } ], "option_major": [ "biology" ], "doi": "10.7907/RGPT-RS80", "abstract": "Notch signaling acts in many diverse developmental spatial patterning processes. To better understand why this particular pathway is employed where it is and how downstream feedbacks interact with the signaling system to drive patterning, we have pursued three aims: (i) to quantitatively measure the Notch system's signal input/output (I/O) relationship in cell culture, (ii) to use the quantitative I/O relationship to computationally predict patterning outcomes of downstream feedbacks, and (iii) to reconstitute a Notch-mediated lateral induction feedback (in which Notch signaling upregulates the expression of Delta) in cell culture. The quantitative Notch I/O relationship revealed that in addition to the trans-activation between Notch and Delta on neighboring cells there is also a strong, mutual cis-inactivation between Notch and Delta on the same cell. This feature tends to amplify small differences between cells. Incorporating our improved understanding of the signaling system into simulations of different types of downstream feedbacks and boundary conditions lent us several insights into their function. The Notch system converts a shallow gradient of Delta expression into a sharp band of Notch signaling without any sort of feedback at all, in a system motivated by the Drosophila wing vein. It also improves the robustness of lateral inhibition patterning, where signal downregulates ligand expression, by removing the requirement for explicit cooperativity in the feedback and permitting an exceptionally simple mechanism for the pattern. When coupled to a downstream lateral induction feedback, the Notch system supports the propagation of a signaling front across a tissue to convert a large area from one state to another with only a local source of initial stimulation. It is also capable of converting a slowly-varying gradient in parameters into a sharp delineation between high- and low-ligand populations of cells, a pattern reminiscent of smooth muscle specification around artery walls. Finally, by implementing a version of the lateral induction feedback architecture modified with the addition of an autoregulatory positive feedback loop, we were able to generate cells that produce enough cis ligand when stimulated by trans ligand to themselves transmit signal to neighboring cells, which is the hallmark of lateral induction." }, { "name": "Lee, Toni Marie", "degree": "PhD", "year": "2014", "title": "Computationally-Guided Thermostabilization of the Primary Endoglucanase from Hypocrea jerorina for Cellulosic Biofuel Production", "advisor": "Mayo, Stephen L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:11172013-001549340", "creators": [ { "name": { "family": "Lee", "given": "Toni Marie" }, "id": "Lee-Toni-Marie", "display_name": "Lee, Toni Marie" } ], "advisors": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "advisor", "display_name": "Mayo, Stephen L." } ], "committee": [ { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "chair", "display_name": "Rees, Douglas C." }, { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "member", "display_name": "Arnold, Frances Hamilton" }, { "name": { "family": "Tirrell", "given": "David A." }, "id": "Tirrell-D-A", "orcid": "0000-0003-3175-4596", "role": "member", "display_name": "Tirrell, David A." }, { "name": { "family": "Shan", "given": "Shu-ou" }, "id": "Shan-Shu-ou", "orcid": "0000-0002-6526-1733", "role": "member", "display_name": "Shan, Shu-ou" }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." } ], "option_major": [ "biochem" ], "doi": "10.7907/Z9S180G0", "abstract": "The creation of thermostable enzymes has wide-ranging applications in industrial, scientific, and pharmaceutical settings. As various stabilization techniques exist, it is often unclear how to best proceed. To this end, we have redesigned Cel5A (HjCel5A) from Hypocrea jecorina (anamorph Trichoderma reesei) to comparatively evaluate several significantly divergent stabilization methods: 1) consensus design, 2) core repacking, 3) helix dipole stabilization, 4) FoldX \u0394\u0394G approximations, 5) Triad \u0394\u0394G approximations, and 6) entropy reduction through backbone stabilization. As several of these techniques require structural data, we initially solved the first crystal structure of HjCel5A to 2.05 \u00c5. Results from the stabilization experiments demonstrate that consensus design works best at accurately predicting highly stabilizing and active mutations. FoldX and helix dipole stabilization, however, also performed well. Both methods rely on structural data and can reveal non-conserved, structure-dependent mutations with high fidelity. HjCel5A is a prime target for stabilization. Capable of cleaving cellulose strands from agricultural waste into fermentable sugars, this protein functions as the primary endoglucanase in an organism commonly used in the sustainable biofuels industry. Creating a long-lived, highly active thermostable HjCel5A would allow cellulose hydrolysis to proceed more efficiently, lowering production expenses. We employed information gleaned during the survey of stabilization techniques to generate HjCel5A variants demonstrating a 12-15 \u00b0C increase in the temperature at which 50% of the total activity persists, an 11-14 \u00b0C increase in optimal operating temperature, and a 60% increase over the maximal amount of hydrolysis achievable using the wild type enzyme. We anticipate that our comparative analysis of stabilization methods will prove useful in future thermostabilization experiments.
" }, { "name": "Loson, Oliver Calvin", "degree": "PhD", "year": "2014", "title": "Regulation of Mitochondrial Division by the Drp1 Receptors", "advisor": "Chan, David C.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05072014-160207088", "creators": [ { "name": { "family": "Loson", "given": "Oliver Calvin" }, "id": "Loson-Oliver-Calvin", "display_name": "Loson, Oliver Calvin" } ], "advisors": [ { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "advisor", "display_name": "Chan, David C." } ], "committee": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "chair", "display_name": "Lester, Henry A." }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Shan", "given": "Shu-ou" }, "id": "Shan-Shu-ou", "role": "member", "display_name": "Shan, Shu-ou" }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "member", "display_name": "Chan, David C." } ], "option_major": [ "bioch", "biology" ], "doi": "10.7907/J23G-KQ18", "abstract": "Mitochondria can remodel their membranes by fusing or dividing. These processes are required for the proper development and viability of multicellular organisms. At the cellular level, fusion is important for mitochondrial Ca2+ homeostasis, mitochondrial DNA maintenance, mitochondrial membrane potential, and respiration. Mitochondrial division, which is better known as fission, is important for apoptosis, mitophagy, and for the proper allocation of mitochondria to daughter cells during cellular division.
\r\n\r\nThe functions of proteins involved in fission have been best characterized in the yeast model organism Sarccharomyces cerevisiae. Mitochondrial fission in mammals has some similarities. In both systems, a cytosolic dynamin-like protein, called Dnm1 in yeast and Drp1 in mammals, must be recruited to the mitochondrial surface and polymerized to promote membrane division. Recruitment of yeast Dnm1 requires only one mitochondrial outer membrane protein, named Fis1. Fis1 is conserved in mammals, but its importance for Drp1 recruitment is minor. In mammals, three other receptor proteins\u2014Mff, MiD49, and MiD51\u2014play a major role in recruiting Drp1 to mitochondria. Why mammals require three additional receptors, and whether they function together or separately, are fundamental questions for understanding the mechanism of mitochondrial fission in mammals.
\r\n\r\nWe have determined that Mff, MiD49, or MiD51 can function independently of one another to recruit Drp1 to mitochondria. Fis1 plays a minor role in Drp1 recruitment, suggesting that the emergence of these additional receptors has replaced the system used by yeast. Additionally, we found that Fis1/Mff and the MiDs regulate Drp1 activity differentially. Fis1 and Mff promote constitutive mitochondrial fission, whereas the MiDs activate recruited Drp1 only during loss of respiration.
\r\n\r\nTo better understand the function of the MiDs, we have determined the atomic structure of the cytoplasmic domain of MiD51, and performed a structure-function analysis of MiD49 based on its homology to MiD51. MiD51 adopts a nucleotidyl transferase fold, and binds ADP as a co-factor that is essential for its function. Both MiDs contain a loop segment that is not present in other nucleotidyl transferase proteins, and this loop is used to interact with Drp1 and to recruit it to mitochondria.
\r\n" }, { "name": "Lovely, Geoffrey A.", "degree": "PhD", "year": "2014", "title": "Biophysics of V(D)J Recombination and Genome Packaging: In Singulo Studies on RAG, HMGB1, and TFAM", "advisor": "Baltimore, David L.; Phillips, Robert B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06072014-140700155", "creators": [ { "name": { "family": "Lovely", "given": "Geoffrey A." }, "id": "Lovely-Geoffrey-A", "display_name": "Lovely, Geoffrey A." } ], "advisors": [ { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "orcid": "0000-0001-8723-8190", "role": "co-advisor", "display_name": "Baltimore, David L." }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "co-advisor", "display_name": "Phillips, Robert B." } ], "committee": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "chair", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Cai", "given": "Long" }, "id": "Cai-Long", "orcid": "0000-0002-7154-5361", "role": "member", "display_name": "Cai, Long" }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "orcid": "0000-0001-8723-8190", "role": "member", "display_name": "Baltimore, David L." }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." } ], "option_major": [ "biochem" ], "doi": "10.7907/Z9W9573H", "abstract": "The recombination-activating gene products, RAG1 and RAG2, initiate V(D)J recombination during lymphocyte development by cleaving DNA adjacent to conserved recombination signal sequences (RSSs). The reaction involves DNA binding, synapsis, and cleavage at two RSSs located on the same DNA molecule and results in the assembly of antigen receptor genes. Since their discovery full-length, RAG1 and RAG2 have been difficult to purify, and core derivatives are shown to be most active when purified from adherent 293-T cells. However, the protein yield from adherent 293-T cells is limited. Here we develop a human suspension cell purification and change the expression vector to boost RAG production 6-fold. We use these purified RAG proteins to investigate V(D)J recombination on a mechanistic single molecule level. As a result, we are able to measure the binding statistics (dwell times and binding energies) of the initial RAG binding events with or without its co-factor high mobility group box protein 1 (HMGB1), and to characterize synapse formation at the single-molecule level yielding insights into the distribution of dwell times in the paired complex and the propensity for cleavage upon forming the synapse. We then go on to investigate HMGB1 further by measuring it compact single DNA molecules. We observed concentration dependent DNA compaction, differential DNA compaction depending on the divalent cation type, and found that at a particular HMGB1 concentration the percentage of DNA compacted is conserved across DNA lengths. Lastly, we investigate another HMGB protein called TFAM, which is essential for packaging the mitochondrial genome. We present crystal structures of TFAM bound to the heavy strand promoter 1 (HSP1) and to nonspecific DNA. We show TFAM dimerization is dispensable for DNA bending and transcriptional activation, but is required for mtDNA compaction. We propose that TFAM dimerization enhances mtDNA compaction by promoting looping of mtDNA.\r\n" }, { "name": "Marinov, Georgi Kolev", "degree": "PhD", "year": "2014", "title": "Functional Genomic Studies of the Structure and Regulation of Eukaryotic Transcriptomes", "advisor": "Wold, Barbara J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05122014-102729631", "creators": [ { "name": { "family": "Marinov", "given": "Georgi Kolev" }, "id": "Marinov-Georgi-Kolev", "orcid": "0000-0003-1822-7273", "display_name": "Marinov, Georgi Kolev" } ], "advisors": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "advisor", "display_name": "Wold, Barbara J." } ], "committee": [ { "name": { "family": "Aravin", "given": "Alexei A." }, "id": "Aravin-A-A", "orcid": "0000-0002-6956-8257", "role": "chair", "display_name": "Aravin, Alexei A." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/1BXM-ZM46", "abstract": "The main focus of this thesis is the use of high-throughput sequencing technologies in functional genomics (in particular in the form of ChIP-seq, chromatin immunoprecipitation coupled with sequencing, and RNA-seq) and the study of the structure and regulation of transcriptomes. Some parts of it are of a more methodological nature while others describe the application of these functional genomic tools to address various biological problems. A significant part of the research presented here was conducted as part of the ENCODE (ENCyclopedia Of DNA Elements) Project.
\r\n\r\nThe first part of the thesis focuses on the structure and diversity of the human transcriptome. Chapter 1 contains an analysis of the diversity of the human polyadenylated transcriptome based on RNA-seq data generated for the ENCODE Project. Chapter 2 presents a simulation-based examination of the performance of some of the most popular computational tools used to assemble and quantify transcriptomes. Chapter 3 includes a study of variation in gene expression, alternative splicing and allelic expression bias on the single-cell level and on a genome-wide scale in human lymphoblastoid cells; it also brings forward a number of critical to the practice of single-cell RNA-seq measurements methodological considerations.
\r\n\r\nThe second part presents several studies applying functional genomic tools to the study of the regulatory biology of organellar genomes, primarily in mammals but also in plants. Chapter 5 contains an analysis of the occupancy of the human mitochondrial genome by TFAM, an important structural and regulatory protein in mitochondria, using ChIP-seq. In Chapter 6, the mitochondrial DNA occupancy of the TFB2M transcriptional regulator, the MTERF termination factor, and the mitochondrial RNA and DNA polymerases is characterized. Chapter 7 consists of an investigation into the curious phenomenon of the physical association of nuclear transcription factors with mitochondrial DNA, based on the diverse collections of transcription factor ChIP-seq datasets generated by the ENCODE, mouseENCODE and modENCODE consortia. In Chapter 8 this line of research is further extended to existing publicly available ChIP-seq datasets in plants and their mitochondrial and plastid genomes.
\r\n\r\nThe third part is dedicated to the analytical and experimental practice of ChIP-seq. As part of the ENCODE Project, a set of metrics for assessing the quality of ChIP-seq experiments was developed, and the results of this activity are presented in Chapter 9. These metrics were later used to carry out a global analysis of ChIP-seq quality in the published literature (Chapter 10). In Chapter 11, the development and initial application of an automated robotic ChIP-seq (in which these metrics also played a major role) is presented.
\r\n\r\nThe fourth part presents the results of some additional projects the author has been involved in, including the study of the role of the Piwi protein in the transcriptional regulation of transposon expression in Drosophila (Chapter 12), and the use of single-cell RNA-seq to characterize the heterogeneity of gene expression during cellular reprogramming (Chapter 13).
\r\n\r\nThe last part of the thesis provides a review of the results of the ENCODE Project and the interpretation of the complexity of the biochemical activity exhibited by mammalian genomes that they have revealed (Chapters 15 and 16), an overview of the expected in the near future technical developments and their impact on the field of functional genomics (Chapter 14), and a discussion of some so far insufficiently explored research areas, the future study of which will, in the opinion of the author, provide deep insights into many fundamental but not yet completely answered questions about the transcriptional biology of eukaryotes and its regulation.
" }, { "name": "Nichols, Weston A.", "degree": "PhD", "year": "2014", "title": "Lynxl and the \u03b22V287L Mutation Affect the Stoichiometry of the \u03b14\u03b22 Nicotinic Acetylcholine Receptor", "advisor": "Lester, Henry A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05292014-190146869", "creators": [ { "name": { "family": "Nichols", "given": "Weston A." }, "id": "Nichols-Weston-A", "display_name": "Nichols, Weston A." } ], "advisors": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "advisor", "display_name": "Lester, Henry A." } ], "committee": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "chair", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Dougherty", "given": "Dennis A." }, "id": "Dougherty-D-A", "role": "member", "display_name": "Dougherty, Dennis A." }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "role": "member", "display_name": "Prober, David A." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "member", "display_name": "Lester, Henry A." } ], "option_major": [ "neurobio" ], "doi": "10.7907/2S9B-R910", "abstract": "GPI-anchored neurotoxin-like receptor binding proteins, such as lynx modulators, are topologically positioned to exert pharmacological effects by binding to the extracellular portion of nAChRs. These actions are generally thought to proceed when both lynx and the nAChRs are on the plasma membrane. Here, we demonstrate that lynx1 also exerts effects on \u03b14\u03b22 nAChRs within the endoplasmic reticulum. Lynx affects assembly of nascent \u03b14 and \u03b22 subunits, and alters the stoichiometry of the population that reaches the plasma membrane. Additionally, these data suggest that lynx1 alters nAChR stoichiometry primarily through this intracellular interaction, rather than via effects on plasma membrane nAChRs. To our knowledge, these data represent the first test of the hypothesis that a lynx family member, or indeed any GPI-anchored protein, could act within the cell to alter assembly of multi-subunit protein." }, { "name": "Ohayon, Shay S.", "degree": "PhD", "year": "2014", "title": "Dissecting Neural Circuits for Vision in Nonhuman Primates using fMRI-Guided Electrophysiology and Optogenetics", "advisor": "Tsao, Doris Y.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05162014-072424521", "creators": [ { "name": { "family": "Ohayon", "given": "Shay S." }, "id": "Ohayon-Shay-S", "display_name": "Ohayon, Shay S." } ], "advisors": [ { "name": { "family": "Tsao", "given": "Doris Y." }, "id": "Tsao-D-Y", "role": "advisor", "display_name": "Tsao, Doris Y." } ], "committee": [ { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "role": "chair", "display_name": "Perona, Pietro" }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "role": "member", "display_name": "Andersen, Richard A." }, { "name": { "family": "Tsao", "given": "Doris Y." }, "id": "Tsao-D-Y", "role": "member", "display_name": "Tsao, Doris Y." } ], "option_major": [ "cns" ], "doi": "10.7907/WD1D-W918", "abstract": "The visual system is a remarkable platform that evolved to solve difficult computational\r\nproblems such as detection, recognition, and classification of objects. Of great interest is the\r\nface-processing network, a sub-system buried deep in the temporal lobe, dedicated for\r\nanalyzing specific type of objects (faces). In this thesis, I focus on the problem of face\r\ndetection by the face-processing network. Insights obtained from years of developing\r\ncomputer-vision algorithms to solve this task have suggested that it may be efficiently and\r\neffectively solved by detection and integration of local contrast features. Does the brain use\r\na similar strategy? To answer this question, I embark on a journey that takes me through the\r\ndevelopment and optimization of dedicated tools for targeting and perturbing deep brain\r\nstructures. Data collected using MR-guided electrophysiology in early face-processing\r\nregions was found to have strong selectivity for contrast features, similar to ones used by\r\nartificial systems. While individual cells were tuned for only a small subset of features, the\r\npopulation as a whole encoded the full spectrum of features that are predictive to the presence\r\nof a face in an image. Together with additional evidence, my results suggest a possible\r\ncomputational mechanism for face detection in early face processing regions. To move from\r\ncorrelation to causation, I focus on adopting an emergent technology for perturbing brain\r\nactivity using light: optogenetics. While this technique has the potential to overcome\r\nproblems associated with the de-facto way of brain stimulation (electrical microstimulation),\r\nmany open questions remain about its applicability and effectiveness for\r\nperturbing the non-human primate (NHP) brain. In a set of experiments, I use viral vectors\r\nto deliver genetically encoded optogenetic constructs to the frontal eye field and faceselective\r\nregions in NHP and examine their effects side-by-side with electrical microstimulation\r\nto assess their effectiveness in perturbing neural activity as well as behavior.\r\nResults suggest that cells are robustly and strongly modulated upon light delivery and that\r\nsuch perturbation can modulate and even initiate motor behavior, thus, paving the way for\r\nfuture explorations that may apply these tools to study connectivity and information flow in\r\nthe face processing network." }, { "name": "Rome, Michael Evan", "degree": "PhD", "year": "2014", "title": "The Get3 ATPase Drives Unidirectional Targeting of Tail-Anchored Membrane Proteins", "advisor": "Shan, Shu-ou", "url": "https://resolver.caltech.edu/CaltechTHESIS:04162014-173759568", "creators": [ { "name": { "family": "Rome", "given": "Michael Evan" }, "id": "Rome-Michael-Evan", "display_name": "Rome, Michael Evan" } ], "advisors": [ { "name": { "family": "Shan", "given": "Shu-ou" }, "id": "Shan-Shu-ou", "role": "advisor", "display_name": "Shan, Shu-ou" } ], "committee": [ { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "chair", "display_name": "Chan, David C." }, { "name": { "family": "Clemons", "given": "William M." }, "id": "Clemons-W-M", "role": "member", "display_name": "Clemons, William M." }, { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "role": "member", "display_name": "Campbell, Judith L." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Shan", "given": "Shu-ou" }, "id": "Shan-Shu-ou", "role": "member", "display_name": "Shan, Shu-ou" } ], "option_major": [ "biology" ], "doi": "10.7907/Z9DZ068N", "abstract": "Efficient and accurate localization of membrane proteins is essential to all cells and requires a complex cascade of interactions between protein machineries. This is exemplified in the recently discovered Guided Entry of Tail-anchored protein pathway, in which the central targeting factor Get3 must sequentially interact with three distinct binding partners (Get4, Get1 and Get2) to ensure the targeted delivery of Tail-anchored proteins to the endoplasmic reticulum membrane. To understand the molecular and energetic principles that provide the vectorial driving force of these interactions, we used a quantitative fluorescence approach combined with mechanistic enzymology to monitor the effector interactions of Get3 at each stage of Tail-anchored protein targeting. We show that nucleotide and membrane protein substrate generate a gradient of interaction energies that drive the cyclic and ordered transit of Get3 from Get4 to Get2 and lastly to Get1. These data also define how the Get3/Tail-anchored complex is captured, handed over, and disassembled by the Get1/2 receptor at the membrane, and reveal a novel role for Get4/5 in recycling Get3 from the endoplasmic reticulum membrane at the end of the targeting reaction. These results provide general insights into how complex cascades of protein interactions are coordinated and coupled to energy inputs in biological systems." }, { "name": "Shocklee, Miceala Marie", "degree": "Senior Thesis", "year": "2014", "title": "The Art of Madness", "advisor": "Weinstein, Cindy A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05222014-111111602", "creators": [ { "name": { "family": "Shocklee", "given": "Miceala Marie" }, "id": "Shocklee-Miceala-Marie", "display_name": "Shocklee, Miceala Marie" } ], "advisors": [ { "name": { "family": "Weinstein", "given": "Cindy A." }, "id": "Weinstein-C", "orcid": "0009-0006-0352-2981", "role": "advisor", "display_name": "Weinstein, Cindy A." } ], "committee": [ { "name": { "family": "None", "given": "None" }, "display_name": "None, None" } ], "option_major": [ "biology" ], "doi": "10.7907/043M-TF33", "abstract": "The nineteenth century was not an entirely kind time for the female artist. Coming of age as the 1800\u2019s bridged into its latter half, literary artists Elizabeth Stuart Phelps Ward, Charlotte Perkins Gilman, and Kate Chopin were all well aware of their uncharitable culture. Equipped with firm feminist bents and creative visions, each of three women produced a seminal work \u2013 The Story of Avis, \u201cThe Yellow Wallpaper,\u201d and The Awakening, respectively \u2013 taking that atmosphere to task. In these stories, each of the three women produces a female protagonist who struggles for having been born simultaneously an artist and a woman. The writers pit their women\u2019s desires against the restrictive latitude of their time and show how such conditions drive women to madness, as a result of which they are forced to either escape into the blind mind of insanity or deal daily with their pain and inescapable societal condemnation. In an age where \u201chysteria\u201d was a frequent hit in the vernacular, Phelps, Gilman and Chopin use art and literature as mediums to show that, indeed, there is a method behind the madness." }, { "name": "Sosa Padilla Araujo, Bernardo", "degree": "PhD", "year": "2014", "title": "Computational Enzyme Design", "advisor": "Mayo, Stephen L.; Miller, Thomas F.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05052014-122744794", "creators": [ { "name": { "family": "Sosa Padilla Araujo", "given": "Bernardo" }, "id": "Sosa-Padilla-Araujo-Bernardo", "display_name": "Sosa Padilla Araujo, Bernardo" } ], "advisors": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "advisor", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Miller", "given": "Thomas F." }, "id": "Miller-T-F", "role": "co-advisor", "display_name": "Miller, Thomas F." } ], "committee": [ { "name": { "family": "Shan", "given": "Shu-ou" }, "id": "Shan-Shu-ou", "orcid": "0000-0002-6526-1733", "role": "chair", "display_name": "Shan, Shu-ou" }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Miller", "given": "Thomas F." }, "id": "Miller-T-F", "orcid": "0000-0002-1882-5380", "role": "member", "display_name": "Miller, Thomas F." }, { "name": { "family": "Clemons", "given": "William M." }, "id": "Clemons-W-M", "orcid": "0000-0002-0021-889X", "role": "member", "display_name": "Clemons, William M." } ], "option_major": [ "biochem" ], "doi": "10.7907/Z9KP805N", "abstract": "Computational protein design (CPD) is the automated identification of amino acid sequences that will fold into a specified three-dimensional structure. This method has emerged as a promising tool for engineering enzymes. In this thesis, I describe my efforts at improving and applying CPD methods to the design of enzymes.
\r\n\r\nChapter II describes the development and benchmark results for a molecular dynamics (MD) protocol to prescreen enzyme designs. Results indicate that the MD protocol is successful in screening enzymes for enzymatic activity. The protocol is general, reproducible, and excels at discarding false positive designs while predicting the most active enzymes correctly.
\r\n\r\nConformational changes are part of the repertoire that natural enzymes use to catalyze reactions. Chapter III comprises the computational design and experimental characterization of triosephosphate isomerase (TIM), a model enzyme for the study of catalytic activity in the context of conformational changes. Using a novel multi-state design (MSD) method that can consider multiple states in a single protein sequence optimization calculation, we designed the flexible hinges in TIM\u2019s active site loop.
\r\n\r\nA central challenge in the CPD field is the reliable engineering of an enzyme for any desired reaction. In Chapter IV, I describe the conceptualization and application of a high-throughput computational framework for the de novo design of enzymatic activity into inert scaffolds. TIM is used as a model system.
\r\n" }, { "name": "Su, Tsu-Te Judith", "degree": "PhD", "year": "2014", "title": "Label-Free Detection of Single Molecule Using Microtoroid Optical Resonators", "advisor": "Rees, Douglas C.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05302014-140200007", "creators": [ { "name": { "family": "Su", "given": "Tsu-Te Judith" }, "id": "Su-Tsu-Te-Judith", "display_name": "Su, Tsu-Te Judith" } ], "advisors": [ { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "advisor", "display_name": "Rees, Douglas C." } ], "committee": [ { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "chair", "display_name": "Rees, Douglas C." }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." }, { "name": { "family": "Vahala", "given": "Kerry J." }, "id": "Vahala-K-J", "orcid": "0000-0003-1783-1380", "role": "member", "display_name": "Vahala, Kerry J." }, { "name": { "family": "Davis", "given": "Mark E." }, "id": "Davis-M-E", "orcid": "0000-0001-8294-1477", "role": "member", "display_name": "Davis, Mark E." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." } ], "option_major": [ "biochem" ], "doi": "10.7907/EHWP-DH17", "abstract": "Being able to detect a single molecule without the use of labels has been a long standing goal of bioengineers and physicists. This would simplify applications ranging from single molecular binding studies to those involving public health and security, improved drug screening, medical diagnostics, and genome sequencing. One promising technique that has the potential to detect single molecules is the microtoroid optical resonator. The main obstacle to detecting single molecules, however, is decreasing the noise level of the measurements such that a single molecule can be distinguished from background. We have used laser frequency locking in combination with balanced detection and data processing techniques to reduce the noise level of these devices and report the detection of a wide range of nanoscale objects ranging from nanoparticles with radii from 100 to 2.5 nm, to exosomes, ribosomes, and single protein molecules (mouse immunoglobulin G and human interleukin-2). We further extend the exosome results towards creating a non-invasive tumor biopsy assay. Our results, covering several orders of magnitude of particle radius (100 nm to 2 nm), agree with the 'reactive' model prediction for the frequency shift of the resonator upon particle binding. In addition, we demonstrate that molecular weight may be estimated from the frequency shift through a simple formula, thus providing a basis for an ``optical mass spectrometer'' in solution. We anticipate that our results will enable many applications, including more sensitive medical diagnostics and fundamental studies of single receptor-ligand and protein-protein interactions in real time. The thesis summarizes what we have achieved thus far and shows that the goal of detecting a single molecule without the use of labels can now be realized." }, { "name": "Tan, Alison", "degree": "Senior Thesis", "year": "2014", "title": "Eavan Boland and Paula Meehan : Irish Voices of the Past", "advisor": "Gilmartin, Kevin M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05232014-004544454", "creators": [ { "name": { "family": "Tan", "given": "Alison" }, "id": "Tan-Alison", "display_name": "Tan, Alison" } ], "advisors": [ { "name": { "family": "Gilmartin", "given": "Kevin M." }, "id": "Gilmartin-K-M", "role": "advisor", "display_name": "Gilmartin, Kevin M." } ], "committee": [ { "name": { "family": "None", "given": "None" }, "display_name": "None, None" } ], "option_major": [ "bioeng" ], "doi": "10.7907/7928-NF27", "abstract": "History, myth, exile, identity\u2014for generations those have been the themes of Irish poetry, an Irish poetry written almost exclusively by male poets. As women moved in to claim a voice the themes were often the same, though reworked in essential ways. The key to that reworking, the pivot for an Irish women\u2019s poetry, was the development of a female poetic identity. Eavan Boland led the way. In particular, Boland\u2019s struggles as the first prominent female poet of modern Irish Literature emphasize a search for self-identity. At the forefront of this movement and a precedent for those around her, she establishes themes that pave the way for Irish women writers. With Boland, comes a hopeful recovery of the contemporary female literary experience, with the perspective and approach towards self-identity endlessly evolving over time with each new poet. Inspired by Boland, but a generation younger, Paula Meehan explores similar themes of female constraint, yet raises her own distinctive concerns, in particular the division of male and female roles and generational conflict, exploring what is real and ordinary." }, { "name": "Trudeau, Devin Lee", "degree": "PhD", "year": "2014", "title": "Engineering Enzyme Systems by Recombination", "advisor": "Arnold, Frances Hamilton", "url": "https://resolver.caltech.edu/CaltechTHESIS:01202014-144143175", "creators": [ { "name": { "family": "Trudeau", "given": "Devin Lee" }, "id": "Trudeau-Devin-Lee", "display_name": "Trudeau, Devin Lee" } ], "advisors": [ { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "advisor", "display_name": "Arnold, Frances Hamilton" } ], "committee": [ { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "chair", "display_name": "Arnold, Frances Hamilton" }, { "name": { "family": "Tirrell", "given": "David A." }, "id": "Tirrell-D-A", "orcid": "0000-0003-3175-4596", "role": "member", "display_name": "Tirrell, David A." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Miller", "given": "Thomas F." }, "id": "Miller-T-F", "orcid": "0000-0002-1882-5380", "role": "member", "display_name": "Miller, Thomas F." }, { "name": { "family": "Shapiro", "given": "Mikhail G." }, "id": "Shapiro-M-G", "orcid": "0000-0002-0291-4215", "role": "member", "display_name": "Shapiro, Mikhail G." } ], "option_major": [ "bioeng" ], "doi": "10.7907/F8T8-3S80", "abstract": "Homologous recombination is a source of diversity in both natural and directed evolution. Standing genetic variation that has passed the test of natural selection is combined in new ways, generating functional and sometimes unexpected changes. In this work we evaluate the utility of homologous recombination as a protein engineering tool, both in comparison with and combined with other protein engineering techniques, and apply it to an industrially important enzyme: Hypocrea jecorina Cel5a.
\r\n\r\nChapter 1 reviews work over the last five years on protein engineering by recombination. Chapter 2 describes the recombination of Hypocrea jecorina Cel5a endoglucanase with homologous enzymes in order to improve its activity at high temperatures. A chimeric Cel5a that is 10.1 \u00b0C more stable than wild-type and hydrolyzes 25% more cellulose at elevated temperatures is reported. Chapter 3 describes an investigation into the synergy of thermostable cellulases that have been engineered by recombination and other methods. An engineered endoglucanase and two engineered cellobiohydrolases synergistically hydrolyzed cellulose at high temperatures, releasing over 200% more reducing sugars over 60 h at their optimal mixture relative to the best mixture of wild-type enzymes. These results provide a framework for engineering cellulolytic enzyme mixtures for the industrial conditions of high temperatures and long incubation times.
\r\n\r\nIn addition to this work on recombination, we explored three other problems in protein engineering. Chapter 4 describes an investigation into replacing enzymes with complex cofactors with simple cofactors, using an E. coli enolase as a model system. Chapter 5 describes engineering broad-spectrum aldehyde resistance in Saccharomyces cerevisiae by evolving an alcohol dehydrogenase simultaneously for activity and promiscuity. Chapter 6 describes an attempt to engineer gene-targeted hypermutagenesis into E. coli to facilitate continuous in vivo selection systems.
" }, { "name": "Walkup, Ward Gale IV", "degree": "PhD", "year": "2014", "title": "Biochemical Studies of Postsynaptic Density Signaling Proteins With a Focus on synGAP and PDZ Domains", "advisor": "Kennedy, Mary B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:12182013-191308687", "creators": [ { "name": { "family": "Walkup", "given": "Ward Gale IV" }, "id": "Walkup-Ward-Gale-IV", "orcid": "0000-0002-0385-6256", "display_name": "Walkup, Ward Gale IV" } ], "advisors": [ { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "advisor", "display_name": "Kennedy, Mary B." } ], "committee": [ { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "chair", "display_name": "Rees, Douglas C." }, { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "member", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." } ], "option_major": [ "biochem" ], "doi": "10.7907/Z9N877R8", "abstract": "Memory storage in the brain involves adjustment of the strength of existing synapses and formation of new neural networks. A key process underlying memory formation is synaptic plasticity, the ability of excitatory synapses to strengthen or weaken their connections in response to patterns of activity between their connected neurons. Synaptic plasticity is governed by the precise pattern of Ca²⁺ influx through postsynaptic N-methyl-D-aspartate-type glutamate receptors (NMDARs), which can lead to the activation of the small GTPases Ras and Rap. Differential activation of Ras and Rap acts to modulate synaptic strength by promoting the insertion or removal of 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid receptors (AMPARs) from the synapse. Synaptic GTPase activating protein (synGAP) regulates AMPAR levels by catalyzing the inactivation of GTP-bound (active) Ras or Rap. synGAP is positioned in close proximity to the cytoplasmic tail regions of the NMDAR through its association with the PDZ domains of PSD-95. SynGAP\u2019s activity is regulated by the prominent postsynaptic protein kinase, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (CDK5), a known binding partner of CaMKII. Modulation of synGAP\u2019s activity by phosphorylation may alter the ratio of active Ras to Rap in spines, thus pushing the spine towards the insertion or removal of AMPARs, subsequently strengthening or weakening the synapse. To date, all biochemical studies of the regulation of synGAP activity by protein kinases have utilized impure preparations of membrane bound synGAP. Here we have clarified the effects of phosphorylation of synGAP on its Ras and Rap GAP activities by preparing and utilizing purified, soluble recombinant synGAP, Ras, Rap, CaMKII, CDK5, PLK2, and CaM. Using mass spectrometry, we have confirmed the presence of previously identified CaMKII and CDK5 sites in synGAP, and have identified novel sites of phosphorylation by CaMKII, CDK5, and PLK2. We have shown that the net effect of phosphorylation of synGAP by CaMKII, CDK5, and PLK2 is an increase in its GAP activity toward HRas and Rap1. In contrast, there is no effect on its GAP activity toward Rap2. Additionally, by assaying the GAP activity of phosphomimetic synGAP mutants, we have been able to hypothesize the effects of CDK5 phosphorylation at specific sites in synGAP. In the course of this work, we also found, unexpectedly, that synGAP is itself a Ca²⁺/CaM binding protein. While Ca²⁺/CaM binding does not directly affect synGAP activity, it causes a conformational change in synGAP that increases the rate of its phosphorylation and exposes additional phosphorylation sites that are inaccessible in the absence of Ca²⁺/CaM.
\r\n\r\nThe postsynaptic density (PSD) is an electron-dense region in excitatory postsynaptic neurons that contains a high concentration of glutamate receptors, cytoskeletal proteins, and associated signaling enzymes. Within the PSD, three major classes of scaffolding molecules function to organize signaling enzymes and glutamate receptors. PDZ domains present in the Shank and PSD-95 scaffolds families serve to physically link AMPARs and NMDARs to signaling molecules in the PSD. Because of the specificity and high affinity of PDZ domains for their ligands, I reasoned that these interacting pairs could provide the core components of an affinity chromatography system, including affinity resins, affinity tags, and elution agents. I show that affinity columns containing the PDZ domains of PSD-95 can be used to purify active PDZ domain-binding proteins to very high purity in a single step. Five heterologously expressed neuronal proteins containing endogenous PDZ domain ligands (NMDAR GluN2B subunit Tail, synGAP, neuronal nitric oxide synthase PDZ domain, cysteine rich interactor of PDZ three and cypin) were purified using PDZ domain resin, with synthetic peptides having the sequences of cognate PDZ domain ligands used as elution agents. I also show that conjugation of PDZ domain-related affinity tags to Proteins Of Interest (POIs) that do not contain endogenous PDZ domains or ligands does not alter protein activity and enables purification of the POIs on PDZ domain-related affinity resins.
" }, { "name": "Wang, Yun Elisabeth", "degree": "PhD", "year": "2014", "title": "Characterizing the Regulation of Mitochondrial Nucleoids", "advisor": "Chan, David C.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05282014-004119763", "creators": [ { "name": { "family": "Wang", "given": "Yun Elisabeth" }, "id": "Wang-Yun-Elisabeth", "display_name": "Wang, Yun Elisabeth" } ], "advisors": [ { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "advisor", "display_name": "Chan, David C." } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "member", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "member", "display_name": "Chan, David C." } ], "option_major": [ "biology" ], "doi": "10.7907/1MN9-DR40", "abstract": "Mitochondria contain a 16.6 kb circular genome encoding 13 proteins as well as mitochondrial tRNAs and rRNAs. Copies of the genome are organized into nucleoids containing both DNA and proteins, including the machinery required for mtDNA replication and transcription. Although mtDNA integrity is essential for cellular and organismal viability, regulation of proliferation of the mitochondrial genome is poorly understood. To elucidate the mechanisms behind this, we chose to study the interplay between mtDNA copy number and the proteins involved in mitochondrial fusion, another required function in cells. Strikingly, we found that mouse embryonic fibroblasts lacking fusion also had a mtDNA copy number deficit. To understand this phenomenon further, we analyzed the binding of mitochondrial transcription factor A, whose role in transcription, replication, and packaging of the genome is well-established and crucial for cellular maintenance. Using ChIP-seq, we were able to detect largely uniform, non-specific binding across the genome, with no occupancy in the known specific binding sites in the regulatory region. We did detect a single binding site directly upstream of a known origin of replication, suggesting that TFAM may play a direct role in replication. Finally, although TFAM has been previously shown to localize to the nuclear genome, we found no evidence for such binding sites in our system.
\r\n\r\nTo further understand the regulation of mtDNA by other proteins, we analyzed publicly available ChIP-seq datasets from ENCODE, modENCODE, and mouseENCODE for evidence of nuclear transcription factor binding to the mitochondrial genome. We identified eight human transcription factors and three mouse transcription factors that demonstrated binding events with the classical strand asymmetrical morphology of classical binding sites. ChIP-seq is a powerful tool for understanding the interactions between proteins and the mitochondrial genome, and future studies promise to further the understanding of how mtDNA is regulated within the nucleoid.
" }, { "name": "Wolfe, Brian Robert", "degree": "PhD", "year": "2014", "title": "Design and Analysis of Nucleic Acid Reaction Pathways", "advisor": "Pierce, Niles A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03272014-193400848", "creators": [ { "name": { "family": "Wolfe", "given": "Brian Robert" }, "id": "Wolfe-Brian-Robert", "display_name": "Wolfe, Brian Robert" } ], "advisors": [ { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "role": "advisor", "display_name": "Pierce, Niles A." } ], "committee": [ { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "role": "chair", "display_name": "Winfree, Erik" }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Miller", "given": "Thomas F." }, "id": "Miller-T-F", "role": "member", "display_name": "Miller, Thomas F." }, { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "role": "member", "display_name": "Pierce, Niles A." } ], "option_major": [ "bioeng" ], "doi": "10.7907/G4XZ-ZF47", "abstract": "Nucleic acids are a useful substrate for engineering at the molecular level. Designing the detailed energetics and kinetics of interactions between nucleic acid strands remains a challenge. Building on previous algorithms to characterize the ensemble of dilute solutions of nucleic acids, we present a design algorithm that allows optimization of structural features and binding energetics of a test tube of interacting nucleic acid strands. We extend this formulation to handle multiple thermodynamic states and combinatorial constraints to allow optimization of pathways of interacting nucleic acids. In both design strategies, low-cost estimates to thermodynamic properties are calculated using hierarchical ensemble decomposition and test tube ensemble focusing. These algorithms are tested on randomized test sets and on example pathways drawn from the molecular programming literature. To analyze the kinetic properties of designed sequences, we describe algorithms to identify dominant species and kinetic rates using coarse-graining at the scale of a small box containing several strands or a large box containing a dilute solution of strands." }, { "name": "Yu, Jiun-Yann", "degree": "PhD", "year": "2014", "title": "Innovations of Wide-Field Optical-Sectioning Fluorescence Microscopy: Toward High-Speed Volumetric Bio-Imaging with Simplicity", "advisor": "Guo, Chin-Lin", "url": "https://resolver.caltech.edu/CaltechTHESIS:05102014-143921707", "creators": [ { "name": { "family": "Yu", "given": "Jiun-Yann" }, "id": "Yu-Jiun-Yann", "display_name": "Yu, Jiun-Yann" } ], "advisors": [ { "name": { "family": "Guo", "given": "Chin-Lin" }, "id": "Guo-Chin-Lin", "role": "advisor", "display_name": "Guo, Chin-Lin" } ], "committee": [ { "name": { "family": "Yang", "given": "Changhuei" }, "id": "Yang-Changhuei", "role": "chair", "display_name": "Yang, Changhuei" }, { "name": { "family": "Blake", "given": "Geoffrey A." }, "id": "Blake-G-A", "role": "member", "display_name": "Blake, Geoffrey A." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Guo", "given": "Chin-Lin" }, "id": "Guo-Chin-Lin", "role": "member", "display_name": "Guo, Chin-Lin" } ], "option_major": [ "bioeng" ], "doi": "10.7907/4V14-HW42", "abstract": "Optical microscopy has become an indispensable tool for biological researches since its invention, mostly owing to its sub-cellular spatial resolutions, non-invasiveness, instrumental simplicity, and the intuitive observations it provides. Nonetheless, obtaining reliable, quantitative spatial information from conventional wide-field optical microscopy is not always intuitive as it appears to be. This is because in the acquired images of optical microscopy the information about out-of-focus regions is spatially blurred and mixed with in-focus information. In other words, conventional wide-field optical microscopy transforms the three-dimensional spatial information, or volumetric information about the objects into a two-dimensional form in each acquired image, and therefore distorts the spatial information about the object. Several fluorescence holography-based methods have demonstrated the ability to obtain three-dimensional information about the objects, but these methods generally rely on decomposing stereoscopic visualizations to extract volumetric information and are unable to resolve complex 3-dimensional structures such as a multi-layer sphere.
\r\n\r\nThe concept of optical-sectioning techniques, on the other hand, is to detect only two-dimensional information about an object at each acquisition. Specifically, each image obtained by optical-sectioning techniques contains mainly the information about an optically thin layer inside the object, as if only a thin histological section is being observed at a time. Using such a methodology, obtaining undistorted volumetric information about the object simply requires taking images of the object at sequential depths.
\r\n\r\nAmong existing methods of obtaining volumetric information, the practicability of optical sectioning has made it the most commonly used and most powerful one in biological science. However, when applied to imaging living biological systems, conventional single-point-scanning optical-sectioning techniques often result in certain degrees of photo-damages because of the high focal intensity at the scanning point. In order to overcome such an issue, several wide-field optical-sectioning techniques have been proposed and demonstrated, although not without introducing new limitations and compromises such as low signal-to-background ratios and reduced axial resolutions. As a result, single-point-scanning optical-sectioning techniques remain the most widely used instrumentations for volumetric imaging of living biological systems to date.
\r\n\r\nIn order to develop wide-field optical-sectioning techniques that has equivalent optical performance as single-point-scanning ones, this thesis first introduces the mechanisms and limitations of existing wide-field optical-sectioning techniques, and then brings in our innovations that aim to overcome these limitations. We demonstrate, theoretically and experimentally, that our proposed wide-field optical-sectioning techniques can achieve diffraction-limited optical sectioning, low out-of-focus excitation and high-frame-rate imaging in living biological systems. In addition to such imaging capabilities, our proposed techniques can be instrumentally simple and economic, and are straightforward for implementation on conventional wide-field microscopes. These advantages together show the potential of our innovations to be widely used for high-speed, volumetric fluorescence imaging of living biological systems.
" }, { "name": "Bialecka-Fornal, Maja I.", "degree": "PhD", "year": "2013", "title": "Single-Cell Analysis of the Physiology of Mechanosensation in Bacteria", "advisor": "Phillips, Robert B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05142013-213725125", "creators": [ { "name": { "family": "Bialecka-Fornal", "given": "Maja I." }, "id": "Bialecka-Fornal-Maja-I", "display_name": "Bialecka-Fornal, Maja I." } ], "advisors": [ { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "advisor", "display_name": "Phillips, Robert B." } ], "committee": [ { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "chair", "display_name": "Rees, Douglas C." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Jensen", "given": "Grant J." }, "id": "Jensen-G-J", "orcid": "0000-0003-1556-4864", "role": "member", "display_name": "Jensen, Grant J." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." } ], "option_major": [ "biochem" ], "doi": "10.7907/7SRD-WS94", "abstract": "Escherichia coli is one of the best studied living organisms and a model system for many biophysical investigations. Despite countless discoveries of the details of its physiology, we still lack a holistic understanding of how these bacteria react to changes in their environment. One of the most important examples is their response to osmotic shock. One of the mechanistic elements protecting cell integrity upon exposure to sudden changes of osmolarity is the presence of mechanosensitive channels in the cell membrane. These channels are believed to act as tension release valves protecting the inner membrane from rupturing. This thesis presents an experimental study of various aspects of mechanosensation in bacteria. We examine cell survival after osmotic shock and how the number of MscL (Mechanosensitive channel of Large conductance) channels expressed in a cell influences its physiology. We developed an assay that allows real-time monitoring of the rate of the osmotic challenge and direct observation of cell morphology during and after the exposure to osmolarity change. The work described in this thesis introduces tools that can be used to quantitatively determine at the single-cell level the number of expressed proteins (in this case MscL channels) as a function of, e.g., growth conditions. The improvement in our quantitative description of mechanosensation in bacteria allows us to address many, so far unsolved, problems, like the minimal number of channels needed for survival, and can begin to paint a clearer picture of why there are so many distinct types of mechanosensitive channels." }, { "name": "Bower, Danielle Vera Brown", "degree": "PhD", "year": "2013", "title": "Chemical and Neural Regulation of Embryonic Branching Morphogenesis", "advisor": "Fraser, Scott E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05302013-135915010", "creators": [ { "name": { "family": "Bower", "given": "Danielle Vera Brown" }, "id": "Bower-Danielle-Vera-Brown", "display_name": "Bower, Danielle Vera Brown" } ], "advisors": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "advisor", "display_name": "Fraser, Scott E." } ], "committee": [ { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "chair", "display_name": "Patterson, Paul H." }, { "name": { "family": "Warburton", "given": "David" }, "id": "Warburton-D", "role": "member", "display_name": "Warburton, David" }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biology" ], "doi": "10.7907/Z9SX6B6R", "abstract": "Lung development is a complex process orchestrated by as many as 40 different types of cells and many signaling and regulatory factors. The spatial sequence of branching of bronchial epithelial tubes is stereotyped. However, it remains unknown how the timing of branch formation is encoded and whether this branching clock function is unique for different tissue types or is conserved across species and lineages that undergo iterative branching. Investigations of the function of the sarcoplasmic-endoplasmic reticulum calcium ATP-ase (SERCA) reveal that protein kinase C (PKC)-modulated SERCA activity controls branch formation across tissues and species.
\r\n\r\nSERCA controls the rate of intersomitic blood vessel sprouting and branching in zebrafish embryos in a dose-dependent manner. Vessel sprouting recovers upon removal of inhibition and restoration of pump activity. Regulation of cell motility is responsible for these effects.
\r\n\r\nSimilarly, during Drosophila embryonic development, SERCA activity is required for the proper formation of both the central nervous system axon tracts and the network of tracheal tubules that deliver oxygen to tissues. SERCA blockade results in breaks in the tracheal structure and displaced axons. Removal of inhibitor partially rescues these defects, while simultaneous treatment with both SERCA inhibitor and PKC activator remarkably rescues tracheal and neural development. Dynamic imaging of Drosophila embryonic tracheal morphogenesis demonstrates that SERCA's principal function is to govern cell migration. Together, these finding reveal that SERCA regulates cell migration, and this serves as a conserved mechanism that governs branch formation in various cell types and species during development.
\r\n\r\nOn the other hand, morphogens and cell-cell interactions are critical to form a complex, specialized organ such as the mammalian lung. Nerves are known to be present from the early stages of lung branching. Yet a role for nerves in modulating epithelial branching remains to be discerned. Denervation of embryonic mouse lung explants reveals that lung branching requires nerves. Targeted neural ablation, but not inhibition of acetylcholine receptors, halts lung branching and causes a reduction in endothelial cells and epithelial and mesenchymal proliferation. Likewise, ablation of nerves in Drosophila embryos derails tracheal morphogenesis. Therefore, nerves play a conserved role in directing epithelial airway branching.
" }, { "name": "Cho, Julie YoungHee", "degree": "PhD", "year": "2013", "title": "State-Dependent Modulation of Neuronal Circuits in C. elegans Sleep", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05312013-162338827", "creators": [ { "name": { "family": "Cho", "given": "Julie YoungHee" }, "id": "Cho-Julie-YoungHee", "display_name": "Cho, Julie YoungHee" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "chair", "display_name": "Anderson, David J." }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "role": "member", "display_name": "Prober, David A." }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "role": "member", "display_name": "Siapas, Athanassios G." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/Z9028PF1", "abstract": "C. elegans is a compact system of 302 neurons with identifiable and mapped connections that makes it ideal for systems analysis. This work is a demonstration of what I have been able to learn about the nature of state-specific modulation and reversibility during a state called lethargus, a sleep-like state in the worm. I begin with description about the nervous system of the worm, the nature of sleep in the worm, the questions about behavior and its apparent circuit properties, the tools available and used to manipulate the nervous system, and what I have been able to learn from these studies. I end with clues that the physiology helps to teach us about the dynamics of state specific modulation, what makes sleep so different from other states, and how we can use these measurements to understand which modulators, neurotransmitters, and channels can be used to create different dynamics in a simple model system." }, { "name": "Del Real, Marissa Morales", "degree": "PhD", "year": "2013", "title": "Analysis of a Transcriptional Network Involving PU.1, Notch, and Gata3 in the Lymphomyeloid Lineage Decision during Early T-cell Development", "advisor": "Rothenberg, Ellen V.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05022013-181603910", "creators": [ { "name": { "family": "Del Real", "given": "Marissa Morales" }, "id": "Del-Real-Marissa-Morales", "display_name": "Del Real, Marissa Morales" } ], "advisors": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "role": "advisor", "display_name": "Rothenberg, Ellen V." } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "member", "display_name": "Mazmanian, Sarkis K." }, { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "member", "display_name": "Stathopoulos, Angelike" } ], "option_major": [ "biology" ], "doi": "10.7907/PEWS-KM18", "abstract": "Hematopoiesis is a well-established system used to study developmental choices amongst cells with multiple lineage potentials, as well as the transcription factor network interactions that drive these developmental paths. Multipotent progenitors travel from the bone marrow to the thymus where T-cell development is initiated and these early T-cell precursors retain lineage plasticity even after initiating a T-cell program. The development of these early cells is driven by Notch signaling and the combinatorial expression of many transcription factors, several of which are also involved in the development of other cell lineages. The ETS family transcription factor PU.1 is involved in the development of progenitor, myeloid, and lymphoid cells, and can divert progenitor T-cells from the T-lineage to a myeloid lineage. This diversion of early T-cells by PU.1 can be blocked by Notch signaling. The PU.1 and Notch interaction creates a switch wherein PU.1 in the presence of Notch promotes T-cell identity and PU.1 in the absence of Notch signaling promotes a myeloid identity. Here we characterized an early T-cell cell line, Scid.adh.2c2, as a good model system for studying the myeloid vs. lymphoid developmental choice dependent on PU.1 and Notch signaling. We then used the Scid.adh.2c2 system to identify mechanisms mediating PU.1 and Notch signaling interactions during early T-cell development. We show that the mechanism by which Notch signaling is protecting pro-T cells is neither degradation nor modification of the PU.1 protein. Instead we give evidence that Notch signaling is blocking the PU.1-driven inhibition of a key set of T-regulatory genes including Myb, Tcf7, and Gata3. We show that the protection of Gata3 from PU.1-mediated inhibition, by Notch signaling and Myb, is important for retaining a T-lineage identity. We also discuss a PU.1-driven mechanism involving E-protein inhibition that leads to the inhibition of Notch target genes. This is mechanism may be used as a lockdown mechanism in pro-T-cells that have made the decision to divert to the myeloid pathway. " }, { "name": "Dillman, Adler Ray", "degree": "PhD", "year": "2013", "title": "Host Seeking and the Genomic Architecture of Parasitism among Entomopathogenic Nematodes", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:11062012-124428507", "creators": [ { "name": { "family": "Dillman", "given": "Adler Ray" }, "id": "Dillman-Adler-Ray", "display_name": "Dillman, Adler Ray" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "chair", "display_name": "Hay, Bruce A." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "role": "member", "display_name": "Dickinson, Michael H." }, { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "role": "member", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "role": "member", "display_name": "Prober, David A." }, { "name": { "family": "Baldwin", "given": "James G." }, "id": "Baldwin-J-G", "role": "member", "display_name": "Baldwin, James G." } ], "option_major": [ "biology" ], "doi": "10.7907/H7Z3-5A73", "abstract": "Nematodes represent an especially abundant and species-rich phylum, with many free-living and parasitic species. Among the diversity of parasitic species is a guild of specialists known as entomopathogenic nematodes due to their unusual ability to quickly kill their hosts with the aid of pathogenic bacteria. Herein I discuss in detail the hallmarks of entomopathogenic nematodes and how they are different from other insect parasites. Further I explore their host-seeking behaviors, demonstrating their ability to detect insect hosts in complex soil environments and assess their odor preference profiles. I show that CO2 is a major driver of host seeking and that entomopathogenic nematodes detect CO2 using the same pair of conserved neurons that the fruit-dwelling Caenorhabditis elegans uses to detect and respond to CO2. I demonstrate dramatic differences in odor preference profiles and virulence capabilities, even between closely related nematodes. I discuss the role of genomic sequencing generally and more specifically in nematology, including how genomes are sequenced and analyzed and the types of characteristics that are most prominently assessed. This thesis concludes with a discussion of the genomic sequencing of entomopathogenic nematodes in the genus Steinernema and the clues these genomes provide regarding the genomic architecture of parasitism." }, { "name": "Hernandez, Gilberto", "degree": "PhD", "year": "2013", "title": "Building a Gene Regulatory Network in Adult Mouse Skeletal Muscle Following Nerve Injury: Transcriptome Characterization and New Model for Functional cis-Regulatory Analysis In Vivo", "advisor": "Wold, Barbara J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:09092012-232337189", "creators": [ { "name": { "family": "Hernandez", "given": "Gilberto" }, "id": "Hernandez-Gilberto", "display_name": "Hernandez, Gilberto" } ], "advisors": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "advisor", "display_name": "Wold, Barbara J." } ], "committee": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "chair", "display_name": "Davidson, Eric H." }, { "name": { "family": "Varshavsky", "given": "Alexander J." }, "id": "Varshavsky-A-J", "role": "member", "display_name": "Varshavsky, Alexander J." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." } ], "option_major": [ "biology" ], "doi": "10.7907/Z9833Q0J", "abstract": "The essential functional linkages of gene regulatory networks (GRNs) consist of the interactions between cis-regulatory DNA sequences and trans-acting regulatory factors. These genomically encoded regulatory interactions govern the differential gene expression programs which direct specific biological processes during development and adulthood. Detailed analysis of GRNs during development has yielded important insights regarding the structural and functional dynamics of cis-regulatory modules (CRMs) and cis-regulatory elements (CREs). Indeed, the comprehensive GRNs that have been characterized for various developmental processes provide a model for both the methodological approach and the intellectual understanding required to explore cis-regulatory architecture in other biological contexts. The present study focuses on the physiological context, investigating the GRNs that govern the molecular response to nerve injury in adult mouse skeletal muscle. Until now, high-quality GRN investigations in this context have been hampered by the absence of two fundamental components: a comprehensive catalog of genes differentially expressed after nerve injury, and an effective in vivo gene transfer technique to functionally test putative cis-regulatory modules. Using RNAseq, we have compiled a comprehensive list of all differentially expressed genes at 6.0, 12.0, 24.0, and 168.0 hours following nerve injury. This data has validated previously known differentially expressed genes, as well as identified novel candidates for cis-regulatory analysis. The in vivo gene transfer technique I have adapted and advanced targets an easily accessible muscle group for minimally invasive injection and electroporation of DNA; with it, I demonstrate highly efficient, reproducible, and stable gene transfer in mouse skeletal muscle. In addition, I have optimized the gene transfer technique not only for plasmid DNA reporter vectors, but also for BAC DNA reporter vectors, thus enabling cis-regulatory modules to be tested in a broad chromosomal environment. Finally, I have validated the capacity of this gene transfer method to functionally test CRMs, by identifying a nerve injury-associated CRM of the skeletal muscle-specific myogenin gene. The enhanced resolution provided by this technique allowed for qualitative and quantitative detection of increased reporter signal from a mutated version the nerve injury-associated CRM at ten days following denervation, when compared to the wild-type CRM, implicating it as the cis-acting regulatory sequence responsible for mediating the down-regulation of myogenin during late phase neurogenic skeletal muscle atrophy. This work lays the foundation from which a high-quality adult skeletal muscle GRN can be constructed for nerve injury and other muscle-associated disease states.\r\n" }, { "name": "Hsiao, Elaine Yih-Nien", "degree": "PhD", "year": "2013", "title": "Brain, Gut and Immune Interactions in Autism Spectrum Disorder", "advisor": "Patterson, Paul H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:12172012-110932095", "creators": [ { "name": { "family": "Hsiao", "given": "Elaine Yih-Nien" }, "id": "Hsiao-Elaine-Yih-Nien", "display_name": "Hsiao, Elaine Yih-Nien" } ], "advisors": [ { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "advisor", "display_name": "Patterson, Paul H." } ], "committee": [ { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "chair", "display_name": "Mazmanian, Sarkis K." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." } ], "option_major": [ "biology" ], "doi": "10.7907/DEVQ-1P16", "abstract": "Autism spectrum disorder (ASD) is a class of complex neurodevelopmental disabilities that are characterized by the presence and severity of stereotyped behaviors, impaired communication, and abnormal social interactions. The incidence of autism has rapidly increased to 1 in 88 children in the United States, making ASD one of the most significant medical and social burdens of our time. However, drugs are often used to treat autism-related conditions, including anxiety, hyperactivity, epilepsy, and obsessive-compulsive behaviors, and therapies for treating the core symptoms of autism are limited. Moreover, molecular diagnostics are not available for the reproducible identification of ASD; as yet, the disorder is diagnosed based on standardized behavioral assessments. Much research into ASD has focused on genetic, behavioral, and neurological aspects of the illness. However, primary roles for environmental risk factors and peripheral disruptions, such as immune dysregulation and gastrointestinal distress, have gained significant attention.
\r\n\r\nThe work described in this thesis uncovers molecular mechanisms involved in the pathogenesis of autism-related endophenotypes in a mouse model of a primary autism risk factor, maternal immune activation (MIA). MIA is founded upon the strong epidemiological link between maternal infection and increased autism risk in the offspring. This risk factor can be translated to a mouse model with face and construct validity for autism, wherein pregnant mice injected with the immunogenic, double-stranded RNA poly(I:C) yield offspring with the core behavioral and neuropathological features of autism. Specifically, we report that MIA critically alters placental immune status and endocrine function, reflecting a key pathway by which fetal development may be disrupted to manifest in ASD-related phenotypes. We identify signature changes to the fetal brain transcriptome in response to multiple modes of MIA, highlighting a converging pathway involved in the development of autism-related behaviors and neuropathologies. We characterize peripheral, neural, and enteric immune alterations in MIA offspring and uncover an immune contribution to autism-related behavioral abnormalities. Finally we demonstrate that a microbe-based therapeutic can ameliorate intestinal pathology, metabolic function, and autism-related behaviors in MIA mice, which supports a role for the gut-immune-brain axis in ASD.
\r\n" }, { "name": "Hu, Na", "degree": "PhD", "year": "2013", "title": "Epigenetic Regulation in Neural Crest Development: Role of DNA Methyltransferases 3A and 3B", "advisor": "Bronner, Marianne E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04202013-232100847", "creators": [ { "name": { "family": "Hu", "given": "Na" }, "id": "Hu-Na", "display_name": "Hu, Na" } ], "advisors": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "advisor", "display_name": "Bronner, Marianne E." } ], "committee": [ { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "chair", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "role": "member", "display_name": "Prober, David A." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." } ], "option_major": [ "biology" ], "doi": "10.7907/Z99021R1", "abstract": "The neural crest is a group of migratory, multipotent stem cells that play a crucial role in many aspects of embryonic development. This uniquely vertebrate cell population forms within the dorsal neural tube but then emigrates out and migrates long distances to different regions of the body. These cells contribute to formation of many structures such as the peripheral nervous system, craniofacial skeleton, and pigmentation of the skin. Why some neural tube cells undergo a change from neural to neural crest cell fate is unknown as is the timing of both onset and cessation of their emigration from the neural tube. In recent years, growing evidence supports an important role for epigenetic regulation as a new mechanism for controlling aspects of neural crest development. In this thesis, I dissect the roles of the de novo DNA methyltransferases (DNMTs) 3A and 3B in neural crest specification, migration and differentiation. First, I show that DNMT3A limits the spatial boundary between neural crest versus neural tube progenitors within the neuroepithelium. DNMT3A promotes neural crest specification by directly mediating repression of neural genes, like Sox2 and Sox3. Its knockdown causes ectopic Sox2 and Sox3 expression at the expense of neural crest territory. Thus, DNMT3A functions as a molecular switch, repressing neural to favor neural crest cell fate. Second, I find that DNMT3B restricts the temporal window during which the neural crest cells emigrate from the dorsal neural tube. Knockdown of DNMT3B causes an excess of neural crest emigration, by extending the time that the neural tube is competent to generate emigrating neural crest cells. In older embryos, this resulted in premature neuronal differentiation. Thus, DNMT3B regulates the duration of neural crest production by the neural tube and the timing of their differentiation. My results in avian embryos suggest that de novo DNA methylation, exerted by both DNMT3A and DNMT3B, plays a dual role in neural crest development, with each individual paralogue apparently functioning during a distinct temporal window. The results suggest that de novo DNA methylation is a critical epigenetic mark used for cell fate restriction of progenitor cells during neural crest cell fate specification. Our discovery provides important insights into the mechanisms that determine whether a cell becomes part of the central nervous system or peripheral cell lineages. " }, { "name": "Kolawa, Natalie J.", "degree": "PhD", "year": "2013", "title": "Proteomic Analysis of the Cdc48/Ubx Network Identifies a Role for Ubx2 in the Regulation of Lipid Biosynthesis", "advisor": "Deshaies, Raymond Joseph", "url": "https://resolver.caltech.edu/CaltechTHESIS:05282013-142345187", "creators": [ { "name": { "family": "Kolawa", "given": "Natalie J." }, "id": "Kolawa-Natalie-J", "display_name": "Kolawa, Natalie J." } ], "advisors": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "advisor", "display_name": "Deshaies, Raymond Joseph" } ], "committee": [ { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "chair", "display_name": "Chan, David C." }, { "name": { "family": "Shan", "given": "Shu-ou" }, "id": "Shan-Shu-ou", "role": "member", "display_name": "Shan, Shu-ou" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "member", "display_name": "Deshaies, Raymond Joseph" } ], "option_major": [ "biology" ], "doi": "10.7907/FN9G-KV67", "abstract": "Cdc48/p97 is an essential, highly abundant hexameric member of the AAA (ATPase associated with various cellular activities) family. It has been linked to a variety of processes throughout the cell but it is best known for its role in the ubiquitin proteasome pathway. In this system it is believed that Cdc48 behaves as a segregase, transducing the chemical energy of ATP hydrolysis into mechanical force to separate ubiquitin-conjugated proteins from their tightly-bound partners.
\r\n\r\nCurrent models posit that Cdc48 is linked to its substrates through a variety of adaptor proteins, including a family of seven proteins (13 in humans) that contain a Cdc48-binding UBX domain. As such, due to the complexity of the network of adaptor proteins for which it serves as the hub, Cdc48/p97 has the potential to exert a profound influence on the ubiquitin proteasome pathway. However, the number of known substrates of Cdc48/p97 remains relatively small, and smaller still is the number of substrates that have been linked to a specific UBX domain protein. As such, the goal of this dissertation research has been to discover new substrates and better understand the functions of the Cdc48 network. With this objective in mind, we established a proteomic screen to assemble a catalog of candidate substrate/targets of the Ubx adaptor system.
\r\n\r\nHere we describe the implementation and optimization of a cutting-edge quantitative mass spectrometry method to measure relative changes in the Saccharomyces cerevisiae proteome. Utilizing this technology, and in order to better understand the breadth of function of Cdc48 and its adaptors, we then performed a global screen to identify accumulating ubiquitin conjugates in cdc48-3 and ubx\u0394 mutants. In this screen different ubx mutants exhibited reproducible patterns of conjugate accumulation that differed greatly from each other, pointing to various unexpected functional specializations of the individual Ubx proteins.
\r\n\r\nAs validation of our mass spectrometry findings, we then examined in detail the endoplasmic-reticulum bound transcription factor Spt23, which we identified as a putative Ubx2 substrate. In these studies ubx2\u0394 cells were deficient in processing of Spt23 to its active p90 form, and in localizing p90 to the nucleus. Additionally, consistent with reduced processing of Spt23, ubx2\u0394 cells demonstrated a defect in expression of their target gene OLE1, a fatty acid desaturase. Overall, this work demonstrates the power of proteomics as a tool to identify new targets of various pathways and reveals Ubx2 as a key regulator lipid membrane biosynthesis.
\r\n \r\n" }, { "name": "Lee, Sung-Eun Melanie", "degree": "PhD", "year": "2013", "title": "Mechanism of Intestinal Colonization by Symbiotic Bacteria", "advisor": "Mazmanian, Sarkis K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:12122012-183251952", "creators": [ { "name": { "family": "Lee", "given": "Sung-Eun Melanie" }, "id": "Lee-Sung-Eun-Melanie", "display_name": "Lee, Sung-Eun Melanie" } ], "advisors": [ { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "advisor", "display_name": "Mazmanian, Sarkis K." } ], "committee": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "role": "chair", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "member", "display_name": "Baltimore, David L." }, { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "role": "member", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "member", "display_name": "Mazmanian, Sarkis K." } ], "option_major": [ "biology" ], "doi": "10.7907/24H5-8V73", "abstract": "All animals live in symbiosis. Shaped by eons of co-evolution, host-bacterial associations have developed into prosperous relationships creating mechanisms for mutual benefits to both microbe and host. No better example exists in biology than the astounding numbers of bacteria harbored by the lower gastrointestinal tract of mammals. This community of symbionts establishes a life-long habitat within the distal gut and profoundly impacts host health. Although many recent investigations have led to determination of the microbiota composition, molecular mechanisms mediating establishment and maintenance of the microbial community within the gut is poorly described.
\r\n\r\nWe use gnotobiotic mice to elucidate mechanisms of colonization by Bacteroides, one of the most numerically prominent genera in the intestine. We generate mutant strains of Bacteroides fragilis that lack the ability to express multiple capsular polysaccharides and demonstrate defect in colonization in competition with wild-type strain, suggesting a role for surface sugar architecture during host-symbiont mutualism. Through a functional in vivo genetic screen of colonization, we identify a novel operon from the genome of B. fragilis that is highly conserved among many sequenced intestinal Bacteroides and that mediate a species-specific colonization profile. We have named this genetic locus the commensal colonization factor (ccf). B. fragilis deleted in the ccf genes exhibit colonization defects in both germ-free and complex microbiota harboring mice. The ccf genes of B. fragilis are up-regulated during gut colonization, preferentially at the mucosal surface, supporting an in vivo function. Indeed, deletion of ccf genes leads to reduced mucosal association and a defect in bacterial occupation of colonic crypts of mice. The ability of B. fragilis to repopulate the gut after antibiotic perturbation or gastroenteritis requires expression of ccf, suggesting the niche within colonic crypts represents a colonization reservoir for the gut microbiota following environmental stress. These findings suggest a novel and evolutionarily conserved mechanism for persistent gut colonization by the Bacteroides species.
\r\n" }, { "name": "Nawroth, Janna C.", "degree": "PhD", "year": "2013", "title": "Conceptual Framework and Physical Implementation of a Systematic Design Strategy for Tissue-Engineered Devices", "advisor": "Dabiri, John O.", "url": "https://resolver.caltech.edu/CaltechTHESIS:12172012-143708325", "creators": [ { "name": { "family": "Nawroth", "given": "Janna C." }, "id": "Nawroth-Janna-C", "display_name": "Nawroth, Janna C." } ], "advisors": [ { "name": { "family": "Dabiri", "given": "John O." }, "id": "Dabiri-J-O", "role": "advisor", "display_name": "Dabiri, John O." } ], "committee": [ { "name": { "family": "Dabiri", "given": "John O." }, "id": "Dabiri-J-O", "role": "chair", "display_name": "Dabiri, John O." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biology" ], "doi": "10.7907/5ZTQ-2J09", "abstract": "Tissue-engineered and biologically inspired devices promise to advance medical implants, robotic devices and diagnostic tools. Ideally, biohybrid constructs combine the versatility and fine control of traditional building substrates with dynamic properties of living tissues including sensory modalities and mechanisms of repair, plasticity and self-organization. These dynamic properties also complicate the design process as they arise from, and act upon, structure-function relationships across multiple spatiotemporal scales that need to be recapitulated in the engineered tissue. Biomimetic designs merely copying the structure of native organs and organisms, however, are likely to reflect evolutionary constraints, phenotypic variability and environmental factors rather than rendering optimal engineering solutions.
\r\n\r\nThis thesis describes an alternative to biomimetic design, i.e., a systematic approach to tissue engineering based on mechanistic analysis and a focus on functional, not structural, approximation of native and engineered system. As proof of concept, the design, fabrication and evaluation of a tissue-engineered jellyfish medusa with biomimetic propulsion and feeding currents is presented with an emphasis on reasoning and strategy of the iterative design process. A range of experimental and modeling approaches accomplishes mechanistic analysis at multiple scales, control of individual and emergent cell behavior, and quantitative testing of functional performance. The main achievement of this thesis lies in presenting both conceptual framework and physical implementation of a systematic design strategy for muscular pumps and other bioinspired and tissue-engineered applications.
" }, { "name": "Pham, Anh Hoang", "degree": "PhD", "year": "2013", "title": "Tools for Assessing Mitochondrial Dynamics in Mouse Tissues and Neurodegenerative Models", "advisor": "Chan, David C.", "url": "https://resolver.caltech.edu/CaltechTHESIS:09012012-181931478", "creators": [ { "name": { "family": "Pham", "given": "Anh Hoang" }, "id": "Pham-Anh-Hoang", "display_name": "Pham, Anh Hoang" } ], "advisors": [ { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "advisor", "display_name": "Chan, David C." } ], "committee": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "chair", "display_name": "Fraser, Scott E." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "member", "display_name": "Chan, David C." } ], "option_major": [ "biology" ], "doi": "10.7907/E61Z-9C26", "abstract": "Mitochondria are dynamic organelles that undergo membrane fusion and fission and transport. The dynamic properties of mitochondria are important for regulating mitochondrial function. Defects in mitochondrial dynamics are linked to neurodegenerative diseases and affect the development of many tissues. To investigate the role of mitochondrial dynamics in diseases, versatile tools are needed to explore the physiology of these dynamic organelles in multiple tissues. Current tools for monitoring mitochondrial dynamics have been limited to studies in cell culture, which may be inadequate model systems for exploring the network of tissues. Here, we have generated mouse models for monitoring mitochondrial dynamics in a broad spectrum of tissues and cell types. The photoactivatable mitochondria (PhAMfloxed) line enables Cre-inducible expression of a mitochondrial targeted photoconvertible protein, Dendra2 (mito-Dendra2). In the PhAMexcised line, mito-Dendra2 is ubiquitously expressed to facilitate broad analysis of mitochondria at various developmental processes.
\r\n\r\nWe have utilized these models to study mitochondrial dynamics in the nigrostriatal circuit of Parkinson\u2019s disease (PD) and in the development of skeletal muscles. Increasing evidences implicate aberrant regulation of mitochondrial fusion and fission in models of PD. To assess the function of mitochondrial dynamics in the nigrostriatal circuit, we utilized transgenic techniques to abrogate mitochondrial fusion. We show that deletion of the Mfn2 leads to the degeneration of dopaminergic neurons and Parkinson\u2019s-like features in mice. To elucidate the dynamic properties of mitochondria during muscle development, we established a platform for examining mitochondrial compartmentalization in skeletal muscles. This model system may yield clues to the role of mitochondrial dynamics in mitochondrial myopathies.
" }, { "name": "Saxena, Abigail Green", "degree": "PhD", "year": "2013", "title": "Sulfur-Cycling in Methane-Rich Ecosystems: Uncovering Microbial Processes and Novel Niches", "advisor": "Orphan, Victoria J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05282013-062935856", "creators": [ { "name": { "family": "Saxena", "given": "Abigail Green" }, "id": "Saxena-Abigail-Green", "orcid": "0000-0002-8502-6589", "display_name": "Saxena, Abigail Green" } ], "advisors": [ { "name": { "family": "Orphan", "given": "Victoria J." }, "id": "Orphan-V-J", "role": "advisor", "display_name": "Orphan, Victoria J." } ], "committee": [ { "name": { "family": "Jensen", "given": "Grant J." }, "id": "Jensen-G-J", "role": "chair", "display_name": "Jensen, Grant J." }, { "name": { "family": "Orphan", "given": "Victoria J." }, "id": "Orphan-V-J", "role": "member", "display_name": "Orphan, Victoria J." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "member", "display_name": "Mazmanian, Sarkis K." }, { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "role": "member", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." } ], "option_major": [ "biology" ], "doi": "10.7907/Z9125QKD", "abstract": "Microbial sulfur cycling communities were investigated in two methane-rich ecosystems, terrestrial mud volcanoes (TMVs) and marine methane seeps, in order to investigate niches and processes that would likely be central to the functioning of these crucial ecosystems. Terrestrial mud volcanoes represent geochemically diverse habitats with varying sulfur sources and yet sulfur-cycling in these environments remains largely unexplored. Here we characterized the sulfur-metabolizing microorganisms and activity in 4 TMVs in Azerbaijan, supporting the presence of active sulfur-oxidizing and sulfate-reducing guilds in all 4 TMVs across a range of physiochemical conditions, with diversity of these guilds being unique to each TMV. We also found evidence for the anaerobic oxidation of methane coupled to sulfate reduction, a process which we explored further in the more tractable marine methane seeps. Diverse associations between methanotrophic archaea (ANME) and sulfate-reducing bacterial groups (SRB) often co-occur in marine methane seeps, however the ecophysiology of these different symbiotic associations has not been examined. Using a combination of molecular, geochemical and fluorescence in situ hybridization coupled to nano-scale secondary ion mass spectrometry (FISH-NanoSIMS) analyses of in situ seep sediments and methane-amended sediment incubations from diverse locations, we show that the unexplained diversity in SRB associated with ANME cells can be at least partially explained by preferential nitrate utilization by one particular partner, the seepDBB. This discovery reveals that nitrate is likely an important factor in community structuring and diversity in marine methane seep ecosystems. The thesis concludes with a study of the dynamics between ANME and their associated SRB partners. We inhibited sulfate reduction and followed the metabolic processes of the community as well as the effect of ANME/SRB aggregate composition and growth on a cellular level by tracking 15N substrate incorporation into biomass using FISH-NanoSIMS. We revealed that while sulfate-reducing bacteria gradually disappeared over time in incubations with an SRB inhibitor, the ANME archaea persisted in the form of ANME-only aggregates, which are capable of little to no growth when sulfate reduction is inhibited. These data suggest ANME are not able to synthesize new proteins when sulfate reduction is inhibited.
" }, { "name": "Schwarzkopf, Ma'ayan", "degree": "PhD", "year": "2013", "title": "Engineering Nucleic Acid Mechanisms for Regulation and Readout of Gene Expression: Conditional Dicer Substrate Formation and Sensitive Multiplexed Northern Blots", "advisor": "Pierce, Niles A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05212013-161536447", "creators": [ { "name": { "family": "Schwarzkopf", "given": "Ma'ayan" }, "id": "Schwarzkopf-Ma'ayan", "display_name": "Schwarzkopf, Ma'ayan" } ], "advisors": [ { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "role": "advisor", "display_name": "Pierce, Niles A." } ], "committee": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "chair", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "member", "display_name": "Baltimore, David L." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "role": "member", "display_name": "Pierce, Niles A." } ], "option_major": [ "biology" ], "doi": "10.7907/Z94Q7S0V", "abstract": "Nucleic acids are most commonly associated with the genetic code, transcription and gene expression. Recently, interest has grown in engineering nucleic acids for biological applications such as controlling or detecting gene expression. The natural presence and functionality of nucleic acids within living organisms coupled with their thermodynamic properties of base-pairing make them ideal for interfacing (and possibly altering) biological systems. We use engineered small conditional RNA or DNA (scRNA, scDNA, respectively) molecules to control and detect gene expression. Three novel systems are presented: two for conditional down-regulation of gene expression via RNA interference (RNAi) and a third system for simultaneous sensitive detection of multiple RNAs using labeled scRNAs.
\r\n\r\nRNAi is a powerful tool to study genetic circuits by knocking down a gene of interest. RNAi executes the logic: If gene Y is detected, silence gene Y. The fact that detection and silencing are restricted to the same gene means that RNAi is constitutively on. This poses a significant limitation when spatiotemporal control is needed. In this work, we engineered small nucleic acid molecules that execute the logic: If mRNA X is detected, form a Dicer substrate that targets independent mRNA Y for silencing. This is a step towards implementing the logic of conditional RNAi: If gene X is detected, silence gene Y. We use scRNAs and scDNAs to engineer signal transduction cascades that produce an RNAi effector molecule in response to hybridization to a nucleic acid target X. The first mechanism is solely based on hybridization cascades and uses scRNAs to produce a double-stranded RNA (dsRNA) Dicer substrate against target gene Y. The second mechanism is based on hybridization of scDNAs to detect a nucleic acid target and produce a template for transcription of a short hairpin RNA (shRNA) Dicer substrate against target gene Y. Test-tube studies for both mechanisms demonstrate that the output Dicer substrate is produced predominantly in the presence of a correct input target and is cleaved by Dicer to produce a small interfering RNA (siRNA). Both output products can lead to gene knockdown in tissue culture. To date, signal transduction is not observed in cells; possible reasons are explored.
\r\n \r\nSignal transduction cascades are composed of multiple scRNAs (or scDNAs). The need to study multiple molecules simultaneously has motivated the development of a highly sensitive method for multiplexed northern blots. The core technology of our system is the utilization of a hybridization chain reaction (HCR) of scRNAs as the detection signal for a northern blot. To achieve multiplexing (simultaneous detection of multiple genes), we use fluorescently tagged scRNAs. Moreover, by using radioactive labeling of scRNAs, the system exhibits a five-fold increase, compared to the literature, in detection sensitivity. Sensitive multiplexed northern blot detection provides an avenue for exploring the fate of scRNAs and scDNAs in tissue culture.
\r\n" }, { "name": "Shemorry, Anna", "degree": "PhD", "year": "2013", "title": "Studies of the N-End Rule Pathway in Saccharomyces cerevisiae", "advisor": "Varshavsky, Alexander J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01282013-132123935", "creators": [ { "name": { "family": "Shemorry", "given": "Anna" }, "id": "Shemorry-Anna", "display_name": "Shemorry, Anna" } ], "advisors": [ { "name": { "family": "Varshavsky", "given": "Alexander J." }, "id": "Varshavsky-A-J", "role": "advisor", "display_name": "Varshavsky, Alexander J." } ], "committee": [ { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "role": "chair", "display_name": "Campbell, Judith L." }, { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "role": "member", "display_name": "Dunphy, William G." }, { "name": { "family": "Shan", "given": "Shu-ou" }, "id": "Shan-Shu-ou", "role": "member", "display_name": "Shan, Shu-ou" }, { "name": { "family": "Varshavsky", "given": "Alexander J." }, "id": "Varshavsky-A-J", "role": "member", "display_name": "Varshavsky, Alexander J." } ], "option_major": [ "biology" ], "doi": "10.7907/09SW-FR88", "abstract": "Many intracellular proteins are either conditionally or constitutively short-lived, with in vivo half-lives that can be as brief as a few minutes. The regulated and processive degradation of intracellular proteins is carried out largely by the ubiquitin (Ub)-proteasome system (UPS). In eukaryotes, the N-end rule pathway is a part of the UPS. The N-end rule relates the regulation of the in vivo half-life of a protein to the identity of its N-terminal residue. Degradation signals (degrons) that are targeted by the N-end rule pathway include a set called N-degrons. E3 Ub ligases of the N-end rule pathway are called N-recognins. They bind to primary destabilizing N-terminal residues of N-end rule substrates. The N-end rule pathway comprises two major branches, the Arg/N-end rule pathway and the Ac/N-end rule pathway.
\r\n\r\nThe Arg/N-end rule branch involves the N-terminal arginylation of protein substrates and also the targeting of specific unmodified N-terminal residues by E3 N-recognins. The S. cerevisiae Arg/N-end rule pathway contains a single N-recognin, Ubr1. The Ub-fusion degradation (UFD) pathway is also a part of the UPS. This pathway recognizes a \"nonremovable\" N-terminal Ub moiety of a Ub fusion as a primary degron. My collaborator, Cheol-Sang Hwang, and I demonstrated that the RING-type Ubr1 E3 and the HECT-type Ufd4 E3 interact, both physically and functionally. We showed that the Ubr1-Ufd4 complex targets the S. cerevisiae Mgt1 DNA repair enzyme through a degron near its N-terminus, in addition to mediating the Arg/N-end rule pathway and a part of the UFD pathway as well. We also further characterized the physical interaction between Ubr1 and Ufd4.
\r\n\r\nI also report the discovery of the other branch of the N-end rule pathway, the Ac/N-end rule pathway, which recognizes N-terminally acetylated residues as N-degrons, termed Ac/N-degrons. We showed that Ac/N-degrons are recognized by the Doa10 E3 Ub ligase and apparently by other E3s as well. Given the prevalence of Ac/N-degrons, as nearly 90% of human proteins are Nt-acetylated, we also demonstrated the physiological role of Ac/N-degrons in protein quality, including the regulation of input stoichiometries of subunits in oligomeric proteins.
\r\n" }, { "name": "Tetreault, Nicole Anne", "degree": "PhD", "year": "2013", "title": "Microglia in the Cerebral and Cerebellar Cortices in Individuals with Autism", "advisor": "Allman, John Morgan; Wold, Barbara J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06072013-005232013", "creators": [ { "name": { "family": "Tetreault", "given": "Nicole Anne" }, "id": "Tetreault-Nicole-Anne", "display_name": "Tetreault, Nicole Anne" } ], "advisors": [ { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "advisor", "display_name": "Allman, John Morgan" }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "co-advisor", "display_name": "Wold, Barbara J." } ], "committee": [ { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "role": "chair", "display_name": "Adolphs, Ralph" }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" } ], "option_major": [ "biology" ], "doi": "10.7907/VGPA-4D87", "abstract": "In this thesis, we explore the density of the microglia in the cerebral and cerebellar cortices of individuals with autism to investigate the hypothesis that neuroinflammation is involved in autism. We describe in our findings an increase in microglial density in two disparate cortical regions, frontal insular cortex and visual cortex, in individuals with autism (Tetreault et al., 2012). Our results imply that there is a global increase in the microglial density and neuroinflammation in the cerebral cortex of individuals with autism.
\r\n\r\nWe expanded our cerebellar study to additional neurodevelopmental disorders that exhibit similar behaviors to autism spectrum disorder and have known cerebellar pathology. We subsequently found a more than threefold increase in the microglial density specific to the molecular layer of the cerebellum, which is the region of the Purkinje and parallel fiber synapses, in individuals with autism and Rett syndrome. Moreover, we report that not only is there an increase in microglia density in the molecular layer, the microglial cell bodies are significantly larger in perimeter and area in individuals with autism spectrum disorder and Rett syndrome compared to controls that implies that the microglia are activated. Additionally, an individual with Angelman syndrome and the sibling of an individual with autism have microglial densities similar to the individuals with autism and Rett syndrome. By contrast, an individual with Joubert syndrome, which is a developmental hypoplasia of the cerebellar vermis, had a normal density of microglia, indicating the specific pathology in the cerebellum does not necessarily result in increased microglial densities. We found a significant decrease in Purkinje cells specific to the cerebellar vermis in individuals with autism.
\r\n\r\nThese findings indicate the importance for investigation of the Purkinje synapses in autism and that the relationship between the microglia and the synapses is of great utility in understanding the pathology in autism. Together, these data provide further evidence for the neuroinflammation hypothesis in autism and a basis for future investigation of neuroinflammation in autism. In particular, investigating the function of microglia in modifying synaptic connectivity in the cerebellum may provide key insights into developing therapeutics in autism spectrum disorder.
" }, { "name": "Venturelli, Ophelia Shalini", "degree": "PhD", "year": "2013", "title": "Role of Feedback and Dynamics of a Gene Regulatory Network", "advisor": "Murray, Richard M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06072013-095239958", "creators": [ { "name": { "family": "Venturelli", "given": "Ophelia Shalini" }, "id": "Venturelli-Ophelia-Shalini", "display_name": "Venturelli, Ophelia Shalini" } ], "advisors": [ { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "advisor", "display_name": "Murray, Richard M." } ], "committee": [ { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "chair", "display_name": "Phillips, Robert B." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "orcid": "0000-0002-3671-9354", "role": "member", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "El-Samad", "given": "Hana" }, "id": "El-Samad-H", "role": "member", "display_name": "El-Samad, Hana" }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "orcid": "0000-0002-5785-7481", "role": "member", "display_name": "Murray, Richard M." } ], "option_major": [ "biochem" ], "doi": "10.7907/WGK3-Y839", "abstract": "Cells exhibit a diverse repertoire of dynamic behaviors. These dynamic functions are implemented by circuits of interacting biomolecules. Although these regulatory networks function deterministically by executing specific programs in response to extracellular signals, molecular interactions are inherently governed by stochastic fluctuations. This molecular noise can manifest as cell-to-cell phenotypic heterogeneity in a well-mixed environment. Single-cell variability may seem like a design flaw but the coexistence of diverse phenotypes in an isogenic population of cells can also serve a biological function by increasing the probability of survival of individual cells upon an abrupt change in environmental conditions. Decades of extensive molecular and biochemical characterization have revealed the connectivity and mechanisms that constitute regulatory networks. We are now confronted with the challenge of integrating this information to link the structure of these circuits to systems-level properties such as cellular decision making. To investigate cellular decision-making, we used the well studied galactose gene-regulatory network in Saccharomyces cerevisiae. We analyzed the mechanism and dynamics of the coexistence of two stable on and off states for pathway activity. We demonstrate that this bimodality in the pathway activity originates from two positive feedback loops that trigger bistability in the network. By measuring the dynamics of single-cells in a mixed sugar environment, we observe that the bimodality in gene expression is a transient phenomenon. Our experiments indicate that early pathway activation in a cohort of cells prior to galactose metabolism can accelerate galactose consumption and provide a transient increase in growth rate. Together these results provide important insights into strategies implemented by cells that may have been evolutionary advantageous in competitive environments. \r\n" }, { "name": "Zhao, Jimmy Liu", "degree": "PhD", "year": "2013", "title": "Function of MicroRNA-146a and NF-\u03baB in Physiologic and Pathologic Hematopoiesis", "advisor": "Baltimore, David L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04122013-165433477", "creators": [ { "name": { "family": "Zhao", "given": "Jimmy Liu" }, "id": "Zhao-Jimmy-Liu", "display_name": "Zhao, Jimmy Liu" } ], "advisors": [ { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "advisor", "display_name": "Baltimore, David L." } ], "committee": [ { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "chair", "display_name": "Patterson, Paul H." }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "member", "display_name": "Mazmanian, Sarkis K." }, { "name": { "family": "Rao", "given": "Dinesh S." }, "id": "Rao-Dinesh-S", "role": "member", "display_name": "Rao, Dinesh S." }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "member", "display_name": "Baltimore, David L." } ], "option_major": [ "biology" ], "doi": "10.7907/99A5-DR49", "abstract": "During inflammation and infection, hematopoietic stem and progenitor cells (HSPCs) are stimulated to proliferate and differentiate into mature immune cells, especially of the myeloid lineage. MicroRNA-146a (miR-146a) is a critical negative regulator of inflammation. Deletion of the gene encoding miR-146a\u2014expressed in all blood cell types\u2014produces effects that appear as dysregulated inflammatory hematopoiesis, leading to a decline in the number and quality of hematopoietic stem cells (HSCs), excessive myeloproliferation, and, ultimately, to exhaustion of the HSCs and hematopoietic neoplasms. Six-week-old deleted mice are normal, with no effect on cell numbers, but by 4 months bone marrow hypercellularity can be seen, and by 8 months marrow exhaustion is becoming evident. The ability of HSCs to replenish the entire hematopoietic repertoire in a myelo-ablated mouse also declines precipitously as miR-146a-deficient mice age. In the absence of miR-146a, LPS-mediated serial inflammatory stimulation accelerates the effects of aging. This chronic inflammatory stress on HSCs in deleted mice involves a molecular axis consisting of upregulation of the signaling protein TRAF6 leading to excessive activity of the transcription factor NF-\u03baB and overproduction of the cytokine IL-6. At the cellular level, transplant studies show that the defects are attributable to both an intrinsic problem in the miR-146a-deficient HSCs and extrinsic effects of miR-146a-deficient lymphocytes and non-hematopoietic cells. This study has identified a microRNA, miR-146a, to be a critical regulator of HSC homeostasis during chronic inflammatory challenge in mice and has provided a molecular connection between chronic inflammation and the development of bone marrow failure and myeloproliferative neoplasms. This may have implications for human hematopoietic malignancies, such as myelodysplastic syndrome, which frequently displays downregulated miR-146a expression. " }, { "name": "Bryan, Ronald Edward", "degree": "PhD", "year": "2012", "title": "Distance Based Visual Cues to Interpersonal Trust", "advisor": "Adolphs, Ralph; Perona, Pietro", "url": "https://resolver.caltech.edu/CaltechTHESIS:06042012-095743080", "creators": [ { "name": { "family": "Bryan", "given": "Ronald Edward" }, "id": "Bryan-Ronald-Edward", "display_name": "Bryan, Ronald Edward" } ], "advisors": [ { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "advisor", "display_name": "Adolphs, Ralph" }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "co-advisor", "display_name": "Perona, Pietro" } ], "committee": [ { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "chair", "display_name": "Perona, Pietro" }, { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "member", "display_name": "Adolphs, Ralph" }, { "name": { "family": "Camerer", "given": "Colin F." }, "id": "Camerer-C-F", "orcid": "0000-0003-4049-1871", "role": "member", "display_name": "Camerer, Colin F." }, { "name": { "family": "Tsao", "given": "Doris Y." }, "id": "Tsao-D-Y", "orcid": "0000-0003-1083-1919", "role": "member", "display_name": "Tsao, Doris Y." }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "member", "display_name": "Koch, Christof" } ], "option_major": [ "cns" ], "doi": "10.7907/Z2N2-4C56", "abstract": "This thesis examines the role of interpersonal spacing in determining the visual appearance and emotional response to images of faces. We present new methods for isolating the distance-dependent perspective projection as a visual feature, while controlling for confounding variables such as emotional expression. In behavioral experiments, we demonstrate the relevance of viewing distance to implicit social judgments, notably trust behavior in which real money was at stake. Finally, we provide tools for classifying face images according to viewing distance, and manipulating face images to simulate their appearance at different distances and different levels of trustworthiness. " }, { "name": "Chang, Kuang-Jung", "degree": "PhD", "year": "2012", "title": "A Role for Protein Kinase Dbf2-Mob1 in Mitotic Exit", "advisor": "Deshaies, Raymond Joseph", "url": "https://resolver.caltech.edu/CaltechTHESIS:06112012-092401902", "creators": [ { "name": { "family": "Chang", "given": "Kuang-Jung" }, "id": "Chang-Kuang-Jung", "display_name": "Chang, Kuang-Jung" } ], "advisors": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "advisor", "display_name": "Deshaies, Raymond Joseph" } ], "committee": [ { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "chair", "display_name": "Zinn, Kai George" }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Varshavsky", "given": "Alexander J." }, "id": "Varshavsky-A-J", "role": "member", "display_name": "Varshavsky, Alexander J." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "member", "display_name": "Deshaies, Raymond Joseph" } ], "option_major": [ "biology" ], "doi": "10.7907/Z92F7KFT", "abstract": "Exit from mitosis is characterized by precise control of the cyclin-dependent kinase complex (Cdk) activity, breaking down mitotic structures, and completing cytokinesis. In Saccaromyces cerevisiae, protein phosphotase Cdc14 is involved in counteracting mitotic-Cdk activity by promoting the degradation of mitotic cyclin Clb2, and stabilizing the Cdc28 inhibitor Sic1. The activity and the cellular localization of Cdc14 are tightly regulated by the cell cycle. Cdc14 is sequestered and inhibited in the nucleolus by forming the RENT (Regulator of Nucleolar Silencing and Teleophase) complex for most of the cell cycle. It is released and distributed into the cytoplasm in anaphase and telophase, and then returns to the nucleolus in G1 phase. Activation of Cdc14 is achieved via multi-site phosphorylation of Net1 leading to the release of Cdc14. Net1 is first phosphorylated by the Fourteen Early Anaphase Release (FEAR) network, and then by the Mitotic Exit Network (MEN).
\r\n \r\nIn this thesis, we show that a MEN component, protein kinase Dbf2-Mob1, plays a role in phosphorylating Net1 in late anaphase. We identified the effective Dbf2-Mob1 phosphorylation sites in the N-terminal of Net1 by in vitro kinase reaction assay. We found that cells that express mutant Net1 show growth defects and chain-like terminal morphology under restrictive temperatures. Genetic interactions suggested that the MEN kinases and Cdc14 are related to the cell cycle defects caused by the phosphosite mutated Net1. Analyzing the phosphosite mutants with Fluorecence-activated cell sorting (FACS) and immunofluorescence assay, we found that the growth defects and the abnormal cell morphology are due to a defect in releasing Cdc14 in late anaphase, leading to disruption of mitotic exit. This result is further confirmed by western blot assay and the beads releasing assay.
\r\n\r\nIn summary, the regulation of Cdc14 release in late anaphase via phosphorylation of its inhibitor Net1 by Dbf2-Mob1 is demonstrated in this work. This thesis provides a crucial piece of information that furthers our understanding of the mechanism of mitotic exit. It also points to a fascinating mechanism of controlling cytokinesis and meiosis by regulating Net1 phosphorylation by the MEN.
\r\n" }, { "name": "Chaudhuri, Aadel Ahmed", "degree": "PhD", "year": "2012", "title": "MicroRNAs 155 and 125b Physiologically and Pathologically Regulate Hematopoiesis and Immunity", "advisor": "Baltimore, David L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:09042011-230002591", "creators": [ { "name": { "family": "Chaudhuri", "given": "Aadel Ahmed" }, "id": "Chaudhuri-Aadel-Ahmed", "display_name": "Chaudhuri, Aadel Ahmed" } ], "advisors": [ { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "advisor", "display_name": "Baltimore, David L." } ], "committee": [ { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "chair", "display_name": "Zinn, Kai George" }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "member", "display_name": "Mazmanian, Sarkis K." }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." }, { "name": { "family": "Shan", "given": "Shu-ou" }, "id": "Shan-Shu-ou", "role": "member", "display_name": "Shan, Shu-ou" }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "member", "display_name": "Baltimore, David L." } ], "option_major": [ "biology" ], "doi": "10.7907/HFSQ-8278", "abstract": "MicroRNAs are a class of ~22 nucleotide RNA molecules with roles in diverse biological processes. Here I focus on two microRNAs, miR-155 and miR-125b, and reveal pathways by which their dysregulation leads to myeloproliferative disorder (MPD) and leukemia, respectively. I begin by searching for miR-155 target genes relevant to MPD. By writing an algorithm to search microarray data for predicted microRNA target genes, I identified 89 candidate target genes for miR-155 in myeloid cells. Literature search whittled this list down to 11, and one gene among them, SHIP1, turned out to be largely responsible for miR-155\u2019s ability to cause MPD. My focus shifted to miR-125b when I noticed that miR-125b was enriched in macrophages and thus might play important roles in that cell type. Indeed, gain- and loss-of-function experiments indicated that miR-125b is a potent activator of macrophage activation, and I identified IRF4 as the primary target gene in this process. Finally I asked whether miR-125b plays pathophysiological roles in the development of the hematopoietic system. Thus I overexpressed miR-125b in the hematopoietic system and, to my surprise, observed a very aggressive myeloid leukemia capable of infiltrating peripheral organs including the lungs, liver, kidneys and brain. To determine whether miR-125b is physiologically necessary for normal hematopoietic development, I designed a loss-of-function sponge vector that acts as a decoy, attracting the microRNA away from its normal targets. Use of the sponge in the mouse hematopoietic system led to significantly decreased overall hematopoietic ouput, indicating that miR-125b is physiologically required for normal hematopoiesis. Next, I assayed in vitro a panel of miR-125b target genes and saw that one, Lin28, was superior to the rest. Indeed, Lin28 gain- and loss-of-function in vivo recapitulated major aspects of miR-125b loss- and gain-of-function, respectively. Thus I identified Lin28 as a primary target of miR-125b in the hematopoietic system. In summary, my work shows that two microRNAs, miR-155 and miR-125b, physiologically and pathologically control hematopoietic development. I also identify important target genes for each of these microRNAs in their respective disease processes. Indeed, therapeutic targeting of these pathways may prove useful in the treatment of cancer." }, { "name": "Choe, Andrea", "degree": "PhD", "year": "2012", "title": "Pheromones in Free-Living and Parasitic Nematodes", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:07042011-125318322", "creators": [ { "name": { "family": "Choe", "given": "Andrea" }, "id": "Choe-Andrea", "display_name": "Choe, Andrea" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "chair", "display_name": "Wold, Barbara J." }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "member", "display_name": "Mazmanian, Sarkis K." }, { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "role": "member", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Platzer", "given": "Edward G." }, "id": "Platzer-E-G", "role": "member", "display_name": "Platzer, Edward G." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/DX46-C462", "abstract": "Nematodes are among the most diverse phyla of animals, occupying almost every ecological niche available. Their ubiquity has led to a number of problems for civilization, including the loss of crops and the spread of neglected tropical diseases. Because they are responsible for a broad range of agricultural and human diseases, many pheromone-mediated nematode behaviors have been described but very few pheromones have been identified.
\r\n\r\nWe report, via high-performance liquid chromatography electrospray ionization mass spectrometry, the discovery that many free-living and parasitic nematodes secrete small-molecule pheromones called ascarosides. These pheromones, called ascarosides, were first found to play a role in sex attraction and induction into a stress-resistant diapausal life stage in the free-living organism, Caenorhabditis elegans. We have performed a double-blind purification of the female sex pheromone in the sour paste nematode Panagrellus redivivus and report that the female sex pheromone is composed of at least two ascarosides. We have also found that both free-living and parasitic nematodes respond to different concentrations of ascarosides through attraction or repulsion, demonstrating cross-species communication. These results suggest that ascarosides could be a universal nematode cue, similar to the role of N-Acyl homoserine lactones in bacteria quorum sensing.
\r\n\r\nBecause ascarosides are nonvolatile, they can only mediate close-range communication. Nematodes have a well-characterized capacity for long-range chemoattraction to a range of volatile cues. However, no studies have been done towards characterizing natural volatile cues derived from nematodes. Here I describe the discovery of volatile cues are produced by male-female species in the genus Caenorhabditis, but are lacking in the hermaphroditic species C. elegans, C. briggsae, and C. sp11. These volatile cues attract males (and sometimes females) from other Caenorhabditis species, demonstrating a cross-species gonochoristic cue.
\r\n" }, { "name": "Dixson, Alana Doreen", "degree": "PhD", "year": "2012", "title": "Zebrafish Magnetite and Long-lived Rohon-Beard Neurons: Expanding Our View of Two Zebrafish Sensory Systems in Development and Adulthood", "advisor": "Kirschvink, Joseph L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02252012-093603144", "creators": [ { "name": { "family": "Dixson", "given": "Alana Doreen" }, "id": "Dixson-Alana-Doreen", "display_name": "Dixson, Alana Doreen" } ], "advisors": [ { "name": { "family": "Kirschvink", "given": "Joseph L." }, "id": "Kirschvink-J-L", "orcid": "0000-0001-9486-6689", "role": "advisor", "display_name": "Kirschvink, Joseph L." } ], "committee": [ { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "chair", "display_name": "Zinn, Kai George" }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "orcid": "0000-0002-7371-4675", "role": "member", "display_name": "Prober, David A." }, { "name": { "family": "Kirschvink", "given": "Joseph L." }, "id": "Kirschvink-J-L", "orcid": "0000-0001-9486-6689", "role": "member", "display_name": "Kirschvink, Joseph L." }, { "name": { "family": "Raub", "given": "Timothy D." }, "id": "Raub-T-D", "orcid": "0000-0002-7471-0246", "role": "member", "display_name": "Raub, Timothy D." } ], "option_major": [ "biology" ], "doi": "10.7907/Y9K0-NE54", "abstract": "During embryogenesis, the central nervous system (CNS) transforms from what seems like an amorphous mass of cells to a rod-like structure, and then to a fully functional and complex system of tissues composed of multiple cell types. Using confocal laser scanning microscopy (CLSM), I demonstrate a modified version of in toto imaging to track normal spinal cord organization in zebrafish from bud stage, ~ 11 hours post-fertilization (hpf), to 48 hpf. I also assisted in identifying several transgenic lines using a gene trap vector, the FlipTrap (FT), which creates a normally localized and functional fluorescent fusion protein for in vivo analysis of gene expression throughout development. I used two FT lines along with a modified version of in toto imaging to study sensory cells in the dorsal spinal cord.
\r\n\r\nWith the FT tool, I discovered a subset of Rohon-Beard (RB) neurons that perdures into the adult. These uniquely transient chemo- and mechano-sensory cells have been well characterized in the dorsal spinal cord of lower vertebrates; however, the notion of persistent RBs contrasts with dogma suggesting that the entire population disappears during the early larval period. The coexistence of RB-like neurons with dorsal root ganglia (DRG) suggests that zebrafish have two post-embryonic sensory systems, challenging the previous notion that only peripheral sensory neurons survive.
\r\n\r\nIn the second part of my dissertation I describe my studies of biogenic magnetite which has been detected in a broad range of organisms, including magnetotactic bacteria, migratory fish and birds, invertebrates, and humans. Magnetite mediates magnetosensation in many species through the effects of pulse-remagnetization on behavior. The mechanisms of magnetite biomineralization are not well characterized in higher organisms. Previous studies have shown deposits of magnetite in projections of the trigeminal nerve, alongside behavioral evidence suggesting that both optical pumping and magnetite-based mechanisms may operate simultaneously. Subsequent efforts to identify the anatomical seat of magnetoreceptors have focused on the same locations in new organisms, excluding other areas. Here I report the unexpected presence of biogenic magnetite in the lateral line region of the genetically and physiologically tractable vertebrate model organism, Danio rerio.
\r\n" }, { "name": "Dobro, Megan J.", "degree": "PhD", "year": "2012", "title": "The Structural Biology of HIV Budding and Maturation", "advisor": "Jensen, Grant J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06012012-133153407", "creators": [ { "name": { "family": "Dobro", "given": "Megan J." }, "id": "Dobro-Megan-J", "display_name": "Dobro, Megan J." } ], "advisors": [ { "name": { "family": "Jensen", "given": "Grant J." }, "id": "Jensen-G-J", "role": "advisor", "display_name": "Jensen, Grant J." } ], "committee": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "role": "chair", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "member", "display_name": "Chan, David C." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Jensen", "given": "Grant J." }, "id": "Jensen-G-J", "role": "member", "display_name": "Jensen, Grant J." } ], "option_major": [ "biology" ], "doi": "10.7907/X6XW-5303", "abstract": "The Human Immunodeficiency Virus (HIV) depends on the ability to exit infected cells, mature into an infectious state, and infect new host cells. The structural details of exiting and maturation (known as the \"late stage events\") remain elusive, but further understanding could lead to new therapies. HIV exits cells by hijacking a host cellular complex called ESCRT (Endosomal Sorting Complex Required for Transport), which evolved to constrict membranes in multivesicular body formation and cytokinesis. Electron cryotomography (ECT) was used to gain three-dimensional images of ESCRT in several contexts, including the physiological system of archaeal cell division. This study provided insight into the monomer interactions in the complex and led to a molecular mechanism of membrane constriction.
\r\n\r\nHIV is released from the cell as an immature particle with the main structural protein, Gag, forming a spherical shell around the RNA genome and enzymes. Gag is then cleaved into several proteins that refold and assemble into the conical capsid that is characteristic of the mature, infectious particle. The capsid is typically a closed cone, but unclosed varieties provide insight to the mechanism of assembly. By combining ECT, computer simulations, and fluorescence light microscopy, we analyzed features of unclosed capsids that suggest a \"curling sheet\" model of capsid assembly. These studies not only provided novel insight into the late stages of the HIV life cycle, but also contributed to the methods used by electron microscopists and researchers of HIV.
\r\n" }, { "name": "Frazier, Shawnalea Jimee", "degree": "PhD", "year": "2012", "title": "Optimization of the GluC1/IVM Neuronal Silencing Tool via Protein Engineering", "advisor": "Lester, Henry A.; Dougherty, Dennis A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05252012-121406958", "creators": [ { "name": { "family": "Frazier", "given": "Shawnalea Jimee" }, "id": "Frazier-Shawnalea-Jimee", "display_name": "Frazier, Shawnalea Jimee" } ], "advisors": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "advisor", "display_name": "Lester, Henry A." }, { "name": { "family": "Dougherty", "given": "Dennis A." }, "id": "Dougherty-D-A", "role": "co-advisor", "display_name": "Dougherty, Dennis A." } ], "committee": [ { "name": { "family": "Shan", "given": "Shu-ou" }, "id": "Shan-Shu-ou", "role": "chair", "display_name": "Shan, Shu-ou" }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Dougherty", "given": "Dennis A." }, "id": "Dougherty-D-A", "role": "member", "display_name": "Dougherty, Dennis A." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." } ], "option_major": [ "biochem" ], "doi": "10.7907/4AGK-FP05", "abstract": "A variety of genetically encoded tools have been developed for deciphering the neural circuitry of the brain. Such tools allow physical manipulation of neuronal excitability in a reversible, cell-specific manner, enabling researchers to establish how electrical activity and connectivity facilitate the information processing that mediates perception and drives behavior. An expanding toolkit of engineered neuroreceptors, particularly those actuated by orthogonal pharmacological ligands, provide noninvasive manipulation of regional or disperse neuronal populations with adequate spatiotemporal precision and great potential for multiplexing. We previously engineered an invertebrate glutamate-gated chloride channel (GluCl \u03b1\u03b2) that enabled pharmacologically induced silencing of electrical activity in targeted CNS neurons in vivo by the anthelmintic drug compound ivermectin (IVM; Lerchner et al., 2007). With this receptor, GluCl opt \u03b1-CFP + opt \u03b2-YFP Y182F, the concentration of IVM necessary to elicit a consistent silencing phenotype was higher than expected, raising concern about its potential side effects. Considerable variability in the extent of spike suppression was also apparent and was attributed to variable co-expression levels of \u03b1 and \u03b2 subunits. Thus, a rational protein engineering strategy was employed to optimize the GluCl/IVM tool. To increase agonist sensitivity, a gain-of-function gating mutation involving the highly conserved leucine 9\u2019 residue of the \u03b1 pore-lining M2 transmembrane domain was introduced. Various mutations at this position facilitate channel opening in the absence and presence of ligand. Analysis of side chain properties revealed that helix-destabilizing energy correlated with increases in agonist sensitivity. One mutation, L9\u2019F, enhances \u03b2 subunit incorporation to substantially increase IVM sensitivity without permitting unliganded channel opening. Removal of an arginine-based ER retention motif (RSR_AAA) from the intracellular loop of \u03b2 promoted plasma membrane expression of heteromeric GluCl \u03b1\u03b2 by preventing ER-associated degradation of the \u03b2 subunit. An additional monomeric XFP mutation complements these effects. The newly engineered GluCl opt \u03b1-mXFP L9\u2019F + opt \u03b2-mXFP Y182F RSR_AAA receptor significantly increases conductance and reduces variability in evoked spike generation in vitro using a lower concentration of IVM. This receptor, dubbed \u2018GluClv2.0\u2019, is an improved tool for IVM-induced silencing." }, { "name": "Garcia, Mayra", "degree": "PhD", "year": "2012", "title": "Dorsal-Ventral Patterning and Gene Regulation in the Early Embryo of Drosophila melanogaster", "advisor": "Stathopoulos, Angelike", "url": "https://resolver.caltech.edu/CaltechTHESIS:04142011-122818148", "creators": [ { "name": { "family": "Garcia", "given": "Mayra" }, "id": "Garcia-Mayra", "display_name": "Garcia, Mayra" } ], "advisors": [ { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "advisor", "display_name": "Stathopoulos, Angelike" } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Parker", "given": "Carl Stevens" }, "id": "Parker-C-S", "role": "member", "display_name": "Parker, Carl Stevens" }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "member", "display_name": "Stathopoulos, Angelike" } ], "option_major": [ "biology" ], "doi": "10.7907/GFQK-GE02", "abstract": "In order for an embryo to develop and form properly, the anterior-posterior and dorsal-ventral axes must be specified. This is accomplished by controlled regulation of gene expression that allows for the activation and repression of tissue specific genes. Patterning of the Drosophila dorsal-ventral axis is an excellent model for understanding how axis specification is controlled. The dorsal-ventral axis is patterned by a nuclear gradient of Dorsal that is highest in ventral regions of the embryo. Dorsal activates genes in a concentration dependent manner to establish early patterning of the embryo. The patterns are refined by interactions between Dorsal and other activators as well as repressors in both dorsal and ventral regions of the embryo. Until recently there was only evidence for repressors acting in ventral regions of the embryo but our studies and other recent studies have provided evidence to suggest that several repressors act in dorsal regions of the embryo to refine Dorsal target genes. Here we show that an element, the A-box, previously identified in the cis-regulatory module (CRM) of the gene intermediate neuroblast defective (ind), is necessary and sufficient to mediate dorsal repression and is also involved in activation of ind. We conducted an affinity chromatography assay and identified factors that bound the A-box element. One of the factors that bound to this element, Grh, activates ind. We also identified several chromatin-remodeling factors that may function to silence ind in dorsal regions of the embryo. Our results also indicate that a second tier of repression that is independent of the A-box element, mediates repression of ind via Dpp-signaling. We extended our studies to the CRMs of ventral neuroblast defective (vnd) and short gastrulation (sog). Using a chimeric CRM repression assay, we found that strong and weak dorsal repression are also mediated by the vnd and sog CRMs, respectively. This suggests that limiting amounts of Dorsal are not sufficient to establish the dorsal borders of dorsal-ventral patterning genes as was previously believed, and rather, repressors are used to establish these borders. " }, { "name": "Hinz, Flora Irma", "degree": "PhD", "year": "2012", "title": "Genetically Restricted Metabolic Labeling in Danio rerio, a Simple Vertebrate Capable of Protein Synthesis-Dependent Memory Formation", "advisor": "Schuman, Erin Margaret", "url": "https://resolver.caltech.edu/CaltechTHESIS:04182012-045031338", "creators": [ { "name": { "family": "Hinz", "given": "Flora Irma" }, "id": "Hinz-Flora-Irma", "display_name": "Hinz, Flora Irma" } ], "advisors": [ { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "advisor", "display_name": "Schuman, Erin Margaret" } ], "committee": [ { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "orcid": "0000-0002-7371-4675", "role": "chair", "display_name": "Prober, David A." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "member", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "member", "display_name": "Schuman, Erin Margaret" } ], "option_major": [ "biology" ], "doi": "10.7907/P3VP-B991", "abstract": "Determining which neural circuits and proteins are involved in encoding memories is a central goal in neuroscience. Protein expression in the nervous system is known to undergo regulated changes in response to changes in behavioral states, in particular long-term memory formation. In this study we developed tools to investigate protein synthesis in an intact organism, the larval zebrafish, capable of simple learning behavior. Methods have recently been developed (BONCAT and FUNCAT), which introduce noncanonical amino acids bearing small bioorthogonal functional groups into proteins using the cells\u2019 own translational machinery. Using the selective \u2018click reaction\u2019, this allows for the identification and visualization of newly synthesized proteins in vitro.
\r\n\r\nHere we demonstrate that noncanonical amino acid labeling can be achieved in vivo in the larval zebrafish. We show that azidohomoalanine is metabolically incorporated into newly synthesized proteins, in a time- and concentration-dependent manner, but has no apparent toxic effect and does not influence simple behaviors such as spontaneous swimming and escape responses. This enables fluorescent labeling of newly synthesized proteins in whole mount larval zebrafish. Furthermore, we demonstrate that genetically restricted expression of a mutant methionyl-tRNA synthetase permits cell-specific metabolic labeling with the larger noncanonical amino acid, azidonorleucine, both in vitro and in vivo. Finally, we present an associative conditioning paradigm for larval zebrafish. During a three-hour training period, 6-8dpf larvae learn to associate the social reward of visual access to a group of conspecifics with a dark environment. The memory formed during this place-conditioning paradigm undergoes rapid extinction, but is extremely stable, lasting for up to 36h. Furthermore, memory formation is both protein synthesis- and partially NMDAR-dependent. Together, the techniques developed in this study will enable the investigation of protein synthesis during long-term memory formation in the larval zebrafish.
\r\n" }, { "name": "Johnson, Stephanie Lynn", "degree": "PhD", "year": "2012", "title": "DNA Mechanics and Transcriptional Regulation in the E. coli lac Operon", "advisor": "Phillips, Robert B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05112012-140027276", "creators": [ { "name": { "family": "Johnson", "given": "Stephanie Lynn" }, "id": "Johnson-Stephanie-Lynn", "display_name": "Johnson, Stephanie Lynn" } ], "advisors": [ { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "advisor", "display_name": "Phillips, Robert B." } ], "committee": [ { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "chair", "display_name": "Rees, Douglas C." }, { "name": { "family": "Wang", "given": "Zhen-Gang" }, "id": "Wang-Zhen-Gang", "orcid": "0000-0002-3361-6114", "role": "member", "display_name": "Wang, Zhen-Gang" }, { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "member", "display_name": "Pierce, Niles A." }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." } ], "option_major": [ "biochem" ], "doi": "10.7907/W40T-PD39", "abstract": "Many gene regulatory motifs in both prokaryotes and eukaryotes involve physical manipulations of the genetic material, often on length scales short enough that the mechanical properties of the DNA significantly impact gene expression. One class of such manipulations, called \u201caction at a distance\u201d, includes transcription factor-mediated DNA looping, in which a binding site some distance away on the DNA is brought into close proximity with the transcription machinery at the promoter. DNA looping is a key component of several important regulatory systems in bacteria, and is crucial to the combinatorial control that is common at eukaryotic promoters regulated by more transcription factors than can physically bind adjacent to the promoter. Here we use a prototypical DNA looping protein, the Lac repressor from E. coli, to explore questions regarding the role of DNA mechanics in DNA looping and combinatorial control, particularly concerning the role of sequence flexibility in short-length-scale looping. We combine a statistical mechanical model of looping by the Lac repressor with a single-molecule technique called tethered particle motion that allows us to quantify this looping, and the systematic tuning of four biologically relevant and experimentally tractable parameters: loop length, loop sequence, repressor-DNA affinity, and repressor concentration. We show that this combination is a powerful approach to measuring repressor-DNA binding affinities and sequence-dependent DNA flexibilities in a way that is orthogonal, and therefore complementary, to conventional ensemble assays. Our results show that the sequence dependence to looping is more complicated than has been observed in other contexts, suggesting that \u201csequence flexibility\u201d as a general term is misleading, and, we argue, that the measurement of sequence flexibilities depend more strongly than previously appreciated on the shape of the deformation used to make the measurement. Finally, we present preliminary results with a more complicated system that is a case study for broader issues in combinatorial control, and a new hidden Markov model approach, based on variational Bayesian inference, to analyze these more complicated systems, which we hope will allow more precise dissections of, and more robust extraction of kinetic parameters from, tethered particle motion assays." }, { "name": "Liu, Cambrian Yangshao", "degree": "PhD", "year": "2012", "title": "Characterization of an Unusual Collection of Olfactory Neurons in the Nose", "advisor": "Fraser, Scott E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05062012-022516031", "creators": [ { "name": { "family": "Liu", "given": "Cambrian Yangshao" }, "id": "Liu-Cambrian-Yangshao", "display_name": "Liu, Cambrian Yangshao" } ], "advisors": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "advisor", "display_name": "Fraser, Scott E." } ], "committee": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "chair", "display_name": "Lester, Henry A." }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biochem" ], "doi": "10.7907/47P6-2H66", "abstract": "We have used a combination of histochemical, electrophysiological, and behavioral approaches to study signal transduction, membrane biophysics, and chemosensory function in the neurons of the mouse Grueneberg ganglion (GG) olfactory subsystem. The GG is a recently appreciated collection of ~1,000 clustered primary olfactory neurons located at the anterior tip of the mammalian nasal cavity. Despite their far-forward position, GG neurons are fully trapped beneath a keratinized epithelium and are wrapped by glial cells. This raises the question of how they contribute to the sense of smell. We found that GG neurons have key components of cGMP signal transduction pathway and are molecularly similar to GC-D neurons, which project to the enigmatic necklace glomeruli in the olfactory bulb. In electrophysiological analyses, individual GG neurons spontaneously discharged action potentials in one of three distinct temporal patterns that were stable for >20 min. An auxiliary fast-inactivating Na+ current accounted for the various discharge patterns in computer simulations of the neuronal ionic currents. Despite differences in baseline activity, the majority of GG neurons responded to specific mammalian pheromones. In behavioral experiments, we found that the weaning of adolescent mice induced GG activity; however, the effects did not depend on ambient temperature or the presence of other animals. Because GG neurons reside on a dense vascular bed, have specialized access to serum contents, and directly responded to pressure ejections of serum, their activity can likely be modulated by internally circulating hormones or proteins associated with specific physiological states such as stress. Taken together, our results demonstrate unusual molecular and functional aspects of a morphologically and anatomically atypical olfactory nerve." }, { "name": "Materna, Stefan Christian", "degree": "PhD", "year": "2012", "title": "The Regulatory Origin of Oral and Aboral Mesoderm in Sea Urchin Embryos", "advisor": "Davidson, Eric H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05122012-162210497", "creators": [ { "name": { "family": "Materna", "given": "Stefan Christian" }, "id": "Materna-Stefan-Christian", "display_name": "Materna, Stefan Christian" } ], "advisors": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "advisor", "display_name": "Davidson, Eric H." } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "member", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Goentoro", "given": "Lea A." }, "id": "Goentoro-L-A", "role": "member", "display_name": "Goentoro, Lea A." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." } ], "option_major": [ "biochem" ], "doi": "10.7907/77VQ-8A52", "abstract": "Gene regulatory networks (GRN) underlie the control processes that are executed during embryonic development. Their constituents are transcription factors that regulate downstream targets, including other transcription factors. The regulatory architecture of a GRN reveals how discrete developmental tasks, such as cell specification, are implemented.
\r\n\r\nWe here expand the GRN underlying development of sea urchin non-skeletogenic mesoderm (NSM). NSM cells are the offspring of the inner ring of the veg2 cells that lie adjacent to the skeletogenic mesoderm (SM) and receive the Delta signal presented by these cells. Perturbation of Delta reveals that all NSM-specific genes are activated by Delta, but also indicate that Delta has few direct targets. A large number of genes are activated only after delta expression in the SM disappears, thus indicating that these genes are indirect targets and downstream of early NSM transcription factors. We show that the second phase of delta expresion (in the NSM) activates the foxY gene; loss of NSM Delta does not interfere with early NSM specification but instead abolishes development of late mesoderm derivates.
\r\n\r\nNSM is partitioned into an oral and an aboral segment as a consequence of Nodal signaling. However, Nodal activates the homeobox gene not, which represses early NSM genes on the oral side, causing them to become restricted to the aboral side. This allows oral NSM genes to be activated. Oral NSM genes can be expressed throughout the entire NSM if aboral NSM specification is perturbed. This shows that the driver of NSM genes is present throughout the NSM, making SM Delta a likely candidate. We examine the regulatory state of oral and aboral NSM segments and show that a GRN subcircuit on the aboral side locks down its expression. The sets of regulatory genes on both sides of the NSM are entirely distinct and mutually exclusive.
\r\n" }, { "name": "Matzen, Kelly Jean", "degree": "PhD", "year": "2012", "title": "Engineering of Dengue Virus Refractoriness in Aedes aegypti and Development of an Underdominant Gene Drive System in Drosophila melanogaster", "advisor": "Hay, Bruce A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05192012-130917004", "creators": [ { "name": { "family": "Matzen", "given": "Kelly Jean" }, "id": "Matzen-Kelly-Jean", "display_name": "Matzen, Kelly Jean" } ], "advisors": [ { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "advisor", "display_name": "Hay, Bruce A." } ], "committee": [ { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "chair", "display_name": "Strauss, James H." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "member", "display_name": "Baltimore, David L." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." } ], "option_major": [ "biochem" ], "doi": "10.7907/YNFQ-WM56", "abstract": "Vector-borne diseases have a profound impact on world health. The two most well-known and costly diseases are dengue fever and malaria, both spread by mosquito vectors. In the last decade, many new solutions to halting the spread of these diseases have been sought, including vector-mediated disease suppression. The work presented here seeks to generate alleles to effect this suppression, and engineer a drive system to replace the native population. Additional work on systems to keep engineered organisms genetically isolated from native populations has also been carried out. Initial studies in C. elegans investigated use of the transitive nature of RNAi in this species to genetically isolate one population from another. This type of speciation could be used in plant populations to limit gene flow of engineered crops into local environments.
\r\n\r\nThe next series of studies details work on engineering of refractoriness alleles. Dengue virus has several enzymatic activities that are essential for its replicative cycle, including an RNA-dependednt RNA polymerase (RdRp) responsible for synthesizing both the sense and antisense viral genomes and a protease responsible for several essential cleavages of the viral polyprotein. Artificial substrates for these proteins were created to act as sensors, triggering an apoptotic response when viral infection occurs. Several generations of constructs were tested, but so far no completely functional sensor has been generated.
\r\n\r\nLastly, a series of underdominant gene drive architectures were built and tested in Drosophila melanogaster. Initial systems utilized a Drosophila cell death protein, Hid, as toxin, and engineered microRNAs designed to target the Hid proteins as antidote. Two toxin-antidote pairs were mismatched and positioned on separate chromosomes so that an organism carrying both chromosomes survives, but an organism carrying only a single chromosome is unviable. Construction of a proof-of-principle in the eye was successful, but work in essential tissues is ongoing. Systems using engineered microRNAs as toxins and resupply of the native protein as antidote were tested in essential tissues. Testing of many components has contributed to the development of these systems, but a complete system has not yet been constructed.
" }, { "name": "Ng, Thomas Sheung Chee", "degree": "PhD", "year": "2012", "title": "Non-Invasive in vivo Molecular Imaging of Cancer Nanotherapy Uptake and Response with PET/MRI", "advisor": "Jacobs, Russell E.; Fraser, Scott E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05282012-135618123", "creators": [ { "name": { "family": "Ng", "given": "Thomas Sheung Chee" }, "id": "Ng-Thomas-Sheung-Chee", "display_name": "Ng, Thomas Sheung Chee" } ], "advisors": [ { "name": { "family": "Jacobs", "given": "Russell E." }, "id": "Jacobs-R-E", "role": "advisor", "display_name": "Jacobs, Russell E." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "co-advisor", "display_name": "Fraser, Scott E." } ], "committee": [ { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "chair", "display_name": "Patterson, Paul H." }, { "name": { "family": "Jacobs", "given": "Russell E." }, "id": "Jacobs-R-E", "role": "member", "display_name": "Jacobs, Russell E." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Davis", "given": "Mark E." }, "id": "Davis-M-E", "role": "member", "display_name": "Davis, Mark E." }, { "name": { "family": "Jensen", "given": "Grant J." }, "id": "Jensen-G-J", "role": "member", "display_name": "Jensen, Grant J." } ], "option_major": [ "biology" ], "doi": "10.7907/53JJ-MG42", "abstract": "Although researchers have made great strides toward understanding the biological processes underlying cancer pathology, this has not led to major improvements in the management of the disease. Development of new treatments to combat cancer remains imperative. Nanosized therapies show promise to improve tumor treatment response by localizing therapy while reducing treatment-related toxicity. Understanding how nanotherapies are taken up and cause their effects at the intact tumor level in vivo will complement ex vivo histological and in vitro biochemical studies and facilitate the translation of nanotherapy treatments to the clinic. Currently, few in vivo methods exist to study nanotherapy uptake and response at the intact tumor scale. Magnetic resonance imaging (MRI) and positron emission tomography (PET) are imaging methods that provide different but complementary information about the tumor microenvironment and nanotherapy uptake/response. Direct spatiotemporal correlation of PET and MRI data via their simultaneous acquisition has the potential to be powerfully synergistic, especially for the study of physiological processes that are time sensitive or where good spatial coregistration of the multimodal data is important. As the field of hybrid PET/MRI is still in its infancy, with only a handful of active systems worldwide, it is vital that continued PET/MRI technology development be pursued to realize its full potential.
\r\n\r\nThe objective of this thesis is to develop noninvasive, multimodal PET/MRI methods to study the uptake and response of cancer nanotherapies. Three studies were pursued toward this goal. First, we describe the development of a quantitative, small animal simultaneous PET/MRI system that is capable of dynamic, intratumoral imaging. The results show that the system provides quantitative images that are highly correlated with ex vivo autoradiography. The system was able to follow the uptake of a radiolabelled antibody inside the tumor over time, visualizing antibody movement from the vascular space to the tumor mass. Second, we adapted a functional MRI technique, diffusion MRI, to monitor treatment response of the cancer nanotherapy CRLX101. CRLX101-treated animals showed a significant diffusion MRI response within 2 days of treatment, before significant size changes were observed. Modeling of the diffusion MRI data was able to predict the potent antiproliferative effect of CRLX101, commensurate with histological data. Finally, we developed MRI and PET/MRI methods to study the tumor response to the tumor-penetrating peptide iRGD, which has shown good potential to improve cancer nanotherapy uptake. The results show that iRGD can have a variable tumor response, which may be dependent on the tumor microenvironment.
\r\n\r\nThe primary contributions of this thesis work is the development of small animal hybrid PET/MRI technology to enable multimodal intratumoral studies and the development of clinically-applicable imaging methods to monitor the uptake and response of cancer nanotherapies.
" }, { "name": "Nisthal, Alex", "degree": "PhD", "year": "2012", "title": "Accelerating the Interplay Between Theory and Experiment in Protein Design", "advisor": "Mayo, Stephen L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02062012-154120957", "creators": [ { "name": { "family": "Nisthal", "given": "Alex" }, "id": "Nisthal-Alex", "display_name": "Nisthal, Alex" } ], "advisors": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "advisor", "display_name": "Mayo, Stephen L." } ], "committee": [ { "name": { "family": "Tirrell", "given": "David A." }, "id": "Tirrell-D-A", "orcid": "0000-0003-3175-4596", "role": "chair", "display_name": "Tirrell, David A." }, { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "member", "display_name": "Arnold, Frances Hamilton" }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." } ], "option_major": [ "biochem" ], "doi": "10.7907/Z9833Q2F", "abstract": "Protein engineering techniques such as directed evolution and structure-based design aim to improve the properties of natural proteins. The next step, the de novo insertion of function into previously inert protein scaffolds, is the lofty promise of computational protein design. In order to achieve this goal reliably and efficiently, computational methods can be iteratively improved by cycling between theory and experiment.
\r\nEfforts to both accelerate the rate and broaden the information exchanged within protein design cycles form the core of this thesis. Improvements in the throughput of experimental stability determination allowed the thorough assessment of new multi-state and library design tools. Intending to alleviate the fixed backbone, single native state design approximation, the study found constrained molecular dynamics ensembles useful for core repacking applications. The subsequent development of automated liquid handling protocols for common molecular biology techniques brings design experiments to new levels of sample throughput. This technology facilitated the creation of a stability database encompassing every single mutant in a small protein domain. Although constructed to facilitate future computational training efforts, we answer a multitude of questions pertaining to mutational outcomes, distributions, positional sensitivity, tolerance, and additivity in the context of a protein domain.
\r\nBy expanding the constraints of experimental molecular biology, this work opens up new possibilities in the efforts to train and assay new computational methodologies for protein engineering applications.
\r\n" }, { "name": "Pierce, Nathan William", "degree": "PhD", "year": "2012", "title": "Sequential Processivity and CAND1 Regulate SCF Ubiquitin Ligases", "advisor": "Deshaies, Raymond Joseph", "url": "https://resolver.caltech.edu/CaltechTHESIS:11282011-135435821", "creators": [ { "name": { "family": "Pierce", "given": "Nathan William" }, "id": "Pierce-Nathan-William", "display_name": "Pierce, Nathan William" } ], "advisors": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "advisor", "display_name": "Deshaies, Raymond Joseph" } ], "committee": [ { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "role": "chair", "display_name": "Dunphy, William G." }, { "name": { "family": "Shan", "given": "Shu-ou" }, "id": "Shan-Shu-ou", "role": "member", "display_name": "Shan, Shu-ou" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "member", "display_name": "Deshaies, Raymond Joseph" } ], "option_major": [ "biology" ], "doi": "10.7907/ME1H-AK32", "abstract": "The modular design of the multi-subunit SCF ubiquitin ligases allows for recognition of a diverse set of target proteins. However, the speed and complexity of the SCF ubiquitylation reaction have precluded direct experimental tests to understand how SCF complex formation is regulated and the pathway by which ubiquitin chains are generated. Herein we introduce new theoretical and experimental methodologies to address both limitations. First, a quantitative framework based on product distribution predicts that the really interesting new gene (RING) E3s SCFCdc4 and SCF\u03b2-TrCP work with the E2 Cdc34 to build polyubiquitin chains on substrates by sequential transfers of single ubiquitins. Measurements with millisecond time resolution directly demonstrate that substrate polyubiquitylation proceeds sequentially. Second, we present a novel FRET assay that enables real-time measurements of binding dynamics of the SCFFbxw7 complex. We find that the Cul1-associated protein CAND1 is able to actively remove Fbxw7/Skp1 from Cul1/Rbx1 by changing the dissociation rate of the complex a million-fold, yet CAND1 does not affect the assembly rate of SCFFbxw7. This activity is abolished when Cul1 is neddylated. Experiments show that CAND1 accelerates the rate at which multiple SCF complexes can form. Thus, CAND1 appears to function as an exchange factor. Lastly, several measurements reveal an extra step in the ubiquitylation pathway for yeast SCF that implies a substrate induced conformational change exists for Fbox proteins. These results present an unprecedented glimpse into the mechanism of RING ubiquitin ligases and their regulation by CAND1.
" }, { "name": "Shen, Yue", "degree": "PhD", "year": "2012", "title": "Interkingdom Communication of a Bacterial Mutualist and its Mammalian Host", "advisor": "Mazmanian, Sarkis K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06062012-145546209", "creators": [ { "name": { "family": "Shen", "given": "Yue" }, "id": "Shen-Yue", "display_name": "Shen, Yue" } ], "advisors": [ { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "advisor", "display_name": "Mazmanian, Sarkis K." } ], "committee": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "chair", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "member", "display_name": "Chan, David C." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Grant", "given": "Jensen" }, "id": "Jensen-G-J", "role": "member", "display_name": "Grant, Jensen" }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "member", "display_name": "Mazmanian, Sarkis K." } ], "option_major": [ "biology" ], "doi": "10.7907/56YV-8J33", "abstract": "Microbial molecules have evolved to promote transient and/or permanent associations with mammals. Although numerous examples of secretion systems employed by pathogens during infection have been described, mechanisms by which commensal bacteria export molecules during symbiosis remain unknown. The human gut mutualist Bacteroides fragilis produces a capsular polysaccharide (PSA) that directs host immune development. We reveal herein that outer membrane vesicles (OMVs) deliver PSA to dendritic cells (DCs), promoting development of regulatory T cells and inducing anti-inflammatory cytokines during in vivo protection of intestinal disease. OMV mediated regulatory responses required the Growth Arrest and DNA-Damage-Inducible protein (Gadd45\u03b1) in DCs. DCs treated with OMVs containing PSA protect mice from experimental colitis, whereas Gadd45\u03b1-/- DCs are unable to support T cell regulatory response and are defective in suppressing proinflammatory cytokine production and host pathology. Our findings demonstrate DC-induced protection from disease via interaction with a beneficial microbial molecule delivered by OMVs, uncovering a novel paradigm for interkingdom communication between the microbiota and mammals. \r\nIn another effort to test the immunomodulatory activity of PSA outside of the gut, we found systemic treatment with PSA protects animals from experimental sepsis, a model for systemic inflammatory disease. More interestingly, this protection is mediated by B cells but not T cells because Rag-/- mice reconstituted with B cells gained the protection by PSA while those reconstituted with T cells were not protected. We further showed that a subset of B cells, marginal B cells, which are known to produce natural antibodies against bacterial antigens, were sufficient in mediating this protection. Preliminary data also suggested that secretion of IgM and/ or expression of type II Interleukin 1 receptor (IL-1R2) from marginal zone B cells might be critical for the suppression of the excessive inflammation during disease. This study will help to uncover the systemic effect of PSA, a microbial molecule from a gut commensal, and its potential as a novel therapy for human sepsis." }, { "name": "Suloway, Christian J. M.", "degree": "PhD", "year": "2012", "title": "Structural Insights into Tail-Anchored Protein Targeting by Get3", "advisor": "Clemons, William M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02282012-151846967", "creators": [ { "name": { "family": "Suloway", "given": "Christian J. M." }, "id": "Suloway-Christian-J-M", "display_name": "Suloway, Christian J. M." } ], "advisors": [ { "name": { "family": "Clemons", "given": "William M." }, "id": "Clemons-W-M", "orcid": "0000-0002-0021-889X", "role": "advisor", "display_name": "Clemons, William M." } ], "committee": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "chair", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Clemons", "given": "William M." }, "id": "Clemons-W-M", "orcid": "0000-0002-0021-889X", "role": "member", "display_name": "Clemons, William M." }, { "name": { "family": "Jensen", "given": "Grant J." }, "id": "Jensen-G-J", "orcid": "0000-0003-1556-4864", "role": "member", "display_name": "Jensen, Grant J." }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." } ], "option_major": [ "biology" ], "doi": "10.7907/AGES-WJ42", "abstract": "Translocation of membrane proteins from the point of synthesis to their integration in the membrane is critical to the function of the cell. Tail-anchored (TA) proteins are an important class of membrane proteins with a single transmembrane domain (TMD) close to the carboxyl-terminus. They are defined topologically by having their amino-terminus in the cytosol and their carboxyl-terminus on the exterior side of the membrane. Since the TMD is sequestered by the ribosome during translation, co-translational translocation of TA proteins by the SRP-dependent pathway is not possible. The Guided Entry of Tail-anchored proteins (GET) pathway post-translationally targets TA proteins to the endoplasmic reticulum (ER) membrane. The conserved nucleotide hydrolase Get3 is the central protein in the pathway that specifically binds the TMD of TA proteins to chaperone them from a sorting complex of Get4, Get5, Sgt2 and other chaperones to an ER membrane receptor formed by Get1 and Get2. We have created a model for the mechanism of Get3 TA protein binding coupled to nucleotide state using X-ray crystallography, structural modeling and mutagenesis experiments. We then demonstrate expression, purification and crystallization of complexes of Get3 with TA proteins for structural studies. Finally, we present a crystal structure of a tetrameric archaeal Get3 homologue that forms a central hydrophobic chamber and is capable of binding TA proteins. Using small-angle X-ray scattering, the structure is comparable to a tetrameric fungal Get3 complex with TA protein, which is capable of TA protein membrane integration in vitro. This suggests a model in which a tetramer of Get3 binds TA proteins for delivery to the membrane." }, { "name": "Young, Jonathan Wan", "degree": "PhD", "year": "2012", "title": "Architecture, Dynamics, and Function of the General Stress Response System in B. subtilis", "advisor": "Elowitz, Michael B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05292012-120719001", "creators": [ { "name": { "family": "Young", "given": "Jonathan Wan" }, "id": "Young-Jonathan-Wan", "display_name": "Young, Jonathan Wan" } ], "advisors": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "advisor", "display_name": "Elowitz, Michael B." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "orcid": "0000-0002-1221-0967", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biology" ], "doi": "10.7907/93MF-Q808", "abstract": "Cells exhibit diverse and dynamic responses to stress. However, in many cases it remains unclear what the dynamics are, how they are generated, and why they are beneficial to the cell or organism. To investigate these issues we studied the General Stress Response in B. subtilis, a critical, conserved stress signaling pathway, mediated by the alternative sigma factor, \u03c3B. First, we find that \u03c3B activates with stochastic, frequency modulated pulses in response to energy stress. We explore the mechanism behind this striking response and find that a small, compact circuit facilitates this behavior. Second, we find that \u03c3B activates with a single-homogenous pulse of activity exposed to environmental stress, in contrast to energy stress dynamics. We also find that activation is rate-responsive, and show how this property may separate broad and specific regulatory modes. Lastly, we present some preliminary work toward a synthetic sigma factor activation circuit. Combined, these results present a comprehensive study of \u03c3B activation and generate a platform by which other dynamic stress response systems can be understood." }, { "name": "Yu, Kenneth Kwok-Chang", "degree": "PhD", "year": "2012", "title": "Engineering Immunity Against HIV\r ", "advisor": "Baltimore, David L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04162012-151100376", "creators": [ { "name": { "family": "Yu", "given": "Kenneth Kwok-Chang" }, "id": "Yu-Kenneth-Kwok-Chang", "display_name": "Yu, Kenneth Kwok-Chang" } ], "advisors": [ { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "advisor", "display_name": "Baltimore, David L." } ], "committee": [ { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "chair", "display_name": "Mazmanian, Sarkis K." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "member", "display_name": "Baltimore, David L." } ], "option_major": [ "biology" ], "doi": "10.7907/GFQ8-M763", "abstract": "An effective vaccine against the human immunodeficiency virus (HIV)-1 has so far been elusive. Anti-viral vaccines against other viruses work by stimulating the production of neutralizing antibodies that block infection. To be useful, an anti-HIV vaccine preparation needs to elicit potent neutralizing antibody response with sufficient breadth to cover the diversity of HIV variants. Despite sustained research efforts, such an immunogen has been difficult to develop. We could overcome this difficulty by using gene therapy to directly instruct the body to produce anti-HIV broadly neutralizing antibodies (bNAbs). In this thesis, I describe a technology I developed termed the \u201cMolecular Rheostat\u201d for directing the simultaneous expression of anti-HIV surface and secreted immunoglobulins using mutant 2A \u201cself-cleaving\u201d peptides. I describe the application of this system to the programming of hematopoeitic stem cells to generate anti-HIV B cells as a strategy to \u201cvaccinate\u201d against HIV infection. I then pivot to consider alternatives to B-cell programming to produce antibodies against HIV. I investigate the modification of non-lymphoid hematopoietic cells to produce antibodies using retroviral vectors and describe the use of lentiviral vectors to program muscle to produce anti-HIV broadly neutralizing antibodies. In addition to presenting a novel tool for controlling the simultaneous expression of full-length and truncated proteins, the work described here furnishes a foundation for future development into potential gene-therapeutic prophylaxis against HIV." }, { "name": "Zhang, Jingli A.", "degree": "PhD", "year": "2012", "title": "Global Analysis of Dynamic Epigenetic Marking and Transcriptional Regulation Underlying T-Cell Lineage Commitment\r ", "advisor": "Rothenberg, Ellen V.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01262012-055600494", "creators": [ { "name": { "family": "Zhang", "given": "Jingli A." }, "id": "Zhang-Jingli-A", "display_name": "Zhang, Jingli A." } ], "advisors": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "advisor", "display_name": "Rothenberg, Ellen V." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "member", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "member", "display_name": "Mazmanian, Sarkis K." } ], "option_major": [ "biology" ], "doi": "10.7907/9Q4G-E674", "abstract": "T-cell lineage specification and commitment success depends on precise temporal induction of T-lineage specific genes, as well as repression of lineage-inappropriate programs. After entry into the thymus, T-cell progenitors still retain inherited lineage plasticity, reflected by the mixed-lineage pattern of gene expression and the abilities to give rise to alternative lineages. Although Notch-Delta signaling is an essential force to trigger and sustain T-lineage differentiation, it does not appear to be the only requirement for this process. Successful commitment also depends on additional transcription factors, which often cooperatively interact with Notch-Delta signaling. However, the molecular mechanism by which pro-T cells are advanced to become committed T cells, in particular how the alternative lineage potentials are eliminated, is not fully understood. Using the genome-wide high-throughput sequencing, we track global shifts in gene expression pattern and transcriptional activity associated histone modifications in five successive stages of T-cell differentiation that span the commitment process. Our results show that T-lineage commitment is defined by the surprisingly complex downregulation of progenitor- and/or alternative lineage-associated programs, with relatively few regulatory genes are substantially upregulated. Rather than being silenced by a single global repression event, progenitor- and/or alternative lineage-associated genes are regulated by individual gene-specific mechanisms, indicated by the unsynchronized epigenetic transformations at discrete cis-elements of genes loci linked to progenitor and/or alternative lineage programs. We also investigate the genome-wide occupancies of PU.1 and GATA-3, two regulatory factors that have critical but complementary roles in early T-cell development. Binding sites choices of these two factors imply that transcriptional regulation by one particular factor is developmental context as well as dosage dependent. Furthermore, We combine this genome-wide approach with gene perturbation to study the function of Bcl11b, a transcription factor required for the completion of T-cell lineage commitment. Our analyses reveal that, in part through directly or indirectly regulation of Notch1 and GATA-3, Bcl11b mediates the modulation of T-cell lineage specification and commitment." }, { "name": "Ballor, Nicholas R.", "degree": "PhD", "year": "2011", "title": "Hydrogenases and Hydrogen Sensors in the Symbiotic Microbial Communities of Wood-Feeding Termites", "advisor": "Leadbetter, Jared R.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08182010-104705359", "creators": [ { "name": { "family": "Ballor", "given": "Nicholas R." }, "id": "Ballor-Nicholas-R", "display_name": "Ballor, Nicholas R." } ], "advisors": [ { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "role": "advisor", "display_name": "Leadbetter, Jared R." } ], "committee": [ { "name": { "family": "Orphan", "given": "Victoria J." }, "id": "Orphan-V-J", "role": "chair", "display_name": "Orphan, Victoria J." }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Mazmanian", "given": "Sarkis" }, "id": "Mazmanian-S-K", "role": "member", "display_name": "Mazmanian, Sarkis" }, { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "role": "member", "display_name": "Leadbetter, Jared R." } ], "option_major": [ "biochem" ], "doi": "10.7907/621E-9221", "abstract": "The termite gut is an ideal ecosystem for studying hydrogen ecophysiology. Hydrogen is central to the obligate mutualism between termites and their gut microbes and is turned over at rates as high as 33 m3 H2 per m3 hindgut volume daily and maintained near saturation in some species. Acetogenic bacteria use hydrogen to produce up to 1/3 of the total flux of the termite\u2019s primary carbon and energy source, acetate. We have taken a three-fold approach to investigate the hydrogen ecophysiology of the termite gut. In our first approach (Chapter 2) we completed a bioinformatic analysis of [FeFe] hydrogenase-like (H domain) proteins encoded in the genomes of three termite gut treponemes. Treponemes are among the most highly represented groups of gut bacteria. The remarkable diversity of H domain proteins encoded accentuates the importance of hydrogen to their physiology. Moreover, they encoded a poorly understood class hydrogen sensing H domain proteins and thereby present a unique opportunity for their further study. In our second approach (Chapters 3 and 4) we analyzed molecular inventories prepared from termite gut microbiomes of a class of [FeFe] hydrogenases found highly represented in a termite hindgut metagenome. The libraries of peptide sequences clustered with one another in a manner congruent with termite host phylogeny suggesting co-evolution. Interestingly, we observed that higher termite guts may harbor higher sequence diversity than lower termites. In our third approach (Chapter 5) we used microfluidic digital PCR to identify bacteria in the gut of Reticulitermes tibialis encoding [FeFe] hydrogenases. The majority of the 16S rRNA gene phylotypes observed to co-amplify with hydrogenase sequences were treponemal, and the only observed instances of the same 16S rRNA-hydrogenase gene pair co-amplifying in multiple microfluidic chambers corresponded to treponemal phylotypes. Therefore, treponemes may be an important or predominant bacterial group encoding an important family of [FeFe] hydrogenases in the termite gut. The above results provide support for an important role for treponemes in mediating hydrogen metabolism in the termite gut and accentuate the intimacy and stability of the association termites have maintained over the course of their evolution with their gut microbial communities. " }, { "name": "Barnet, Matthew Edward", "degree": "PhD", "year": "2011", "title": "Dynamics of Sea Urchin Gastrulation Revealed by Tracking Cells of Diverse Lineage and Regulatory State", "advisor": "Fraser, Scott E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10152010-141750757", "creators": [ { "name": { "family": "Barnet", "given": "Matthew Edward" }, "id": "Barnet-Matthew-Edward", "display_name": "Barnet, Matthew Edward" } ], "advisors": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "advisor", "display_name": "Fraser, Scott E." } ], "committee": [ { "name": { "family": "Parker", "given": "Carl Stevens" }, "id": "Parker-C-S", "role": "chair", "display_name": "Parker, Carl Stevens" }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "member", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biochem" ], "doi": "10.7907/9MJ2-7Q87", "abstract": "During gastrulation in the sea urchin embryo the archenteron, or primitive gut, is formed by an initial process of invagination at the vegetal pole of the embryo, followed by extension across the blastocoel toward the future mouth. Neither the genetic basis of gastrulation nor the detailed movement of the cells involved in archenteron formation is well understood. This thesis describes a new 4D imaging methodology by which embryonic lineage and gene regulatory states can be connected to cell behavior by tracking individual cells of the living embryo through developmental time. The work presented in this thesis shows directly the dramatic cellular rearrangement that comprises gastrulation. Furthermore, it shows that this rearrangement occurs in the veg2 lineage of cells expressing foxa, and not in the adjacent veg1 lineage of cells expressing brachyury. Very late in gastrulation some veg1 cells move in as a coherent truncated cone to produce the hindgut. However, veg1 cells located outside the vegetal ring of brachyury expression prior to gastrulation never contribute to the archenteron. " }, { "name": "Bugg, Charles Walter", "degree": "PhD", "year": "2011", "title": "Domain Organization of Mutant Huntingtin Fibrils", "advisor": "Patterson, Paul H.; Langen, Ralf", "url": "https://resolver.caltech.edu/CaltechTHESIS:09192010-204534950", "creators": [ { "name": { "family": "Bugg", "given": "Charles Walter" }, "id": "Bugg-Charles-Walter", "display_name": "Bugg, Charles Walter" } ], "advisors": [ { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "advisor", "display_name": "Patterson, Paul H." }, { "name": { "family": "Langen", "given": "Ralf" }, "id": "Langen-R", "role": "co-advisor", "display_name": "Langen, Ralf" } ], "committee": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "chair", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Langen", "given": "Ralf" }, "id": "Langen-R", "role": "member", "display_name": "Langen, Ralf" }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." } ], "option_major": [ "biochem" ], "doi": "10.7907/XAYY-9E20", "abstract": "Huntington\u2019s disease is a progressive, fatal neurodegenerative disorder caused by a polyglutamine (polyQ) expansion in exon 1 of the huntingtin gene (HDx1). A hallmark of the disease is the formation of fibrillar aggregates within cells. In vitro, HDx1 with a polyQ expansion forms fibrils that have a cross beta structure common to amyloid fibrils, but little else is definitively known about HDx1 fibril structure. We used electron paramagnetic resonance spectroscopy to study the organization of the major domains (N-terminus, polyQ, C-terminus) of HDx1 with 46Q within the fibril. Our data show that HDx1 fibrils do not have a parallel, in-register structure like most other disease-associated amyloid fibrils. The C-terminus is highly dynamic and is attached like a tail to the polyQ domain, which is mostly immobilized and forms the core of the fibril. However, the C-terminal portion of the polyQ lies outside the core and has a mobility similar to the C-terminus. The N-terminus produced heterogeneous spectra, indicating that it is able to sample multiple conformations. In sum, our study excluded the parallel, in-register arrangement of beta strands within HDx1 fibrils and represents a first step toward a high-resolution structure of HDx1 fibrils." }, { "name": "Chow, Janet", "degree": "PhD", "year": "2011", "title": "A Pathobiont of the Mammalian Microbiota Balances Intestinal Inflammation and Colonization", "advisor": "Mazmanian, Sarkis K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05022011-211415511", "creators": [ { "name": { "family": "Chow", "given": "Janet" }, "id": "Chow-Janet", "display_name": "Chow, Janet" } ], "advisors": [ { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "advisor", "display_name": "Mazmanian, Sarkis K." } ], "committee": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "chair", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "role": "member", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "member", "display_name": "Mazmanian, Sarkis K." } ], "option_major": [ "biology" ], "doi": "10.7907/4FZ1-A113", "abstract": "Humans and mammals are colonized by a multitude of microbial organisms that have co\u2010evolved with their hosts for millions of years. The majority of these microbes reside in the gastrointestinal (GI) tract as a complex and dynamic consortium. Though most associations with the host are symbiotic or commensal, some resident bacteria have the potential to cause disease under certain conditions. We refer to these bacteria as \u2018pathobionts.\u2019 Pathobionts are distinct from opportunistic pathogens, which are often acquired from the environment and cause acute infections. Bacterial type VI secretion systems (T6SSs) are one mechanism for mediating close host\u2010microbial interactions. Herein we report that the T6SS of H. hepaticus, a pathobiont of the murine intestinal microbiota, mediates critical protective functions during association with its mammalian host. In cell cultures, infection of intestinal epithelial cells (IECs) with H. hepaticus T6SS mutants results in increased bacterial association compared to wild\u2010type bacteria. In animals, T6SS mutants colonize the lower GI tract to a higher degree. Most importantly, H. hepaticus defective in type VI secretion is unable to restrain potent innate and adaptive immune responses in an animal model of experimental colitis. In addition, the H. hepaticus T6SS directs an anti\u2010inflammatory gene expression profile in IECs, and CD4+ T cells from mice colonized with T6SS mutants produce increased proinflammatory interleukin\u201017 cytokine in response to IECs presenting H. hepaticus antigens. Thus, our findings reveal that H. hepaticus has evolved a T6SS as a mechanism to actively maintain a non\u2010pathogenic, symbiotic relationship in the GI tract by regulating bacterial colonization and host inflammation. Disturbances in the dynamic interaction between gut bacteria and the intestinal immune system may lead to exacerbated host inflammation. As intestinal bacteria profoundly influence host biology, our findings support an emerging hypothesis that alterations in the composition of the microbiota, known as dysbiosis, is a critical factor in various human disorders such as inflammatory bowel disease and colon cancer." }, { "name": "Damle, Sagar S.", "degree": "PhD", "year": "2011", "title": "A Study of Information Processing in the Sea Urchin Embryo by Rewiring Mesodermal Gene Regulatory Networks and cis-Regulatory Analysis of Skeletogenic Regulators", "advisor": "Davidson, Eric H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01202011-110522273", "creators": [ { "name": { "family": "Damle", "given": "Sagar S." }, "id": "Damle-Sagar-S", "display_name": "Damle, Sagar S." } ], "advisors": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "advisor", "display_name": "Davidson, Eric H." } ], "committee": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "chair", "display_name": "Wold, Barbara J." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." } ], "option_major": [ "biology" ], "doi": "10.7907/186Z-VJ85", "abstract": "This work focuses on the GRNs specifying embryonic skeletogenesis and the pigment cell differentiation in the purple sea urchin, Strongylocentrotus purpuratus. These networks make predictions about the necessity of regulatory gene expression and cis-regulatory wiring for directing development. Here, these predictions are tested in a novel way, by add regulatory linkages to the GRN, and effectively rewiring development at the level of genomic DNA. The rewiring experiment presented here showed a previously unknown repression function for the pigment-cell terminal differentiation regulator, gcm, on an important regulator of skeletogenesis, alx1. This result motivated a complete cis-regulatory analysis of alx1 that identified a potential mechanism for gcm repression. Finally, this work describes a method for measuring GFP reporter activity in live sea-urchin embryos that will permit real-time cis-regulatory analysis." }, { "name": "Fortini, Barbara Karmen Kraatz", "degree": "PhD", "year": "2011", "title": "Biochemical and Genetic Studies of Genomic Stability", "advisor": "Campbell, Judith L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05172011-123048725", "creators": [ { "name": { "family": "Fortini", "given": "Barbara Karmen Kraatz" }, "id": "Fortini-Barbara-Karmen-Kraatz", "display_name": "Fortini, Barbara Karmen Kraatz" } ], "advisors": [ { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "role": "advisor", "display_name": "Campbell, Judith L." } ], "committee": [ { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "role": "chair", "display_name": "Dunphy, William G." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "member", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Jensen", "given": "Grant J." }, "id": "Jensen-G-J", "role": "member", "display_name": "Jensen, Grant J." }, { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "role": "member", "display_name": "Campbell, Judith L." } ], "option_major": [ "biology" ], "doi": "10.7907/ZH26-B677", "abstract": "Genomes face a constant barrage of threats from endogenous and exogenous sources. The need to maintain fidelity while replicating the entire genome during each cell division necessitates a dynamic cadre of proteins and protein complexes that participate in the DNA replication process. Furthermore, DNA can be damaged during all phases of the cell\u2019s life and that damage must be recognized and repaired in a way that preserves genetic information. These studies focus on one enzyme at the nexus of DNA replication and DNA repair, the helicase/nuclease Dna2. We show that Dna2 possesses a novel ATP/Mn2+ dependent flap endo/exonuclease activity and a DNA end-independent endonuclease activity that is inhibited by Replication Protein A. The regulation of Dna2 activity in the context of the global DNA damage response is of great interest. To that end, we explored the relationship of Dna2 and the DNA damage sensor kinase Mec1. We find that Dna2 is phosphorylated by Mec1 following DNA damage in its N-terminal domain. We then extended these studies from yeast to higher eukaryotes utilizing the Xenopus cell free extract system. Using simulated double strand breaks (DSBs), we constructed a timeline of protein processing steps required for homologous recombination mediated repair. This strategy using Xenopus extracts also place Dna2 on chromatin during DNA replication, physically interacting with other proteins involved in lagging strand replication. Taken together, these biochemical and genetic studies elucidate the multiple roles the Dna2 enzyme plays in order to ensure genomic stability. " }, { "name": "Gomez, Tara Adele", "degree": "PhD", "year": "2011", "title": "Mutational Analysis of Ubiquitin Shuttle Receptor Docking Sites on the 26S Proteasome", "advisor": "Deshaies, Raymond Joseph", "url": "https://resolver.caltech.edu/CaltechTHESIS:05232011-170710428", "creators": [ { "name": { "family": "Gomez", "given": "Tara Adele" }, "id": "Gomez-Tara-Adele", "display_name": "Gomez, Tara Adele" } ], "advisors": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "advisor", "display_name": "Deshaies, Raymond Joseph" } ], "committee": [ { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "chair", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "role": "member", "display_name": "Campbell, Judith L." }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "member", "display_name": "Chan, David C." }, { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "role": "member", "display_name": "Dunphy, William G." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "member", "display_name": "Deshaies, Raymond Joseph" } ], "option_major": [ "biology" ], "doi": "10.7907/K9K3-0105", "abstract": "Protein degradation is essential for many basic cellular functions. Most intracellular protein degradation occurs via the ubiquitin proteasome system. Cellular proteins are marked for degradation by the appendage of an ubiquitin chain. Ubiquitin receptor proteins recognize the ubiquitin chains and play a \"garbage man\" function in ensuring delivery of the protein trash to the cell\u2019s degradation machinery, the proteasome.
\r\n\r\nOne such class of ubiquitin receptor proteins, known as UBA-UBL proteins, recognizes ubiquitylated substrates and shuttles them to the proteasome. These shuttle receptors include Rad23, Dsk2, and Ddi1. The goal of this dissertation research has been to understand how these UBA-UBL proteins interact with the proteasome. In budding yeast, Saccharomyces cerevisiae, Rpn1 has been proposed to be the major docking site for UBL-containing proteins. More recent studies suggested that proteasome subunits Rpn10 and Rpn13, may also bind UBA-UBL proteins. However, no cis proteasome mutants existed to address these plausible redundant modes of delivery to the proteasome.
\r\n\r\nThe specific aims of this proposal were to: identify the sites on the proteasome that are necessary for specific UBA-UBL receptor docking, to study the consequence of the deletion of these sites (such as the effects on protein turnover), and to assess if the elimination of these sites is the same as elimination of the receptor proteins themselves.
\r\n\r\nHere, I describe a two-pronged genetic screen I conducted to identify a specific docking site within Rpn1 for UBA-UBL proteins. I uncover a highly conserved residue, D517A that appears to impinge on the ability for both Ddi1 and\u2013in the absence of Rpn13 or the dual absence of the ubiquitin interaction motifs of Rpn10 and Rpn13\u00ac\u2013Dsk2 to interact with the proteasome. However, under no set of genetic conditions does a mutation at Rpn1-D517 have any effect on Rad23 or the UBL-containing ubiquitin isopeptidase Ubp6. Taken together, my observations point to unanticipated diversity and complexity in the mechanisms underlying the recruitment of UBA-UBL proteins to the proteasome.
\r\n \r\nHence, I show that docking sites on the proteasome are not completely exclusive, both Ddi1 and Dsk2 share the D517 residue of Rpn1. However, there may be exclusive sites for docking Rad23 and Ubp6. There are also appears to be a layer of complexity and redundancy in docking UBA-UBL proteins to the proteasome. Follow-up studies on these proteasome cis mutatnts have also uncovered roles of the proteasome in regulating mitochondrial protein import and the methyl cycle.
\r\n" }, { "name": "Hergarden, Anne Christina", "degree": "PhD", "year": "2011", "title": "The Role of Peptidergic Neurons in the Regulation of Satiety in Drosophila", "advisor": "Anderson, David J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02272011-173157140", "creators": [ { "name": { "family": "Hergarden", "given": "Anne Christina" }, "id": "Hergarden-Anne-Christina", "display_name": "Hergarden, Anne Christina" } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "advisor", "display_name": "Anderson, David J." } ], "committee": [ { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "chair", "display_name": "Zinn, Kai George" }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "role": "member", "display_name": "Prober, David A." } ], "option_major": [ "biology" ], "doi": "10.7907/BSHW-AP17", "abstract": "Understanding the neural mechanisms that motivate us to eat is important because of the increasing rates of obesity and the consequential increasing rates of diabetes and cardiovascular disease in our society. The aim of this dissertation is to gain insight into the neuromodulators and neural mechanisms that regulate satiety. To do this, we turned to Drosophila melanogaster, which has been a powerful model organism to study the molecular mechanisms underlying innate animal behaviors and which exhibits many conserved elements of feeding regulation and energy homeostasis found in mammals. A common theme in animal behavior is that food deprivation modifies behavioral responses, e.g., the likelihood that an animal will accept a low-nutrient food. I manipulated the parameters of a feeding assay to screen for animals that lacked several starvation-induced feeding behaviors: increased foraging for food, increased acceptance of low-nutrient food, and increased ingestion of low-quality food. Using this feeding assay, I identified a neuronal circuit manipulation that inhibits several starvation-induced behaviors. Activation of a subset of Allatostatin-A-expressing neurons, using a novel transgenic tool that we generated, inhibits starvation-induced changes in both the acceptance and the ingestion of low-quality foods. In contrast, this circuit manipulation did not affect starvation-induced metabolic changes or foraging behavior. This suggests that we tapped into a mechanism that regulates a specific subset of starvation-induced changes in feeding behavior that is independent from general starvation-induced behavioral responses and energy metabolism. Studies in blowflies have revealed that the primary mechanism that promotes satiety is inhibitory proprioceptive feedback from the gut, but whether such a mechanism operates in Drosophila is unclear. While Allatostatin A has been implicated as a satiety factor and as a myoinhibitor in several other insects, it has no known function in Drosophila. A mechanism that promotes satiety but that does not alter energy metabolism has not previously been identified in Drosophila. I have used this circuit manipulation to better understand how a state of satiety is achieved in Drosophila, by integrating the knowledge acquired from studies in other insects with the knowledge acquired from molecular genetic manipulations in Drosophila." }, { "name": "Kuntz, Steven Gregory", "degree": "PhD", "year": "2011", "title": "hlh-1 and the C. elegans Body Wall Muscle Transcriptional Differentiation Network", "advisor": "Wold, Barbara J.; Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10212010-102536869", "creators": [ { "name": { "family": "Kuntz", "given": "Steven Gregory" }, "id": "Kuntz-Steven-Gregory", "display_name": "Kuntz, Steven Gregory" } ], "advisors": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "co-advisor", "display_name": "Wold, Barbara J." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "co-advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "chair", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "member", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biochem" ], "doi": "10.7907/18XS-YM65", "abstract": "To understand the structure and function of gene regulatory networks, it is important to first catalogue the components. Measurable constituents of networks include cis-regulatory elements, identified by their conservation and ability to drive expression; transcription factor binding motifs, identified by protein binding; transcription factors, identified by their necessity in network function; and target genes, identified by their conditional expression. The heart of a regulatory network is the transcription factor, which is dedicated to its role in the network. Transcription factors must be activated and regulate downstream targets in a discrete and reproducible fashion. Any deviation in network function may result in the collapse of the network and death of the animal. Thus, a network must be robust enough to function under a variety of biological conditions. However, network redundancies are inefficient in terms of fitness and lost during the course of evolution. The network structure and function reflects these evolutionary realities: strong sequence conservation of cis-regulatory elements coupled with widespread stochastic transcription factor binding, and ancient transcription factor conservation coupled with overlapping activation of targets. The evolution of functional transcription factor networks therefore must be a balance between conservation and flexibility. " }, { "name": "Schaedel, Oren Noah", "degree": "PhD", "year": "2011", "title": "Dynamic Regulation of the Dauer Decision", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04272011-021639361", "creators": [ { "name": { "family": "Schaedel", "given": "Oren Noah" }, "id": "Schaedel-Oren-Noah", "display_name": "Schaedel, Oren Noah" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "chair", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "co-chair", "display_name": "Fraser, Scott E." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Guo", "given": "Chin-Lin" }, "id": "Guo-Chin-Lin", "role": "member", "display_name": "Guo, Chin-Lin" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/JZ9E-AD68", "abstract": "Many animals can choose between different developmental fates to maximize fitness. Despite the complexity of environmental cues and life history, different developmental fates are executed in a robust fashion. The mechanisms that guarantee robust execution of a development choice in such environments remain unknown. The nematode Caenorhabditis elegans serves as a powerful model to examine this phenomenon because it has an advanced toolkit for cellular and genetic manipulations, and can adopt one of two developmental fates depending on environmental conditions. Nematodes grown in favorable conditions (sufficient food, low population density) develop into adults, whereas nematodes grown in unfavorable conditions (insufficient food, high population density) arrest development as a stress-resistant diapause form called dauer.
\r\n\r\nThe steroid hormone dafachronic acid (DA), product of DAF-9/cytochrome P450, directs development to adulthood by regulating the transcriptional activity of the nuclear hormone receptor DAF-12. The known role of DA suggests that it may be the molecular mediator of environmental condition effects on the developmental fate decision, although the mechanism is yet unknown. We hypothesize that information from the environment is integrated and reduced to a single cell nonautonomous environmental integrator, thereby explaining the tight binary nature of the developmental fate decision. We propose a fate coordination mechanism in which production of a small amount of DA is amplified, locking in the adult fate. Using a combination of laser ablations and time lapse image analysis, we demonstrate that upon the decision to become an adult, the XXX neuroendocrine cells act as a source releasing DA. As a result, DAF-12 dependent expression of daf-9 in the epidermis is amplified and propagated from anterior to posterior, dispersing high amounts of DA throughout the body. This dispersion of DA drives adult programs in the gonad, epidermis and vulva. Furthermore, we demonstrate that the XXX cells are not necessary for maintaining the adult fate after the signal amplification has started. This indicates that the epidermal amplification also confers the irreversibility of the decision by uncoupling the execution of the decision from the environmental integrator. We propose that this relay serves as a robust fate-locking mechanism to enforce an organism wide binary decision, despite noisy and complex environmental cues.
\r\n" }, { "name": "Tadmor, Arbel David", "degree": "PhD", "year": "2011", "title": "Phage-Host Interaction in Nature", "advisor": "Phillips, Robert B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05252011-233927917", "creators": [ { "name": { "family": "Tadmor", "given": "Arbel David" }, "id": "Tadmor-Arbel-David", "display_name": "Tadmor, Arbel David" } ], "advisors": [ { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "advisor", "display_name": "Phillips, Robert B." } ], "committee": [ { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "orcid": "0000-0002-7033-0844", "role": "chair", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Orphan", "given": "Victoria J." }, "id": "Orphan-V-J", "orcid": "0000-0002-5374-6178", "role": "member", "display_name": "Orphan, Victoria J." }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "orcid": "0000-0001-8723-8190", "role": "member", "display_name": "Baltimore, David L." }, { "name": { "family": "Phillips", "given": "Robert B." }, "id": "Phillips-R", "orcid": "0000-0003-3082-2809", "role": "member", "display_name": "Phillips, Robert B." } ], "option_major": [ "biochem" ], "doi": "10.7907/68Q5-D532", "abstract": "Though viruses may be the most abundant biological entities on the planet, very little is known about phage-host interaction in the wild due to the absence of proper experimental tools. In the present work we report of a method to pair environmental phages with their bacterial hosts at the single-cell level without having to culture either host or virus. The method utilizes microfluidic digital PCR in conjunction with a metagenome data mining tool that was developed to find a viral marker gene in an unknown environment. We implemented this technique on the microbial community residing in the hindgut of termites. Consequently, we discovered genus-wide infection patterns displaying remarkable intra-genus selectivity, with viral alleles displaying limited lateral gene transfer and/or host switching despite host proximity. To try and explain phage-host interactions from a theoretical perspective, we formulated a simple biophysical model describing the interaction of bacteria and viruses in aqueous environments. We predict that the radius r of a bacterium is the most critical parameter determining its fixed point concentration, which scales as r-4. Given the hypothesis that there is no selection pressure on bacterial radii, our model predicts that the size spectrum of marine bacteria follows a power law with slope -1, close to the observed average spectrum. Moreover, given the total concentration of bacteria in the ocean, our model enables us to estimate the total number of bacterial \u201cspecies\u201d per volume of water providing a lower and upper bound on the total number of species in the oceans. To elucidate the concept of a \u201cspecies\u201d, we consider a bacterial-viral co-speciation model, which is consistent with the observed narrow host range of phages. Our model hints that the bacterial-viral \u201carms race\u201d may be a critical component in the process of co-speciation. We suggest further experiments to test both models. Finally, we consider a recent high resolution measurement of the force as a function of time generated by stress fibers within a single fibroblast cell and suggest a stochastic model that is capable of accounting for the observed data." }, { "name": "Tulin, Sarah Lynn", "degree": "PhD", "year": "2011", "title": "Analysis of Drosophila Fibroblast Growth Factor Functional Domains", "advisor": "Stathopoulos, Angelike", "url": "https://resolver.caltech.edu/CaltechTHESIS:11052010-143655093", "creators": [ { "name": { "family": "Tulin", "given": "Sarah Lynn" }, "id": "Tulin-Sarah-Lynn", "display_name": "Tulin, Sarah Lynn" } ], "advisors": [ { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "advisor", "display_name": "Stathopoulos, Angelike" } ], "committee": [ { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "chair", "display_name": "Zinn, Kai George" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "member", "display_name": "Stathopoulos, Angelike" } ], "option_major": [ "biology" ], "doi": "10.7907/CJDY-ST97", "abstract": "The exciting Fibroblast Growth Factor (FGF) field lies at the crossroads of cell signaling, development, evolution, trafficking, physiology and human disease. A current challenge is to understand the mechanisms used by this signaling pathway to accomplish its myriad tasks in patterning the embryo, forming organs, and maintaining systems in the adult animal. My thesis work has focused on tackling this challenge in the model system of Drosophila melanogastor, the vinegar fly. By examining functional domains of Thisbe and Pyramus, FGF ligands in the fly, we have begun to understand the properties of Drosophila FGFs and the way in which they may contribute to regulation of FGF signaling.
\r\n\r\nFGF ligands in vertebrates are small molecules that bind to a corresponding receptor through two immunoglobulin domains. The FGF ligands in Drosophila are predicted to be much larger molecules than their vertebrate homologs. Whether Drosophila FGFs bind to the receptor as full-length proteins or are first cleaved to smaller molecules was previously unknown. My thesis work addressed this question through experiments in Drosophila embryos and Drosophila cell culture. I found evidence that the N-terminal FGF-domain alone is capable of signaling by itself in the embryo. In addition, experiments in cell culture showed that Thisbe and Pyramus are secreted as small forms, presumably as a result of intracellular proteolytic cleavage. Cleaved forms for Thisbe and Pyramus were detected in embryonic extracts as well. The Ths ligand is also present outside the cell as a full-length form and this form may act to regulate the diffusion or activity of the ligand. Addition of the Thisbe C-terminus to the Pyramus N-terminus to make a Pyramus-Thisbe chimeric protein creates a protein that has reduced activity compared to Thisbe alone. The opposite Thisbe-Pyramus chimera creates a protein that has increased activity compared to Ths alone.
\r\n\r\nOver the course of animal evolution the FGF superfamily has diversified in many ways. Understanding the mechanism of FGF signaling in Drosophila and comparing this to other Drosophilids, insects, and more distantly related animals will reveal the likely makeup of the ancestral FGF signaling system.
\r\n" }, { "name": "Wade, Lawrence A.", "degree": "PhD", "year": "2011", "title": "An Evanescent Perspective on Cells", "advisor": "Fraser, Scott E.; Lester, Henry A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:09102010-145946974", "creators": [ { "name": { "family": "Wade", "given": "Lawrence A." }, "id": "Wade-Lawrence-A", "display_name": "Wade, Lawrence A." } ], "advisors": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "advisor", "display_name": "Fraser, Scott E." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "co-advisor", "display_name": "Lester, Henry A." } ], "committee": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "role": "chair", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Jensen", "given": "Grant J." }, "id": "Jensen-G-J", "role": "member", "display_name": "Jensen, Grant J." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "member", "display_name": "Lester, Henry A." } ], "option_major": [ "molbiochem" ], "doi": "10.7907/AGAJ-PE93", "abstract": "We have optically sectioned living cells to a maximum depth of ~250 nm using a Variable Angle-Total Internal Reflection Fluorescence Microscope (VA-TIRFM). This yields 3D images of cell membranes and nearby organelles similar to that gained by confocal microscopes but with at least an order-of-magnitude greater depth resolution. It also enables cellular membranes to be imaged in near isolation from cell organelles. Key to achieving this resolution was integration of a controllable excitation laser micropositioner into a standard through-the-lens TIRF illuminator and development of a custom culture dish for re-use of expensive high index of refraction cover slips. Images are acquired at several penetration depths by varying the excitation laser illumination angles. At the shallowest penetration depth (~46 nm) just the membrane and a few internal puncta are imaged. As the penetration depth is increased up to 250 nm organelles near the membrane, such as the ER, are imaged as well. The sequence of images from shallow deep is processed to yield a z-stack of images of approximately constant thickness at increasing distance from the coverslip. We employ this method to distinguish membrane-localized fluorophores (\u03b14 GFP \u03b22 nicotinic acetylcholine receptors and pCS2:lyn-mCherry) at the plasma membrane (PM) from those in near-PM endoplasmic reticulum (ERTracker green, \u03b14 GFP \u03b22 nicotinic acetylcholine receptors), on a z-axis distance scale of ~45 to ~250 nm in N2a cells. In doing so we observe occasional smooth ER structures that cannot be resolved as being distinct from the membrane.
\r\n\r\nIn a second project substantial progress has been made towards developing a Tip Enhanced Fluorescence Microscope (TEFM) capable of imaging wet biological samples with ~10 nm resolution. A TEFM combines a TIRFM with an Atomic Force Microscope (AFM) to modulate sample fluorescence through near-field dipole-dipole coupling.
\r\n\r\nIn the third project the capability to consistently produce high quality nanotube AFM probes was developed and a technique for chemically functionalizing the tip of a nanotube AFM probe was invented.
\r\n" }, { "name": "Wang, Liming", "degree": "PhD", "year": "2011", "title": "Genetic and Neural Regulation of Aggressive Behavior in Drosophila melanogaster", "advisor": "Anderson, David J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05152011-122043232", "creators": [ { "name": { "family": "Wang", "given": "Liming" }, "id": "Wang-Liming", "display_name": "Wang, Liming" } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "advisor", "display_name": "Anderson, David J." } ], "committee": [ { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "chair", "display_name": "Zinn, Kai George" }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "role": "member", "display_name": "Prober, David A." } ], "option_major": [ "biology" ], "doi": "10.7907/T6WZ-BV58", "abstract": "Aggression is an evolutionarily conserved behavior across the animal kingdom. Aggressive behavior among conspecifics is critical for the acquisition and defense of important resources including food, mates, and shelter, hence contributing to the survival and reproduction of animals. Therefore, it is of particular interest to understand how this behavior is regulated.
\r\n\r\nWe use the fruit fly Drosophila melanogaster as a model system to understand the regulation of aggression. We identify Cyp6a20, a cytochrome P450, as a gene mediating the suppressive effect of social experience on the intensity of male-male aggression. Notably, Cyp6a20 has been previously identified by profiling Drosophila strains subjected to genetic selection for differences in aggressiveness. Therefore our findings reveal a common genetic target for environmental and heritable influences on aggressiveness. Interestingly, Cyp6a20 is expressed in a subset of non-neuronal support cells associated with pheromone-sensing olfactory sensilla, suggesting that olfactory pheromone(s) may contribute to the regulation of aggression. Consistent with this idea, we find that cis-11-vaccenyl acetate (cVA), a previously identified olfactory pheromone, promotes male-male aggression via a group of olfactory receptor neurons expressing Or67d.
\r\n\r\nDespite its robust behavioral effect, cVA is not required for baseline male-male aggression, and exogenous cVA does not induce male-female aggression, suggesting that sex specificity of male aggression is independent of cVA. Our subsequent studies show that the sex specificity of male social behaviors is determined by a different class of pheromones, named male cuticular hydrocarbons. Male flies perform significantly less aggression and more courtship towards male flies lacking male CHs, both of which can be rescued by synthetic (Z)-7-tricosene (7-T), the most abundant male cuticular hydrocarbon. The opposite influences of 7-T on aggression and courtship are independent, but both require the gustatory receptor Gr32a. Surprisingly, sensitivity to 7-T is required for the aggression-promoting effect of cVA, but not vice versa. Furthermore, the increased courtship in the absence of male cuticular hydrocarbons is induced by pheromone(s) detected by an olfactory receptor Or47b. Thus, male social behaviors are controlled by gustatory pheromones that promote and suppress aggression and courtship, respectively, and whose influences are dominant to olfactory pheromones that enhance these behaviors.
" }, { "name": "Ward, Catherine Marie", "degree": "PhD", "year": "2011", "title": "Medea Selfish Genetic Elements as Tools for Altering Traits of Wild Populations: A Theoretical Analysis", "advisor": "Hay, Bruce", "url": "https://resolver.caltech.edu/CaltechTHESIS:05312011-123357339", "creators": [ { "name": { "family": "Ward", "given": "Catherine Marie" }, "id": "Ward-Catherine-Marie", "display_name": "Ward, Catherine Marie" } ], "advisors": [ { "name": { "family": "Hay", "given": "Bruce" }, "id": "Hay-B-A", "role": "advisor", "display_name": "Hay, Bruce" } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." } ], "option_major": [ "biochem" ], "doi": "10.7907/T656-ZN91", "abstract": "Insect-borne diseases kill millions of people annually. One strategy for controlling transmission of insect-borne disease involves replacing the native insect population with transgenic animals unable to transmit disease. Population replacement requires a drive mechanism to ensure the rapid spread of linked transgenes conferring disease refractoriness. Medea selfish genetic elements have the feature that when present in a female, only offspring that inherit the element survive, a behavior that can lead to spread. Here we use modeling to identify conditions under which Medea elements spread. We derive equations describing the allele frequencies required for spread of Medea elements with a fitness cost, and the equilibrium allele frequencies attained. We validate our model against a synthetic Medea element created in Drosophila and find that the model fits the data without parameter fitting. We show that when Medea spreads, it drives the non-Medea genotype out of the population, and we provide estimates of the number of generations required to achieve this goal. We also characterize two contexts in which Medea elements with fitness costs drive the non-Medea allele from the population: an autosomal element in which zygotic rescue is incomplete and an X-linked element in species in which X/Y individuals are male. Finally, we explore costs and benefits associated with the introduction of multiple Medea elements. Our results suggest that Medea elements can drive population replacement under a wide range of conditions, potentially reducing disease burden." }, { "name": "Beale, Holly C.", "degree": "PhD", "year": "2010", "title": "Synaptic Signal Transduction and Transcriptional Control", "advisor": "Kennedy, Mary B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05212010-144632355", "creators": [ { "name": { "family": "Beale", "given": "Holly C." }, "id": "Beale-Holly-C", "display_name": "Beale, Holly C." } ], "advisors": [ { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "role": "advisor", "display_name": "Kennedy, Mary B." } ], "committee": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "chair", "display_name": "Lester, Henry A." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." }, { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "role": "member", "display_name": "Kennedy, Mary B." } ], "option_major": [ "biology" ], "doi": "10.7907/5NQ9-KX48", "abstract": "Synaptic signal transduction regulates synaptic plasticity, and, on a larger scale, memory itself. The aim of this dissertation is to elucidate some of the mechanisms that control synaptic plasticity in the short term by modulating synaptic morphology and in the long term by controlling gene expression.
\r\n\r\nOne modification associated with synaptic plasticity is the change in the size of the spine, the micron-scale structure on the dendrite which supports the synapse. The size and shape of the spine are controlled by the actin cytoskeleton. I studied how stimulation of synaptic receptors drives changes in activation of proteins that regulate actin polymerization. We identified neuron-specific aspects of a canonical actin regulation pathway and characterized activity-regulated phosphatase activity.
\r\n\r\nChanges in spine size and other events associated with synaptic plasticity can begin within seconds of synaptic stimuli, but persistent changes require gene expression. For example, Arc, an immediate early gene required for changes in synaptic strength to persist, is the only transcript known to be both transcribed in response to synaptic stimulation and translocated specifically to the site of the stimulation. However, the role of Arc in promoting the plasticity of the synapse is still under investigation. We studied its binding partners and found that an interaction demonstrated in non-neuronal cells was not evident in neurons.
\r\n\r\nWe also studied changes in transcription over longer time periods. In order to identify pathways involving the postsynaptic protein densin, we assessed global changes in transcription with RNA-Seq, which uses ultra-high-throughput, short-read sequencing to measure transcript abundance. Compared to wild-type mice, densin knockout mice exhibit increased abundance of CaMKII\u03b1 (a densin binding partner), increased abundance of immediate early gene expression including Arc, and downregulated GABA_AR subunits.
\r\n\r\nIn summary, we investigated posttranslational modifications that take place within seconds of stimulation, binding interactions occurring in steady-state conditions in wild-type mice, and homeostatic adaptations to the chronic absence of a gene. These investigations into synaptic signaling illustrate not only the complexity of synapse-related regulatory networks but also the range of time scales they span.
" }, { "name": "Betancur, Paola A.", "degree": "PhD", "year": "2010", "title": "Cis-Regulatory Analysis of the Key Developmental Gene, SOX1O, in Neural Crest and Ear", "advisor": "Sauka-Spengler, Tatjana; Bronner, Marianne E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02252010-145712126", "creators": [ { "name": { "family": "Betancur", "given": "Paola A." }, "id": "Betancur-Paola-A", "display_name": "Betancur, Paola A." } ], "advisors": [ { "name": { "family": "Sauka-Spengler", "given": "Tatjana" }, "id": "Sauka-Spengler-T", "role": "co-advisor", "display_name": "Sauka-Spengler, Tatjana" }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "advisor", "display_name": "Bronner, Marianne E." } ], "committee": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "chair", "display_name": "Fraser, Scott E." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "member", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Sauka-Spengler", "given": "Tatjana" }, "id": "Sauka-Spengler-T", "role": "member", "display_name": "Sauka-Spengler, Tatjana" }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." } ], "option_major": [ "biology" ], "doi": "10.7907/FVX3-ES42", "abstract": "In vertebrates, Sox10 is a transcription factor essential for the formation of neural crest cells and their derivatives as well as placode-derived inner ear structures. At gastrula stages, both presumptive neural crest and placode cells reside at the neural plate border, between the non-neural ectoderm and the neural plate. Despite their common site of origin, these two cell populations have different characteristics. Neural crest cells are a multipotent, stem cell-like population that arises at all axial levels, migrates extensively in the embryo and forms a wide array of derivatives ranging from neurons to melanocytes and cartilage. On the other hand, placode cells are restricted to the cranial region, have limited migratory capacity and contribute only to sensory structures such as the eye, ear, olfactory epithelium and distal portions of the cranial sensory ganglia. Interestingly, I have identified cis-regulatory modules responsible for the regulation of Sox10 in these two different embryonic regions and at different times. Among these modules, I found a downstream novel cis-regulatory region, Sox10E2,that mediates initial Sox10 expression in cranial neural crest cells and otic placode cells. Through a combination of computational analysis, experimental perturbation of putative upstream transcription factors and their binding sites within the Sox10E2 regulatory module, plus chromatin immunoprecipitation, I revealed a set of direct inputs into Sox10E2 regulatory region. The results show that cMyb, Sox9 and Ets1 are responsible for the initial Sox10 expression in delaminating cranial neural crest cells, whereas cMyb, Sox8 and Pea3 regulate Sox10 expression in the otic placode. Analyzing Sox10 regulation through the enhancer Sox10E2 has helped unravel gene regulatory inputs contributing to both neural crest formation and otic placode development. The finding that paralogous factors activate the same regulatory module in these two populations suggests the intriguing possibility of an ancient cooption of regulatory function and/or a common ancestral crest-placode origin.\t " }, { "name": "Carvalho, Gil Bastos de", "degree": "PhD", "year": "2010", "title": "Drosophila Feeding Behavior and Demographic Mechanisms of Lifespan Extension", "advisor": "Anderson, David J.; Benzer, Seymour", "url": "https://resolver.caltech.edu/CaltechTHESIS:06022010-183440587", "creators": [ { "name": { "family": "Carvalho", "given": "Gil Bastos de" }, "id": "Carvalho-Gil-Bastos-de", "display_name": "Carvalho, Gil Bastos de" } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "advisor", "display_name": "Anderson, David J." }, { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "advisor", "display_name": "Benzer, Seymour" } ], "committee": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "chair", "display_name": "Anderson, David J." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "member", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "member", "display_name": "Baltimore, David L." } ], "option_major": [ "biology" ], "doi": "10.7907/NMN1-N566", "abstract": "Why do we age? Most organisms undergo senescence, a process involving progressive functional decline culminating in death, yet this widespread phenomenon remains largely mysterious. A number of genetic and environmental factors affect longevity, the best conserved and most widely studied of which is dietary restriction (DR), a reduction of nutrient intake short of malnutrition. Since nutrient ingestion determines lifespan, any factor affecting longevity\u2014particular food components, genetic pathways or drugs\u2014may do so indirectly, by altering feeding behavior. This is particularly true in Drosophila, which is normally kept in conditions where food is present in excess. Moreover, since DR is applied by aging flies on two different food concentrations\u2014diluted media are associated with an extended lifespan\u2014animals may alter their intake in response to the change in nutrient content. Since the medium is also the only water source, this compensatory feeding would result in changes in hydration, introducing a second experimental variable. Despite these issues, Drosophila feeding behavior has classically been ignored or superficially characterized in the context of aging research, partly due to the absence of appropriate methodology. We developed two complementary assays allowing long-term measurement of food intake. Using these techniques, we present the first characterization of real-time Drosophila feeding behavior. Our results reveal gender-specific feeding trends and show that mating stimulates female appetite via the seminal Sex Peptide. Additionally, we show that ingestion is dramatically affected by food dilution or dietary additives. Animals fed concentrated media restrict their intake and are chronically thirsty. We have found that lifespan extension by classical DR paradigms is abolished in the presence of ad libitum water, challenging the long-held assumption that DR affects longevity by altering nutrient intake. We characterize a new regime that robustly prolongs lifespan irrespective of water availability, and thus likely represents a more relevant model for mammalian DR. In contrast to previous claims, demographic analysis using this paradigm indicates that DR acts not by reducing the immediate risk of death, but by slowing the accumulation of age-related damage. Our findings directly challenge current views on the mechanistic basis of DR and have broad implications for the study of aging and nutrition in model organisms. \r\n\r\n\r\n" }, { "name": "Dalal, Chiraj Kiran", "degree": "PhD", "year": "2010", "title": "Causes and Consequences of Gene Expression Noise", "advisor": "Elowitz, Michael B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03312010-150904525", "creators": [ { "name": { "family": "Dalal", "given": "Chiraj Kiran" }, "id": "Dalal-Chiraj-Kiran", "display_name": "Dalal, Chiraj Kiran" } ], "advisors": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "advisor", "display_name": "Elowitz, Michael B." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "member", "display_name": "Elowitz, Michael B." } ], "option_major": [ "biochem" ], "doi": "10.7907/MHSR-9875", "abstract": "Genetically identical cells harvested in the same environment exhibit heterogeneity in gene expression. This phenomenon, termed gene expression noise, has been measured in several model organisms under various conditions. However, we still do not have a clear understanding of (1) the factors responsible for generating gene expression noise, or (2) the potential consequences noise can have on cellular processes. In an attempt to investigate these issues, we have determined the effects of 1) directional selection, 2) promoter mutation and 3) fluctuations in transcription factor localization on gene expression noise. First, we have used analytic and computational modeling of the effects of directional selection on gene expression noise to discover that, assuming expression can be described by up to two independent parameters, \u03bc, mean, and \u03c3, noise, strong directional selection yields an increase in noise. Next, we generated mutant promoter libraries and measured gene expression to determine the effects of cis-regulatory mutations on gene expression noise. Here we found that the expression noise can indeed be modulated by mutation independent of mean expression levels, lending credence to the previously mentioned analytical result. Based on this result, that mutations can harness noise, we wanted to determine whether the binding and unbinding of transcription factors to promoter regions also contributed to gene expression noise. To do so, we analyzed the localization dynamics of a transcription factor Crz1. We determined that Crz1 translocates to the nucleus in coherent bursts of localization in response to calcium. The frequency, but not the duration, of these bursts increases with the concentration of extracellular calcium. This frequency modulation propagates downstream of Crz1, enabling proportional regulation of target genes. Intrigued by this result, we characterized different types of localization dynamics used by the yeast proteome. We have found several classes of localization behavior, including proteins that burst on several timescales, exhibit static heterogeneity, and show amplitude modulation. Strikingly, several of these dynamic localization systems must co-exist in the same cell under the same conditions. Amongst the proteins that burst on a fast timescale like Crz1, Msn2 and Mig1 are transcription factors that both burst when deprived of glucose. Furthermore, both regulate a common set of target genes. Interestingly, when imaged together, the proteins exhibit correlations on two timescales, a positive correlation that typically lasts for an hour and an anti-correlation that lasts a few minutes. We are continuing to investigate the potential regulatory impact of these correlations by measuring the expression of their combinatorial target genes." }, { "name": "Gold, Daniel A.", "degree": "PhD", "year": "2010", "title": "Molecular Characterization of the Dbf4/Drf1-Dependent Kinase (DDK) and the DNA Replication Checkpoint Mediator Claspin in Xenopus Egg Extracts", "advisor": "Dunphy, William G.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10232009-160656505", "creators": [ { "name": { "family": "Gold", "given": "Daniel A." }, "id": "Gold-Daniel-A", "display_name": "Gold, Daniel A." } ], "advisors": [ { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "role": "advisor", "display_name": "Dunphy, William G." } ], "committee": [ { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "role": "chair", "display_name": "Campbell, Judith L." }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "member", "display_name": "Chan, David C." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "role": "member", "display_name": "Dunphy, William G." } ], "option_major": [ "biology" ], "doi": "10.7907/XG90-5475", "abstract": "The integrity of DNA replication control and checkpoint mechanisms is essential for preventing tumorigenesis. DNA replication is initiated by the S-CDK and DDK kinases which mediate the unwinding of the replication fork. Genotoxic stress which specifically affects cells in S-phase is detected by the replication checkpoint. Replication blockages activate the ATR kinase which, in turn, activates the downstream effector kinase Chk1 through the mediator protein, Claspin. Chk1 facilitates the arrest of cell cycle progression and the inhibition of replication origin firing.
\r\n\r\nClaspin has two broadly defined roles, one to mediate Chk1 activation and the other as a component of the replication fork. We have endeavored to study a link between these two facets of Claspin function. Here, we show that Claspin associates with several core replication fork proteins in Xenopus egg extracts. We identified a replication fork-interacting domain on Claspin that associates with the replication fork proteins and is required for Claspin association with chromatin. However, chromatin binding-deficient Claspin proteins can still mediate Chk1 activation in Claspin-depleted extracts, albeit with reduced efficiency. Thus, the localization of Claspin to the replication fork is not required for mediation of Chk1 activation but it does potentiate this process.
\r\n\r\nAnother focus of this study, DDK, is composed of the catalytic subunit Cdc7 and one of two distinct adaptor proteins, Drf1 or Dbf4. Drf1 forms a stable, active complex with Cdc7, even after replication arrest in egg extracts. Accumulation of Drf1 on chromatin in the presence of replication blocks is dependent upon ATR and Claspin but not Chk1. We characterized Xenopus Claspin as a kinase substrate of DDK which forms a stable nuclear complex with Cdc7 and Drf1 under both arrested and unperturbed replication conditions. Moreover, we identified a region of Claspin required for association with DDK that lies within the Chk1-binding domain, which contains a series of repeat sequences. This DDK-associating region is the first, but not the second of these repeat sequences. Furthermore, we have identified two evolutionarily conserved residues within this region required for DDK interaction. Claspin mutant proteins unable to interact with DDK still bind to Chk1 and rescue Chk1 activation in Claspin-depleted extracts. Therefore, we conclude that DDK regulates a largely checkpoint-independent role of Claspin function.
\r\n" }, { "name": "Hodas, Jennifer Jin Lee", "degree": "PhD", "year": "2010", "title": "Elucidating the Hippocampal Dopaminergic Subproteome with Novel Bioorthogonal Techniques", "advisor": "Schuman, Erin Margaret", "url": "https://resolver.caltech.edu/CaltechTHESIS:10012009-223603699", "creators": [ { "name": { "family": "Hodas", "given": "Jennifer Jin Lee" }, "id": "Hodas-Jennifer-Jin-Lee", "display_name": "Hodas, Jennifer Jin Lee" } ], "advisors": [ { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "advisor", "display_name": "Schuman, Erin Margaret" } ], "committee": [ { "name": { "family": "Bjorkman", "given": "Pamela Jane" }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "chair", "display_name": "Bjorkman, Pamela Jane" }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "orcid": "0000-0002-3671-9354", "role": "member", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "member", "display_name": "Schuman, Erin Margaret" } ], "option_major": [ "biochem" ], "doi": "10.7907/DBGH-J143", "abstract": "Both synaptic and behavioral plasticity require de novo protein synthesis. Dopamine is a critical neuromodulator, and abnormalities in dopaminergic regulation underlie disorders like Parkinson\u2019s disease, Alzheimer\u2019s disease, and schizophrenia\u2014 diseases that impair the ability of the brain to perform complex processes, including the formation and retrieval of memories. The stimulation of D1/D5 dopaminergic receptors in the hippocampus is critical for protein synthesis-dependent long-term potentiation (LTP), a process important for long-term synaptic plasticity and memory. The proteins synthesized upon activation of dopaminergic pathways, the dopaminergic subproteome, however, remain unknown.
\r\n\r\nHere, we describe the development of two sister technologies that employ bioorthogonal chemistry to effectively and specifically identify and visualize a proteome in an unbiased, nontoxic manner. In both bioorthogonal noncanonical amino acid tagging (BONCAT) and fluorescent noncanonical amino acid tagging (FUNCAT), we utilize methionine surrogates, either azidohomoalanine (AHA) or homopropargylglycine (HPG), which are conjugated via [3+2] copper (I)-catalyzed cycloaddition to either a biotin or fluorescent molecule-bearing probe. We demonstrate the utility of these methods by showing that both AHA and HPG can be used to examine two temporally distinct protein populations. Furthermore, we visualize the dendrite-specific contribution to the neuronal proteome by taking advantage of the spatial control achievable by FUNCAT. We then combine these techniques to address the question of the identity of the proteins in the specifically dendritic subproteome of the hippocampus. We confirm that upon stimulation with a D1/D5 dopamine receptor-specific agonist, there are significantly increased levels of protein synthesis in dendrites when compared to unstimulated dendrites. By utilizing a combination of these novel methods and more traditional techniques, we are able to provide the first comprehensive list of the dopaminergic dendritic subproteome of the hippocampus. These data suggest that the initial stages of D1/D5 receptor activation lead to the translation of proteins that may play a role in synaptic strengthening.
\r\n" }, { "name": "Ito, Hiroshi", "degree": "PhD", "year": "2010", "title": "Neuromodulator-Mediated Control of Spatial and Nonspatial Information Processing in the Hippocampus", "advisor": "Schuman, Erin Margaret", "url": "https://resolver.caltech.edu/CaltechTHESIS:10152009-180857846", "creators": [ { "name": { "family": "Ito", "given": "Hiroshi" }, "id": "Ito-Hiroshi", "orcid": "0000-0001-7726-0781", "display_name": "Ito, Hiroshi" } ], "advisors": [ { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "advisor", "display_name": "Schuman, Erin Margaret" } ], "committee": [ { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "chair", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "orcid": "0000-0001-8837-678X", "role": "member", "display_name": "Siapas, Athanassios G." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "member", "display_name": "Schuman, Erin Margaret" } ], "option_major": [ "biology" ], "doi": "10.7907/3ZPG-0E52", "abstract": "How the brain implements learning is a long-standing question in neuroscience research. Many studies have indicated a critical role of the hippocampus in establishing memories of facts and episodes. As episodic memories require the association of many different sensory events in the environment, the hippocampus integrates multimodal information acquired from sensory systems. The brain area that sends major afferent inputs to the hippocampus, the entorhinal cortex, can be further divided into two subareas, the medial and lateral entorhinal cortex, each of which primarily transfers either spatial or nonspatial information to the hippocampus. The proper control of these two information streams is essential for constructing neuronal representations of the environment in hippocampus. To understand this process, my studies have focused primarily on the projection from the entorhinal cortex to area CA1, the temporoammonic pathway. Although this pathway has been relatively unexplored, recent studies have suggested that it plays a unique role in hippocampal function. I investigated how the temporoammonic synapses influence hippocampal function from three different perspectives; in single-neuron studies, local-circuit analyses, and behavioral manipulations. I propose that the temporoammonic pathway gives rise to a unique functional circuit in the hippocampus, which allows for the independent control of spatial and nonspatial information processing. Neuromodulators are a key component to this control as they differentially influence two streams of information from the entorhinal cortex. Finally, I describe my studies on the pathophysiology of schizophrenia-like behaviors at a neuronal circuit level. A mouse model of schizophrenia, generated by maternal immune activation, displays several behavioral abnormalities relevant to schizophrenia patients. We found that hippocampal slices prepared from these mice exhibit altered synaptic properties in the temporoammonic pathway. The mice also exhibit behavioral abnormality in novel object recognition. Taken together, my studies shed light on two information streams in hippocampal circuits. Anatomical or neuromodulatory-based disturbance of this control may underlie some of the behavioral abnormalities observed in several mental disorders." }, { "name": "Luong, Tinh Nghi", "degree": "PhD", "year": "2010", "title": "Signaling Proteins in the Post-Synaptic Density", "advisor": "Kennedy, Mary B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06012010-124413933", "creators": [ { "name": { "family": "Luong", "given": "Tinh Nghi" }, "id": "Luong-Tinh-Nghi", "display_name": "Luong, Tinh Nghi" } ], "advisors": [ { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "advisor", "display_name": "Kennedy, Mary B." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "orcid": "0000-0002-3671-9354", "role": "member", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "member", "display_name": "Kennedy, Mary B." } ], "option_major": [ "biology" ], "doi": "10.7907/GQ1D-CY75", "abstract": "The ability of an organism to respond to its environment stems from synapses and signaling in the post-synaptic density (PSD). Neurological disorders often occur at the level of faulty signal transduction in the PSD. Here we describe the behavioral characterization of Densin 180, a PSD-enriched scaffold protein. We also report on the regulation of Ras guanine release factor1 (RasGRF1), a guanine exchange factor that promotes activation of Ras and thus the ERK pathway as part of an NMDA (N-methyl-D-aspartate) receptor complex with CaMKII (Ca2+/calmodulin-dependent kinase). The Densin KO exhibits severe nest building deficits, elevated anxiety and aggressiveness, impaired sensorimotor gating, hyperlocomotion to novel objects, and short-term memory (hippocampal- and cortical- dependent) deficits. These behavioral abnormalities resemble schizophrenia and autism. Decreases in the schizophrenia susceptibility gene products, DISC1 and mGluR5, are observed in the KO relative to the WT and may be a result of a decrease in their common binding partner \u03b1-actinin. \u03b1-actinin is known to regulate mGluR5 surface levels. Cross-linking and stabilization of PSD protein architecture by scaffold proteins like Densin may contribute to some of the observed behavioral abnormalities. The Densin KO also has blunted activity-dependent gene expression. Steady-state levels of the immediate early genes Arc and c-fos are decreased in the hippocampus and cortex of brain sections. Levels of Arc induced in response to stimulation by the neurotrophin BDNF is significantly decreased in the Densin KO neurons after 8 hours of treatment. Impairments in BDNF signaling can lead to affective and cognitive disorder and has a role in cortical inhibition. Dysfunction in BDNF signaling and DISC1 signaling have been previously implicated in autism. The Densin KO also is susceptible to seizures, in particular when injected with Nembutal, a GABA(A) agonist. One point of intersection between signaling pathways that involve DISC1, mGluR, and BDNF is at the level of ERK signaling, which if impaired may establish a hypoglutamatergic state in the Densin KO.
\r\n\r\nIn addition to characterizing the Densin KO, we studied possible interactions of RasGRF1 and CaMKII with the NR2B subunit tail of the NMDA receptor. CaMKII phosphorylation sites on RasGRF1 were identified, including Ser916, by mass spectrometry. Immunoprecipitation from HEK cells revealed that RasGRF1 enhances CaMKII association with NR2B.
" }, { "name": "McMahon, Amy Jeanette", "degree": "PhD", "year": "2010", "title": "Fibroblast Growth Factors Influence Collective Cell Behavior During Mesoderm Migration", "advisor": "Stathopoulos, Angelike", "url": "https://resolver.caltech.edu/CaltechTHESIS:03122010-112724332", "creators": [ { "name": { "family": "McMahon", "given": "Amy Jeanette" }, "id": "McMahon-Amy-Jeanette", "display_name": "McMahon, Amy Jeanette" } ], "advisors": [ { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "advisor", "display_name": "Stathopoulos, Angelike" } ], "committee": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "chair", "display_name": "Fraser, Scott E." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "member", "display_name": "Stathopoulos, Angelike" } ], "option_major": [ "biology" ], "doi": "10.7907/G4RJ-7158", "abstract": "Collective cell migration is a complex process that occurs in development and disease. As a result, understanding migration has become an important topic in biology. Several models have been developed over the last decade, but these models lack enough diversity to encompass the many different types of migration. Therefore, we propose to add mesoderm migration in Drosophila melanogaster as a model for collective migration. Mesoderm migration involves the movement of hundreds of cells in concert, a process that occurs in many developing animals especially during gastrulation. We have developed a technique for studying mesoderm migration in vivo using two-photon microscopy and subsequent quantitative analyses. Using this technique, we explored the role of fibroblast growth factors (FGFs) during migration of the mesoderm. Drosophila embryos exhibit a simplified FGF signaling pathway, with two ligands interacting with one receptor, making it an ideal system for addressing two complementary questions. Firstly, we investigated what role FGF signaling plays in collective cell migration. At the same time, we were able to ask whether both FGF ligands are required for mesoderm migration, as it is an unanswered question in the FGF field whether FGF ligands function redundantly. We found that during mesoderm migration FGF signaling is required for movement of mesoderm cells toward the ectoderm, and that both ligands are involved. We found some evidence of functional redundancy, but also found that each ligand tended to play a dominant role during different developmental events.\tIn addition, we discovered that mesoderm migration is a multistep process, with only a subset of steps requiring FGF signaling. As a result, we have established the role of FGF during mesoderm migration and opened up many interesting avenues for further study." }, { "name": "Papadopoulou, Maria", "degree": "PhD", "year": "2010", "title": "Gain Control and Sparse Representations in the Olfactory System of the Locust and Fly", "advisor": "Laurent, Gilles J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05292010-035212206", "creators": [ { "name": { "family": "Papadopoulou", "given": "Maria" }, "id": "Papadopoulou-Maria", "display_name": "Papadopoulou, Maria" } ], "advisors": [ { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "advisor", "display_name": "Laurent, Gilles J." } ], "committee": [ { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "orcid": "0000-0001-8837-678X", "role": "chair", "display_name": "Siapas, Athanassios G." }, { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "member", "display_name": "Schuman, Erin Margaret" }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "member", "display_name": "Laurent, Gilles J." } ], "option_major": [ "biology" ], "doi": "10.7907/WSYW-KA90", "abstract": "The giant GABAergic neuron (GGN) is a single, paired, non-spiking neuron that arborizes extensively in the mushroom body (MB) of the locust (Leitch and Laurent, 1996), where it overlaps with the dendrites and the axons of Kenyon cells (KCs). KCs are the intrinsic neurons of the MB and are thought to be important for olfactory learning and memory (Davis, 2004). We are interested in understanding the function of GGN in olfactory processing: in particular, its pattern of arborization makes it an attractive candidate for controlling or modulating KC responses to odors, with potential implications for learning and recall. Physiological recordings of KCs in the locust show that these neurons respond sparsely to odors, in marked contrast to their excitatory inputs from the antennal lobe (projection neurons or PNs) (Laurent and Naraghi, 1994; Perez-Orive et al., 2002; Stopfer et al., 2003; Mazor and Laurent, 2005). Inhibition appears to be critical to control KC response threshold, probability and duration during odor stimulation (Perez-Orive et al., 2002). We show that there exists a feedback loop whereby KCs provide excitatory input to GGN , which in return provides inhibitory control of KC excitability. We further demonstrate that manipulating GGN during olfactory stimulation affects odor-evoked subthreshold oscillations, as measured in individual intracellularly recorded KCs, or by a more global measurement of the local field potential. We also assess the influence of GGN by recording from a population of extrinsic MB neurons that receive input from KCs in the \u03b2-lobe of the MB (\u03b2-LNs).We show that GGN can suppress KC activity to such an extent as to eliminate all spiking in the downstream neurons. With these experiments in the locust, we show that GGN controls the gain of PN-to-KC information transfer and normalizes the output of the KC-population, much reducing its dependence on input strength.
\r\n\r\nWith experiments in Drosophila Melanogaster we try to extend the generality of GGN as a solution for gain control in the MB. Specifically, we carry out intracellular recordings from a Drosophila neuron discovered by Greg Jefferis, which has extensive arborizations throughout the MB calyx and lobes, resembling the locust GGN. We show that this cell is GABAergic, that it is a non-spiking neuron, and that its response to olfactory stimulation is very similar to that of the locust GGN, including a graded response to increasing odor concentration.
" }, { "name": "Ram\u00edrez-Lugo, Juan S.", "degree": "PhD", "year": "2010", "title": "The Activation of ATR in Response to Double-Stranded DNA Breaks", "advisor": "Dunphy, William G.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05262010-155732939", "creators": [ { "name": { "family": "Ram\u00edrez-Lugo", "given": "Juan S." }, "id": "Ramirez-Lugo-Juan-S", "display_name": "Ram\u00edrez-Lugo, Juan S." } ], "advisors": [ { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "role": "advisor", "display_name": "Dunphy, William G." } ], "committee": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "chair", "display_name": "Wold, Barbara J." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "role": "member", "display_name": "Campbell, Judith L." }, { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "role": "member", "display_name": "Dunphy, William G." } ], "option_major": [ "biology" ], "doi": "10.7907/EG9Z-0H60", "abstract": "The cellular response to the presence of double-stranded DNA breaks (DSBs) is primarily mediated by the ATM protein kinase. A related kinase, ATR, regulates the responses to dysfunctional DNA replication and is also activated, in an ATM-dependent manner, when breaks occur during S-phase. The latter is achieved by the ability of ATM to interact with TopBP1, an inducer of ATR activity. Additionally, in Xenopus egg extracts, the Mre11-Rad50-Nbs1 (MRN) complex is required to bridge ATM and TopBP1 together. With our current work, we show that CtIP, a known MRN-interacting protein, is recruited to DSB-containing chromatin and interacts with TopBP1 in a damage-dependent manner. A region containing the first two BRCT repeats of TopBP1 is essential for this interaction. Furthermore, two distinct regions of CtIP participate in mediating the association between CtIP and TopBP1. The first region includes two putative ATM/ATR phosphorylation sites. Secondly, an MRN-binding region in the N-terminal region of CtIP is involved. In addition, the binding between CtIP and TopBP1 is diminished in Nbs1-depleted extracts and, reciprocally, the binding of Nbs1 to TopBP1 decreases in the absence of CtIP. This suggests the formation of a complex containing CtIP, TopBP1 and the MRN complex. When CtIP is removed from egg extracts, the levels of TopBP1 and Nbs1 in damaged nuclei are reduced, thereby compromising the activation of the damage response. Thus, CtIP interacts with TopBP1 in a damage-stimulated, MRN-dependent manner to mediate the activation of ATR in response to DSBs. \r\n
\r\nWe additionally explore the involvement of the chromatin remodeling ATPase ISWI in the responses to DNA damage. We find that ISWI associates with ATR, ATRIP, and TopBP1 on DNA in the presence of damage. In addition, ISWI is a substrate of both ATM and ATR in vitro. Furthermore, the activities of ATM and ATR stimulate an increase in the levels of ISWI on chromatin that contains DSBs. Finally, we assessed the role of ISWI in the activation of multiple damage responses in Xenopus egg extracts. Taken together, our work describes several previously uncharacterized features of ISWI with implications in the response to damaged and incompletely replicated DNA.\r\n
" }, { "name": "Rao, Dinesh Subba", "degree": "PhD", "year": "2010", "title": "Small RNAs Play Big Roles in Hematopoietic Development", "advisor": "Baltimore, David L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05142010-184014918", "creators": [ { "name": { "family": "Rao", "given": "Dinesh Subba" }, "id": "Rao-Dinesh-Subba", "display_name": "Rao, Dinesh Subba" } ], "advisors": [ { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "advisor", "display_name": "Baltimore, David L." } ], "committee": [ { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "chair", "display_name": "Patterson, Paul H." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "member", "display_name": "Baltimore, David L." } ], "option_major": [ "biology" ], "doi": "10.7907/D6GJ-4057", "abstract": "The relatively recent identification of microRNAs (miRNAs) has added a new layer of complexity to our understanding of gene regulation. These small noncoding RNAs cause the downregulation of target mRNAs, leading to downstream effects. We have studied how particular miRNAs can impact hematopoietic development in mice and correlated these findings with miRNA deregulation in human disease. Specifically, we studied the role of miR-155 in myeloid cells, finding that it promoted myeloid cell expansions when induced by lipopolysaccharide and a myeloproliferative disease when constitutively expressed. Interestingly, miR-155 is overexpressed in acute myeloid leukemia, a human disease that has the proliferation of myeloid cells in common with our mouse model. miR-155 is a growth-promoting factor in cells and acts in early hematopoiesis by repressing the inositol phosphatase, SHIP1. We have also studied the role of miR-34a, a putative growth-suppressing miRNA, in hematopoietic development, finding a specific perturbation in B-cell development by gain- and loss of function analyses. In this case, the majority of the findings are attributable to repression of Foxp1, a transcription factor involved in regulation of immunoglobulin gene V(D)J recombination. Our findings show how miRNAs are integrated into developmental pathways that control hematopoiesis, and suggest that they may act as nodes of regulation during specific hematopoietic developmental processes." }, { "name": "Robie, Alice Anne Pennoyer", "degree": "PhD", "year": "2010", "title": "Multimodal Sensory Control of Exploration by Walking Drosophila melanogaster", "advisor": "Dickinson, Michael H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05242010-123734162", "creators": [ { "name": { "family": "Robie", "given": "Alice Anne Pennoyer" }, "id": "Robie-Alice-Anne-Pennoyer", "display_name": "Robie, Alice Anne Pennoyer" } ], "advisors": [ { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "role": "advisor", "display_name": "Dickinson, Michael H." } ], "committee": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "chair", "display_name": "Anderson, David J." }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Frye", "given": "Mark" }, "id": "Frye-M", "role": "member", "display_name": "Frye, Mark" }, { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "role": "member", "display_name": "Dickinson, Michael H." } ], "option_major": [ "biology" ], "doi": "10.7907/GN01-P258", "abstract": "Walking fruit flies, Drosophila melanogaster, use visual information to orient towards salient objects in their environment, presumably as a search strategy for finding food, shelter or other resources. Less is known about the role of vision or other sensory modalities in the evaluation of objects once they have been reached. In order to study these behaviors, I developed a large arena in which I could track individual fruit flies as they walk through either simple or more topologically complex landscapes. Flies use visual cues from the distant background to stabilize their walking trajectories. When exploring an arena containing objects, flies actively orient towards, climb onto, and explore the objects, spending most of their time on the tallest, steepest object. A fly\u2019s behavioral response to an object\u2019s geometry depends upon the intrinsic properties of each object and not an assessment relative to other nearby objects. Further, the preference is due to a change in locomotor behavior once a fly reaches and explores the object\u2019s surface. Specifically, flies are much more likely to stop walking for long periods on tall, steep objects. Both the visual and the antennal mechanosensory systems provide sufficient information about an object\u2019s geometry to elicit the observed change in locomotor behavior. Only when both these sensory systems are impaired do flies not show the behavioral preference for the tall, steep objects. Additionally, I examined the locomotor and social behaviors of large groups of flies. In order to do these studies, I assisted in the development of automated software for tracking and maintaining the individual identity of large groups of flies and for the quantification of individual flies\u2019 locomotor and social behaviors. Behavioral differences between individuals are consistent over the time of the trials and are sufficient to predict a fly\u2019s gender (male vs. female), genotype (wild type vs. fruitless), or sensory environment (with vs. without visual cues). During encounters, males approach other flies more closely than do females and are most often located behind the other fly. The software developed is publicly available and represents a new level of automated quantification in behavioral studies of flies." }, { "name": "Simon, Jasper Chen", "degree": "PhD", "year": "2010", "title": "Behavioral Analysis of Exploration and Dispersal in Drosophila", "advisor": "Dickinson, Michael H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:11122009-111028665", "creators": [ { "name": { "family": "Simon", "given": "Jasper Chen" }, "id": "Simon-Jasper-Chen", "display_name": "Simon, Jasper Chen" } ], "advisors": [ { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "orcid": "0000-0002-8587-9936", "role": "advisor", "display_name": "Dickinson, Michael H." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "member", "display_name": "Benzer, Seymour" }, { "name": { "family": "Greenspan", "given": "Ralph" }, "id": "Greenspan-R", "role": "member", "display_name": "Greenspan, Ralph" }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "member", "display_name": "Perona, Pietro" }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "member", "display_name": "Koch, Christof" }, { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "orcid": "0000-0002-8587-9936", "role": "member", "display_name": "Dickinson, Michael H." } ], "option_major": [ "biology" ], "doi": "10.7907/P7PH-5K38", "abstract": "A fundamentally important decision for all animals is whether to utilize a particular resource or to disperse elsewhere in search of potentially superior resources. Within this dissertation, I present results from laboratory experiments carried out using the experimental genetic workhorse, Drosophila melanogaster, to identify and quantify various causal factors contributing to an animal's decision to disperse from food.
\r\n\r\nWith the set of experiments described within the second chapter, I studied the influence of mating experience on the movement priorities of Drosophila. From these experiments, I suggest that prior mating experience is a significant and likely an important factor modulating the dispersal of Drosophila, and that the change in dispersal results from a change in the fly's priorities rather than simply a change in the general levels of activity. In chapter three, using methods similar to those used to assess the modulatory effects of mating, I explored how the amount and accessibility of food affects the dispersal of hungry Drosophila. From these experiments, I suggest that the hunger state of flies can override the visual and olfactory cues from food, and I hypothesize that the observed increase in dispersal resulting from hunger is due to a qualitative change in locomotor behavior related to food search.
\r\n\r\nWith a new machine-vision tracking strategy discussed within the fourth chapter, I studied the exploratory behaviors of individual flies within the environmental chambers discussed in Chapters 2 and 3. I introduced single flies that had recently consumed food into chambers and tracked their walking and monitored their flying movements as they became hungry. In collaboration, I have attempted to use learning algorithms based on the statistics of each fly's behavior during short windows of time to predict the fly's behavior during the rest of their experimental trial.
\r\n\r\nI conclude with chapter five by describing a new experimental chamber that I have developed to complement machine-vision methods for tracking individuals within large groups. The motivation behind developing the chamber was to study the changes of social interaction, e.g., courtship and aggressive posturing, of flies near food.
" }, { "name": "Wawrousek, Karen Elizabeth", "degree": "PhD", "year": "2010", "title": "Contributions of Dna2 and the Tim/Tipin Complex to Genomic Stability", "advisor": "Dunphy, William G.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01032010-225423037", "creators": [ { "name": { "family": "Wawrousek", "given": "Karen Elizabeth" }, "id": "Wawrousek-Karen-Elizabeth", "display_name": "Wawrousek, Karen Elizabeth" } ], "advisors": [ { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "role": "advisor", "display_name": "Dunphy, William G." } ], "committee": [ { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "role": "chair", "display_name": "Campbell, Judith L." }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "role": "member", "display_name": "Dunphy, William G." } ], "option_major": [ "biochem" ], "doi": "10.7907/QYSH-WH15", "abstract": "This thesis describes the essential roles of Dna2 and the Tim/Tipin complex in the maintenance of genomic stability. Dna2 participates in DNA replication and double-strand break repair by homologous recombination. Meanwhile, the Tim/Tipin complex is required for efficient checkpoint activation upon replication stress, which can be caused by stalled DNA replication forks.
\r\n\r\nWhile yeast genetics and experiments with purified proteins have revealed much about yeast Dna2, we chose to pursue characterization of metazoan Dna2 using Xenopus cell-free extracts. We show that binding of Dna2 to origins of replication is dependent upon formation of pre-replication complexes but independent of CDK2 activity. Upon initiation of DNA replication, Dna2 travels with replication forks. Physical interactions with Mcm10 and And-1, proteins involved in lagging strand DNA replication, are indicative of a role in replication of the lagging strand; this result is consistent with genetic results in yeast and in vitro biochemical experiments.
\r\n\r\nDna2 also participates in the response to double-strand breaks and accumulates on chromatin containing double-strand breaks. We show that Dna2 binds to free DNA ends after the Mre11-Rad50-Nbs1 complex and ATM, but before RPA. Dna2-depleted extracts exhibit delayed processing of DNA ends, indicating that other nucleases do not easily compensate for the lack of Dna2. Consistent with genetic results in yeast, we find that the Mre11-Rad50-Nbs1 protein complex is essential for the processing of free DNA ends, but inhibition of Mre11 nuclease activity only slows processing. This observation indicates that other nucleases, possibly Dna2, can compensate for loss of Mre11 nuclease activity. Despite the role of Dna2 in double-strand break processing, Dna2 is not required for checkpoint activation.
\r\n\r\nTimeless (Tim) and Tipin participate in the checkpoint response to stalled replication forks. We demonstrate here that Tim and Tipin form a complex, associate with chromatin in S phase, and physically interact with many proteins at the replication fork. Human cells lacking the Tim/Tipin complex do not exhibit robust checkpoint activation in response to stalled replication forks. Finally, we show that Tipin is also a target of both the ATR and Cdc7 kinases, which respond to stalled replication forks.
\r\n" }, { "name": "Wright, Ashley Palani", "degree": "PhD", "year": "2010", "title": "Genetic Analysis of Axon Guidance in Drosophila melanogaster", "advisor": "Zinn, Kai George", "url": "https://resolver.caltech.edu/CaltechTHESIS:05252010-112354381", "creators": [ { "name": { "family": "Wright", "given": "Ashley Palani" }, "id": "Wright-AshleyPalani", "display_name": "Wright, Ashley Palani" } ], "advisors": [ { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "advisor", "display_name": "Zinn, Kai George" } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "member", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "role": "member", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Prober", "given": "David A." }, "id": "Prober-D-A", "role": "member", "display_name": "Prober, David A." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" } ], "option_major": [ "biology" ], "doi": "10.7907/FTNQ-S839", "abstract": "Due to its genetic manipulability and relatively short reproductive cycle, genetic screens are often carried out in the fruit fly, Drosophila melanogaster. Deficiency \u201ckits\u201d that cover the Drosophila genome with a minimum number of lines have been established by other groups to facilitate gene mapping. These kits cannot be systematically analyzed for many phenotypes, however, because embryos homozygous for many deficiencies fail to develop due to the loss of key gene products. To create new kits that can be screened for more phenotypes, we have examined the development of the nervous system in embryos homozygous for more than 700 distinct deficiency mutations. A kit of ~400 deficiency lines for which homozygotes have a recognizable nervous system and intact body walls encompasses >80% of the genome. Here we show examples of screens of this kit for orphan receptor ligands and neuronal antigen expression. Screens of this kit can also be used to find genes involved in expression, patterning, and subcellular localization of any protein that can be visualized by antibody staining. A subset kit of 233 deficiency lines, for which homozygotes develop relatively normally to late stage 16 (thus allowing for central nervous system development), covers ~50% of the genome. We have screened this smaller kit for motor axon guidance phenotypes, and we present examples of new axon guidance phenotypes in the central nervous system and neuromuscular system. Through screening of these kits, we also found deficiencies that fail to stain with monoclonal antibody BP102, which recognizes an unknown epitope on the proximal segments of central nervous system axons. In addition, we have found a deficiency that exhibits ectopic BP102 staining on peripheral sensory neurons. By defining the single genes under these deficiencies, we have obtained evidence that BP102 may recognize a chondroitin sulfate proteoglycan and that BP102 epitope expression is regulated by matrix metalloproteinase 1. Thus, in addition to this screen providing information about motor axon guidance in the embryo, we have also been able to further characterize an antibody that is frequently used by the Drosophila community.\r\n" }, { "name": "Yorozu, Suzuko", "degree": "PhD", "year": "2010", "title": "Distinct Sensory Representations of Wind and Near-Field Sound in the Drosophila Brain", "advisor": "Anderson, David J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05282010-134450366", "creators": [ { "name": { "family": "Yorozu", "given": "Suzuko" }, "id": "Yorozu-Suzuko", "display_name": "Yorozu, Suzuko" } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "advisor", "display_name": "Anderson, David J." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "role": "member", "display_name": "Dickinson, Michael H." }, { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "member", "display_name": "Benzer, Seymour" }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." } ], "option_major": [ "biology" ], "doi": "10.7907/8H1W-RW51", "abstract": "Behavioral responses to wind are thought to play a critical role in controlling the dispersal and population genetics of wild Drosophila species, as well as their navigation in flight, but their underlying neurobiological basis is unknown. I show that Drosophila melanogaster, like wild-caught Drosophila strains, exhibits robust wind-induced suppression of locomotion (WISL), in response to air currents delivered at speeds normally encountered in nature. Furthermore, I identify wind-sensitive neurons in the Johnston\u2019s organ (JO), an antennal mechanosensory structure previously implicated in near-field sound detection. Using Gal4 lines targeted to different subsets of JO neurons, and a genetically encoded calcium indicator, I show that wind and near-field sound (courtship song) activate distinct JO populations, which project to different regions of the antennal and mechanosensory motor center (AMMC) in the central brain. Selective genetic ablation of wind-sensitive JO neurons in the antenna abolishes WISL behavior, without impairing hearing. Different neuronal sub-populations within the wind-sensitive population, moreover, respond to different directions of arista deflection caused by airflow and project to different regions of the AMMC, providing a rudimentary map of wind direction in the brain. Importantly, sound- and wind-sensitive JO neurons exhibit different intrinsic response properties: the former are phasically activated by small, bidirectional displacements of the aristae, while the latter are tonically activated by unidirectional, static deflections of larger magnitude. These different intrinsic properties are well suited to the detection of oscillatory pulses of near-field sound and laminar airflow, respectively. These data identify wind-sensitive neurons in JO, a structure that has been primarily associated with hearing, and reveal how the brain can distinguish different types of air particle movements, using a common sensory organ." }, { "name": "Zarnegar, Mark Andrew", "degree": "PhD", "year": "2010", "title": "Investigating the Transcriptional Mechanisms Controlling Sfpi1, a Critical Regulatory Node Within Multiple Lineage Specifying Subcircuits of the Hematopoietic Gene Regulatory Network", "advisor": "Rothenberg, Ellen V.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05062010-152920403", "creators": [ { "name": { "family": "Zarnegar", "given": "Mark Andrew" }, "id": "Zarnegar-Mark-Andrew", "display_name": "Zarnegar, Mark Andrew" } ], "advisors": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "advisor", "display_name": "Rothenberg, Ellen V." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." } ], "option_major": [ "biology" ], "doi": "10.7907/ST6P-C529", "abstract": "The Sfpi1 gene encodes PU.1, a critical transcription factor in multiple hematopoietic lineages. PU.1 expression is upregulated as hematopoietic stem cells become granulocyte-macrophage progenitors. In contrast, Sfpi1 must be silenced after progenitors undergo T-lineage specification. If unrestrained in early T-lineage cells, PU.1 can both block developmental progress and induce diversion to a myeloid fate. When PU.1 expression is not sufficiently increased or maintained in myeloid lineage cells, myeloid hyperproliferation and cancer can result. In mouse DN thymocytes, PU.1 mRNA begins at high levels in early T-cell progenitors, but drops about fivefold as cells enter the T-cell program (DN2) and then falls tenfold further as the cells reach T-lineage commitment (DN3). This implies operation of a stage-specific repression mechanism correlated with commitment. Only one major cis-regulatory element has previously been described for Sfpi1, which is a compound conserved region around -14 kb that is thought to mediate activation as well as some repression. However, it cannot account for all PU.1 regulation in early T-lineage cells nor in myeloid cells. In particular, that -14 kb element can show strong enhancer activity in an immature T-cell line in which the endogenous Sfpi1 gene is profoundly repressed. Additionally, absence of the -14 kb element does not abolish PU.1 expression in myeloid lineages. We now present evidence for another complex of conserved noncoding elements that appear to mediate several cell-type-specific regulatory features, including cell-type-specific repression in early T-cells. We describe fine mapping of a T-cell specific bipartite silencer and show that the T lineage specific repressive activity requires Runx1. We also describe additional regulatory complexes that may contribute to lineage specific regulation of PU.1 in early hematopoietic progenitors, including a myeloid specific enhancer. We provide evidence of lineage restricted occupancy of these additional regulatory elements and show that the novel enhancer elements are additional sites of PU.1 auto regulation." }, { "name": "Adams, Meghan Sara", "degree": "PhD", "year": "2009", "title": "Uncovering Molecular Properties of Neural Crest Cells", "advisor": "Bronner, Marianne E.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05292009-111417", "creators": [ { "name": { "family": "Adams", "given": "Meghan Sara" }, "id": "Adams-Meghan-Sara", "display_name": "Adams, Meghan Sara" } ], "advisors": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "advisor", "display_name": "Bronner, Marianne E." } ], "committee": [ { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "chair", "display_name": "Zinn, Kai George" }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biology" ], "doi": "10.7907/KYEQ-T802", "abstract": "The neural crest is a transient population of cells that arises at the border between the neural and non-neural ectoderm. These cells are induced, undergo an epithelial-to-mesenchymal transition, and then migrate along stereotypical pathways to form a wide array of derivatives. While these cells have long been studied, much about these cells and their interactions is still not understood. In order to better define these cells, we performed a screen for genes involved in neural crest cell development based on an in vitro culture system that produces neural crest cells. This highly successful screen resulted in a large number of candidates to examine, and we performed in situ hybridization to define the mRNA expression of 112 these genes. Moreover, we performed QPCR on several transcription factors that resulted from this screen to determine the level at which they were upregulated in our in vitro culture system. We also present loss-of-function analyses of two different genes that were discovered in our screen for neural crest effectors. These genes, Adh5 and Ccar1, are both functionally relevant in neural crest cells and the loss of either one through morpholino knockdown significantly decreases the mRNA of Sox10 on the injected side. Furthermore, we also show that Adh5 morpholino knockdown also results in a reduction of Snail2 and FoxD3 mRNA. Taken as a whole, this body of work represents the discovery of many new genes involved neural crest cell development, and the demonstration that at least two of these genes are functionally important for neural crest cells. " }, { "name": "Anderson, Megan Jo", "degree": "PhD", "year": "2009", "title": "Microfluidics-Based Strategies for Protein Crystallography", "advisor": "Quake, Stephen R.", "url": "https://resolver.caltech.edu/CaltechETD:etd-10172008-222221", "creators": [ { "name": { "family": "Anderson", "given": "Megan Jo" }, "id": "Anderson-Megan-Jo", "display_name": "Anderson, Megan Jo" } ], "advisors": [ { "name": { "family": "Quake", "given": "Stephen R." }, "id": "Quake-S-R", "orcid": "0000-0002-1613-0809", "role": "advisor", "display_name": "Quake, Stephen R." } ], "committee": [ { "name": { "family": "Quake", "given": "Stephen R." }, "id": "Quake-S-R", "orcid": "0000-0002-1613-0809", "role": "chair", "display_name": "Quake, Stephen R." }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "co-chair", "display_name": "Rees, Douglas C." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." } ], "option_major": [ "biochem" ], "doi": "10.7907/P5ZD-0S21", "abstract": "Protein crystallography is an invaluable tool for the study of biological processes at the molecular level. While several crystallization techniques are actively pursued in both academic and industrial laboratories to produce high-quality protein crystals, the use of microfluidic technology for structural biology was previously shown to improve protein crystallization over more traditional methods. This thesis describes a microfluidics-based crystallization strategy that was developed to increase the success rate of crystallizing challenging proteins. The crystallization strategy involves using multiple microfluidic devices to characterize the solubility trends of the crystallization target, to perform nanoliter volume free interface diffusion crystallization experiments designed around the solubility trends, and to enable in situ diffraction analysis of crystals grown in microfluidic devices. The crystallization strategy was applied to the crystallization of a dozen challenging proteins and increased the overall crystallization and diffraction success rates compared with conventional automation. The crystallization strategy was also utilized to crystallize four metabolic proteins and provides the first demonstration of in situ structure determination for novel crystallization targets using a microfluidic crystallization platform. Additional technological advances were accomplished by the development of a novel microfluidic device designed to address the specific challenges of membrane protein crystallography. To date, this microfluidic crystallization strategy has produced four novel protein structures and holds great promise for future work in the field of protein crystallography.\r\n" }, { "name": "Escobedo, Jessica Rose", "degree": "PhD", "year": "2009", "title": "Investigating Moral Events: Characterization and Structure of Autobiographical Moral Memories", "advisor": "Adolphs, Ralph", "url": "https://resolver.caltech.edu/CaltechETD:etd-11112008-122002", "creators": [ { "name": { "family": "Escobedo", "given": "Jessica Rose" }, "id": "Escobedo-Jessica-Rose", "display_name": "Escobedo, Jessica Rose" } ], "advisors": [ { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "role": "advisor", "display_name": "Adolphs, Ralph" } ], "committee": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "role": "chair", "display_name": "Andersen, Richard A." }, { "name": { "family": "Woodward", "given": "Jim" }, "id": "Woodward-J", "role": "member", "display_name": "Woodward, Jim" }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "role": "member", "display_name": "Adolphs, Ralph" } ], "option_major": [ "biology" ], "doi": "10.7907/M8XP-KT54", "abstract": "Moral events and the actions, decisions and people they involve, are judged as right or wrong, and the moral responsibility associated with them generates further judgments, often legal in nature, of blame and punishment or praise. Not only do moral events and the normative judgments they presuppose define essential aspects of human nature, they are also ubiquitous at the level of society as well as the individual. Despite their importance, characterizing the sociological, psychological, and neurological features of moral events is in its infancy. Much of the recent research has focused on a priori philosophical frameworks and has used artificial events as probes, in part because collecting, characterizing and analyzing real-life moral events is a major undertaking. This dissertation attempts such an undertaking.
\r\n\r\n758 autobiographical memories of personal moral events were collected from a well-characterized and representative sample of 100 healthy Californian adults. Transcriptions of the events were further characterized and all data were entered into a large, searchable database. An initial set of results provides a detailed description of the participants and the memories of moral events they generated. This description showed that participants were highly representative of the general population of California; that the overall amount and patterns of moral events recollected was relatively universal and not influenced by gender, ethnicity, IQ, or personality; and that the moral events produced could generally be judged quite reliably both by the participants themselves as well as by independent raters.
\r\n\r\nThe database was further analyzed with respect to three specific aims: (1) to study the semantic structure of real-life moral events; (2) to study the effects of focal lesions to emotion-related brain regions on recollection of moral events; (3) to study the temporal distribution of autobiographical moral events. We found that real-life moral events have a hierarchical structure, with two broad categories of \u201cgood\u201d and \u201cbad\u201d, and subordinate categories of \u201cgood\u201d, \u201clying\u201d, \u201cstealing\u201d, and \u201churting another person\u201d. These categories define the most common scripts encountered in real life that have strong moral value. In studying neurological patients with focal lesions to the ventromedial prefrontal cortex or the amygdala, we found no evidence for a notable skew in the moral events that were recollected, further evidence for the universality and robustness of such events in our autobiography. Finally, we found that positively valenced moral events were systematically recalled as being more recent in time than negatively valenced moral events, a temporal bias that was independent of absolute participant age. The methods used here, the database that was constructed, and the scientific questions that were analyzed constitute the first comprehensive investigation of a large number of real-life moral events and provide a rich resource for future studies.
\r\n" }, { "name": "Gordon, Sean Patrick", "degree": "PhD", "year": "2009", "title": "Hormone and Gene Feedback during Development and Regeneration in Arabidopsis thaliana", "advisor": "Meyerowitz, Elliot M.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05022009-103703", "creators": [ { "name": { "family": "Gordon", "given": "Sean Patrick" }, "id": "Gordon-Sean-Patrick", "orcid": "0000-0003-3431-5804", "display_name": "Gordon, Sean Patrick" } ], "advisors": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "advisor", "display_name": "Meyerowitz, Elliot M." } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "chair", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "member", "display_name": "Deshaies, Raymond Joseph" } ], "option_major": [ "biochem" ], "doi": "10.7907/T3CR-TC54", "abstract": "Higher plants maintain continuous development throughout their life by closely regulating the process of cell differentiation (Clark, 2001; Sablowski, 2007). In plants, the balance between undifferentiated and differentiated cell fate is managed within a stem cell niche termed the meristem. Cell differentiation in the meristem is in part controlled by genetic mechanisms. For example, mutations in CLAVATA (CLV) genes increase the number of undifferentiated cells within shoot and floral meristems leading to supernumerary organs (Clark, 2001). In contrast, mutations in genes of the homeodomain transcription factors WUSCHEL (WUS) and SHOOT-MERISTEMLESS (STM) lead to the absence of the shoot or floral meristem or its early termination through differentiation (Laux et al., 1996; Long et al., 1996).
\r\n\r\nCell differentiation in the meristem is also controlled by hormonal cues, which interfaces with gene function. For example, cytokinin treatment leads to phenotypes resembling clv mutants (Lindsay et al., 2006). Furthermore, exogenous cytokinin treatment has been shown to rescue the stm mutant phenotype and WUS protein has been shown to repress transcription of genes that act in the negative feedback pathway of cytokinin signaling (Leibfried et al., 2005; Yanai et al., 2005). The plant hormone auxin also plays a role in regulating differentiation. Auxin is thought to stimulate the initiation, development and differentiation of cells specified into organs (Teale et al., 2006). Disruption of auxin transport leads to a reduction in organ initiation and differentiation (Okada et al., 1991).
\r\n\r\nIn this thesis we investigate spatially regulated signaling and action of auxin and cytokinin which regulate patterning of gene expression and cell differentiation. To this end, we employed two model systems of shoot meristem initiation and development in the model plant Arabidopsis thaliana: shoot and floral meristem development and de novo shoot meristem initiation from tissue culture. Based on characterization of hormone signaling and patterning of gene expression during de novo shoot meristem initiation from tissue culture we propose a novel Turing-like model by which auxin and cytokinin interact to regulate patterning of cell differentiation. In this model, the activity of auxin, the activator of cell differentiation, is regulated by cytokinin, an inhibitor of cell differentiation. Computational models of these interactions lead to self organizing patterning of hormone response and cell differentiation as observed in experiments.
\r\n\r\nIn our second investigation, we show that cytokinin signaling regulates the spatial patterning of the homeodomain transcription factor WUS within the shoot meristem. We demonstrate that WUS misregulation after cytokinin treatment is mediated by both CLAVATA-dependent and independent mechanisms leading to multiple feedback loops. We reveal the presence of a cytokinin perception and signaling gradient within the shoot meristem, which spatially influences size and position of the WUS domain.
\r\n\r\nFinally, we have begun to identify the molecular components required for cytokinin activation of WUS expression. Of the three characterized cytokinin receptors, only Arabidopsis Histidine Kinase 2 (AHK2) is required for WUS induction in the presence of cytokinin. In contrast, the AHK3 receptor is required for negative feedback on cytokinin signaling and thus WUS. These data reveal an unappreciated specificity in cytokinin signaling in regulating downstream targets which may be important for eliciting different cell behaviors depending on the threshold of signaling and the ratio of the three cytokinin receptors within a given cell.
\r\n" }, { "name": "Khudyakov, Jane Igor", "degree": "PhD", "year": "2009", "title": "Role of Bmi-1 in Epigenetic Regulation during Early Neural Crest Development", "advisor": "Bronner, Marianne E.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05192009-141110", "creators": [ { "name": { "family": "Khudyakov", "given": "Jane Igor" }, "id": "Khudyakov-Jane-Igor", "orcid": "0000-0001-7038-102X", "display_name": "Khudyakov, Jane Igor" } ], "advisors": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "advisor", "display_name": "Bronner, Marianne E." } ], "committee": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "chair", "display_name": "Fraser, Scott E." }, { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "member", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/6G1J-PT32", "abstract": "The neural crest is a transient, multipotent cell population in the developing vertebrate embryo that migrates extensively and contributes to a staggering diversity of cell lineages. Neural crest progenitors are specified at the neural plate border during gastrulation; however, commitment to the neural crest lineage does not occur for some time. I find that the chick neural plate border is characterized by co-expression of many neural crest specifier genes, previously considered \u201clate\u201d signals, which often overlap with \u201cearly\u201d neural plate border genes. This suggested that continuously expressed members of the neural crest gene regulatory network may be modulated or repressed for proper maintenance of the multipotent state. Consistent with this possibility, several members of the Polycomb Group of epigenetic repressors are expressed at these early stages. For example, the stem cell factor Bmi-1 is expressed at the neural plate border, dorsal neural folds, and migrating neural crest, but is extinguished in differentiated derivatives. Morpholino-mediated knock-down of Bmi-1 causes early upregulation of Msx1, FoxD3, and Sox9 in the chick neurula without affecting cell proliferation. Conversely, Bmi-1 over-expression causes a downregulation of Msx1, suggesting that it negatively regulates neural crest network genes. I find that several alternative splice variants of Bmi-1 are expressed in the developing chick and that an N-terminal variant, V4, acts as a dominant-negative regulator of the full-length version, up-regulating Msx1 expression. Taken together, these results suggest that neural crest progenitors are exposed to numerous specification signals during gastrulation, some of which are regulated by Polycomb Group factors such as Bmi-1. The activity of Bmi-1, in turn, is modulated by alternatively spliced variants, demonstrating an additional level of regulatory complexity acting during early neural crest development.\r\n" }, { "name": "Klein, Joshua Simon", "degree": "PhD", "year": "2009", "title": "Investigations in the Design and Characterization of HIV-1 Neutralizing Molecules", "advisor": "Bjorkman, Pamela J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06042009-131723", "creators": [ { "name": { "family": "Klein", "given": "Joshua Simon" }, "id": "Klein-Joshua-Simon", "display_name": "Klein, Joshua Simon" } ], "advisors": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "advisor", "display_name": "Bjorkman, Pamela J." } ], "committee": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "chair", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "orcid": "0000-0001-8723-8190", "role": "member", "display_name": "Baltimore, David L." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." } ], "option_major": [ "biochem" ], "doi": "10.7907/QPEC-8Y25", "abstract": "Human Immunodeficiency Virus (HIV) is a T-lymphotrophic retrovirus that is the causative agent of Acquired Immunodeficiency Syndrome and is estimated to currently infect approximately 40 million people worldwide. Life-extending therapies are credited for the precipitous drop in HIV-related mortality in developed countries, but their high costs prevent widespread distribution in developing countries. To date, all attempts to produce a vaccine capable of preventing or controlling an HIV infection have failed, but a comprehensive explanation for these failures has yet to emerge from the available data. In this thesis the first chapter provides an overview of the pandemic, the antigenic properties of gp120 and gp41, which are the two glycoproteins that comprise the outer envelope spike of the virus, and the broadly neutralizing antibodies that have been isolated against them. The second and third chapters discuss biophysical characterizations of these monoclonal antibodies and newly designed molecules derived from them. Based on a comparison of these data with pre-existing research, a novel hypothesis called the \"island effect\" was developed and is presented as a possible explanation for the consistent failure of the human immune system to respond to infection or vaccination with an effective humoral response. The final chapter summarizes ongoing investigations in the capacities of broadly neutralizing monoclonal antibodies to recruit antibody-dependent cellular cytotoxicity, a mechanism by which antibodies can trigger the lysis of HIV-infected cells by the innate immune system. " }, { "name": "Liberman, Louisa M.", "degree": "PhD", "year": "2009", "title": "Regulation of Neurogenic Ectoderm Specification in Drosophila melanogaster", "advisor": "Stathopoulos, Angelike", "url": "https://resolver.caltech.edu/CaltechETD:etd-05292009-123115", "creators": [ { "name": { "family": "Liberman", "given": "Louisa M." }, "id": "Liberman-Louisa-M", "orcid": "0000-0002-1315-0097", "display_name": "Liberman, Louisa M." } ], "advisors": [ { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "advisor", "display_name": "Stathopoulos, Angelike" } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "member", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biology" ], "doi": "10.7907/RNSK-G868", "abstract": "Creating a functional organism requires reproducible developmental patterning. A nuclear gradient of the NF-\u03baB transcription factor, Dorsal, provides positional information necessary to specify the mesoderm, neurogenic ectoderm, dorsal ectoderm and amnioserosa along the dorsal-ventral axis in Drosophila melanogaster embryos. In this work we investigate the role that Dorsal and other transcription factors play in these crucial patterning events. We focus primarily on the gene regulation that controls patterning of the presumptive neurogenic ectoderm that is specified in lateral regions of the embryo. We investigate this early patterning event in two ways: first, by studying a known regulatory element for this region, and second, by examining the levels of Dorsal in the nuclei. We find that Dorsal can function with Zelda, a maternally deposited ubiquitous activator, to specify the neurogenic ectoderm. We then ask if the levels of Dorsal in wild type embryos are predictive of the gene expression outputs, as suggested by existing models. We measure the amount of Dorsal protein able to activate target gene expression in mutants, where the levels of Dorsal protein have been genetically manipulated. Our measurements indicate that Dorsal does not regulate gene expression in a concentration-dependent fashion. Instead, our data support the idea that Dorsal functions with other proteins to establish gene expression boundaries. These studies jointly suggest that regulation of differential gene expression requires combinatorial interactions between spatially localized and uniformly distributed transcription factors. " }, { "name": "Medina-Marino, Andrew G. A.", "degree": "PhD", "year": "2009", "title": "Construction and Initial Characterization of the Densin Knockout Mouse", "advisor": "Kennedy, Mary B.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06022009-194041", "creators": [ { "name": { "family": "Medina-Marino", "given": "Andrew G. A." }, "id": "Medina-Marino-Andrew-G-A", "orcid": "0000-0002-2691-982X", "display_name": "Medina-Marino, Andrew G. A." } ], "advisors": [ { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "role": "advisor", "display_name": "Kennedy, Mary B." } ], "committee": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "chair", "display_name": "Wold, Barbara J." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "role": "member", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "role": "member", "display_name": "Bjorkman, Pamela J." } ], "option_major": [ "biology" ], "doi": "10.7907/HYHX-A061", "abstract": "Densin-180 is a core protein of postsynaptic densities (PSDs) in excitatory neurons. Densin is known to interact with Maguin-1 and PSD-95, suggesting that it plays a role in the NMDA receptor complex. Densin also interacts with \u03b4-Catenin and N-Cadherin, an adhesion complex known to play a role in spine morphology. A ternary complex of Densin, CaMKII, and alpha-actinin suggests that Densin plays a key role in cytoskeleton dynamics. Finally, Densin can directly bind to shank, a core scaffolding molecule of the postsynaptic density. The association of Densin with such diverse complexes of proteins suggests that it acts as an integrator of numerous signaling cascades. Here I describe the construction and initial characterization of a Densin knockout mouse. Mice homozygous for the Densin deletion are prone to seizures induced by barbiturates. Also, Densin^-/- animals have altered spine morphologies and show changes in the expression levels of other core PSD proteins. Densin^-/- neurons in culture exhibit an overall decrease in their dendritic complexity. Furthermore, we show that in the absence of the NMDA receptor, Densin can act to bind CaMKII in the PSD. A new high-throughput method for studying changes in gene transcription, RNA seq, was also used to study the effect of the Densin deletion on the forebrain and the hippocampus. This work represents the first time RNA seq has been used to study an animal with a knockout mutation. Two candidate genes that may mediate the seizure sensitivity, Npas4 and GABAA\u03b12, were identified by this method. Npas4 is known to directly affect the number of inhibitory synapses formed by neurons, and GABAA\u03b12 is a major GABA receptor subunit that mediates the effects of Nembutal. These results suggest that Densin may play a role in maintaining the balance between inhibitory and excitatory networks. Together, our results demonstrate that Densin is important for dendritic arbor formation, spine morphology, CaMKII localization in the PSD, and seizure susceptibility.\r\n" }, { "name": "Muffat, Julien A.", "degree": "PhD", "year": "2009", "title": "Role of Apolipoprotein D and Its Homologs, in Normal and Pathological Aging, in Drosophila melanogaster", "advisor": "Benzer, Seymour; Patterson, Paul H.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05182009-232337", "creators": [ { "name": { "family": "Muffat", "given": "Julien A." }, "id": "Muffat-J-A", "orcid": "0000-0003-1889-7023", "display_name": "Muffat, Julien A." } ], "advisors": [ { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "advisor", "display_name": "Benzer, Seymour" }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "advisor", "display_name": "Patterson, Paul H." } ], "committee": [ { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "chair", "display_name": "Patterson, Paul H." }, { "name": { "family": "Varshavsky", "given": "Alexander J." }, "id": "Varshavsky-A-J", "role": "member", "display_name": "Varshavsky, Alexander J." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Walker", "given": "David" }, "id": "Walker-D", "role": "member", "display_name": "Walker, David" }, { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "member", "display_name": "Benzer, Seymour" } ], "option_major": [ "biology" ], "doi": "10.7907/H1ST-ZY74", "abstract": "The free radical theory of aging is probably the most enduring one to date. It stipulates that, in the process of normal cellular function, in particular due to oxygen-based respiration, reactive oxygen species are formed. These constantly put a strain on the cell, damaging lipids in the membranes, causing protein aggregation and loss-of-function, or mutations in the genome. Over time, this accumulated damage overcomes the repair potential of a given cell, and scaled up to an entire organism, results in the deterioration seen in normal aging. Under these assumptions, age-related pathologies are only an acceleration of the process in a given tissue, leading to the emergence of the pathology over the noise of normal aging. In the past decade, invertebrates such as Drosophila melanogaster and C. elegans have provided invaluable insight into these processes and the canonical pathways that regulate them. This success is owed in part to the power of the genetic tools available, to their relatively short lifespans, and to the wide arrays of phenotypes that can be studied. We set out to study a particular protein, Glaz, whose overexpression enables fruitflies to live 30% longer than normal. This protein was identified as a hit from a screen looking at an accelerated aging paradigm, placing fruitflies in 100% oxygen. While an interesting protein in its own right, it was through its homology with mammalian Apolipoprotein D (ApoD), that our interest was truly piqued. ApoD and its homologs turn out to be fascinating proteins, upregulated by various stresses and in age-related diseases such as cancers and Alzheimer\u2019s. We showed, in our model organism of choice, that this upregulation is part of a beneficial stress response. Understanding and harnessing its functions can only help provide therapeutic approaches for a wide range of disorders.\r\n" }, { "name": "Ottesen, Elizabeth Ann", "degree": "PhD", "year": "2009", "title": "The Biology and Community Structure of CO\u2082-Reducing Acetogens in the Termite Hindgut", "advisor": "Leadbetter, Jared R.", "url": "https://resolver.caltech.edu/CaltechETD:etd-10032008-170935", "creators": [ { "name": { "family": "Ottesen", "given": "Elizabeth Ann" }, "id": "Ottesen-Elizabeth-Ann", "orcid": "0000-0002-7898-2425", "display_name": "Ottesen, Elizabeth Ann" } ], "advisors": [ { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "role": "advisor", "display_name": "Leadbetter, Jared R." } ], "committee": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "chair", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "role": "member", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Mazmanian", "given": "Sarkis K." }, "id": "Mazmanian-S-K", "role": "member", "display_name": "Mazmanian, Sarkis K." } ], "option_major": [ "biology" ], "doi": "10.7907/V57J-FB39", "abstract": "In the guts of wood-feeding termites, CO\u2082-reductive acetogenesis serves as the dominant sink for H\u2082 generated during the fermentation of wood polysaccharides. This activity can generate up to 1/3 of the acetate that powers the energy metabolism of the host insect. The gene for formyl-tetrahydrofolate synthetase (FTHFS), a key gene in the acetyl-CoA pathway, can be used as a genetic marker of acetogenic capability. The dominant FTHFS types in the guts of wood-feeding termites are known to cluster phylogenetically with those from acetogenic Treponemes. In this work, we present the discovery that the guts of wood-feeding roaches are also dominated by Treponeme-like sequences. Phylogenetic analysis of roach-derived FTHFS sequences reveals a cluster that forms a basal radiation of the termite Treponeme cluster. This suggests that the Treponemes found in roach guts represent an ancient divergence, present in the last common ancestor of these insects, rather than a modern lineage acquired by cross-species symbiont transfer. The FTHFS sequences present in the guts of higher termites were also examined. Wood-, palm-, and litter-feeding termites were found to be dominated by acetogenic Treponemes, while subterranean soil/grass feeders were found to be dominated by a novel cluster of Firmicute-like FTHFS types. Also presented herein is the development of microfluidic digital PCR for molecular characterization of individual bacteria from environmental samples. We used this technique to retrieve FTHFS and 16S rRNA gene sequences from single bacterial cells, thereby discovering the 16S rRNA sequences of uncultured acetogens in the termite gut. This technique should provide a valuable tool for molecular analyses of termite gut acetogens, and can potentially be adapted for the characterization of uncultured bacteria that carry any metabolic gene of interest.\r\n" }, { "name": "Price-Whelan, Alexa Mari", "degree": "PhD", "year": "2009", "title": "Physiology and Mechanisms of Pyocyanin Reduction in Pseudomonas aeruginosa", "advisor": "Newman, Dianne K.", "url": "https://resolver.caltech.edu/CaltechETD:etd-02182009-100346", "creators": [ { "name": { "family": "Price-Whelan", "given": "Alexa Mari" }, "id": "Price-Whelan-Alexa-Mari", "orcid": "0000-0001-7587-7534", "display_name": "Price-Whelan, Alexa Mari" } ], "advisors": [ { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "advisor", "display_name": "Newman, Dianne K." } ], "committee": [ { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "orcid": "0000-0002-7033-0844", "role": "chair", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Barton", "given": "Jacqueline K." }, "id": "Barton-J-K", "orcid": "0000-0001-9883-1600", "role": "member", "display_name": "Barton, Jacqueline K." } ], "option_major": [ "biology" ], "doi": "10.7907/N42E-M534", "abstract": "The opportunistic pathogen Pseudomonas aeruginosa excretes redox-active small molecules called phenazines. This thesis addresses the possibility that the phenazine pyocyanin acts as an electron acceptor for energy metabolism and exerts beneficial effects on P. aeruginosa physiology. The effects of phenazine production and exposure on P. aeruginosa strain PA14 were examined by comparing the physiological status of the wild type to a mutant defective in phenazine production. Quantification of intracellular NADH and NAD+ pools revealed a more reduced intracellular redox state in the phenazine-null mutant compared to the wild type, consistent with the capacity of P. aeruginosa to reduce pyocyanin. High-performance liquid chromatography of culture metabolites showed that the wild type excreted pyruvate in late stationary phase, indicating that pyocyanin alters flux through central metabolic pathways.
\r\n\r\nWe set out to identify mechanisms allowing P. aeruginosa to catalyze pyocyanin redox cycling. Through a genetic screen, we found two loci required for full pyocyanin-dependent ferric citrate reduction activity in P. aeruginosa PA14: (1) the gene gpsA, encoding the soluble glycerol-3-phosphate dehydrogenase (GpsA), and (2) the operon fbcFBC, encoding the respiratory cytochrome bc1 complex. Mutants lacking functional GpsA had oxidized cytoplasms and may be defective in pyocyanin reduction due to a lack of sufficient NADH. In contrast, mutants lacking a functional cytochrome bc1 complex produced ample reducing power for pyocyanin reduction, raising the possibility that the cytochrome bc1 complex directly catalyzes pyocyanin reduction.
\r\n\r\nPyocyanin has previously been shown to affect the development of P. aeruginosa colonies on agar surfaces: phenazine-null mutants form wrinkled (rugose) colonies, while the wild type forms smooth colonies. Using this colony biofilm assay, we showed that the \u0394gpsA mutant forms rugose colonies, consistent with a role for pyocyanin reduction in stimulating smooth colony formation. Modulation of electron acceptor availability through nitrate addition to the medium promoted smooth colony formation in rugose mutants. These results imply that rugosity is an adaptation to electron acceptor limitation.
\r\n\r\nThe work in this thesis has provided insight into the physiological relevance of pyocyanin reduction in P. aeruginosa, mechanisms controlling intracellular redox state in bacteria, and mechanisms that may contribute to P. aeruginosa virulence.
\r\n" }, { "name": "Rice, Adrian Edward", "degree": "PhD", "year": "2009", "title": "Biophysical and Cell Biological Studies Characterizing the Vertebrate Iron Exporter Ferroportin", "advisor": "Bjorkman, Pamela J.; Rees, Douglas C.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05192009-163326", "creators": [ { "name": { "family": "Rice", "given": "Adrian Edward" }, "id": "Rice-Adrian-Edward", "display_name": "Rice, Adrian Edward" } ], "advisors": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "co-advisor", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "role": "co-advisor", "display_name": "Rees, Douglas C." } ], "committee": [ { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "chair", "display_name": "Chan, David C." }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Jensen", "given": "Grant J." }, "id": "Jensen-G-J", "orcid": "0000-0003-1556-4864", "role": "member", "display_name": "Jensen, Grant J." }, { "name": { "family": "Gray", "given": "Harry B." }, "id": "Gray-H-B", "orcid": "0000-0002-7937-7876", "role": "member", "display_name": "Gray, Harry B." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." } ], "option_major": [ "biochem" ], "doi": "10.7907/2P16-3X70", "abstract": "Mammalian iron homeostasis is maintained by an intricate network of diverse proteins that constantly survey systemic iron levels and carefully regulate the uptake of iron from the diet. Control of this uptake is critically important because once iron is absorbed, mammals have no regulated mechanism for its removal. The portal through which iron enters the body is ferroportin, a multipass membrane protein expressed on the basolateral membrane of epithelial cells in the duodenum. The iron export function of ferroportin is primarily regulated by the serum peptide hormone hepcidin, which is secreted from the liver when systemic iron levels are high. Hepcidin acts as a negative regulator of iron uptake by binding to ferroportin at the cell surface and inducing its internalization and degradation. Genetic defects in ferroportin, hepcidin, or the proteins involved with sensing systemic iron levels lead to iron overload diseases known as hereditary hemochromatosis. Using the tools of biophysics and cell biology, we sought to study ferroportin and its interaction with hepcidin in order to better understand this critical bottleneck in iron uptake and how genetic defects within ferroportin might lead to disease. We developed the first protocols for the overexpression, detergent-solubilization, and purification of recombinant ferroportin. We determined that detergent-solubilized ferroportin is a monomer capable of binding hepcidin in vitro. We characterized the expression and subcellular localization of ferroportin in mammalian tissue culture and determined that both the amino- and carboxy-termini of ferroportin are cytosolic. We developed cell-based assays for the hepcidin-induced internalization of ferroportin and used these to characterize the route of internalization from the plasma membrane through early endosomes to degradative lysosomal compartments. Using live-cell imaging techniques, we showed that this internalization depended on intact microtubules. We expanded this cell-biological study to include sixteen disease-related ferroportin mutants and reported that each mutant was expressed on the plasma membrane like wild-type ferroportin, but that only a subset of the mutants were capable of being internalized by hepcidin. These studies form a foundation for future biophysical and cell-biological studies of ferroportin function. " }, { "name": "Ririe, Ted Olin", "degree": "PhD", "year": "2009", "title": "A Multipartite Approach to Mapping the Gene Network Directing Caenorhabditis elegans Vulval Organogenesis", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05292009-162749", "creators": [ { "name": { "family": "Ririe", "given": "Ted Olin" }, "id": "Ririe-Ted-Olin", "display_name": "Ririe, Ted Olin" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "member", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biology" ], "doi": "10.7907/XY2E-3T19", "abstract": "The complex interactions of signaling molecules, transcription factors, and target genes that direct the development of diverse organisms and their component parts are known as gene regulatory networks (GRNs). The Caenorhabditis elegans vulva, with its invariant lineage, 22 cells of seven types (vulA, vulB1, vulB2, vulC, vulD, vulE, and vulF), and availability of 30 reporter constructs exhibiting a variety of differentiated vulval cell expression patterns, provides us with an elegant system for studying a GRN directing organogenesis. I took several approaches for identifying additional aspects of the vulval gene network. First, I conducted a screen in a zmp-1::GFP (zinc metalloproteinase) background by disrupting activity of 836 transcription factors by RNA interference (RNAi). zmp-1 is a readout for differentiation of the vulA, vulD, and vulE cells. This screen identified nhr-67 (ortholog of Drosophila tailless) and nhr-113 (orphan nuclear hormone receptor) as positive regulators of zmp-1 expression in the vulA cells. nhr-113 appears to have a narrow role in vulval organogenesis; perhaps only as a partial regulator of vulA cell differentiation. Furthering the analysis of zmp-1, I utilized a phylogenetic footprinting approach for analyzing the upstream region of C. elegans zmp-1, and its homologs in the related species C. briggsae and C. remanei. This analysis identified four conserved motifs upstream of the ZMP-1 translational start site. Characterization of these motifs, by deleting them from a zmp-1::GFP transgene, revealed that three of these four motifs exhibit cis-regulatory activity. In addition to investigation of zmp-1 regulation, we undertook a parallel study where we performed sequence alignment analysis on 17 genes with reported vulval expression in combination with in vivo testing. This identified nine ~ 200-bp vulva-specific enhancer elements associated with six of these genes. Yeast one-hybrid analysis of six of these enhancer elements isolated six protein-DNA interactions. Further characterization of these interactions uncovered the role of the zinc finger transcription factor, ZTF-16, in vulval induction and differentiation. These results show that by taking multiple approaches, including the use of post-genome technologies, we can expand our understanding of a gene regulatory network.\r\n\r\n" }, { "name": "Seah, Adeline", "degree": "PhD", "year": "2009", "title": "EGF, WNT & HOX Interactions during Patterning of Caenorhabditis elegans Equivalence Groups", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05242009-123432", "creators": [ { "name": { "family": "Seah", "given": "Adeline" }, "id": "Seah-Adeline", "orcid": "0000-0003-0935-8044", "display_name": "Seah, Adeline" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "chair", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/N559-AE78", "abstract": "During development, as a single-cell zygote divides multiple times to generate a complete organism, previously undifferentiated cells somehow acquire the correct fates. A group of cells that shares the same developmental potential is called an equivalence group. In Caenorhabditis elegans, the most well-characterized equivalence group is the hermaphroditic Vulval Precursor Cell (VPC) group. Epidermal Growth Factor (EGF) signaling specifies VPC fate partly by upregulation of lin-39/SexcombsReduced/Hox5, while Wnt signaling plays a minor role in vulval induction. EGF and Wnt signaling also act together to pattern the P11/12 equivalence group, present in both C. elegans hermaphrodites and males, by upregulating a different Hox gene, egl-5/Antennapedia/Ultrabithorax/Hox6/8, to specify P12 fate. Previous observations suggest that EGF or Wnt signaling may act through Hox genes to specify fate in two other C. elegans equivalence groups: the Hook Competence Group (HCG) and \u03b3/\u03b4 pair. I characterized the roles of EGF and Wnt signaling in the HCG and \u03b3/\u03b4 pair and found that upregulation of Hox genes is controlled by either pathway in each group.
\r\n\r\nI showed that the major hook inductive pathway involves the Wnt ligands and LIN-17/Fz, which specify the 1\u00b0 and 2\u00b0 HCG fates. Also, I identified a role for EGF signaling in specifying the 1\u00b0 fate, although its role is only revealed when Wnt activity is compromised. I provided a link between mab-5/Hox6/8 and Wnt signaling during normal hook development by determining that LIN-17 is required for mab-5/Hox6/8 expression in P11.p.
\r\n\r\nIn the \u03b3/\u03b4 pair, I demonstrated that EGF signaling (through the LIN-31/Forkhead and LIN-1/ETS transcription factors) controls ceh-13/Hox1 expression in \u03b3. I did not find any evidence that Wnt signaling specifies the \u03b3 fate. Instead, I observed that lin-44/Wnt, mom-2/Wnt and lin-17/Fz are required to orient the \u03b3 mitotic spindle. In addition, TGF-\u03b2 signaling (by dbl-1/Dpp) was previously reported to control \u03b3 expression of ceh-13/Hox1. I showed that dbl-1 acts either downstream or in parallel to EGF signaling to specify the \u03b3 fate. I also found that dbl-1/Dpp does not appear to specify fates in the VPC and P11/12 equivalence groups, in which EGF signaling plays an important role, suggesting that TGF-\u03b2 signaling contributes to the specificity of the \u03b3 fate.
\r\n" }, { "name": "Shiau, Celia Eenjing", "degree": "PhD", "year": "2009", "title": "Formation of Cranial Sensory Ganglia: Role of Neural Crest-Placode Interactions, Slit-Robo, and Cadherins", "advisor": "Bronner, Marianne E.", "url": "https://resolver.caltech.edu/CaltechETD:etd-03142009-001526", "creators": [ { "name": { "family": "Shiau", "given": "Celia Eenjing" }, "id": "Shiau-Celia-Eenjing", "orcid": "0000-0002-9347-9158", "display_name": "Shiau, Celia Eenjing" } ], "advisors": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "advisor", "display_name": "Bronner, Marianne E." } ], "committee": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "chair", "display_name": "Fraser, Scott E." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/7E6J-0P43", "abstract": "In vertebrates, the peripheral nervous system is derived from two distinct embryonic cell populations, the neural crest and ectodermal placodes. Neural crest cells arise from the hinges of the invaginating neural plate, while ectodermal placodes form in pairs from discrete, usually thickened, head ectoderm lateral to the neural tube. While these two populations generally contribute to different structures in the nervous system, the exception is where they converge to form the cranial sensory ganglia of the trigeminal (V), facial (VII), glossopharyngeal (IX), and vagal (X) cranial nerves. The dual embryonic origin of cranial sensory ganglia has intrigued investigators for some time, but surprisingly little is known about the neural crest\u2013placode relationship. The process of cranial gangliogenesis exemplifies a fascinating problem on how cell\u2013cell interactions drive assembly of complex structures in the developing embryo.
\r\n\r\nTo investigate this process, I have combined gene expression characterizations, embryological experiments, in vitro culture studies, and in vivo molecular perturbations (gain- and loss-of-functions by electroporation mediated DNA and oligos transfer) to uncover the cellular and molecular events underlying neural crest\u2013placode ganglion assembly in the trigeminal region of the chick embryo. The results show that these cells are in contact and highly intermingled throughout ganglion formation. Ablation of either precursor tissue results in severe ganglion defects. Taken together, the data demonstrate the essential role of neural crest\u2013placode interactions for proper gangliogenesis and the reciprocal nature of their relationship. Their interactions likely involve bi-directional signaling by which each population affects and coordinates with one another.
\r\n\r\nAs candidate mediators, I investigated the potential role of Slits and Robos. The concurrent expression of Slit1 on migratory neural crest cells and its cognate receptor Robo2 on placodal cells raised an intriguing possibility that this ligand\u2013receptor pair may mediate signaling from neural crest to placodal cells. This would represent one form of their cell\u2013cell interactions. Loss of function of either the ligand Slit1 or its cognate receptor Robo2 in vivo resulted in severely disorganized placodal ganglia that were similar phenotypically to the effects of neural crest ablation. More specifically, inhibition of Robo2 resulted in aberrant placodal ingression, axonal projections, and ganglion organization and coalescence. The results suggest that neural crest cells regulate and coordinate assembly of placodal cells for proper trigeminal gangliogenesis through Slit1\u2013Robo2 signaling in chick.
\r\n\r\nA striking defect in ganglion coalescence by blocking Slit1\u2013Robo2 function suggested that cell adhesion may have been affected. Thus, as a possible downstream mechanism, I next tested the function of the cell adhesion molecule N-cadherin. The results show that N-cadherin is expressed by placodal neurons, and its function is required for placodal aggregation and may be regulated by Slit1\u2013Robo2. N-cadherin expression is modulated by Slit1\u2013Robo2 and also can partially rescue Robo2 loss-of-function. Moreover, since neural crest and placodal neurons are highly intermixed, condensation of ganglia may require adhesion of not only placode\u2013placode, but also crest\u2013crest and crest\u2013placode cells. The data suggest that another adhesion molecule Cadherin-7 may complement the role of N-cadherin in driving ganglion coalescence. Finally, the similar expression and function of these molecules in the epibranchial regions suggest that the mechanisms of Slit1\u2013Robo2 and N-cadherin may be general for all cranial ganglia of dual origin.
\r\n\r\nIn summary, the results from my thesis establish a critical role for Slit1\u2013Robo2 signaling and cadherin-mediated cell adhesion for cranial ganglia formation. They provide the first molecular basis for neural crest\u2013placode cell\u2013cell signaling during cranial gangliogenesis, and highlight the critical interplay of cell\u2013cell communication and cell adhesion in animal development.
\r\n" }, { "name": "Southwell, Amber L.", "degree": "PhD", "year": "2009", "title": "Intrabodies as Therapeutics for Huntington\u2019s Disease\r ", "advisor": "Patterson, Paul H.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06082009-164212", "creators": [ { "name": { "family": "Southwell", "given": "Amber L." }, "id": "Southwell-Amber-L", "orcid": "0000-0002-6353-5008", "display_name": "Southwell, Amber L." } ], "advisors": [ { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "advisor", "display_name": "Patterson, Paul H." } ], "committee": [ { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "chair", "display_name": "Schuman, Erin Margaret" }, { "name": { "family": "Khoshnan", "given": "Ali" }, "id": "Khoshnan-A", "role": "member", "display_name": "Khoshnan, Ali" }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." } ], "option_major": [ "neurobio" ], "doi": "10.7907/DDSD-7K09", "abstract": "Huntington\u2019s disease (HD) is a devastating, genetic, neurodegenerative disease for which there is currently no effective therapy. The polyglutamine (polyQ) expansion that causes HD is in the first exon (HDx1) of huntingtin (Htt). However, other parts of the protein, including the 17 N-terminal amino acids (AAs) and two proline (polyP) repeat domains, modulate the toxicity of mutant Htt (mHtt). The role of the P-rich domain that is flanked by the polyP domains has not been explored. Using highly specific intracellular antibodies (intrabodies; iAbs), we tested various epitopes for their roles in mHDx1 toxicity, aggregation, localization and turnover. Three domains in the P-rich region (PRR) of HDx1 are defined by iAbs: MW7 binds the two polyP domains, and Happs 1 and 3, two new iAbs, bind the unique, P-rich epitope located between the two polyP epitopes. In cultured cells, we find that the three PRR-binding iAbs, as well as VL12.3, which binds an epitope in the N-terminal 17 AA segment, decrease the toxicity and aggregation of mHDx-1, but they do so by different mechanisms. The PRR-binding iAbs have no effect on Htt localization, but they cause a significant increase in the turnover rate of mHtt, which VL12.3 does not change. In contrast, expression of VL12.3 increases nuclear Htt. These results suggest that the PRR domain regulates mHtt stability and toxicity. Thus, compromising this pathogenic epitope by iAb binding represents a novel therapeutic strategy for treating HD.
\r\n\r\nWe have tested this hypothesis by delivering both VL12.3 and Happ1 to the brains of HD model mice using an AAV2/1 viral vector with a modified CBA promoter. VL12.3 treatment, while beneficial in a lentiviral model of HD, has no effect on the YAC128 HD model and actually increases severity of phenotype and mortality in the R6/2 HD model. In contrast, Happ1 treatment confers significant beneficial effects in assays of motor and cognitive deficits as well as in the neuropathology found in the lentiviral, R6/2, N171-82Q, YAC128 and BACH models of HD. These results indicate that increasing the turnover of mHtt using AAV-Happ1 gene therapy represents a highly specific and effective treatment possibility for HD.
\r\n" }, { "name": "Tesar, Devin Brent", "degree": "PhD", "year": "2009", "title": "Investigations of the Mechanisms of Receptor-Mediated Immunoglobulin Transport in Mammals and Birds", "advisor": "Bjorkman, Pamela J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-11122008-131536", "creators": [ { "name": { "family": "Tesar", "given": "Devin Brent" }, "id": "Tesar-Devin-Brent", "display_name": "Tesar, Devin Brent" } ], "advisors": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "advisor", "display_name": "Bjorkman, Pamela J." } ], "committee": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "chair", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Jensen", "given": "Grant J." }, "id": "Jensen-G-J", "orcid": "0000-0003-1556-4864", "role": "member", "display_name": "Jensen, Grant J." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biology" ], "doi": "10.7907/8M42-R866", "abstract": "The neonatal Fc receptor (FcRn) mediates the passive acquisition of humoral immunity in early pre- or post-natal mammals by transferring maternal IgG to the fetus or suckling newborn. In addition, FcRn serves to extend the serum half-life of IgG in adult mammals by protecting it from a default degradative pathway in vascular endothelial cells. For both of these functions, FcRn binds the Fc domain of IgG with high affinity at the acidic pH (\u22646.5) of the intestinal lumen and acidic endosomes, and releases the IgG at the slightly basic pH (~7.4) of blood. While the ability of FcRn to transcytose IgG bidirectionally in polarized cell models has been well documented, the specific mechanism(s) by which endocytosed IgG is sorted from other vesicular cargo and directed through a progression of endosomal compartments ultimately leading to the apical or basolateral membrane are poorly understood.
\r\n\r\nWe wished to develop an in vitro system with which to study the trafficking behavior of rat FcRn in polarized epithelia. I developed such a model system using Madin-Darby Canine Kidney cells stably-transfected with the rat FcRn. The cells bind, endocytose, recycle and bidirectionally transcytose FcRn ligands, faithfully recapitulating the function of FcRn in the neonatal rodent gut. We used these cells to test whether or not the presence of two FcRn binding sites on an Fc ligand are required for transport. The results demonstrated that ligand bivalency is not strictly required for transport but does increase the transcytosis efficiency of the system, an interesting result in light of the fact that FcRn binds and transports a naturally monovalent ligand \u2013 serum albumin.
\r\n\r\nWe have used the FcRn-MDCK cells to study trafficking of the receptor in living cells using spinning disk confocal microscopy. For these studies I developed a recombinant Fc ligand containing a tandem dimer of fluorescent proteins on each chain and demonstrated that this ligand is brighter and more photostable than conventional chemically-labeled Fc molecules used in other confocal studies. Our FcRn-MDCK cells have also been used to examine trafficking events at the plasma membrane leading up to exocytosis though use of total internal reflection microscopy (TIRFM).
\r\n\r\nI also created a model system to study an avian Fc receptor present in yolk sac (FcRY). FcRY has been shown to bind IgY (the avian counterpart of IgG) with the same pH-dependence that FcRn exhibits for IgG. Transcytosis and recycling experiments using polarized rat inner medullary collecting duct (IMCD) cells stably transfected with the FcRY gene demonstrated that this receptor is a true functional equivalent of FcRn despite being structurally distinct from FcRn. This presents a striking example of convergent evolution and demonstrates that certain versatile protein folds can play key roles in more than one functional context within a complex organism.
\r\n" }, { "name": "Cassenaer, Stijn", "degree": "PhD", "year": "2008", "title": "Spike-Timing Dependent Plasticity and Synchronous Oscillations in an Invertebrate Olfactory System", "advisor": "Laurent, Gilles J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-12202007-160330", "creators": [ { "name": { "family": "Cassenaer", "given": "Stijn" }, "id": "Cassenaer-Stijn", "display_name": "Cassenaer, Stijn" } ], "advisors": [ { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "role": "advisor", "display_name": "Laurent, Gilles J." } ], "committee": [ { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "chair", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "role": "member", "display_name": "Siapas, Athanassios G." }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "role": "member", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "role": "member", "display_name": "Perona, Pietro" } ], "option_major": [ "biology" ], "doi": "10.7907/5WNM-SQ93", "abstract": "Sensory systems neuroscience aims to study how patterns of neural activity represent stimuli of the outside world. To this end, the present work addresses how olfactory stimuli are represented by three successive layers in the locust olfactory system. Activation by an odorant of primary sensory neurons in the antenna gives rise to broadly distributed, oscillatory spatiotemporal activity patterns across the antennal lobe (AL). This is in marked contrast to the representation in the mushroom body (MB), where Kenyon cells (KCs) respond very sparsely and very briefly. In the AL, an odor gives rise to a particular trajectory through Projection Neuron (PN) phase space, with individual timepoints representing different aspects of the stimulus; in the MB, very small subsets of KCs respond selectively at particular timepoints along this trajectory. Two mechanisms are identified that contribute to the sparsening across the two structures: an intrinsic voltage dependence in the KCs, which gives rise to a superlinear response to synchronous inputs, and a canonical network motif, feedforward inhibition, which diminishes the KC response to nonsynchronous excitatory inputs. From a decoding perspective, this makes the oscillation cycle the relevant timestep of the AL trajectories, and it demonstrates a role for synchronous oscillations in sensory networks. While broad activation of the AL promotes extensive local interactions, giving rise to dynamic representations and enabling multiple features to be extracted, the sparse representation after decoding by KCs likely facilitates the storage of relevant patterns in memory.
\r\n\r\nA subset of MB extrinsic neurons with dendrites densely invading the \u03b2-lobe (\u03b2LNs) is well placed to decode the KCs\u2019 sparse responses. The synapses formed by KCs onto these cells are powerful and undergo Hebbian spike-timing dependent plasticity (STDP) on a timescale similar to the synchronous oscillations generated in the AL (and propagated through the MB). STDP has a homeostatic effect on the firing phase of \u03b2LNs by fine-tuning the strength of KC-\u03b2LN synapses, contributing to tight locking among subsets of \u03b2LNs during odor stimulation and facilitating the flow of synchronous information.
\r\n\r\nThe facilitation of tight synchrony among \u03b2LNs by STDP further ensures that different odor features computed and formatted as a function of cycle number by the AL, and represented by the sparse representations of KCs, remain segregated between LFP oscillation cycles. This segregation is also sustained by phase locked feedforward inhibition onto \u03b2LNs, which restricts the window of integration for inputs from KCs, and is found to be due to neighboring \u03b2LNs of the same class. The implications of the resultant competition among \u03b2LNs due to this inhibition, and particularly its interaction with STDP at the KC-\u03b2LN synapse are addressed with a network model. The results are considered within the context of the circuit in which the KC-\u03b2LN network is embedded, and a cycle-specific mechanism for learning an arbitrary subset of the odor features computed in the AL is proposed.
\r\n" }, { "name": "Cox, Robert Sidney, III", "degree": "PhD", "year": "2008", "title": "Transcriptional Regulation and Combinatorial Genetic Logic in Synthetic Bacterial Circuits", "advisor": "Elowitz, Michael B.", "url": "https://resolver.caltech.edu/CaltechETD:etd-03042008-130011", "creators": [ { "name": { "family": "Cox", "given": "Robert Sidney, III" }, "id": "Cox-Robert-Sidney-III", "orcid": "0000-0002-2009-6236", "display_name": "Cox, Robert Sidney, III" } ], "advisors": [ { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "advisor", "display_name": "Elowitz, Michael B." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Smolke", "given": "Christina D." }, "id": "Smolke-C-D", "role": "member", "display_name": "Smolke, Christina D." }, { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "role": "member", "display_name": "Winfree, Erik" }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "member", "display_name": "Elowitz, Michael B." } ], "option_major": [ "biology" ], "doi": "10.7907/KRW6-DH88", "abstract": "We engineered several synthetic regulatory circuits to study transcriptional regulation in bacteria. We developed a new technique for DNA construction, built and characterized in vivo a library of genetic logic gates, examined the role of genetic noise transcriptional regulation using a fluorescent multi-reporter system, and characterized a synthetic circuit for the control of population density.
\r\n\r\nWe used synthetic duplex DNA fragments and very short cohesive overhangs to direct ordered assemblies of diverse combinatorial libraries. Multiple DNA fragments were simultaneously ligated in a single step to produce random concatemers, without the need for amplification or product purification. We characterized the assembly process to identify optimal cohesive overhangs. We showed that the method was 97% efficient for assembling 100 base-pair concatemers from three duplex fragments.
\r\n\r\nWe constructed a library of 10,000 prokaryotic promoters, containing over 1,000 unique 100 base-pair sequences. These promoters responded to up to three inputs, and contained diverse architectural arrangements of regulatory sequences. We characterized the logical input functions of 288 promoters in Escherichia coli, and analyzed the relationship between promoter function and architecture. We defined promoter function in terms of regulatory range, logic type, and input symmetry; and identified general rules for combinatorial programming of gene expression.
\r\n\r\nWe built a synthetic three-color fluorescent reporter framework. This construct was non-toxic and extensible for synthetic and systems biology applications. Three spectrally distinct and genetically isolated reporter proteins allowed independent monitoring of genetic signals at the single-cell level. We showed that the simultaneous measurement of multiple genes can exploit genetic noise to sensitively detect transcriptional co-regulation.
\r\n" }, { "name": "Green, Jennifer Leigh", "degree": "PhD", "year": "2008", "title": "The C. elegans ROR Receptor Tyrosine Kinase, CAM-1, Regulates Wnt Signaling by Two Distinct Mechanisms", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05272008-112700", "creators": [ { "name": { "family": "Green", "given": "Jennifer Leigh" }, "id": "Green-Jennifer-Leigh", "display_name": "Green, Jennifer Leigh" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "chair", "display_name": "Hay, Bruce A." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "member", "display_name": "Deshaies, Raymond Joseph" } ], "option_major": [ "biology" ], "doi": "10.7907/3KR8-VM32", "abstract": "The ROR (receptor orphan) family of receptor tyrosine kinase, which is required for human skeletal development and has been implicated in human cancer, is one of the few classes of RTK whose activity remains elusive.
\r\n\r\nAlthough the mechanism is unknown, there is evidence that ROR proteins inhibit signaling by Wnts, a conserved class of secreted glycoproteins. We have studied how ROR proteins interact with Wnt signaling during C. elegans vulval development, a classic model used to study cell-signaling pathways. Wnt/\u03b2-catenin signaling controls cell-fate decisions of the vulval precursor cells (VPCs). We found that loss and over-expression of the C. elegans ROR homolog, cam-1, caused reciprocal defects in Wnt-mediated cell-fate specification. Our molecular and genetic analysis revealed that during vulval induction the CAM-1 extracellular domain (ECD) is sufficient to non-autonomously antagonize multiple Wnts, implying that the CAM-1 ECD sequesters Wnts. Our finding that the CAM-1 ECD specifically binds Wnts in vitro supports a sequestration model. Thus, CAM-1/ROR regulates Wnt signaling by modifying the spatial profile of Wnt activity.
\r\n\r\nIn addition to regulating cell-fate specification, Wnt signaling also determines VPC polarity. The mirror symmetry of the C. elegans vulva is achieved by the opposite division orientation of the VPCs flanking the axis of symmetry. We characterized the molecular mechanisms by which opposing Wnts establish this division pattern and how CAM-1 contributes to VPC orientation. Wnts MOM-2 and LIN-44 are expressed at the axis of symmetry and orient the VPCs towards the center. We show that these Wnts, via Fz/LIN-17 and Ryk/LIN-18, control \u03b2-catenin localization and activate gene transcription. In addition, we found that VPCs on both sides of the axis of symmetry possess a uniform underlying \u201cground\u201d polarity, established by the instructive activity of Wnt/EGL-20. EGL-20 establishes ground polarity via a novel type of signaling involving CAM-1 and the Planar Cell Polarity component Van Gogh/VANG-1. CAM-1 activity during ground polarity signaling requires the CAM-1 intracellular domain and is thus likely to be a cell-autonomous function. Therefore, CAM-1 interacts with Wnts by two distinct mechanisms: it sequesters Wnts and it transmits directional information from Wnts to individual cells.
\r\n\r\n" }, { "name": "Iyer, Asha Muthuraman", "degree": "PhD", "year": "2008", "title": "Planning Goal-Directed Actions: fMRI Correlates in Humans and Monkeys", "advisor": "Andersen, Richard A.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06102008-162347", "creators": [ { "name": { "family": "Iyer", "given": "Asha Muthuraman" }, "id": "Iyer-Asha-Muthuraman", "display_name": "Iyer, Asha Muthuraman" } ], "advisors": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "role": "advisor", "display_name": "Andersen, Richard A." } ], "committee": [ { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "role": "chair", "display_name": "Shimojo, Shinsuke" }, { "name": { "family": "Rangel", "given": "Antonio" }, "id": "Rangel-A", "role": "member", "display_name": "Rangel, Antonio" }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "role": "member", "display_name": "Andersen, Richard A." } ], "option_major": [ "biology" ], "doi": "10.7907/Q6P2-GC02", "abstract": "In performing goal-directed actions, primates (humans and monkeys) flexibly select and plan appropriate behavioral responses. However, while a network of frontoparietal regions are traditionally implicated in the transformation of sensory input from the environment into these spatial, goal-directed movements, the type of information encoded in their activity remains nebulous.
\r\n\r\nThis work first addresses the long-standing query as to whether this activity represents a prospective planning of the upcoming action, or a retrospective sensory representation of goals. In an fMRI experiment, subjects performed delayed-reach tasks, in which mnemonic and attentional demands were held constant. BOLD signals showed that the posterior parietal cortex (PPC) and premotor regions exhibit activity specifically related to the planning of upcoming actions.
\r\n\r\nAdditionally, to select and plan the optimal action, the expected consequences of potential responses need to be assessed. To determine whether and how potential outcomes mold action planning activity, subjects were scanned while they performed a demanding motor task to obtain monetary gains or losses contingent on their performance. Monetary consequences modulated activity throughout the action-planning network, most significantly in PPC, as well as in reward structures. While reward areas reflected the expected value of a trial, frontoparietal activity was greatest for both high expected rewards and losses. Moreover, in frontoparietal areas, subjects\u2019 beliefs about the likelihood of possible outcomes influenced BOLD signals, suggesting that cognitive biases may influence the planning of actions.
\r\n\r\nFinally, to compare human imaging findings to large body of related monkey electrophysiological experiments, humans and monkeys were scanned while performing the same delayed-saccade tasks. Frontoparietal oculomotor-planning areas in monkeys and putative homologs in humans evinced coherent response patterns, though prominent differences in the degree of contralaterality and the hemodynamic responses between the two species emerged.
\r\n\r\nIn sum, these findings help characterize fundamental aspects of goal-directed action planning in both species.
" }, { "name": "Mortazavi, Ali", "degree": "PhD", "year": "2008", "title": "Structure and Evolution of Mammalian Gene Networks", "advisor": "Wold, Barbara J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05292008-140438", "creators": [ { "name": { "family": "Mortazavi", "given": "Ali" }, "id": "Mortazavi-Ali", "orcid": "0000-0002-4259-6362", "display_name": "Mortazavi, Ali" } ], "advisors": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "advisor", "display_name": "Wold, Barbara J." } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "chair", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/FT1Y-M657", "abstract": "Accurate measurements of protein:DNA and RNA expression levels are critical to building meaningful models of gene regulatory networks. We develop here two new techniques doing such measurements using ultra-high-throughput DNA sequencing combined with extensive computational analyses, which we call respectively ChIP-seq and RNA-seq. To show the power and versatility of these techniques, we apply them to the study of two model problems that are representative of the research agenda of regulatory biology. We use ChIP-seq to study the conservation and evolution of the binding repertoire of the transcription factor NRSF/REST in boreoeutherian mammals, whereas we use ChIP-seq of RNA Polymerase II phosphoisoforms and RNA-seq to study a developmental time course of myogenesis in the C2C12 mouse cell line. Together, ChIP-seq and RNA-seq show the promise of ultra-high-throughout sequencing in mapping and studying gene regulatory networks which will likely supplant the previous generation of microarray-based technologies as the new generations of sequencers mature and become more generally available.
" }, { "name": "Salazar, Anna Maria", "degree": "PhD", "year": "2008", "title": "A Pumilio Domain That Forms Heritable Amyloid Aggregates in Yeast Can Regulate Pumillio-Mediated Translational Repression in Drosophila", "advisor": "Zinn, Kai George", "url": "https://resolver.caltech.edu/CaltechETD:etd-10012007-011631", "creators": [ { "name": { "family": "Salazar", "given": "Anna Maria" }, "id": "Salazar-Anna-Maria", "display_name": "Salazar, Anna Maria" } ], "advisors": [ { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "advisor", "display_name": "Zinn, Kai George" } ], "committee": [ { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "role": "chair", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Stathopoulos", "given": "Angelike" }, "id": "Stathopoulos-A", "role": "member", "display_name": "Stathopoulos, Angelike" }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" } ], "option_major": [ "biology" ], "doi": "10.7907/6TNW-AR82", "abstract": "Numerous human diseases have been described in which defects in protein folding pathways play a role in the development of a disease state. A subset of these diseases result in a decrease in the level of the active, native conformation of the protein. This could arise from several mechanisms, including an increase in degradation caused by misfolding, alterations in trafficking of the misfolded protein, or aggregation of the protein with a consequent decrease in the soluble, active form of the protein. At least 40 human diseases that fit into this last category, and are associated with the formation of amyloid fibers, deposits, or inclusions, have been characterized. Domains rich in glutamine (Q) and asparagine (N) are one class of sequences that seem to possess an affinity to form amyloids under native conditions. These domains are present in many metazoan proteins, including 472 in Drosophila and 143 in C. elegans. Q/N domains are found in several yeast prions. It is hypothesized that these domains may have been positively selected during evolution, perhaps in order to allow reversible switching of the functional domain of the protein into an inactive aggregated state. We wondered if this type of selection might also maintain Q/N domains in metazoans. The Drosophila melanogaster and Caenorhabditis elegans proteomes were searched for predicted proteins that contain nucleic acid binding domains (for RNA, DNA or both), and Q/N-rich sequences, using a threshold of 30 Q/Ns in 80 residues. One of the two strong Drosophila candidates is the translational repressor Pumilio (Pum). Earlier work by our group (Menon et al., Neuron 44, 663-676 (2004)) had shown that Pum is localized to the postsynaptic side of the larval neuromuscular junction (NMJ), where it acts as a regulator of local mRNA translation. In pum mutants, synaptic morphology is altered and GluRIIa is dramatically upregulated. This study shows that a Q/N-rich domain (denoted NQ1) from Pum is able to form ordered aggregates in budding yeast and is able to recapitulate the activity of a yeast prion, New1p: including visible aggregates, heritable phenotypic switching, and reversibility of an induced yeast prion state, [Psi+], by guanidine hydrochloride. To test whether NQ1 aggregate formation can perturb Pum's function in the nervous system, transgenic fly lines in which NQ1 expression is driven by GAL4 were created. Postsynaptic NQ1 expression generates alterations in the NMJ that phenocopy the pum loss-of-function phenotype and interact genetically with pum mutations. This is observed through an increase in type 1s and type 1b boutons and an increase in GluRIIa at the NMJ. Postsynaptic Pum overexpression in muscles is lethal, but co-overexpression of NQ1 partially rescues this lethality, resulting in Drosophila that survive until eclosion. Thus, in both the wild-type and overexpression contexts, a domain that forms heritable aggregates acts as a negative regulator of Pum activity.\r\n" }, { "name": "Smith, Stephen Edward Paucha", "degree": "PhD", "year": "2008", "title": "Maternal Immune Activation and Abnormal Behavior in the Adult Offspring: Towards a Mechanism", "advisor": "Patterson, Paul H.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05132008-133518", "creators": [ { "name": { "family": "Smith", "given": "Stephen Edward Paucha" }, "id": "Smith-Stephen-Edward-Paucha", "display_name": "Smith, Stephen Edward Paucha" } ], "advisors": [ { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "advisor", "display_name": "Patterson, Paul H." } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "chair", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "role": "member", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." } ], "option_major": [ "biology" ], "doi": "10.7907/CB5Y-P545", "abstract": "Maternal infection is a risk factor for both schizophrenia and autism. The offspring of women who develop an infection during pregnancy are several times more likely to develop these diseases compared to offspring from uncomplicated pregnancies. Modeling this risk factor in animals, when pregnant rodents are given an influenza infection during pregnancy, their offspring show several behavioral and histological abnormalities consistent with human mental illness. Maternal injection of non-infectious, immune-activating compounds, such as the dsRNA poly(I:C), yields similar results, suggesting that the maternal immune response causes deleterious changes in fetal brain development. The main focus of this thesis is establishing the importance of a single component of the maternal immune response, the cytokine interleukin-6 (IL-6), in mediating the observed changes in the brain development and behavior of the offspring. In addition, I report new observations on the offspring of poly(I:C)-activated pregnant mice that are consistent with findings in autism and schizophrenia: a localized deficit of Purkinje cells in the cerebellum, abnormal eye-blink conditioning, abnormal hippocampal-dependent behaviors and hyper-sensitivity to dopamine in the hippocampus. I also present data on the immune reaction to poly(I:C) in pregnant non-human primates. Finally, I describe preliminary findings on the identification of factors that act down-stream of IL-6. The mechanism through which maternal immune activation causes abnormal behavior in the offspring could illuminate important pathways that contribute to the pathogenesis of schizophrenia and autism." }, { "name": "Warren, Luigi Andrea", "degree": "PhD", "year": "2008", "title": "Single-Cell Gene-Expression Analysis by Quantitative RT-PCR", "advisor": "Quake, Stephen R.", "url": "https://resolver.caltech.edu/CaltechETD:etd-08012007-102846", "creators": [ { "name": { "family": "Warren", "given": "Luigi Andrea" }, "id": "Warren-Luigi-Andrea", "display_name": "Warren, Luigi Andrea" } ], "advisors": [ { "name": { "family": "Quake", "given": "Stephen R." }, "id": "Quake-S-R", "role": "advisor", "display_name": "Quake, Stephen R." } ], "committee": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "chair", "display_name": "Fraser, Scott E." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Heath", "given": "James R." }, "id": "Heath-J-R", "role": "member", "display_name": "Heath, James R." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Quake", "given": "Stephen R." }, "id": "Quake-S-R", "role": "member", "display_name": "Quake, Stephen R." } ], "option_major": [ "biology" ], "doi": "10.7907/PJ42-XB85", "abstract": "The problem of development has long been one of the key issues in biology. With stem-cell therapies on the horizon, the \u201creverse engineering\u201d of developmental programs promises to become a task of great practical significance. We now understand the general schemes by which transcriptional networks regulate cellular differentiation and morphogenesis. These genetic circuits function as complex state machines which, over the course of development, undergo sequenced transitions that bring cells to specific end states. A variety of different gene-expression assays can be used to follow these transitions. The sensitivity of the assays now in common use limits the resolution with which we can follow the activity of genetic-regulatory networks. This thesis describes two projects aimed at refining an established gene-profiling method, quantitative RT-PCR, so that it can be used to profile transcriptional-network states cross-sectionally within developing cell populations at single-cell resolution. Two advanced qRT-PCR protocols were developed to support these projects, one based on microfluidic \u201cdigital PCR,\u201d the other based on multiplexed \u201cpreamplification PCR.\u201d These protocols were used to measure transcription-factor expression in hematopoietic progenitor cells, and to evaluate the effects of aging on the stability of gene regulation. In their current form, the digital PCR and preamplification techniques will permit the analysis of perhaps a few hundred to a few thousand cells in a single-cell survey. By combining microfluidic-chip assays with the preamplification method, it will soon be possible to scale up to the analysis of many thousands of cells, while profiling many different transcription factors in each individual cell. This should facilitate the modeling of the transcriptional networks which control cellular differentiation as we press forward into the era of \u201ctissue engineering.\"" }, { "name": "Zid, Brian Matthew", "degree": "PhD", "year": "2008", "title": "Translational Control Mediates Lifespan Extension Due to Dietary Restriction in Drosophila", "advisor": "Benzer, Seymour", "url": "https://resolver.caltech.edu/CaltechETD:etd-02052008-020618", "creators": [ { "name": { "family": "Zid", "given": "Brian Matthew" }, "id": "Zid-Brian-Matthew", "orcid": "0000-0003-1876-2479", "display_name": "Zid, Brian Matthew" } ], "advisors": [ { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "advisor", "display_name": "Benzer, Seymour" } ], "committee": [ { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "role": "co-chair", "display_name": "Campbell, Judith L." }, { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "co-chair", "display_name": "Benzer, Seymour" }, { "name": { "family": "Varshavsky", "given": "Alexander J." }, "id": "Varshavsky-A-J", "role": "member", "display_name": "Varshavsky, Alexander J." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "member", "display_name": "Deshaies, Raymond Joseph" } ], "option_major": [ "biology" ], "doi": "10.7907/8DZY-WB19", "abstract": "Aging is characterized by the declining ability of an organism to maintain homeostasis, which eventually leads to death. Dietary restriction (DR), the reduction of nutrients without malnutrition, extends lifespan in various organisms, yet its molecular underpinnings are poorly understood. We show that in Drosophila, DR upregulates the translational repressor 4EBP, the eukaryotic translation initiation factor 4E binding protein, and that this upregulation is necessary for the full lifespan extension upon DR and sufficient to extend lifespan on a nutrient rich diet. Investigation of the genome-wide translational changes upon DR using translation state array analysis (TSAA) found that translationally downregulated genes tend to have extensive 5\u2019 untranslated regions (UTR) secondary structures, while those that are upregulated have weakly structured 5\u2019UTRs. Among the translationally upregulated genes, mitochondrial ribosomal proteins and electron transport chain components were overrepresented. Mitochondrial genes were found to have weakly structured 5\u2019UTRs in Drosophila, and this was conserved in Humans. The 5\u2019UTRs of mitochondrial genes were found to be sufficient to confer preferential translation during times of high 4EBP activity in a cap-independent manner to reporter constructs. Upregulation of mitochondrial function was verified and found to be d4EBP dependent, implicating a novel mechanism for regulating mitochondrial function upon DR. These results implicate mRNA translation initiation in modulating lifespan and mitochondrial function upon DR." }, { "name": "Alvizo, Oscar", "degree": "PhD", "year": "2007", "title": "Computational Protein Design Force Field Optimization: A Negative Design Approach", "advisor": "Mayo, Stephen L.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05212007-164114", "creators": [ { "name": { "family": "Alvizo", "given": "Oscar" }, "id": "Alvizo-Oscar", "orcid": "0000-0002-3545-1317", "display_name": "Alvizo, Oscar" } ], "advisors": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "advisor", "display_name": "Mayo, Stephen L." } ], "committee": [ { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "chair", "display_name": "Pierce, Niles A." }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Wang", "given": "Zhen-Gang" }, "id": "Wang-Zhen-Gang", "orcid": "0000-0002-3361-6114", "role": "member", "display_name": "Wang, Zhen-Gang" } ], "option_major": [ "biochem" ], "doi": "10.7907/NYVY-7Z76", "abstract": "An accurate force field is essential to computational protein design and protein folding studies. Proper force field tuning is problematic, however, due in part to the incomplete modeling of the unfolded state. The first part of this thesis discusses the optimization of a protein design force field by constraining the amino acid composition of the designed sequences to that of the wild-type protein. According to the random energy model, the unfolded state energies of amino acid sequences with the same composition are identical. Under these constraints, unfolded state energies are inconsequential and any discrepancies between computational predictions and experimental results can be directly attributed to flaws in the force field\u2019s ability to properly account for folded state sequence energies. This aspect of fixed composition design allows for force field optimization by focusing solely on the interactions in the folded state. In addition, the fixed composition requirement imposes a large negative design constraint that is used to ensure fold specificity. Several rounds of fixed composition optimization of the beta-1 domain of protein G yielded force field parameters with significantly greater predictive power: optimized sequences exhibited higher wild-type sequence identity in critical regions of the structure and the wild-type sequence showed an improved Z score. Experimental studies revealed a 24-fold mutant to be stably folded with a melting temperature comparable to that of the wild-type protein.
\r\n\r\nThe second part of the thesis discusses the optimization of HIV protease substrate specificity using a combination of positive and negative design. HIV protease is a homodimeric protein with a symmetrical binding region that recognizes and cleaves asymmetrical substrates that exhibit little sequence homology. The designs attempt to increase specificity towards one of HIV protease\u2019s wild-type targets by optimizing hydrogen bonds and electrostatic interactions using a positive design approach. Explicit negative design is incorporated by modeling predicted mutations on multiple substrates. A scoring function that selects for mutations that pack favorably with the target substrate but result in large steric clashes in alternate substrates is used. A three point mutant was designed and experimentally shown to have increased specificity towards the target substrate.
" }, { "name": "Ashby, Meredith Howard", "degree": "PhD", "year": "2007", "title": "The Sea Urchin Regulome in Development", "advisor": "Davidson, Eric H.", "url": "https://resolver.caltech.edu/CaltechETD:etd-10192006-114144", "creators": [ { "name": { "family": "Ashby", "given": "Meredith Howard" }, "id": "Ashby-Meredith-Howard", "display_name": "Ashby, Meredith Howard" } ], "advisors": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "advisor", "display_name": "Davidson, Eric H." } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "chair", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biochem" ], "doi": "10.7907/523T-5R49", "abstract": "During development an organism undergoes many rounds of pattern formation, generating ever greater complexity with each ensuing round of cell division and specification. The instructions for executing this process are encoded in the DNA, in cis-regulatory modules that direct the expression of developmental transcription factors and signaling molecules. Each transcription factor binding site within a cis-regulatory module contributes information about when, where or how much a gene is turned on, and by dissecting the modules driving a given gene, all the inputs governing expression of the gene can be accurately identified. Furthermore, by mapping the output of each gene to the inputs of other genes, it is possible to reverse engineer developmental circuits and even whole networks, revealing common bilaterian strategies for specifying progenitor fields, locking down regulatory states, and driving development forward. The S. purpuratus endomesodermal gene network is one of the best-characterized developmental networks, with interactions between over 40 regulatory genes mapped by perturbation experiments. With the sequencing of the sea urchin genome, it is possible to move towards the definitive completion of this network. By identifying all the transcription factors in the genome and determining their expression patterns, any previously unrecognized players can be incorporated into the network. In addition, such a comprehensive examination of transcription factor usage in maximally indirect development has not been done and will itself yield interesting conclusions." }, { "name": "Beierholm, Ulrik Ravnsborg", "degree": "PhD", "year": "2007", "title": "Bayesian Modeling of Sensory Cue Combinations", "advisor": "Quartz, Steven R.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05212007-172639", "creators": [ { "name": { "family": "Beierholm", "given": "Ulrik Ravnsborg" }, "id": "Beierholm-Ulrik-Ravnsborg", "orcid": "0000-0002-7296-7996", "display_name": "Beierholm, Ulrik Ravnsborg" } ], "advisors": [ { "name": { "family": "Quartz", "given": "Steven R." }, "id": "Quartz-S-R", "role": "advisor", "display_name": "Quartz, Steven R." } ], "committee": [ { "name": { "family": "Quartz", "given": "Steven R." }, "id": "Quartz-S-R", "role": "chair", "display_name": "Quartz, Steven R." }, { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "role": "member", "display_name": "Winfree, Erik" }, { "name": { "family": "O'Doherty", "given": "John P." }, "id": "O'Doherty-J-P", "role": "member", "display_name": "O'Doherty, John P." }, { "name": { "family": "Shams", "given": "Ladan" }, "id": "Shams-L", "role": "member", "display_name": "Shams, Ladan" }, { "name": { "family": "Bossaerts", "given": "Peter L." }, "id": "Bossaerts-P-L", "role": "member", "display_name": "Bossaerts, Peter L." } ], "option_major": [ "cns" ], "doi": "10.7907/K89B-XW75", "abstract": "We are constantly bombarded by sensory input, from multiple sources. How does the human brain process all this information? How does it decide what information to combine and what to keep segregated? If a visual and auditory stimulus originates from the same cause, say a baseball hitting a bat, there are advantages to combining the information to a single percept.
\r\n\r\nThe Bayesian framework is an obvious way to approach this problem. Given sensory input, the optimal observer would try to infer what sources created the stimuli.
\r\n\r\nWe developed a model based on Bayesian principles that makes no assumptions about the type of correlations between sources in the world, and found it in excellent correspondence with experiments on human subjects.
\r\n\r\nFurther, it is possible to place this problem in the more specific frame of causal inference, which has previously only been applied to cognitive problems. Causal inference tries to infer the hidden causal structure from the observables, implying that we constantly are inferring the causal structure of the world surrounding us. This can be thought of as a more constrained approach than our previous model with fewer parameters.
\r\n\r\nWe performed a number of psychophysics experiments and found the subjects\u2019 responses to be in excellent accordance with the model\u2019s predictions. Further predictions from the model, such as independence of the likelihoods and priors, were tested and found to be in accordance with data.
\r\n\r\nThese studies show that human perception is in accordance with Bayesian inference and implies a commonality between perceptual and cognitive processing.
" }, { "name": "Bender, John Andrew", "degree": "PhD", "year": "2007", "title": "Elements of Feed-Forward and Feedback Control in Drosophila Body Saccades", "advisor": "Dickinson, Michael H.", "url": "https://resolver.caltech.edu/CaltechETD:etd-03042007-163003", "creators": [ { "name": { "family": "Bender", "given": "John Andrew" }, "id": "Bender-John-Andrew", "display_name": "Bender, John Andrew" } ], "advisors": [ { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "role": "advisor", "display_name": "Dickinson, Michael H." } ], "committee": [ { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "role": "chair", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "role": "member", "display_name": "Dickinson, Michael H." }, { "name": { "family": "Murray", "given": "Richard M." }, "id": "Murray-R-M", "role": "member", "display_name": "Murray, Richard M." }, { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "role": "member", "display_name": "Siapas, Athanassios G." } ], "option_major": [ "biology" ], "doi": "10.7907/HEVK-7T03", "abstract": "I have developed a new experimental preparation of the fruit fly, Drosophila melanogaster. A fly is glued to a steel pin, which is held in the field between two magnets such that the fly is free to rotate about only one axis. Such \"magnetically tethered\" flies perform rapid yaw turns, similar to the behaviors termed \"body saccades\" in free flight. Saccades can be evoked by visual stimulation, in a manner suggesting that the underlying neural circuitry may be performing an angular threshold calculation. Once a saccade is initiated, however, visual feedback has very little effect on its dynamics, but rotational feedback from the haltere system plays an important role in structuring the time course of saccades. Vision is important, though, in maintaining a stable orientation in both intact flies and flies with asymmetrical wing alterations. The halteres are known to mediate responses to Coriolis forces correlated with the fly's rotations in flight, but flies with modified halteres also exhibit distorted saccade dynamics when they are not free to rotate. This suggests that the halteres may be involved in saccade initiation, although the precise mechanisms are not clear. There is preliminary evidence suggesting that the haltere strokes may be actively modulated during flight." }, { "name": "Brown, Charles Titus", "degree": "PhD", "year": "2007", "title": "Tackling the Regulatory Genome", "advisor": "Davidson, Eric H.", "url": "https://resolver.caltech.edu/CaltechETD:etd-12192006-100331", "creators": [ { "name": { "family": "Brown", "given": "Charles Titus" }, "id": "Brown-Charles-Titus", "orcid": "0000-0001-6001-2677", "display_name": "Brown, Charles Titus" } ], "advisors": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "advisor", "display_name": "Davidson, Eric H." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Adami", "given": "Christoph Carl" }, "id": "Adami-C-C", "role": "member", "display_name": "Adami, Christoph Carl" }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biology" ], "doi": "10.7907/STPG-0P56", "abstract": "The structure of the gene regulatory networks that drive animal development is encoded in the genome in cis-regulatory regions. Locating these regions and understanding how they integrate regulatory information to produce specific spatiotemporal patterns of gene expression is a major challenge facing developmental biology. This thesis presents computational and experimental work on finding, dissecting, and understanding regulatory regions. I discuss the use of comparative sequence analysis or \"phylogenetic footprinting\" to locate regulatory regions in animals. I then present experimental work on dissecting the information encoded in the cyIIIa cis-regulatory system of the California purple sea urchin, Strongylocentrotus purpuratus. Finally, I present a computational investigation of binding site validation techniques in E.coli." }, { "name": "Budick, Seth Alexander", "degree": "PhD", "year": "2007", "title": "Resource Localization and Multimodal Flight Control in Drosophila melanogaster", "advisor": "Konishi, Masakazu", "url": "https://resolver.caltech.edu/CaltechETD:etd-05292007-123933", "creators": [ { "name": { "family": "Budick", "given": "Seth Alexander" }, "id": "Budick-Seth-Alexander", "orcid": "0000-0002-5618-8810", "display_name": "Budick, Seth Alexander" } ], "advisors": [ { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "advisor", "display_name": "Konishi, Masakazu" } ], "committee": [ { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "chair", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "role": "member", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "role": "member", "display_name": "Dickinson, Michael H." } ], "option_major": [ "biology" ], "doi": "10.7907/1EYM-SG52", "abstract": "Upwind flight is a common strategy among insects searching for the sources of attractive odors. While much is known about the behavior of male Lepidoptera tracking female pheromone plumes, data on odor localization in other taxa, including the model organism Drosophila melanogaster, have been relatively lacking. The work presented in this thesis provides a description of the multimodal control of forward flight in D. melanogaster, including olfactory mediated flight during the localization of attractive resources.
\r\n\r\nHere it is shown that D. melanogaster responds rapidly to the onset of olfactory stimulation by turning upwind and increasing its airspeed, yielding an upwind surge. Following plume loss, flies, like many moths, often cast\u2014flying perpendicular to the wind while making iterated large-angle turns. Flies, however, are anemotactic even in the absence of odor, and unlike many Lepidoptera, they also fly fast and straight upwind in a homogenous odor cloud. Though they respond rapidly to odor contact and loss, flies thus do not require intermittent olfactory stimulation in order to sustain upwind flight.
\r\n\r\nThe results of tethered-flight experiments are largely in accord with those from free-flight. Pulsed and continuous olfactory stimuli elicit qualitatively similar responses in the wing kinematics of tethered flies, suggesting that the intermittency of the odor plume is not a key parameter in modulating flight behavior. At the same time, a tethered-flight analogue of casting may preferentially follow exposure to brief odor pulses, suggesting that pulse duration is an important factor in shaping flight trajectories.
\r\n\r\nThe role of mechanosensory cues in the orientation of flying insects has been the focus of relatively little research. The results presented here suggest that a strong mechanosensory response orients flying flies into an oncoming wind, as would be experienced during forward flight. This response is mediated by the Johnston\u2019s organs and may play an essential role during forward flight. Expanding visual stimuli, which necessarily accompany forward translation, normally elicit a robust turning response in flies. The wind orientation response described here is sufficient to suppress this visual reflex, however, potentially explaining how flies are able to successfully fly forward while searching for attractive resources.
" }, { "name": "Campos, Michael", "degree": "PhD", "year": "2007", "title": "Eye Movements and Reward, Sequential States, and Context-Dependent Target Selection", "advisor": "Andersen, Richard A.", "url": "https://resolver.caltech.edu/CaltechETD:etd-11082006-132934", "creators": [ { "name": { "family": "Campos", "given": "Michael" }, "id": "Campos-Michael", "display_name": "Campos, Michael" } ], "advisors": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "advisor", "display_name": "Andersen, Richard A." } ], "committee": [ { "name": { "family": "O'Doherty", "given": "John P." }, "id": "O'Doherty-J-P", "orcid": "0000-0003-0016-3531", "role": "chair", "display_name": "O'Doherty, John P." }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "member", "display_name": "Koch, Christof" }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "member", "display_name": "Andersen, Richard A." }, { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "orcid": "0000-0002-1290-5232", "role": "member", "display_name": "Shimojo, Shinsuke" } ], "option_major": [ "cns" ], "doi": "10.7907/5RG8-W844", "abstract": "The eye movement system is a complete sensorimotor loop from sensation to action, which includes a large number of distinct cortical and subcortical regions and participates in both reflexive and voluntary behaviors. This dissertation elucidates some of the functions of three cortical areas known to participate in eye movement behavior: the supplementary eye fields (SEF), motor area (SMA), and the lateral intraparietal area (LIP). In the course of executing eye movements, the eye movement circuitry interfaces with other functional circuits, including the networks of brain structures involved in reward processing, the temporal organization of behavior, target selection, and object perception. Here it is shown how LIP, SEF, and SMA participate in these multiple functional circuits, and complement each other during eye movement tasks. First, it is shown that neurons in the SMA carry a reward expectancy signal in the post-saccadic period of oculomotor tasks. Second, the neurons of SEF, but not LIP, are shown to collectively encode the temporal progression of the task. Third, in a target selection task, most LIP neurons are shown to respond to both cue and distractor stimuli, while most SEF neurons respond selectively only to the cue. Finally, fourth, the spatial tuning of parietal neurons is investigated in more natural circumstances, and the directional tuning preferences of cells in parietal cortex are found to be task dependent. These results extend the understanding of how these cortical brain areas that participate in eye movement behavior specialize and complement each other, and how they interface with other brain circuits, to support the organism in successfully completing a variety of instructed tasks.\r\n" }, { "name": "Detmer, Scott A.", "degree": "PhD", "year": "2007", "title": "The Role of Mitofusin Proteins in Mitochondrial Fusion and Disease", "advisor": "Chan, David C.", "url": "https://resolver.caltech.edu/CaltechETD:etd-04132007-181115", "creators": [ { "name": { "family": "Detmer", "given": "Scott A." }, "id": "Detmer-Scott-A", "display_name": "Detmer, Scott A." } ], "advisors": [ { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "advisor", "display_name": "Chan, David C." } ], "committee": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "role": "chair", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "member", "display_name": "Chan, David C." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "member", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biology" ], "doi": "10.7907/7MXP-WS63", "abstract": "We have investigated the role of mitofusin proteins in mitochondrial fusion and Charcot-Marie-Tooth disease Type 2A (CMT2A). Mitofusins (Mfn1 and Mfn2) are required for mammalian mitochondrial fusion. In structure-function analysis, we have identified loss-of-function mutations in mitofusin GTPase and heptad-repeat domains that disrupt homotypic and heterotypic domain interactions. Mutations in Mfn2 cause CMT2A, a progressive peripheral neuropathy. We have functionally characterized Mfn2 disease mutations and find that wild-type Mfn1, but not Mfn2, can efficiently complement nonfunctional CMT2A alleles to restore mitochondrial fusion. This finding demonstrates the importance of Mfn1-Mfn2 heterooligomers and suggests that Mfn1 expression is important in determining the cell-type specificity of CMT2A. To study the consequences of an Mfn2 CMT2A allele in vivo, we generated transgenic mice that express Mfn2 T105M in motor neurons. These animals demonstrate gait impairments due to distal muscle loss, axonopathy and altered mitochondrial morphology and distribution in motor neurons. In a second approach, we have generated CMT2A knock-in mice by replacing the endogenous genomic Mfn2 with Mfn2 alleles L76P or R94Q. Preliminary characterizations suggest that heterozygous animals have no disease symptoms, but homozygous Mfn2 R94Q animals are severely affected. Together, these mouse models provide means to assess the pathology of Mfn2 CMT2A alleles and the role of mitochondrial dynamics in vivo." }, { "name": "Fernandes, Jolene Sabrina", "degree": "PhD", "year": "2007", "title": "Dissection of Gene Regulatory Networks Underlying Patterning and Morphogenesis in the C. elegans Vulva", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05242007-164706", "creators": [ { "name": { "family": "Fernandes", "given": "Jolene Sabrina" }, "id": "Fernandes-Jolene-Sabrina", "display_name": "Fernandes, Jolene Sabrina" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "chair", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biology" ], "doi": "10.7907/AKNB-Q161", "abstract": "During development, in the course of which the single-celled egg generates a whole organism, cells become different from each other and form patterns of types of cells. It is these spatially defined cell-fate patterns that underlie the generation of complex organs. The mechanisms that establish these precise spatial patterning events depend on the implementation of diverse \u2018gene regulatory networks\u2019 (a consequence of functional interconnections between regulatory genes (transcription factors) and their target genes). Dissection of gene regulatory networks that control patterning of gene expression and differentiation would thus help us understand how cells generate a spatially defined pattern of cell fates during organ formation. Resources such as diverse spatial and temporal cell-fate markers, reverse genetics (RNAi), trans-genesis, and the ease of manipulation at the single-cell level make C. elegans a tractable system for studying the execution of cell-type-specific gene expression programs that occur during organogenesis. Consider the C. elegans vulva, a postembryonically derived organ that invariantly consists of seven distinct vulval cell types (vulA, vulB1, vulB2, vulC, vulD, vulE and vulF), each with its own unique gene expression profile. These features make the C. elegans vulva a particularly attractive model for dissecting the postembryonic gene regulatory networks involved in patterning and organ morphogenesis. This thesis focuses on elucidating the regulatory networks that control gene expression in the seven vulval cell types of C. elegans during organogenesis. The transcription factors lin-11(LIM), cog-1(Nkx6.1/6.2), and egl-38(Pax2/5/8) have been previously implicated as key regulators of gene expression in the vulva. Identification of additional regulatory factors is warranted, so as to rigorously dissect the mechanisms that specify the spatial fate patterns of terminally differentiated cell types. To this end, I systematically disrupted the gene activity of 508 transcription factors via RNAi and assayed the expression of ceh-2, a readout for vulB fate during the L4 stage. From this screen, I identified the tailless ortholog nhr-67 as a novel regulator of vulval gene expression. nhr-67 acts in combination with cog-1, egl-38, and lin-11 to execute accurate patterning of gene expression of their downstream targets. The pair-wise interactions between these regulatory genes are complex and vary among the seven cell types. One of the ways in which nhr-67 maintains cell identity is through restriction of inappropriate cell fusion events in specific vulval cells (namely vulE and vulF). The cell fusion defects observed in an nhr-67 RNAi background can be partially attributed to deregulation of fusogens. cog-1 and lin-11 (but not egl-38) mutants also show heterotypic fusion defects to different degrees. I also discovered a striking regulatory circuit that affects a subset of the vulval lineages: cog-1 and nhr-67 inhibit both one another and themselves. We argue that the 1\u00b0 vulval cells (vulE and vulF) utilize this novel regulatory motif to rapidly switch fates in response to transient inputs. We also speculate that the built-in flexibility of this circuit acts as a failsafe mechanism (in the event of cell damage) in the vulE and vulF cells. We postulate that the differential levels and combinatorial patterns of lin-11, cog-1, egl-38, and nhr-67 expression are a part of a regulatory code for the mature vulval cell types" }, { "name": "Ghaboosi, Nazli", "degree": "PhD", "year": "2007", "title": "Genetic Inhibition of the Ubiquitin-Proteasome Pathway: Insights Into Proteasomal Targeting", "advisor": "Deshaies, Raymond Joseph", "url": "https://resolver.caltech.edu/CaltechETD:etd-05242007-234932", "creators": [ { "name": { "family": "Ghaboosi", "given": "Nazli" }, "id": "Ghaboosi-Nazli", "display_name": "Ghaboosi, Nazli" } ], "advisors": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "advisor", "display_name": "Deshaies, Raymond Joseph" } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Varshavsky", "given": "Alexander J." }, "id": "Varshavsky-A-J", "role": "member", "display_name": "Varshavsky, Alexander J." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "member", "display_name": "Deshaies, Raymond Joseph" } ], "option_major": [ "biology" ], "doi": "10.7907/PNPS-JR40", "abstract": "Regulated proteolysis plays a major role in diverse cellular processes, including cell-cycle progression, endocytosis, apoptosis, transcription, and signal transduction. Proteins destined for proteolysis undergo two key steps in the ubiquitin-proteasome pathway. First, a complex enzymatic cascade controls conjugation of a multiubiquitin chain onto a protein. Once ubiquitinated, protein substrates must be recognized and targeted to the proteasome complex, where they are unfolded and degraded. Receptor proteins, such as Rad23, recognize ubiquitinated proteins and deliver them to the proteasome.
\r\n\r\nWhile the list of enzymes involved in ubiquitination is steadily growing, the first enzymatic reaction required for all ubiquitin-dependent processes is catalyzed by one protein, the ubiquitin-activating enzyme, E1. In this work, we describe a genetic screen that targets the Saccharomyces cerevisiae E1 gene, UBA1. We report the isolation of uba1-204, a temperature-sensitive allele UBA1 that exhibits dramatic inhibition of the ubiquitin-proteasome pathway. Shifting mutant cells to the restrictive temperature results in the depletion of cellular ubiquitin conjugates within minutes, accompanied by stabilization of multiple protein substrates. We have employed the tight phenotype of this mutant to investigate the role ubiquitin conjugates play the recognition and delivery of substrates to the proteasome.
\r\n\r\nIt is possible to purify intact and active proteasomes complexes from uba1-204 cells. In the absence of ubiquitin activation, these proteasome complexes are depleted of ubiquitin conjugates and the ubiquitin-binding receptor proteins Rad23 and Dsk2. Binding of Rad23 to these proteasomes in vitro is enhanced by addition of either free or substrate-linked ubiquitin chains. Moreover, association of Rad23 with proteasomes in mutant and wild-type cells is improved upon stabilizing ubiquitin conjugates with proteasome inhibitor. We propose that recognition of polyubiquitin chains by Rad23 promotes its shuttling to the proteasome in vivo. As an additional example of the value of this novel genetic mutant in the study of the ubiquitin-proteasome system, we present preliminary results from a quantitative mass spectrometric analysis of the proteins associated with proteasome complexes isolated from uba1-204 cells.
\r\n\r\nIn summary, we have created a genetic method of rapidly inhibiting the ubiquitin-proteasome system. This will enable future exploration of the ubiquitin-proteasome system and potentially many other ubiquitin-dependent cellular processes.
" }, { "name": "Gold, Carl", "degree": "PhD", "year": "2007", "title": "Biophysics of Extracellular Action Potentials", "advisor": "Koch, Christof", "url": "https://resolver.caltech.edu/CaltechETD:etd-05312007-210112", "creators": [ { "name": { "family": "Gold", "given": "Carl" }, "id": "Gold-Carl", "display_name": "Gold, Carl" } ], "advisors": [ { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "advisor", "display_name": "Koch, Christof" } ], "committee": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "chair", "display_name": "Andersen, Richard A." }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "member", "display_name": "Koch, Christof" }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "member", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Buzsaki", "given": "Gyorgy" }, "id": "Buzsaki-G", "role": "member", "display_name": "Buzsaki, Gyorgy" }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" } ], "option_major": [ "cns" ], "doi": "10.7907/05XP-DT79", "abstract": "The goal of this thesis is to analyze the generation of single unit extracellular action potentials (EAPs), and to explore pertinent issues in the interpretation of EAP recordings. I use the line source approximation to model the EAP produced by individual neurons. I compare simultaneous intracellular and extracellular recordings of CA1 pyramidal neurons in vivo with simulations using the same cells'reconstructions. The model accurately reproduces both the waveform and the amplitude of the EAPs. The composition of ionic currents is reflected in the features of each cell's EAP, while dendritic morphology has little impact.
\r\n\r\nI compared constraining a compartmental model to fit the EAP with matching the intracellular action potential (IAP). I find that the IAP method underconstrains the parameters. The distinguishing characteristics of the EAP constrain the parameters and are fairly invariant to electrode position and cellular morphology. I conclude that matching EAP recordings are an excellent means of constraining compartmental models.
\r\n\r\nI recorded spikes from cat primary visual cortex (V1) and recreated them in the model. I calculated the distance at which an electrode could record the EAPs given the prevalent background noise. My analysis suggests that in the superficial cortical layers 50%-80% of the neurons were active, while in deeper layers only 10%-20% were active. I analyzed the bias towards recording the large neurons in the deep layers. If the detection and clustering algorithm is sensitive enough to include low-amplitude spikes then bias is moderate. If only high amplitude units (> 0.2 mV) are picked up, then recording will be significantly biased towards the deep layers.
\r\n\r\nThe majority of spikes in cortex had a negative peak with a mean of -0.11 mV, but a minority of units (<10%) had a large positive peak of up to 1.5 mV. Simulations demonstrate that a pyramidal neuron may generate a negative spike with amplitude greater than 1 mV, but a positive spike of at most 0.5 mV. I conclude that high-amplitude positive spikes cannot result from a single neuron EAP. I suggest that they may result from synchronized action potentials in groups of L5 pyramidal neurons.
" }, { "name": "Henderson, Gregory Philip", "degree": "PhD", "year": "2007", "title": "Ultrastructural Studies of Two Model Minimal Cells by Electron Cryotomography", "advisor": "Jensen, Grant J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05142007-131453", "creators": [ { "name": { "family": "Henderson", "given": "Gregory Philip" }, "id": "Henderson-Gregory-Philip", "orcid": "0000-0002-6035-0817", "display_name": "Henderson, Gregory Philip" } ], "advisors": [ { "name": { "family": "Jensen", "given": "Grant J." }, "id": "Jensen-G-J", "role": "advisor", "display_name": "Jensen, Grant J." } ], "committee": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "role": "chair", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Jensen", "given": "Grant J." }, "id": "Jensen-G-J", "role": "member", "display_name": "Jensen, Grant J." }, { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "role": "member", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biology" ], "doi": "10.7907/HK2B-CT58", "abstract": "While most motile bacteria propel themselves with flagella, other mechanisms have been described including retraction of surface-attached pili, secretion of polysaccharides, or movement of motors along surface protein tracks. These have been referred to collectively as forms of \"gliding\" motility. Despite being simultaneously one of the smallest and simplest of all known cells, Mycoplasma pneumoniae builds a surprisingly large and complex cell extension known as the attachment organelle that enables it to glide. Here, three-dimensional images of the attachment organelle were produced with unprecedented clarity and authenticity using state-of-the-art electron cryotomography. The attachment organelle was seen to contain a multi-subunit, jointed, dynamic motor much larger than a flagellar basal body and comparable in complexity. A new model for its function is proposed wherein inchworm-like conformational changes of its electron-dense core are leveraged against a cytoplasmic anchor and transmitted to the surface through layered adhesion proteins.
\r\n\r\nThe hallmark of eukaryotic cells is their segregation of key biological functions into discrete, membrane-bound organelles. Creating accurate models of their ultrastructural complexity has been difficult in part because of the limited resolution of light microscopy and the artifact-prone nature of conventional electron microscopy. Here we explored the potential of the emerging technology electron cryotomography to produce three-dimensional images of an entire eukaryotic cell in a near-native state. Ostreococcus tauri was chosen as the specimen because as a unicellular picoplankton with just one copy of each organelle, it is the smallest known eukaryote and was therefore likely to yield the highest resolution images. Whole cells were imaged at various stages of the cell cycle, yielding 3-D reconstructions of complete chloroplasts, mitochondria, endoplasmic reticula, Golgi bodies, peroxisomes, microtubules, and putative ribosome distributions in-situ. Surprisingly, the nucleus was seen to open long before mitosis, and while one microtubule (or two in some predivisional cells) were consistently present, no mitotic spindle was ever observed, prompting speculation that a single microtubule might be sufficient to segregate multiple chromosomes.
" }, { "name": "Hochstim, Christian John", "degree": "PhD", "year": "2007", "title": "Pax6 Controls Astrocyte Positional Identity in the Spinal Cord", "advisor": "Anderson, David J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-07262008-115538", "creators": [ { "name": { "family": "Hochstim", "given": "Christian John" }, "id": "Hochstim-Christian-John", "orcid": "0000-0001-8335-6392", "display_name": "Hochstim, Christian John" } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "advisor", "display_name": "Anderson, David J." } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "co-chair", "display_name": "Anderson, David J." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/KSPD-J220", "abstract": "Astrocytes constitute the most abundant cell type in the CNS, and play diverse functional roles, but the ontogenetic origins of this phenotypic diversity are poorly understood. We have investigated whether positional identity, a fundamental organizing principle governing the generation of neuronal subtype diversity, is also relevant to astrocyte diversification. We identified three positionally distinct subtypes of white matter astrocytes in the spinal cord, which can be distinguished by the combinatorial expression of Reelin and Slit1. These astrocyte subtypes derive from progenitor domains expressing the homeodomain transcription factors Pax6 and Nkx6.1, respectively. Loss- and gain-of-function experiments indicate that the positional identity of these astrocyte subtypes is controlled by Pax6 and Nkx6.1, in a combinatorial manner. Thus, positional identity is an organizing principle underlying astrocyte, as well as neuronal, subtype diversification, and is controlled by a homeodomain transcriptional code whose elements are re-utilized following the specification of neuronal identity earlier in development." }, { "name": "Huang, Jean Jing", "degree": "PhD", "year": "2007", "title": "Acyl-Homoserine Lactone Quorum Signal Degradation by Soil and Clinical Pseudomonas sp.", "advisor": "Newman, Dianne K.", "url": "https://resolver.caltech.edu/CaltechETD:etd-04162007-092010", "creators": [ { "name": { "family": "Huang", "given": "Jean Jing" }, "id": "Huang-Jean-Jing", "display_name": "Huang, Jean Jing" } ], "advisors": [ { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "advisor", "display_name": "Newman, Dianne K." } ], "committee": [ { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "chair", "display_name": "Newman, Dianne K." }, { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "orcid": "0000-0002-7033-0844", "role": "member", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "member", "display_name": "Simon, Melvin I." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/fjr6-5f51", "abstract": "Acyl-homoserine lactones (AHLs) are signaling molecules that are used by several species of Proteobacteria in a process of cell-to-cell communication known as quorum sensing. The production, secretion, and detection of these signaling molecules are used to regulate a variety of microbial group behaviors, such as motility, the production of extracellular enzymes, antibiotics, and virulence factors. This thesis describes the ability for two Pseudomonas sp., a soil - isolate strain PAI-A and a clinical - isolate Pseudomonas aeruginosa strain PAO1, to degrade long chain acyl-homoserine lactone quorum signaling molecules, and explores the implications for this degradation activity. P. aeruginosa is an opportunistic pathogen that engages in quorum sensing with a long and a short chain AHL: 3OC12HSL and C4HSL and regulates the production of its virulence genes in this way. The soil isolate does not accumulate AHLs, and there is no evidence for its engagement in quorum sensing. Both species degrade long chain AHL via an acylase mechanism in which the molecule is cleaved at the amide bond. Two enzymes, PvdQ and QuiP, encoded by the genes PA2385 and PA1032 of P. aeruginosa, were found sufficient for the degradation of long chain AHL, but only the PA1032 gene is necessary for this process. PA1032 is transcribed and its protein product is present during degradation of long chain AHL. Studies of PAO1 lagless, a variant of P. aeruginosa that always degrades long chain AHL, indicate that this strain is broken in the regulation of PA1032. PAO1 lagless was found to express the PA1032 gene throughout planktonic and biofilm growth states, but wild type PAO1 expressed PA1032 locally in the center of biofilm microcolonies. This finding suggests PAO1 may use its ability to degrade one of its two AHLs during this dynamic growth state. Degenerate primers designed from PA1032 of PAO1 enabled the determination of a 2.5 kb putative AHL acylase of the soil isolate. Collectively, these studies of how Pseudomonas soil and clinical isolates degrade AHL suggest the diverse ways in which the degradation of acyl-homoserine lactone molecules may be used." }, { "name": "Jayaraman, Vivek", "degree": "PhD", "year": "2007", "title": "Neural Circuit Dynamics and Ensemble Coding in the Locust and Fruit Fly Olfactory System", "advisor": "Laurent, Gilles J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05192007-195030", "creators": [ { "name": { "family": "Jayaraman", "given": "Vivek" }, "id": "Jayaraman-Vivek", "orcid": "0000-0003-3680-7378", "display_name": "Jayaraman, Vivek" } ], "advisors": [ { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "advisor", "display_name": "Laurent, Gilles J." } ], "committee": [ { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "orcid": "0000-0002-5899-7523", "role": "chair", "display_name": "Winfree, Erik" }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "member", "display_name": "Koch, Christof" }, { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "member", "display_name": "Schuman, Erin Margaret" }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "member", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "orcid": "0000-0002-8587-9936", "role": "member", "display_name": "Dickinson, Michael H." } ], "option_major": [ "cns" ], "doi": "10.7907/A4VB-6C23", "abstract": "Raw sensory information is usually processed and reformatted by an organism\u2019s brain to carry out tasks like identification, discrimination, tracking and storage. The work presented in this dissertation focuses on the processing strategies of neural circuits in the early olfactory system in two insects, the locust and the fruit fly.
\r\n\r\nProjection neurons (PNs) in the antennal lobe (AL) respond to an odor presented to the locust\u2019s antennae by firing in slow information-carrying temporal patterns, consistent across trials. Their downstream targets, the Kenyon cells (KCs) of the mushroom body (MB), receive input from large ensembles of transiently synchronous PNs at a time. The information arrives in slices of time corresponding to cycles of oscillatory activity originating in the AL.
\r\n\r\nIn the first part of the thesis, ensemble-level analysis techniques are used to understand how the AL-MB system deals with the problem of identifying odors across different concentrations. Individual PN odor responses can vary dramatically with concentration, but invariant patterns in PN ensemble responses are shown to allow odor identity to be extracted across a wide range of intensities by the KCs. Second, the sensitivity of the early olfactory system to stimulus history is examined. The PN ensemble and the KCs are found capable of tracking an odor in most conditions where it is pulsed or overlapping with another, but they occasionally fail (are masked) or reach intermediate states distinct from those seen for the odors presented alone or in a static mixture.
\r\n\r\nThe last part of the thesis focuses on the development of new recording techniques in the fruit fly, an organism with well-studied genetics and behavior. Genetically expressed fluorescent sensors of calcium offer the best available option to study ensemble activity in the fly. Here, simultaneous electrophysiology and two-photon imaging are used to estimate the correlation between G-CaMP, a popular genetically expressible calcium sensor, and electrical activity in PNs. The sensor is found to have poor temporal resolution and to miss significant spiking activity. More generally, this combination of electrophysiology and imaging enables explorations of functional connectivity and calibrated imaging of ensemble activity in the fruit fly.
" }, { "name": "Kim, Jongmin", "degree": "PhD", "year": "2007", "title": "In Vitro Synthetic Transcriptional Networks", "advisor": "Winfree, Erik", "url": "https://resolver.caltech.edu/CaltechETD:etd-12182006-115817", "creators": [ { "name": { "family": "Kim", "given": "Jongmin" }, "id": "Kim-Jongmin", "orcid": "0000-0002-2713-1006", "display_name": "Kim, Jongmin" } ], "advisors": [ { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "role": "advisor", "display_name": "Winfree, Erik" } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "role": "member", "display_name": "Winfree, Erik" } ], "option_major": [ "biology" ], "doi": "10.7907/Q9YA-N192", "abstract": "Information processing using biochemical circuits is essential for survival and reproduction of natural organisms. Construction of synthetic biochemical circuits from simple components provides a useful approach to establish the minimal determinants required for complex logical functions. As stripped-down analogues of genetic regulatory networks in cells, we engineered artificial transcriptional networks consisting of synthetic DNA switches, regulated by RNA signals acting as transcription activators or repressors, and two enzymes, bacteriophage T7 RNA polymerase and Escherichia coli ribonuclease H. The synthetic switch design is modular with programmable connectivity and allows dynamic control of RNA signals through enzyme-mediated production and degradation. The switches support sharp and adjustable thresholds using a competitive hybridization mechanism, analogous to a biological threshold mechanism, \"inhibitor ultrasensitivity,\" thus allowing arbitrary analog or digital circuits to be created in principle. Theoretical correspondence of our biochemical network to neural networks where synaptic weights and thresholds are encoded by concentrations of DNA strands greatly facilitates network design and analysis. Experimentally, we have constructed and analyzed several simple networks: positive and negative autoregulatory circuits, a mutual inhibitory circuit, and oscillators with positive and negative feedback. Reasonable agreement between experimental data and a simple mathematical model was obtained for switch input/output functions, phaseplane trajectories, the bifurcation diagram, and oscillation periods. A systematic quantitative characterization lead to identification of important network properties such as the saturation of degradation machinery and challenges to understand such as the interference by incomplete RNA signals. Construction of larger synthetic circuits provides a unique opportunity for evaluating model inference, prediction, and design of complex biochemical systems and could be used to ontrol nanoscale devices and artificial cells." }, { "name": "Lassila, Jonathan Kyle", "degree": "PhD", "year": "2007", "title": "Methods for Computational Enzyme Design and Application to the Chorismate-Prephenate Rearrangement", "advisor": "Mayo, Stephen L.", "url": "https://resolver.caltech.edu/CaltechETD:etd-12182006-150535", "creators": [ { "name": { "family": "Lassila", "given": "Jonathan Kyle" }, "id": "Lassila-Jonathan-Kyle", "display_name": "Lassila, Jonathan Kyle" } ], "advisors": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "advisor", "display_name": "Mayo, Stephen L." } ], "committee": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "chair", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "member", "display_name": "Arnold, Frances Hamilton" }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." } ], "option_major": [ "biochem" ], "doi": "10.7907/4xm1-h147", "abstract": "The Claisen rearrangement of chorismate to prephenate has become an important model system for developing understanding of enzymatic catalysis as well as for computational treatment of enzyme active sites. This thesis presents general methods for the computational design of enzyme active sites and applies these methods to the design of catalysts for the chorismate-prephenate rearrangement. The computational methods described allow the incorporation of transition-state structures and other small molecules into protein design calculations. These design procedures were tested through redesign of the active site of Escherichia coli chorismate mutase. The six predicted mutations were experimentally characterized and most maintained or increased the catalytic activity of the enzyme. To further investigate the context of the mutations predicted in the calculation and the tolerance of a natural enzyme to secondary active site mutations, extensive substitution experiments were performed. The effect of every amino acid in five active site hydrophobic positions and one N-capping position was evaluated. These experiments clarified some of the strengths and weaknesses of the computational modeling procedure. Finally, attempts to design a completely new enzyme for catalysis of the chorismate-prephenate rearrangement are discussed." }, { "name": "Lee, Pei Yun", "degree": "PhD", "year": "2007", "title": "Function and Regulation of the Strongylocentrotus purpuratus gatae Gene", "advisor": "Davidson, Eric H.", "url": "https://resolver.caltech.edu/CaltechETD:etd-04072007-221302", "creators": [ { "name": { "family": "Lee", "given": "Pei Yun" }, "id": "Lee-Pei-Yun", "display_name": "Lee, Pei Yun" } ], "advisors": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "advisor", "display_name": "Davidson, Eric H." } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." } ], "option_major": [ "biology" ], "doi": "10.7907/bjxm-rh79", "abstract": "The Strongylocentrotus purpuratus gatae is orthologous to vertebrate gata4/5/6 genes. gatae is expressed throughout embryogenesis, beginning in the 15 h blastula in presumptive mesoderm cells, and at mesenchyme blastula, in endoderm and mesoderm cells of the veg2 lineage. During gastrulation, gatae is expressed in the midgut, hindgut and mesoderm, while in the pluteus expression it is limited to the midgut and coelomic pouches. Perturbation of gatae expression resulted in the lowered RNA levels for many endomesoderm transcription factors, including foxA, brachyury, and [beta]1/2-otx, highlighting Gatae\u2019s role as a regulator of transcription factors. gatae occupies an important node in the endomesoderm gene regulatory network, using its cross-regulatory interactions with otx to stabilize the endomesoderm gene expression program. Cis-regulatory analysis of gatae identified two modules responsible for its embryonic expression. Module 10 drives endomesoderm expression in the blastula, while module 24 activates gut expression in the gastrula and pluteus. Deletion of module 10 from a gatae GFP BAC resulted in a complete loss of blastula stage expression, demonstrating its necessity and sufficiency for early activity. Global cis-regulatory analysis of the gatae locus suggests that module usage is exclusionary; only one module can associate with the basal transcriptional apparatus and affect gene transcription at any given time. The endomesoderm gene regulatory network predicts that gatae is downstream of Otx and Notch signaling. Analysis of the sequence of module 10 identified Otx and Suppressor-of-Hairless (Su(H)) binding sites. Injection of Otx-engrailed RNA repressed the expression of module 10:GFP reporter; the effect is abolished when Otx binding sites were mutated. Gel shifts demonstrated that the Otx protein binds to module 10. Module 10 expression was reduced under perturbation of Notch signaling. Mutations of either Otx or Su(H) binding sites resulted in lowered GFP RNA levels with no effect on spatial expression. Mutations of both Otx and Su(H) binding sites led to a further reduction but not elimination of reporter expression, suggesting that another input is involved. This unknown input was determined to be also downstream of Notch signaling and that gatae regulation functions via OR logic." }, { "name": "Lu, Carole Chih-Chen", "degree": "PhD", "year": "2007", "title": "Cranial Neural Crest Cell Migration in the Avian Embryo and the Roles of Eph-A4 and Ephrin-A5", "advisor": "Fraser, Scott E.", "url": "https://resolver.caltech.edu/CaltechETD:etd-10132006-153232", "creators": [ { "name": { "family": "Lu", "given": "Carole Chih-Chen" }, "id": "Lu-Carole-Chih-Chen", "display_name": "Lu, Carole Chih-Chen" } ], "advisors": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "advisor", "display_name": "Fraser, Scott E." } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "member", "display_name": "Chan, David C." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biology" ], "doi": "10.7907/v109-k937", "abstract": "The neural crest is a transient population of cells that migrate away from the dorsal neural tube in the vertebrate embryo. As the developing hindbrain constricts into rhombomeres, cranial neural crest cells migrate in three discrete streams adjacent to even-numbered rhombomeres, rhombomere 2 (r2), r4, and r6.
\r\n\r\nTo test the role of intrinsic versus extrinsic cues in influencing an individual cell\u2019s trajectory, we implanted physical barriers in the chick mesoderm, distal to emerging neural crest cells (NCCs). We analyzed spatio-temporal dynamics as NCCs encountered and responded to the barriers by using time-lapse confocal microscopy and cell tracking analysis. The majority of NCCs were able to overcome physical barriers. Even though the lead cells become temporarily blocked by a barrier, follower cells find a novel pathway around a barrier and become de novo leaders of a new stream. Quantitative analyses of cell trajectories find cells that encounter an r3 barrier migrate significantly faster but less directly than cells that encounter an r4 barrier, which migrate normally. NCCs can also migrate into normally repulsive territory as they reroute. These results suggest that cranial neural crest cell trajectories are not intrinsically determined. NCCs can respond to minor alterations in the environment to retarget a peripheral destination. Both intrinsic and extrinsic cues are important in patterning.
\r\n\r\nWe then tested the role of Eph/ephrin signaling on cranial neural crest migration by ectopically expressing full-length ephrin-A5 ligand; a truncated, constitutively active EphA4 receptor; and a truncated, kinase-dead EphA4 receptor within migratory neural crest cells. Ectopic expression of ephrin-A5 specifically causes the r6 subpopulation of neural crest cells to have truncated migration but does not affect directionality, suggesting that the r6 neural crest cells properly follow guidance cues. Our results support a role for ephrin-A5 in regulating the extent of migration.
\r\n\r\nEctopic expression of constitutively active, truncated EphA4 causes NCCs to migrate aberrantly around the otic vesicle. Pathfinding errors are accompanied by changes in migratory behavior, with the NCCs migrating faster but with less directionality. Expression of a truncated, kinase-dead version of EphA4 also leads to pathfinding errors. Our results suggest Eph activity is involved in guidance and extent of migration.
" }, { "name": "Malasarn, Davin", "degree": "PhD", "year": "2007", "title": "Molecular and Environmental Studies of Bacterial Arsenate Respiration", "advisor": "Newman, Dianne K.", "url": "https://resolver.caltech.edu/CaltechETD:etd-02232007-132917", "creators": [ { "name": { "family": "Malasarn", "given": "Davin" }, "id": "Malasarn-Davin", "display_name": "Malasarn, Davin" } ], "advisors": [ { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "advisor", "display_name": "Newman, Dianne K." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." }, { "name": { "family": "Gray", "given": "Harry B." }, "id": "Gray-H-B", "orcid": "0000-0002-7937-7876", "role": "member", "display_name": "Gray, Harry B." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." } ], "option_major": [ "biology" ], "doi": "10.7907/6PAA-PF90", "abstract": "Arsenate [As(V)]-respiring bacteria that reduce As(V) to arsenite, As(III), for energy production have been implicated as possible catalysts for arsenic mobilization into drinking water supplies. To understand how this metabolism contributes to arsenic geochemistry, this thesis explores the dynamics of As(V)-respiratory gene expression, the impact of As(V) respiration on microbial ferric [Fe(III)] reduction, and biochemical properties of the arsenate respiratory reductase, ARR.
\r\n\r\nUsing sequences for arrA, a gene encoding the terminal reductase involved in As(V) respiration, degenerate PCR primers were designed to amplify a diagnostic region of the gene in multiple As(V)-respiring isolates. These primers were used to track arrA transcription in microcosm studies involving synthetic sediments. arrA was required for As(V) reduction in this context, and the gene was expressed in contaminated sediments at Haiwee Reservoir in Olancha, CA.
\r\n\r\nTo understand the impact of As(V) respiration on Fe(III) reduction, native microbial consortia from Haiwee Reservoir and pure cultures of the genetically tractable Shewanella sp. strain ANA-3 were incubated with As-sorbed hydrous ferric oxide (HFO), and rates of As(V) and Fe(III) reduction were determined. As(V) reduction occurred simultaneously with or prior to Fe(III) reduction, consistent with the idea that electron acceptor utilization is determined by thermodynamic favorability. Furthermore, the presence of sorbed As(III) increased rates of Fe(III) reduction, potentially by increasing HFO surface area.
\r\n\r\nLastly, the expression, assembly, and kinetic properties of ARR from ANA-3 were characterized. ARR is a soluble periplasmic heterodimer that is expressed during early exponential growth and persists into late stationary phase. The enzyme contains molybdenum, Fe, and sulfur cofactors. It has a Km of 5 \u00b5M, a Vmax of 11,111 \u00b5mol As(V) reduced . min-1 . mg protein-1, and reduces only As(V). Mutational analysis of the residues corresponding to the diagnostic region of arrA mentioned above resulted in loss of enzyme activity.
\r\n\r\nThis work brings us closer to being able to quantify and predict the contribution of As(V) respiration to the solubilization of arsenic from sediments. Structural studies, the development of probes to detect ARR, and comparisons of ARR from different bacterial species are now possible.
" }, { "name": "Montgomery, Jennifer Pielstick", "degree": "PhD", "year": "2007", "title": "The Effects of Behavioral Stress and Endothelin Receptor Antagonists on Cancer", "advisor": "Patterson, Paul H.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05222007-105207", "creators": [ { "name": { "family": "Montgomery", "given": "Jennifer Pielstick" }, "id": "Montgomery-Jennifer-Pielstick", "display_name": "Montgomery, Jennifer Pielstick" } ], "advisors": [ { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "advisor", "display_name": "Patterson, Paul H." } ], "committee": [ { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "chair", "display_name": "Zinn, Kai George" }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biology" ], "doi": "10.7907/3FGQ-YS43", "abstract": "This work examines two potential mediators of cancer. Section one considers the putative link between behavioral stress and cancer. We developed a stress paradigm of alternating established stressors that dramatically increases serum corticosterone and causes thymus involution. When this stress paradigm was applied to mice implanted with either melanoma or lymphosarcoma tumors we observed no alteration in the growth of either tumor. In addition, we applied a protocol established in another laboratory that demonstrated a dramatic enhancement of lymphosarcoma tumors following rotational stress (Riley V. (1981). Psychoneuroendocrine influences on immunocompetence and neoplasia. Science 212, 1100-1109). We find no significant effect on lymphosarcoma progression. Therefore, under the conditions used in our studies, strong behavioral stress does not influence tumor growth.
\r\n\r\nSection two considers the effects of endothelin receptor (ETR) antagonists on cancer progression. Their ability to inhibit growth of many cancers is well documented, but similar research on glioma has been limited. We find that two ETRB-specific antagonists, BQ788 and A-192621, reduce the number of viable cells in glioma and melanoma cell lines in a dose- and time-dependent manner. In glioma cells, A-192621 induces a G2/M arrest, decreases the mean number of cell divisions and enhances apoptosis. BQ123, an ETRA-specific antagonist, has no effect on cell viability. A-192621 also up-regulates several DNA damage-inducible genes. Interestingly, reducing ETRB expression with small interfering RNAs does not abrogate the effects of either A-192621 or BQ788 in glioma or melanoma cells. Thus, while ETRB antagonists are effective against glioma, it appears unlikely that their therapeutic effects are mediated by ETRB. Further investigation is needed to define the mechanism by which these compounds decrease cell viability.
" }, { "name": "Moradi, Farshad", "degree": "PhD", "year": "2007", "title": "Conscious Awareness Determined by Selective Gating of Information in Early Visual Areas", "advisor": "Shimojo, Shinsuke; Koch, Christof", "url": "https://resolver.caltech.edu/CaltechETD:etd-05072007-115624", "creators": [ { "name": { "family": "Moradi", "given": "Farshad" }, "id": "Moradi-Farshad", "orcid": "0000-0002-6526-8521", "display_name": "Moradi, Farshad" } ], "advisors": [ { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "orcid": "0000-0002-1290-5232", "role": "advisor", "display_name": "Shimojo, Shinsuke" }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "advisor", "display_name": "Koch, Christof" } ], "committee": [ { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "chair", "display_name": "Adolphs, Ralph" }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "member", "display_name": "Koch, Christof" }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "member", "display_name": "Perona, Pietro" }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "member", "display_name": "Andersen, Richard A." }, { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "orcid": "0000-0002-1290-5232", "role": "member", "display_name": "Shimojo, Shinsuke" } ], "option_major": [ "cns" ], "doi": "10.7907/5r7f-9f33", "abstract": "A number of psychophysical methods that suppress retinal input from reaching awareness have been used to isolate and study the neural correlates of visual consciousness. I describe a novel disappearance phenomenon in which a low-contrast peripheral pattern is vividly erased from awareness: after adapting to the pattern for a few seconds, flashing a high-contrast patch over it can elicit the perceptual disappearance of the stimulus. This finding was explained in terms of nonlinear interaction between adaptation to sustained spatial pattern and rapid gain adjustment to transient change. It was next shown that transient changes contingent upon prior adaptation elicit perceptual alternations in structure from motion, binocular rivalry, Necker cube, and ambiguous apparent motion\u2014linking disappearance phenomena and bistable perception. We next used binocular rivalry and inattentional blindness to examine if invisible inputs influence the neuronal mechanisms that adapt to different aspects of the stimuli. The face identity-specific aftereffect was found to be cancelled by binocular suppression or by inattentional blindness of the inducing face. Conversely, the same suppression did not interfere with the orientation-specific aftereffect. Thus, the competition between incompatible or interfering visual inputs to reach awareness is resolved before those aspects of information that are exploited in face identification are processed. Subsequent experiments showed that face identity aftereffect is invariant to eye movements, but fMRI adaptation in face-selective region of the fusiform cortex did not show such invariance. Therefore identity aftereffect originates either at the same level or subsequent to the level of face processing in the fusiform area. Next, we show that recognition of facial emotional expressions occurs after the level of attentional selection: visual search results were incompatible with preattentive processing of emotional categories. We thus suggest that the invisible or unattended faces are suppressed in early visual areas. This conjecture was experimentally confirmed by showing that when a stimulus is not attended, it evoked a weaker and weaker response in fMRI in subsequent stages of visual processing hierarchy. Thus, attention determines how far the visual input is processed and whether or not a high-level representation of the input would be constructed." }, { "name": "Mosser, Eric Ardon", "degree": "PhD", "year": "2007", "title": "Visualization of Cadherin-Cadherin Association in Living Cells", "advisor": "Schuman, Erin Margaret", "url": "https://resolver.caltech.edu/CaltechETD:etd-02142007-004140", "creators": [ { "name": { "family": "Mosser", "given": "Eric Ardon" }, "id": "Mosser-Eric-Ardon", "display_name": "Mosser, Eric Ardon" } ], "advisors": [ { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "advisor", "display_name": "Schuman, Erin Margaret" } ], "committee": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "chair", "display_name": "Fraser, Scott E." }, { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "member", "display_name": "Schuman, Erin Margaret" }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." } ], "option_major": [ "biology" ], "doi": "10.7907/1we9-7t88", "abstract": "The almost universally accepted model for long-term potentiation (LTP) involves Ca2+ flux through NMDA receptors into dendritic spines. This Ca2+ influx may cause a transient Ca2+ decrease in the synaptic cleft. We hypothesize that this decrease in cleft Ca2+ may destabilize cadherin-cadherin bonds in the synapse and that this conformational change may allow synaptic cadherins to function as synaptic activity sensors.
\r\n\r\nIn the last decade, much effort has been devoted to understanding the structure and molecular associations of classic cadherins. Crystallographic and biophysical studies have yielded somewhat conflicting results and an as yet unclear picture of the homotypic interactions of the cadherin extracellular domain during dimerization. To better understand the dynamics of cadherin interactions we have developed Fluorescence Resonance Energy Transfer (FRET) based sensors to monitor cadherin associations across cellular junctions in a dynamic manner in living cells.
\r\n\r\nHere, we demonstrate the functionality of FRET-based cadherin interaction reporters. FRET is unique in its ability to provide signals that are sensitive to changes in intra- or intermolecular distances in the 1-10 nm range, well below the inherent diffraction limit of conventional fluorescence microscopy. We believe that FRET is a powerful technique to monitor cadherin orientation and interactions in heterologous, living cells.
" }, { "name": "Murphy, Gavin Erick", "degree": "PhD", "year": "2007", "title": "Cryoelectron Tomography of Bacteria and their Macromolecular Machines", "advisor": "Jensen, Grant J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05212007-162731", "creators": [ { "name": { "family": "Murphy", "given": "Gavin Erick" }, "id": "Murphy-Gavin-Erick", "display_name": "Murphy, Gavin Erick" } ], "advisors": [ { "name": { "family": "Jensen", "given": "Grant J." }, "id": "Jensen-G-J", "role": "advisor", "display_name": "Jensen, Grant J." } ], "committee": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "role": "chair", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Jensen", "given": "Grant J." }, "id": "Jensen-G-J", "role": "member", "display_name": "Jensen, Grant J." }, { "name": { "family": "Leadbetter", "given": "Jared R." }, "id": "Leadbetter-J-R", "role": "member", "display_name": "Leadbetter, Jared R." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "member", "display_name": "Elowitz, Michael B." } ], "option_major": [ "biochem" ], "doi": "10.7907/517B-0Z68", "abstract": "Cryoelectron tomography (CET) fills a glaring gap in the imaging capabilities of biology by reconstructing cells to medium resolution. The technique was applied in three areas to understand biology\u2019s macromolecular machines: (1) the quaternary structure of the octahedrally-cored E. coli pyruvate dehydrogenase (PDHC) and 2-oxoglutarate dehydrogenase (OGDHC) complexes in vitro; (2) the ultrastructure of the spirochete Treponema primitia; and (3) the structure of the in situ flagellar motors from T. primitia, Hylemonella gracilis, Caulobacter crescentus, and Vibrio cholerae. Whereas the complexes PDHC and OGDHC were thought to have their subunit proteins E1 and E3 bound directly to the octahedral E2 core\u2014the so-called face/edge model\u2014it was discovered that the subunits are flexibly tethered 11 nm from the corners of the core. Several novel structures were discovered in the spirochete T. primitia. Spirochetes are spiral-shaped cells that propel themselves with periplasmic, not external, flagella. Bowl-shaped structures dot its surface and hook-like appendages that form arcades stripe the length of the cell. Fibrils extend from its cell tips that might help attach the cells to surfaces. Inside the periplasm, porous, cone-shaped structures reside at each cell tip and a second periplasmic layer undergirds its outer membrane, which might prevent the periplasmic flagella from rupturing the cell. Previous imaging of the flagellar motor produced either high-resolution reconstructions of the purified basal body removed from its context or low-resolution images of the in situ motor. Our in situ 3-D reconstructions described for the first time the structure of the stators, the membrane embedded component that spins the rotor. Novel shapes were discovered that indicate there are various attachments and versions of the flagellar motor that were never expected." }, { "name": "Nieman, Dylan Rhichard", "degree": "PhD", "year": "2007", "title": "Postdiction and the Effects of Spatial, Temporal, and Feature Compatibility on Sensory Integration", "advisor": "Shimojo, Shinsuke", "url": "https://resolver.caltech.edu/CaltechETD:etd-01092007-152909", "creators": [ { "name": { "family": "Nieman", "given": "Dylan Rhichard" }, "id": "Nieman-Dylan-Rhichard", "display_name": "Nieman, Dylan Rhichard" } ], "advisors": [ { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "role": "advisor", "display_name": "Shimojo, Shinsuke" } ], "committee": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "role": "chair", "display_name": "Andersen, Richard A." }, { "name": { "family": "Bruck", "given": "Jehoshua" }, "id": "Bruck-J", "role": "member", "display_name": "Bruck, Jehoshua" }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "role": "member", "display_name": "Shimojo, Shinsuke" } ], "option_major": [ "cns" ], "doi": "10.7907/QGYV-PR54", "abstract": "The brain continually integrates stimuli over multiple sensory modalities and reconciles often disparate information into a unified, consistent representation of the surrounding environment. This process must be robust to differential neural latencies and imperfect alignments of spatial reference frames between sensory modalities. Numerous studies have examined the perception of multisensory stimuli with the presumption that multisensory integration is categorically different from within-modality integration. We looked at a variety of issues related to the updating of sensory reference frames and the integration of unimodal and multimodal stimuli over temporal and spatial disparities. Study 1 found simultaneous, opposite gaze-dependent aftereffects at the same retinal location for both depth and color, demonstrating the degree to which visual-coordinate space is gaze-contingent, not merely retinotopic. Study 2 found that the flash-lag effect, in which a flashed target is perceived as lagging behind a smoothly moving target, generalizes to third-order motion perception of cyclopean stimuli. Study 3 introduced a novel motion illusion which we termed the \"turn-point phantom,\" wherein the position of an abrupt orthogonal direction change is mislocalized backwards along the object's subsequent trajectory. This effect, like flash-lag, can only be adequately explained with postdiction. Study 4 explored the effect of passive head or body turns on spatial perception of visual and auditory stimuli and found systematic mislocalization of pre-turn stimuli in the direction of the turn. This mislocalization decayed with added delay between target and turn onset. Study 5 examined spatial and temporal disparity in visual-motor ventriloquism and found that early visual distracters were essentially equivalent, whereas the influence of late visual distracters diminished with increasing asynchrony. Study 6 found suppression of saccade latency induced by stimulus repetition in certain multisensory experimental contexts. Together, these studies provide numerous examples supporting the idea that sensory perception, both unimodal and multimodal, is postdictive in nature, involving integration of sensory information over a time window that includes, but does not end with, task-relevant stimulus presentation. Additionally, these results provide clues to the character and relevant parameters of the integration process." }, { "name": "Preuschoff, Kerstin", "degree": "PhD", "year": "2007", "title": "Neural Representations of Expected Reward and Risk During Gambling", "advisor": "Quartz, Steven R.", "url": "https://resolver.caltech.edu/CaltechETD:etd-12142006-105901", "creators": [ { "name": { "family": "Preuschoff", "given": "Kerstin" }, "id": "Preuschoff-Kerstin", "orcid": "0000-0001-7254-833X", "display_name": "Preuschoff, Kerstin" } ], "advisors": [ { "name": { "family": "Quartz", "given": "Steven R." }, "id": "Quartz-S-R", "role": "advisor", "display_name": "Quartz, Steven R." } ], "committee": [ { "name": { "family": "O'Doherty", "given": "John P." }, "id": "O'Doherty-J-P", "orcid": "0000-0003-0016-3531", "role": "chair", "display_name": "O'Doherty, John P." }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "member", "display_name": "Koch, Christof" }, { "name": { "family": "Bossaerts", "given": "Peter L." }, "id": "Bossaerts-P-L", "orcid": "0000-0003-2308-2603", "role": "member", "display_name": "Bossaerts, Peter L." }, { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "orcid": "0000-0002-1290-5232", "role": "member", "display_name": "Shimojo, Shinsuke" }, { "name": { "family": "Quartz", "given": "Steven R." }, "id": "Quartz-S-R", "role": "member", "display_name": "Quartz, Steven R." } ], "option_major": [ "cns" ], "doi": "10.7907/X8G3-B032", "abstract": "Organisms continuously monitor the stimuli they encounter and the outcome of their actions. To survive in an uncertain world they aim for rewards and try to avoid punishments. Research in neuroscience, ecology, and economics implies that organisms base their decisions in uncertain situations on expected rewards and risk. Neuroscience focuses on reward prediction learning based on reward prediction errors. In contrast, economic studies emphasize risk in addition to expected reward.
\r\n\r\nWe used functional imaging in humans during gambling tasks to understand how the brain represents expected reward and risk. We find that brain activity in subcortical dopaminoceptive structures can be separated, both spatially and temporally, into signals that correlate with (mathematical) expectation of reward, and with reward variance (risk) \u2013 two fundamental parameters in financial decision theory. Our results suggest that the primary function of the dopaminergic system extends beyond its established role in learning, motivation, and salience: it signals different aspects of upcoming stochastic rewards \u2013 expected reward and risk.
\r\n\r\nBased on financial decision theory we then hypothesized neural representations of prediction risk and prediction risk errors. We find that the insula represents both. In analogy with reward representations in subcortical structures, the signals are spatially and temporally differentiated. These findings expand our understanding of the neural basis of decision making under uncertainty by adding prediction risk estimation.
\r\n\r\nFinally, we investigated where and how expected reward and risk are combined into the neural representation of a gamble\u2019s overall value. Using canonical correlation analysis, we find a new predictor that \u2013 contrary to expected utility theory \u2013 adds risk to expected reward. This sum may define a metric of conflict or attention. This metric significantly correlates with activation in the anterior cingulate cortex a structure associated with conflict monitoring.
\r\n\r\nDrawing on financial theories, we show how the brain represents expected reward and risk. Our results suggest that the earlier understanding of decision making under uncertainty needs to be expanded to include (prediction) risk as measured by variance as well as prediction risk errors. Such integration has far-reaching implications, in particular for pathological decision making.
\r\n" }, { "name": "Revilla-i-Domingo, Roger", "degree": "PhD", "year": "2007", "title": "CIS-Regulatory Analysis of the Sea Urchin Delta Gene: Validating the Architecture of the Gene Regulatory Network Model for Early Micromere Lineage Specification", "advisor": "Davidson, Eric H.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06042007-145414", "creators": [ { "name": { "family": "Revilla-i-Domingo", "given": "Roger" }, "id": "Revilla-i-Domingo-Roger", "orcid": "0000-0001-7943-5776", "display_name": "Revilla-i-Domingo, Roger" } ], "advisors": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "advisor", "display_name": "Davidson, Eric H." } ], "committee": [ { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "role": "chair", "display_name": "Campbell, Judith L." }, { "name": { "family": "Parker", "given": "Carl Stevens" }, "id": "Parker-C-S", "role": "member", "display_name": "Parker, Carl Stevens" }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "member", "display_name": "Strauss, James H." } ], "option_major": [ "biochem" ], "doi": "10.7907/G9GQ-H097", "abstract": "During the specification of the endomesoderm of the sea urchin embryo, mesodermal and endodermal cell types derive from common progenitors. The Delta signal, a ligand of the Notch receptor, serves as the spatial cue that triggers the segregation between these two fates. Expression of the delta gene exclusively in the micromere lineage early in development is essential for Delta to be able to correctly serve this role. According to a model of the gene regulatory network (GRN) underlying this process, the mechanism by which the micromere lineage is specified as a distinct domain, and by which the delta gene is expressed exclusively there, depends on a double repression system. A gene encoding a transcriptional repressor, pmar1, is activated specifically in the micromeres, where it represses transcription of a second repressor that is otherwise active globally. Zygotic expression of delta and micromere specific control genes depends on ubiquitous activators, and localization in the micromere lineage depends on repression by the second repressor everywhere else. In this model the second repressor is an unidentified gene, the existence of which is implied by numerous experiments. The work presented in this thesis experimentally validates the double repression architecture for micromere lineage specification and localization of delta expression. To prove the existence of the double repression system a genomic screen was devised to identify the gene playing the role of the second repressor. hesC, a transcription factor of the HES family, was found to be this gene. It is expressed at the right time and place, and its function is to repress micromere specific regulatory genes. To show that expression of delta in the micromere lineage depends on ubiquitous activators and HesC-dependent repression, the relevant cis-regulatory module (CRM) was recovered. This CRM, named R11, is shown to be able to drive the expression of a reporter gene exclusively in the micromere lineage at the right time. Dissection of R11 and its response to blockade of hesC expression show that R11 expression depends on ubiquitously present activators, and on HesC-dependent repression everywhere except the micromere lineage.\r\n" }, { "name": "Teal, Tracy Kristin", "degree": "PhD", "year": "2007", "title": "Studies of the Spatial Organization of Metabolism in Shewanella oneidensis and Pseudomonas aeruginosa Biofilms", "advisor": "Newman, Dianne K.; Wold, Barbara J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05312007-190155", "creators": [ { "name": { "family": "Teal", "given": "Tracy Kristin" }, "id": "Teal-Tracy-Kristin", "orcid": "0000-0002-9180-9598", "display_name": "Teal, Tracy Kristin" } ], "advisors": [ { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "advisor", "display_name": "Newman, Dianne K." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "co-advisor", "display_name": "Wold, Barbara J." } ], "committee": [ { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "orcid": "0000-0002-5899-7523", "role": "chair", "display_name": "Winfree, Erik" }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Adami", "given": "Christoph Carl" }, "id": "Adami-C-C", "orcid": "0000-0002-2915-9504", "role": "member", "display_name": "Adami, Christoph Carl" }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." } ], "option_major": [ "cns" ], "doi": "10.7907/2gdf-yh13", "abstract": "Bacteria grow in the environment as surface-attached microbial communities. These communities are pervasive and resilient in the face of changing and challenging environmental conditions. Because of their community organization and three-dimensional structure, conditions within a biofilm are heterogenous, exposing the bacterial cells to individual microenvironments depending on their location in the biofilm and the biomass of the structure. Communities are therefore thought to be metabolically stratified. To understand how communities are organized with regard to growth activity and metabolic state and what role endogenous compounds might play in this organization, this thesis explores the spatiometabolic organization and dynamics of Shewanella oneidensis biofilms and the roles that acyl-homeserine lactones and phenazines might have in Pseudomonas aeruginosa communities.
\r\n\r\nUsing unstable fluorescent reporters to measure growth activity and protein synthesis and conducting quantitative image analysis, domains of activity were determined for developing S. oneidensis biofilms. Biofilm structures reproducibly stratify with respect to growth activity and metabolism as a function of structure size. Within domains of growth-inactive cells, genes upregulated under anaerobic conditions are expressed demonstrating that cells in the nutrient-limited regions of the biofilm are not dead, but are capable of generating enough energy to persist.
\r\n\r\nTo determine if these growth-inactive cells are able to respond dynamically to changes in environmental conditions and what types of nutrients affect growth activity profiles, S. oneidensis biofilms were exposed to increased concentrations of an electron acceptor and an electron donor. Cells in the growth-inactive regions were able to respond to nutrient changes, but were more affected by a change in electron acceptor than electron donor.
\r\n\r\nTo investigate the role of small molecules in biofilm community organization, the degradation of acyl-homoserine lactone (AHL), was studied. This molecule is an important part of the quorum sensing signaling network in P. aeruginosa, where the bacteria both produce and sense this molecule. When bacteria sense a specific concentration of the AHL, they are induced to form a biofilm or initiate a community wide response. To determine what role AHL degradation has on the community response, a mutant that constitutively degrades the compound was characterized and expression profiles for degradation were compared between this strain and wild type communities. Genes for AHL degradation were expressed in the middle of biofilm colonies suggesting that degradation may be an important part of the community response network. It was also shown that AHLs can be used as a substrate for growth, so nutrient-limited cells might also be able to use AHLs to generate energy.
\r\n\r\nFinally, to investigate whether endogenously produced redox-active small molecules could potentially play a role in energy maintenance in communities, the SoxR sensing system was studied. This system is typically thought to regulate the response to superoxide radicals. In P. aeruginosa and other organisms outside the class of enterics, however, recent evidence suggested that they may instead play a role in the sensing of redox-active small molecules produced under conditions of low nutrients and high cell density. To determine the ubiquity of this response mechanism, bioinformatic analyses were conducted to discover SoxR binding sites across all genomes containing SoxR. Predictions for binding sites and the mechanism of regulation, redox-active molecule induction, were confirmed in the Gram-positive bacterium Streptomyces coelicolor.
\r\n\r\nThis work brings us closer to understanding how cells persist and retain the capacity to dynamically regulate their metabolism in biofilm communities. Using reporter assays and quantitative analyses, studies can be done to determine metabolic organization within communities and further investigate the role that endogenous small molecules can play in community organization.
\r\n" }, { "name": "Benjamin Schooler, Jordan", "degree": "PhD", "year": "2006", "title": "Structural Studies of Human Immunodeficiency Virus Type I by CryoElectron Tomography", "advisor": "Jensen, Grant J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06052006-124843", "creators": [ { "name": { "family": "Benjamin Schooler", "given": "Jordan" }, "id": "Benjamin-Schooler-Jordan", "orcid": "0000-0002-4328-7254", "display_name": "Benjamin Schooler, Jordan" } ], "advisors": [ { "name": { "family": "Jensen", "given": "Grant J." }, "id": "Jensen-G-J", "role": "advisor", "display_name": "Jensen, Grant J." } ], "committee": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "role": "chair", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Jensen", "given": "Grant J." }, "id": "Jensen-G-J", "role": "member", "display_name": "Jensen, Grant J." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biology" ], "doi": "10.7907/HM3M-HZ81", "abstract": "Gag, the major structural component of the type 1 human immunodeficiency virus (HIV-1), comprises the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 proteins, as well as the SP1 and SP2 spacer peptides. In the immature HIV-1 virion, the domains of Gag are arranged radially with the amino-terminus of MA at the membrane. Mature viral particles are formed when Gag is proteolytically cleaved, allowing CA to reassemble into the viral core, which contains NC bound to genomic RNA. While the structures of nearly every HIV-1 protein are known in atomic detail from X-ray crystallography and NMR spectroscopy, many questions remain about the intermolecular interactions in both the immature and mature particles.
\r\n\r\nWe have obtained three-dimensional structures of individual immature and mature HIV-1 virus-like particles by cryoelectron tomography. Reconstructions of the mature particles revealed diverse core morphologies with a preference for conical shapes consistent with 5,7 fullerene cones. Uniform positioning of the wide end of the cores and an internal density likely to be the NC/RNA complex were also observed, as were multiple and nested viral cores. Our results support the fullerene cone model for the core structure and suggest that specific interactions may occur between the CA, MA, and NC layers in the mature virion. These experiments also aided the characterization of a new cryostage allowing routine collection of dual-axis tomograms.
\r\n\r\nTomograms of the immature virions revealed patches of hexagonally ordered Gag molecules interspersed with regions of disordered or absent Gag. We developed novel tools for analysis of locally ordered lattices embedded in curved structures, including a method for locating and averaging the Gag unit cell in situ. The unit cell average revealed that the CA domains and the SP1 spacer peptides were organized in a hexagonal lattice with ring-to-ring spacing of 8 nm in the CA layer and 7.5 nm in SP1. No regular lattice was found in the MA or NC layers. Based on the averaged Gag unit cell, we proposed a pseudoatomic model for the CA and SP1 domains in the immature virion.
\r\n" }, { "name": "Bingol, Baris", "degree": "PhD", "year": "2006", "title": "Ubiquitin-Proteasome System at the Synapse", "advisor": "Schuman, Erin Margaret", "url": "https://resolver.caltech.edu/CaltechETD:etd-05272006-184911", "creators": [ { "name": { "family": "Bingol", "given": "Baris" }, "id": "Bingol-Baris", "orcid": "0000-0002-8225-4367", "display_name": "Bingol, Baris" } ], "advisors": [ { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "advisor", "display_name": "Schuman, Erin Margaret" } ], "committee": [ { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "chair", "display_name": "Zinn, Kai George" }, { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "member", "display_name": "Schuman, Erin Margaret" }, { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "member", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "orcid": "0000-0002-3671-9354", "role": "member", "display_name": "Deshaies, Raymond Joseph" } ], "option_major": [ "biology" ], "doi": "10.7907/qyq5-k147", "abstract": "Each neuron in the mammalian central nervous system makes up to ten thousand synaptic connections with other neurons yet is able to regulate the strength of individual connections locally. Synaptic enhancement or depression induced at one location on the dendritic arbor does not spread through out the entire neuron. This means neurons must be able to regulate the complement and concentration of the synaptic proteins locally, near synapses. The local concentration of synaptic proteins is influenced by many processes, including protein trafficking, buffering and sequestration, and most directly by protein synthesis and degradation. In recent years, it has been shown that neurons can synthesize proteins locally in their dendrites. These studies have suggested that any cellular process that regulates protein availability could be of importance in regulating synaptic function and plasticity. Indeed, the evidence for the contribution of local protein degradation to the regulation of synaptic function and plasticity has started to emerge in recent years.
\r\n\r\nHere, we show that synapses have the machinery required to degrade proteins and local protein degradation occurs in the dendrites. Furthermore, we demonstrate the requirement for protein degradation for one of the main cellular correlates of synaptic plasticity, namely the trafficking of glutamate receptors. In turn, we demonstrate how neuronal activity regulates protein degradation at synapses, specifically by mobilizing the enzymatic machinery for protein degradation. These data show that the interplay between protein degradation and synaptic activity functions to sculpt the protein composition of the synapses.
" }, { "name": "Broome, Bede Michael", "degree": "PhD", "year": "2006", "title": "Population Coding and Reconstruction of Complex Stimuli in the Locust Olfactory System", "advisor": "Laurent, Gilles J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-03142006-171138", "creators": [ { "name": { "family": "Broome", "given": "Bede Michael" }, "id": "Broome-Bede-Michael", "display_name": "Broome, Bede Michael" } ], "advisors": [ { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "advisor", "display_name": "Laurent, Gilles J." } ], "committee": [ { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "chair", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "member", "display_name": "Schuman, Erin Margaret" }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "member", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Dickinson", "given": "Michael H." }, "id": "Dickinson-M-H", "orcid": "0000-0002-8587-9936", "role": "member", "display_name": "Dickinson, Michael H." }, { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "orcid": "0000-0001-8837-678X", "role": "member", "display_name": "Siapas, Athanassios G." } ], "option_major": [ "biology" ], "doi": "10.7907/MR29-EA69", "abstract": "Odors evoke complex sequences of activity in antennal lobe projection neurons (PNs), the insect analogs of mitral/tufted cells. These PN activity patterns evolve over hundreds of milliseconds, are consistent across trials, and contain information about odor identity and concentration. However, in natural settings animals must often identify odor blends or multiple odorants that occur in short temporal succession. We explored the effects of such stimulus history on single cell PN activity, PN ensemble responses, and downstream Kenyon cell (KC) responses by performing two experiments with novel stimulus paradigms. In the first experiment two different odors with variable intervening delays were presented while recording from PN and KC ensembles in the locust. We found that PN ensemble representations rapidly tracked odor stimulus changes and, in conditions of temporary odor overlap, often corresponded to the representation of neither odor alone nor their binary mixture. KC responses tracked the specificity of the instantaneous state of the PN representation: for example, for stimulus conditions that lead to unique and unpredictable PN population activity, we could find KCs with correspondingly unique spiking. These results support the hypotheses that PN population dynamics are history dependent and that PN output is read broadly in space (over the PN population) and piecewise in time by its target population.
\r\n\r\nIn the second experiment two different odors were presented either as single pulses or complex M-sequence odor stimuli while we recorded PN activity and the time-varying stimulus using a specially adapted electronic sensor. Our results describe the numerous similarities between the processing of stimuli with diverse frequencies and highlight several important differences. We found that PN response types are varied, low dimensional, and that individual PNs do not fall into stable functional classes. Also, PN ensembles occupy different regions of high dimensional coding space and follow trajectories that depend on stimulus history. Finally, an observer\u2019s ability to reconstruct either the concentration or identity of an odor improves with PN number. These results add support to a growing body of evidence that Kenyon cells, the targets of PNs in the mushroom bodies, carry out a pattern classification on PN activity vectors sampled over large sets of PNs.
" }, { "name": "Carter, Ronald McKell", "degree": "PhD", "year": "2006", "title": "Explicit and Implicit Processes in Human Aversive Conditioning", "advisor": "Koch, Christof", "url": "https://resolver.caltech.edu/CaltechETD:etd-06022006-124040", "creators": [ { "name": { "family": "Carter", "given": "Ronald McKell" }, "id": "Carter-Ronald-McKell", "orcid": "0000-0003-2235-6529", "display_name": "Carter, Ronald McKell" } ], "advisors": [ { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "advisor", "display_name": "Koch, Christof" } ], "committee": [ { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "chair", "display_name": "Koch, Christof" }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "O'Doherty", "given": "John P." }, "id": "O'Doherty-J-P", "orcid": "0000-0003-0016-3531", "role": "member", "display_name": "O'Doherty, John P." }, { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "member", "display_name": "Adolphs, Ralph" }, { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "orcid": "0000-0002-1290-5232", "role": "member", "display_name": "Shimojo, Shinsuke" } ], "option_major": [ "biology" ], "doi": "10.7907/CS60-1H91", "abstract": "The ability to adapt to a changing environment is central to an organism\u2019s success. The process of associating two stimuli (as in associative conditioning) requires very little in the way of neural machinery. In fact, organisms with only a few hundred neurons show conditioning that is specific to an associated cue. This type of learning is commonly referred to as implicit learning. The learning can be performed in the absence of the subject\u2019s ability to describe it. One example of learning that is thought to be implicit is delay conditioning. Delay conditioning consists of a single cue (a tone, for example) that starts before, and then overlaps with, an outcome (like a pain stimulus).
\r\n\r\nIn addition to associating sensory cues, humans routinely link abstract concepts with an outcome. This more complex learning is often described as explicit since subjects are able to describe the link between the stimulus and outcome. An example of conditioning that requires this type of knowledge is trace conditioning. Trace conditioning includes a separation of a few seconds between the cue and outcome. Explicit learning is often proposed to involve a separate system, but the degree of separation between implicit associations and explicit learning is still debated.
\r\n\r\nWe describe aversive conditioning experiments in human subjects used to study the degree of interaction that takes place between explicit and implicit systems. We do this in three ways. First, if a higher order task (in this case a working memory task) is performed during conditioning, it reduces not only explicit learning but also implicit learning. Second, we describe the area of the brain involved in explicit learning during conditioning and confirm that it is active during both trace and delay conditioning. Third, using functional magnetic resonance imaging (fMRI), we describe hemodynamic activity changes in perceptual areas of the brain that occur during delay conditioning and persist after the learned association has faded.
\r\n\r\nFrom these studies, we conclude that there is a strong interaction between explicit and implicit learning systems, with one often directly changing the function of the other.
" }, { "name": "Choi, Eun Jung", "degree": "PhD", "year": "2006", "title": "Development and Applications of Computational Protein Design", "advisor": "Mayo, Stephen L.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06012006-103350", "creators": [ { "name": { "family": "Choi", "given": "Eun Jung" }, "id": "Choi-Eun-Jung", "display_name": "Choi, Eun Jung" } ], "advisors": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "advisor", "display_name": "Mayo, Stephen L." } ], "committee": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "chair", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "member", "display_name": "Chan, David C." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Goddard", "given": "William A., III" }, "id": "Goddard-W-A-III", "orcid": "0000-0003-0097-5716", "role": "member", "display_name": "Goddard, William A., III" } ], "option_major": [ "biology" ], "doi": "10.7907/ZNED-BV38", "abstract": "Success in computational design of proteins requires a good understanding of the physical properties that determine the protein structure. However, computational protein design also provides us the opportunities to test and improve our current knowledge of protein structure. In our lab, the \"protein design cycle\" is used to improve the energy function and increase our knowledge of protein chemistry. This strategy utilizes a cyclic protocol where first, a modification that is predicted to improve the result is applied to the energy function. Next, proteins are simulated using this modified function. These molecules are then synthesized and analyzed to see if our simulation results correlate with the physical properties of the molecules. The new knowledge from the analysis is incorporated into the next design in the form of a new modification and the whole cycle starts again. Using this technique, an energy function highly successful in designing protein stability has been acquired in our lab.
\r\n\r\nA similar approach was used to determine whether the introduction of a backbone entropy term allows us to incorporate proline into the pool of amino acids we consider in designs. Using ORBIT, several proline mutants of protein G were simulated and synthesized. The correlation between the experimental results and computational results was analyzed. From this analysis, we learned that the entropic benefit of a proline mutation is usually small compared to the enthalpic disadvantages. However, including a backbone entropy term did increase the correlation between the rank order of the experimental stabilities and computational energies.
\r\n\r\nThe ultimate test of our protein design protocol is its application to biological systems of interest. We have applied ORBIT to three different design problems, increasing the affinity of peptide-protein interaction of TRAF6, the elimination of disulfide bonds for biosensor design and enhancement of solubility and stability of an anti-htt antibody fragment. The results provided here show that protein design protocol is a fast and efficient way to manipulate and study biological systems.
" }, { "name": "Cope, Gregory Allan", "degree": "PhD", "year": "2006", "title": "Regulation of SCF Ubiquitin Ligases by Jab1/Csn5 and the Cop9 Signalosome", "advisor": "Deshaies, Raymond Joseph", "url": "https://resolver.caltech.edu/CaltechETD:etd-07222005-141530", "creators": [ { "name": { "family": "Cope", "given": "Gregory Allan" }, "id": "Cope-Gregory-Allan", "display_name": "Cope, Gregory Allan" } ], "advisors": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "advisor", "display_name": "Deshaies, Raymond Joseph" } ], "committee": [ { "name": { "family": "Varshavsky", "given": "Alexander J." }, "id": "Varshavsky-A-J", "role": "chair", "display_name": "Varshavsky, Alexander J." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "member", "display_name": "Chan, David C." }, { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "role": "member", "display_name": "Campbell, Judith L." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "member", "display_name": "Deshaies, Raymond Joseph" } ], "option_major": [ "biochem" ], "doi": "10.7907/43JP-NQ47", "abstract": "SCF ubiquitin ligases regulate the ubiquitin-dependent proteolysis of a myriad of substrate proteins, including p27, Cyclin E, and IkBa. To further gain insight into SCF regulation and function, we purified SCF from mammalian cells and found the Cop9 Signalosome associated with SCF. Interestingly, deletion of the CSN in S. pombe resulted in the hypermodification of Cul1 with Nedd8 in vivo. Furthermore, we found that the CSN can promote the removal of Nedd8 from Cul1 in vitro, suggesting CSN regulates SCF through deneddylation. To investigate the basis of CSN-dependent deneddylation activity, we analyzed the CSN and the 26S proteasome for conserved sequences that could be representative of a catalytic motif. We identified the JAMM motif in Csn5 and Rpn11 of the proteasome. Mutations in JAMM eliminated CSN-dependent deneddylating activity. Moreover, mutations in JAMM reduce the restrictive temperature of several SCF temperature sensitive mutants, suggesting that CSN acts positively on SCF activity. Finally, a JAMM mutant failed to rescue Csn5D defects in drosophila. To investigate the biological effect due to loss of deneddylating activity, we constructed a stable cell line that expresses an inducible siRNA sequence toward CSN5. We found that knock-down of Csn5 results in a dramatic decrease in F-box protein levels. Moreover, this loss correlates with F-box protein substrate accumulation and hyperneddylation of Cul1. We propose an autocatalytic mechanism for the turnover of F-box proteins that is dependent upon the deneddylating activity of CSN." }, { "name": "Graumann, Johannes", "degree": "PhD", "year": "2006", "title": "Implementation of Multidimensional Protein Identification Technology and its Application to the Characterization of Protein Complexes in Bakers Yeast", "advisor": "Deshaies, Raymond Joseph", "url": "https://resolver.caltech.edu/CaltechETD:etd-05262006-095948", "creators": [ { "name": { "family": "Graumann", "given": "Johannes" }, "id": "Graumann-Johannes", "orcid": "0000-0002-3015-5850", "display_name": "Graumann, Johannes" } ], "advisors": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "advisor", "display_name": "Deshaies, Raymond Joseph" } ], "committee": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "role": "chair", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "member", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "role": "member", "display_name": "Dunphy, William G." } ], "option_major": [ "biology" ], "doi": "10.7907/J3V1-QQ44", "abstract": "The analysis of complex polypeptide mixtures poses a central and ubiquitous problem to biochemistry, molecular and cellular biology. Historically the problem has been approached by means of gel electrophoretic separation, coupled to immune-chemistry or Edman degradation (Edman 1949) based identification of separated components. These approaches as well as those based on liquid chromatography are hampered by a central issue: the wide spectrum of polypeptide characteristics that renders their separation difficult. A recent strategy termed multidimensional protein identification technology (MudPIT) tackles this problem by capillary chromatographic separation of not the complete polypeptides, but rather peptides yielded by them through proteolytic digest and analyzing them in-line using ion trap mass spectrometry (Link et al. 1999; Washburn et al. 2001; Wolters et al. 2001).
\r\n\r\nThis work describes the implementation of MudPIT outside of the analytical chemistry environment of its inception. Robustness and generalizability of the technique are tested by analysis of polypeptide complexes copurifyed with 25 selected gene products from Saccharomyces cerevisiae (Graumann et al. 2004). The pilot study reveals MudPIT to be mature enough for use outside of specialized environments and, by yielding with Rtt102p a novel component of the Swi/Snf and RSC chromatin remodelling complexes, to have potential for delivering new insights even into extensively studied systems.
\r\n\r\nSubsequent application of MudPIT to the characterization of components of the ubiquitin-proteasome system (Verma et al. 2004; Mayor et al. 2005) and mitochondrial fission (Griffin et al. 2005) in S. cerevisiae further emphasize its potential to contribute to biochemical research.
" }, { "name": "Green, Harry Miguel", "degree": "PhD", "year": "2006", "title": "Novel Methods for Studying Ras/Erk MAP Kinase Signaling in Developing T Cells", "advisor": "Alberola-Ila, Jose", "url": "https://resolver.caltech.edu/CaltechETD:etd-10242005-165226", "creators": [ { "name": { "family": "Green", "given": "Harry Miguel" }, "id": "Green-Harry-Miguel", "display_name": "Green, Harry Miguel" } ], "advisors": [ { "name": { "family": "Alberola-Ila", "given": "Jose" }, "id": "Alberola-Ila-J", "role": "advisor", "display_name": "Alberola-Ila, Jose" } ], "committee": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "role": "chair", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Alberola-Ila", "given": "Jose" }, "id": "Alberola-Ila-J", "role": "member", "display_name": "Alberola-Ila, Jose" }, { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "role": "member", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biology" ], "doi": "10.7907/8wa9-t690", "abstract": "The Ras/Erk MAPK pathway has been shown to be important in multiple developmental contexts. The development of T cells in the thymus is one such developmental system. Thymocytes undergo positive and negative selection, processes by which they are \"chosen\" for their ability to recognize MHC molecules loaded with peptide on the surface of cells, but only to react when the peptide is foreign. The Ras/Erk cascade has been shown to be indispensable during the onset of positive selection, but the mechanism of Erk signaling in this process is unknown. In addition, it is unclear if the Ras/Erk cascade is involved in the differentiation phase of positive selection called CD4/CD8 lineage determination, where thymocytes either become CD4+ or CD8+ T cells. Furthermore, Erk signaling has been shown to be activated during negative selection, but seems dispensable. In this thesis, we describe novel methods for analyzing Erk signaling by applying new technologies to gain a different perspective on Erk signaling in thymocytes during selection. To this end, we have utilized a technique of intracellular staining to obtain data for single-cell Erk activation in the context of a population of fixed thymocytes. We also pursued the development of FRET-based, genetically-encoded intracellular sensors of Erk activity that could be applied to the analysis of Erk signaling in live thymocytes in vivo. To examine the involvement of Ras/Erk signaling during CD4/CD8 lineage determination, we applied a recently described method of lentiviral transgenesis to examine dose-dependent effects of a dominant negative form of Mek, the Erk MAPK kinase, in a single mouse generation. These studies have yielded insights into Erk signaling events and advanced the development of novel techniques to examine signaling during thymocyte selection." }, { "name": "Griffin, Erik Edmund", "degree": "PhD", "year": "2006", "title": "Mechanisms of Mitochondrial Fusion and Fission", "advisor": "Chan, David C.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05242006-134647", "creators": [ { "name": { "family": "Griffin", "given": "Erik Edmund" }, "id": "Griffin-Erik-Edmund", "orcid": "0000-0001-9958-2466", "display_name": "Griffin, Erik Edmund" } ], "advisors": [ { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "advisor", "display_name": "Chan, David C." } ], "committee": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "chair", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "member", "display_name": "Chan, David C." }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "role": "member", "display_name": "Bjorkman, Pamela J." } ], "option_major": [ "biology" ], "doi": "10.7907/902F-8M09", "abstract": "We have studied the mechanisms of mitochondrial fusion and fission in S. cerevisiae. Using a proteomics-based approach, we have identified the WD domain protein Caf4p as a Fis1p binding partner that, along with its paralog Mdv1p, functions as a molecular adaptor between Fis1p and Dnm1p. This work defines a role for Mdv1p and Caf4p in the recruitment of Dnm1p to mitochondrial fission complexes. In a separate study, we focus on the role of Fzo1p during mitochondrial fusion. Fzo1p and its mammalian homologs, Mfn1 and Mfn2, are conserved transmembrane GTPases that are required for mitochondrial fusion. A structure/function analysis has established an essential role for three Fzo1p heptad repeat regions during mitochondrial fusion. Furthermore, we show that Fzo1p functions as an oligomer and forms critical interactions between the HRN/GTPase and HR1/HR2 regions. Finally, we have identified Om14p as a novel regulator of mitochondrial morphology. Om14p interacts with Fzo1p and Ugo1p and may be the first inhibitor of mitochondrial fusio" }, { "name": "Hemmati, Houman David", "degree": "PhD", "year": "2006", "title": "Neural Stem and Progenitor Cells in Cancer and Development", "advisor": "Bronner, Marianne E.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05232006-140457", "creators": [ { "name": { "family": "Hemmati", "given": "Houman David" }, "id": "Hemmati-Houman-David", "display_name": "Hemmati, Houman David" } ], "advisors": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "advisor", "display_name": "Bronner, Marianne E." } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "chair", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Kornblum", "given": "Harley" }, "id": "Kornblum-H", "role": "member", "display_name": "Kornblum, Harley" }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biology" ], "doi": "10.7907/46NX-E635", "abstract": "Stem cells are unique cells that possess the capacities to both self-renew and give rise to multiple differentiated progeny. There exist two major types of stem cells that help to create the nervous system: CNS stem cells which produce the neurons and glia of the central nervous system and neural crest cells which produce not only the neurons and glia of the peripheral nervous system, but also structures such as the craniofacial skeleton, cardiac outflow tracts, skin pigment cells, and sympathoadrenal cells. The mechanisms of self-renewal, migration, and differentiation of these two stem cell types have been studied in great detail. Yet despite such insight, much remains to be known about key aspects of neural stem cell development. First, it has long been thought that there might be a lineal relationship between CNS stem cells in human embryos or adults and primary brain tumors, particularly those malignancies occurring in children. To earn better insight into this possibility, I examined fresh pediatric brain tumors and found that they contained a subpopulation of cells with characteristics of neural stem cells that, at a clonal level, could recapitulate properties of the parental tumor. These tumor-derived progenitors shared genetic similarities with normal neural stem cells and could migrate and proliferate in vivo. Second, I have studied whether the late-migrating wave of neural crest cells and their derivatives originates from stem or progenitor cells resident in the embryonic spinal cord by culturing quail neural tube cells as neurospheres. I have found that these cells have the potential to generate melanocytes and possibly other neural crest derivatives both in vivo and in vitro after weeks in culture, suggesting that neural crest or melanocytic progenitor cells in the neural tubes of older embryos might contribute to the late-migrating neural crest populations. Taken together, my results in both model systems suggest that neural stem or progenitor cells that persist in the animal beyond early embryonic development play significant roles at later points in development and life, particularly in the continued development of the peripheral nervous system and the development of malignancies of the central nervous system." }, { "name": "Kulkarni, Rajan P.", "degree": "PhD", "year": "2006", "title": "Mechanics of the Cytoskeleton: Examining the Dynamics of Cytoplasmic Transport through Fluorescence Microscopy", "advisor": "Fraser, Scott E.", "url": "https://resolver.caltech.edu/CaltechETD:etd-03112006-214258", "creators": [ { "name": { "family": "Kulkarni", "given": "Rajan P." }, "id": "Kulkarni-Rajan-P", "display_name": "Kulkarni, Rajan P." } ], "advisors": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "advisor", "display_name": "Fraser, Scott E." } ], "committee": [ { "name": { "family": "Davis", "given": "Mark E." }, "id": "Davis-M-E", "role": "chair", "display_name": "Davis, Mark E." }, { "name": { "family": "Gray", "given": "Harry B." }, "id": "Gray-H-B", "role": "member", "display_name": "Gray, Harry B." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biochem" ], "doi": "10.7907/85ea-ed40", "abstract": "The cellular cytoplasmic space contains many different molecules and complexes confined within a small volume. Understanding how objects are transported in this crowded space is important for many potential applications. In this work, we examined various aspects of cytoskeletal mechanics, including microtubule-mediated and diffusive transport using advanced fluorescence microscopy techniques. Spatio-temporal image correlation spectroscopy (ICS) was employed to first examine microtubule-mediated transport of non-viral polyplexes within endosomes through the cytoplasm. ICS analysis of these polyplex-loaded endosomes revealed that they utilized microtubule motors for intracellular trafficking and exhibited different transport behaviors for short (<10 seconds) versus long (~60 seconds) correlation times. These results indicated that, while motor biases may be present for short periods of time, resulting in a net directional velocity, the overall long term motion of the polyplexes is best described as a random walk-like process.
\r\n\r\nMultiple particle tracking (MPT) was next used to independently confirm these results. The labeled endosomes demonstrated enhanced diffusion at short times (t < 7 seconds), with their mean square displacement (MSD) scaling as t1.25. For longer time intervals, their MSD scaled as t0.7. This crossover from an enhanced diffusion to a subdiffusive regime is explained by considering the action of motor proteins and the thermal bending modes of the microtubule network.
\r\n\r\nWe then developed an assay to examine the pH characteristics of the polyplex-loaded endosomes as a function of time and distance from entry. Certain nonviral vectors, including poly-L-lysine (PLL) and cyclodextrin-containing polymers (CDP), cannot buffer the endocytic vesicles, while polyethyleneimine (PEI), CD-PEI, and CDP-imidazole can. When combined with cell uptake and luciferase expression data, we found that there was no correlation between buffering capacity and gene expression.
\r\n\r\nFinally, we developed multi-photon fluorescence recovery after photobleaching (FRAP) to determine diffusion rates in developing zebrafish growth cones in vivo. Leader growth cones had consistently longer recovery times compared to followers. This difference was abolished by perturbing the actin cytoskeleton, thus indicating that diffusion is important during axon navigation. Collectively, these findings reveal important biophysical aspects of intracellular transport that impact diverse physiological processes.
" }, { "name": "Lacenere, Christopher J.", "degree": "PhD", "year": "2006", "title": "Advances in Single Molecule Nucleic Acid Sequencing", "advisor": "Quake, Stephen R.", "url": "https://resolver.caltech.edu/CaltechETD:etd-09302006-140602", "creators": [ { "name": { "family": "Lacenere", "given": "Christopher J." }, "id": "Lacenere-Christopher-J", "display_name": "Lacenere, Christopher J." } ], "advisors": [ { "name": { "family": "Quake", "given": "Stephen R." }, "id": "Quake-S-R", "role": "advisor", "display_name": "Quake, Stephen R." } ], "committee": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "chair", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Stoltz", "given": "Brian M." }, "id": "Stoltz-B-M", "role": "member", "display_name": "Stoltz, Brian M." }, { "name": { "family": "Elowitz", "given": "Michael B." }, "id": "Elowitz-M-B", "role": "member", "display_name": "Elowitz, Michael B." }, { "name": { "family": "Quake", "given": "Stephen R." }, "id": "Quake-S-R", "role": "member", "display_name": "Quake, Stephen R." } ], "option_major": [ "biology" ], "doi": "10.7907/CA4B-S389", "abstract": "The ability to quickly and accurately obtain sequence information from single molecules of DNA and RNA has far-reaching implications for our understanding of biology. In the work presented here, we have made several advances in the area of single-molecule DNA and RNA sequencing. Specifically, in attempting to increase the read length of DNA polymerase, we have assayed several custom synthesized fluorescent nucleotides containing longer dye\u2013base linkers. We have validated the efficacy of these nucleotides at both bulk and single-molecule levels. Furthermore, we have screened several commercially available DNA polymerases for their ability to incorporate these nucleotides. We also show that reverse transcriptase is able to synthesize a complimentary DNA strand of 28 bases in length from an RNA template, using solely fluorescently labeled nucleotides. Additionally, we show that reverse transcriptase is able to incorporate a fluorescently labeled nucleotide into an RNA template at the single-molecule level. Finally, we demonstrate automated reagent exchange for our single-molecule sequencing system." }, { "name": "Lee, Brian", "degree": "PhD", "year": "2006", "title": "Neural Computation of Self-Motion from Optic Flow in Primate Visual Cortex", "advisor": "Andersen, Richard A.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05242006-221959", "creators": [ { "name": { "family": "Lee", "given": "Brian" }, "id": "Lee-Brian", "display_name": "Lee, Brian" } ], "advisors": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "display_name": "Andersen, Richard A." } ], "committee": [ { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "orcid": "0000-0002-1290-5232", "role": "chair", "display_name": "Shimojo, Shinsuke" }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "member", "display_name": "Koch, Christof" }, { "name": { "family": "Burdick", "given": "Joel Wakeman" }, "id": "Burdick-J-W", "orcid": "0000-0002-3091-540X", "role": "member", "display_name": "Burdick, Joel Wakeman" }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "member", "display_name": "Andersen, Richard A." } ], "option_major": [ "biology" ], "doi": "10.7907/ahre-qy74", "abstract": "Area MSTd is involved in the computation of heading direction from the focus of expansion (FOE) of the visual image. Our laboratory previously found that MSTd neurons adjust their focus tuning curves to compensate for shifts in the FOE produced by eye rotation (Bradley et al., 1996) as well as for changes in pursuit speed (Shenoy et al., 2002). The translation speed of an observer also affects the shift of the FOE. To investigate whether MSTd neurons can adjust their focus tuning curves to compensate for varying translation speeds, we recorded extracellular responses from 93 focus-tuned MSTd neurons in two rhesus monkeys (Macaca mulatta) performing pursuit eye movements across displays of varying translation speeds. We found that MSTd neurons had larger shifts in their tuning curves for slow translation speeds and smaller shifts for fast translation speeds. These shifts aligned the focus tuning curves with the true heading direction and not with the retinal position of the FOE. These results indicate that retinal cues related both to translation speed and extraretinal signals from pursuit eye movements are used by MSTd neurons to compute heading direction.
\r\n\r\nAlthough there is much evidence that MSTd neurons are involved in heading computation, it was not known in which coordinate frame the tuning curves were represented. We performed a second set of experiments to determine whether focus tuning curves in MSTd were represented in eye, head, body, or world coordinates. The coordinate frame was determined while the eyes were stationary (fixed gaze, simulated pursuit condition) and while the eyes were moving (real pursuit condition). We recorded extracellular responses from 80 MSTd neurons and found that the FOE tuning curves of the overwhelming majority of neurons were aligned in an eye-centered coordinate frame as opposed to head, body, or world-centered coordinates (fixed gaze: 77/80 (96%); real pursuit: 77/80 (96%); simulated pursuit 74/80 (93%); t-test, p<0.05). We also found that area MSTd demonstrated significant eye position gain modulation much like its posterior parietal neighbors. This gain modulation may be a method of transforming eye coordinates into other coordinate frames at later stations of the nervous system.
" }, { "name": "Livi, Carolina Becker", "degree": "PhD", "year": "2006", "title": "Spblimp1/krox: A Transcriptional Regulator with a Central Role in Endomesoderm Specification in Sea Urchin Embryos", "advisor": "Davidson, Eric H.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06052006-112833", "creators": [ { "name": { "family": "Livi", "given": "Carolina Becker" }, "id": "Livi-Carolina-Becker", "orcid": "0000-0001-8389-2830", "display_name": "Livi, Carolina Becker" } ], "advisors": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "advisor", "display_name": "Davidson, Eric H." } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biology" ], "doi": "10.7907/DZE5-NY77", "abstract": "During cleavage stages Spblimp1/krox is expressed in the large micromeres and veg2 descendents. During later blastula stages, it is expressed in the endoderm precursors of the veg1 ring of cells distal to the vegetal pole of the blastula. Later, its expression is restricted to the blastopore region and the posterior of the invaginating archenteron, and finally to the midgut and hindgut of the pluteus larva. The expression of Spblimp1/krox is thus dynamic, and involves several distinct spatial territories. A GFP recombinant BAC was created to distinguish the expression pattern of the early form from that of the late form. The protein coding sequence of GFP was substituted for that of the second exon, 1b. This construct closely mimics Spblimp1/krox expression during early stages of sea urchin development. Using anti-sense morpholino (MASO) knockdown perturbations we analyzed the downstream targets of Spblimp1/krox. We confirmed previously published data that Spblimp1/krox autoregulates its own expression, and we found that it represses itself. This negative autoregulation is restricted to the mesodermal veg2 territory during the blastula stage as shown by WMISH analysis of MASO injected embryos. Blimp1/Krox inputs expected from the perturbation analysis into other genes have been incorporated in our gene regulatory network.
\r\n\r\nSpblimp1/krox has two isoforms that are alternatively transcribed, 1a and 1b. Spblimp1/krox1a is expressed starting at gastrulation. Phylogenetic footprinting analysis reveals several conserved sequence patches several hundred bp long, in comparisons between Strongylocentrotus purpuratus and Lytechinus variegatus of the genomic region surrounding the blimp1/krox locus. When included in an injected expression construct one of these conserved patches recapitulates the expression of the 1a isoform during embryogenesis. This regulatory module of the Spblimp1/krox gene, which lies immediately upstream of the transcription initiation site for Spblimp1/krox1a, directs expression with great accuracy to the midgut and hindgut of the postgastrular embryo. Work on the regulation of the early 1b isoform will be reported elsewhere.
" }, { "name": "Mao, Jessica", "degree": "PhD", "year": "2006", "title": "Applications of Computational Protein Design", "advisor": "Mayo, Stephen L.", "url": "https://resolver.caltech.edu/CaltechETD:etd-02102006-100744", "creators": [ { "name": { "family": "Mao", "given": "Jessica" }, "id": "Mao-Jessica", "display_name": "Mao, Jessica" } ], "advisors": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "advisor", "display_name": "Mayo, Stephen L." } ], "committee": [ { "name": { "family": "Roberts", "given": "Richard W." }, "id": "Roberts-R-W", "orcid": "0000-0002-8587-5097", "role": "chair", "display_name": "Roberts, Richard W." }, { "name": { "family": "Parker", "given": "Carl Stevens" }, "id": "Parker-C-S", "orcid": "0000-0001-9795-4211", "role": "member", "display_name": "Parker, Carl Stevens" }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." } ], "option_major": [ "biochem" ], "doi": "10.7907/13A4-Z652", "abstract": "Computational protein design determines the amino acid sequence(s) that will adopt a desired fold. It allows the sampling of a large sequence space in a short amount of time compared to experimental methods. Computational protein design tests our understanding of the physical basis of a protein\u2019s structure and function, and over the past decade, has proven to be an effective tool.
\r\n\r\nWe report the diverse applications of computational protein design with ORBIT (Optimization of Rotamers by Iterative Techniques). We successfully utilized ORBIT to construct a reagentless biosensor for nonpolar ligands on the maize non-specific lipid transfer protein, by first removing native disulfide bridges. We identified an important residue position capable of modulating the agonist specificity of the mouse muscle nicotinic acetylcholine receptor (nAChR) for its agonists: acetylcholine, nicotine, and epibatidine. Our efforts on enzyme design produced a lysozyme mutant with ester hydrolysis activity, while progress was made toward the design of a novel aldolase.
\r\n\r\nComputational protein design has proven to be a powerful tool for the development of novel and improved proteins. As we gain a better understanding of proteins and their functions, protein design will find many more exciting applications.
\r\n" }, { "name": "Marcus, Joshua Scott", "degree": "PhD", "year": "2006", "title": "Single Mammalian Cell Gene Expression Analysis Using Microfluidics", "advisor": "Quake, Stephen R.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06282006-103230", "creators": [ { "name": { "family": "Marcus", "given": "Joshua Scott" }, "id": "Marcus-Joshua-Scott", "display_name": "Marcus, Joshua Scott" } ], "advisors": [ { "name": { "family": "Quake", "given": "Stephen R." }, "id": "Quake-S-R", "orcid": "0000-0002-1613-0809", "role": "advisor", "display_name": "Quake, Stephen R." } ], "committee": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "chair", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Barton", "given": "Jacqueline K." }, "id": "Barton-J-K", "orcid": "0000-0001-9883-1600", "role": "member", "display_name": "Barton, Jacqueline K." }, { "name": { "family": "Heath", "given": "James R." }, "id": "Heath-J-R", "orcid": "0000-0001-5356-4385", "role": "member", "display_name": "Heath, James R." }, { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "orcid": "0000-0001-8291-5551", "role": "member", "display_name": "Campbell, Judith L." }, { "name": { "family": "Quake", "given": "Stephen R." }, "id": "Quake-S-R", "orcid": "0000-0002-1613-0809", "role": "member", "display_name": "Quake, Stephen R." } ], "option_major": [ "biochem" ], "doi": "10.7907/M3GA-PT31", "abstract": "Single cell gene expression studies hold great promise for deciphering the ubiquitous heterogeneity present in biological organisms. Although much progress has been made in the field, tools to study gene expression (specific and global) in single cells are generally lacking. This thesis describes the development of novel microfluidic technologies and processes capable of processing single cells to first strand cDNA in a parallel fashion, thereby filling a void in the single cell biology field. The author then utilizes the technology to probe for transcriptional noise in ubiquitous genes present in single mammalian cells. The noise measured far exceeds any measurement reported to this date, and was shown to be attenuated during the G2 stage of the cell cycle. The work presented here is first hand proof that technological innovation is a key component in undertaking novel science." }, { "name": "McGarvey, Raymond Timothy James", "degree": "PhD", "year": "2006", "title": "Ultra-Sensitive Absorption Measurements through Cavity-Enhanced Spectroscopy", "advisor": "Mabuchi, Hideo", "url": "https://resolver.caltech.edu/CaltechETD:etd-09202008-110124", "creators": [ { "name": { "family": "McGarvey", "given": "Raymond Timothy James" }, "id": "McGarvey-Raymond-Timothy-James", "display_name": "McGarvey, Raymond Timothy James" } ], "advisors": [ { "name": { "family": "Mabuchi", "given": "Hideo" }, "id": "Mabuchi-H", "role": "advisor", "display_name": "Mabuchi, Hideo" } ], "committee": [ { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "role": "chair", "display_name": "Rees, Douglas C." }, { "name": { "family": "Yang", "given": "Changhuei" }, "id": "Yang-Changhuei", "role": "member", "display_name": "Yang, Changhuei" }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Mabuchi", "given": "Hideo" }, "id": "Mabuchi-H", "role": "member", "display_name": "Mabuchi, Hideo" } ], "option_major": [ "biochem" ], "doi": "10.7907/CGYD-6J27", "abstract": "The desire to increase the sensitivity of solution-based absorption spectroscopy is motivated by the need for label-free biosensing (which provides a more authentic indication of the state of a biological system) and by the usefulness of characterizing the kinetics of biologically-relevant reactions (which may not be accurately characterizable at reagent concentrations required by standard methods. There are a number of techniques by which such increasingly sensitive measurements have been made, including cavity ringdown spectroscopy, incoherent cavity-enhanced spectroscopy, microsphere-based whispering-gallery mode sensing,and our cavity-enhanced measurements, which are the most sensitive to date and which can be conducted in real time with high bandwidth. Our current device has a demonstrated detection threshold of 1.7x 10^{-7}/sqrt{Hz} (4.36x10^{-6}cm^{-1}), which could with further technical work be improved to a shot-noise limited sensitivity of 1.93x 10^{-10}/sqrt{Hz} (1.06x10^{-8}cm^{-1}). The latter would correspond to an average of 700 strong absorbers (epsilon = 10^5 M^{-1}cm^{-1}) in the optical beam volume. The shot-noise limited detection threshold of our measurement method could potentially be improved by up to two orders of magnitude by incorporating state-of-the-art optical mirrors. With such mirrors, cavity-enhanced absorption experiments performed with gas-phase samples have previously demonstrated single molecule sensitivity. We have established that solution-based cavity-enhanced absorption measurements are more sensitive than standard single-pass measurements by the predicted enhancement factor for our present device (~ 20,000). These measurements provide the proof-of-principle for solution-based, cavity-enhanced spectroscopy and serve as the intermediate step towards the attainment of the theoretical sensitivity of this technique. We believe that this device will be of broad interest to the scientific community, because it is presently the most sensitive solution-based spectroscopic device. It can make real-time absorption measurements which would allow monitoring of the kinetics of chemical reactions in which the spectral properties of reactants change by even a small amount, and, near its theoretical limit of sensitivity (given currently available mirrors), such a device could potentially resolve single-molecule absorption events on the sub-millisecond timescale and below.\r\n" }, { "name": "Neil, Patricia Ann", "degree": "PhD", "year": "2006", "title": "Development of Audiovisual Integration in Human Infants: The Effects of Spatial and Temporal Congruency and Incongruency on Response Latencies", "advisor": "Shimojo, Shinsuke", "url": "https://resolver.caltech.edu/CaltechETD:etd-07102006-114157", "creators": [ { "name": { "family": "Neil", "given": "Patricia Ann" }, "id": "Neil-Patricia-Ann", "display_name": "Neil, Patricia Ann" } ], "advisors": [ { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "role": "advisor", "display_name": "Shimojo, Shinsuke" } ], "committee": [ { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "role": "chair", "display_name": "Adolphs, Ralph" }, { "name": { "family": "Bruck", "given": "Jehoshua" }, "id": "Bruck-J", "role": "member", "display_name": "Bruck, Jehoshua" }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "role": "member", "display_name": "Perona, Pietro" }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "role": "member", "display_name": "Andersen, Richard A." }, { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "role": "member", "display_name": "Shimojo, Shinsuke" } ], "option_major": [ "cns" ], "doi": "10.7907/XPGR-QT95", "abstract": "Every day we are inundated with a mass of sensory inputs providing a continual stream of relevant and irrelevant, redundant and conflicting, information about the external world. Mature brains are very capable in integrating this confusion of input into a unified percept, but this is a non-trivial task for infants, whose brains and sensory systems are still immature at birth and who rely on their current level of integration and interaction of these inputs in order to shape their future development. Failure in being able to properly process basic sensory interactions has been implicated in higher-level developmental problems like attentional or autistic spectrum disorders. Numerous studies have looked at how adults perceive and react to multisensory stimuli, including findings of improved response latencies and target detection for spatially and temporally congruent stimuli, but much less is known about the development of multisensory integration or how spatial or temporal disparities effect sensory interactions in young babies. We examined the role of spatial and temporal congruency and incongruency on the response latencies of infants under ten months of age orienting toward an audiovisual stimulus at +/-25 degrees and/or +/-45 degrees. In Study 1, we found the beginnings of adult-style non-linear integration for spatially and temporally congruent audiovisual targets in 8\u201310 month olds, but not in younger infants, as well as indications of a differential developmental profile for binaural versus monaural processing. In Studies 2 and 3, spatial and temporal disparities were found to significantly lengthen infants\u2019 response latencies to an audiovisual target. We also found clear indications of developmental changes for all three spatial and temporal conditions, as well as key dependencies in relative position, temporal order, and sensory dominance." }, { "name": "Slimko, Eric Michael", "degree": "PhD", "year": "2006", "title": "Selective Silencing of Vertebrate Neurons: Strategies Using Invertebrate Ligand-Gated Ion Channels", "advisor": "Lester, Henry A.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05142008-004518", "creators": [ { "name": { "family": "Slimko", "given": "Eric Michael" }, "id": "Slimko-Eric-Michael", "display_name": "Slimko, Eric Michael" } ], "advisors": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "advisor", "display_name": "Lester, Henry A." } ], "committee": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "chair", "display_name": "Lester, Henry A." }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "member", "display_name": "Koch, Christof" }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "member", "display_name": "Schuman, Erin Margaret" }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "member", "display_name": "Laurent, Gilles J." } ], "option_major": [ "cns" ], "doi": "10.7907/RYJJ-JW20", "abstract": "Selectively reducing the excitability of specific neurons will (1) allow for the creation of animal models of certain human neurological disorders and (2) provide insight into the roles of specific sets of neurons, both in the local circuit and in the behavior of the intact organism. This work focuses on a combined genetic and pharmacological approach to silence neurons electrically. We express invertebrate ivermectin (IVM)-sensitive chloride channels (Caenorhabditis elegans GluCl \u03b1 and \u03b2) in vertebrate neurons first in vitro using viral and tranfection techniques, and then finally in vivo using genetic techniques, to produce inhibition via a Cl- conductance when activated with IVM. We have considerably engineered these two genes by (1) re-coding the genes such that vertebrate-preferred codons are used throughout the sequences, (2) incorporating fluorescent tags within the proteins, and (3) finding a mutation to remove the undesirable glutamate sensitivity of the channel while retaining IVM efficacy. Expression of this new channel does not affect the normal spike activity of the target cell, yet the experimentor can effectively \u201cshut-off\u201d the cell with concentrations of as low as 5 nM IVM. Chapter 1 provides a broad overview of the many \u201cselective silencing\u201d approaches that experimenters have tried. In Chapter 2, the author describes the basic \u201cGluCl/IVM\u201d technique and initial experiments in cultured hippocampal neurons. Chapter 3 refines the technique by describing the strategy and mutation that allowed great reduction in the native glutamate response while maintaining the IVM response. Chapter 4 develops the final engineering of the channel: recoding the sequence for optimal expression and the introduction of fluorescent tags for identification. Finally, Chapters 5 and 6 discuss the successes and failures of in vivo work with what we now call the \u201cGluCl/IVM method.\u201d " }, { "name": "Watson, Karli Kiiko", "degree": "PhD", "year": "2006", "title": "The Von Economo Neurons: From Cells to Behavior", "advisor": "Allman, John Morgan", "url": "https://resolver.caltech.edu/CaltechETD:etd-05252006-224259", "creators": [ { "name": { "family": "Watson", "given": "Karli Kiiko" }, "id": "Watson-Karli-Kiiko", "orcid": "0000-0002-5513-1492", "display_name": "Watson, Karli Kiiko" } ], "advisors": [ { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "advisor", "display_name": "Allman, John Morgan" } ], "committee": [ { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "chair", "display_name": "Koch, Christof" }, { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "member", "display_name": "Schuman, Erin Margaret" }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "O'Doherty", "given": "John P." }, "id": "O'Doherty-J-P", "orcid": "0000-0003-0016-3531", "role": "member", "display_name": "O'Doherty, John P." }, { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "member", "display_name": "Adolphs, Ralph" } ], "option_major": [ "biology" ], "doi": "10.7907/FZ94-DW76", "abstract": "The von Economo neurons are one of the few known specializations to hominoid cortical microcircuitry. The recent emergence of this cell type, as well as its localization to subregions of the frontal cortex, suggest its involvement in sophisticated cognitive behaviors. Studies of this cell may thus provide insights into human uniqueness and origin and may additionally be relevant to the treatment and understanding of mental illness.
\r\n\r\nThe first section of this thesis investigates the anatomical details of these cells, including their structure and surface receptor expression. Using a Golgi preparation of a human postmortem brain, I describe the dendritic architecture of this unique population of neurons. We found that, in contrast to layer 5 pyramidal neurons, the von Economo neurons have sparse dendritic trees with symmetric apical and basal components. This confirms that the von Economo cells in both ACC and FI share the architectural characteristics of a single population, and that this population is distinct from other layer 5 neurons. I additionally used immunohistochemistry to probe the receptor expression on these cells, and found that the von Economo neurons strongly express the dopamine D3 and D5 receptors, as well as serotonin-1b and serotonin-2b receptors. Together, these results provide the first detailed anatomical description of a neuron type unique to great apes and humans.
\r\n\r\nIn the second part of this thesis, I explore whether a behavioral stimulus, humor, activates the regions in which this cell occurs. Humor is a hallmark of social discourse and usually depends on the convergence of fast, intuitive assessments with a slow \"re-interpretation\" of the humor. Because of these characteristics, we thought it likely that humor would activate FI and ACC in addition to other regions in the brain. I used event-related fMRI to differentiate brain activity induced by the hedonic similarities and cognitive differences inherent in cartoons depicting two kinds of humor: visual humor (sight gags) and language-based humor. I found that the brain networks recruited during a humorous experience did indeed include FI and ACC, and that the profile of activation differs according to the type of humor being processed.
\r\n\r\nTaken together, these projects significantly expand on our knowledge of these unusual cells, and provide a basis that allows us to hypothesize about their function. In the conclusion of this paper, we propose that the role of the von Economo neurons is to facilitate fast decision making in the context of high uncertainty, such as during social interaction.
" }, { "name": "Wu, Daw-An", "degree": "PhD", "year": "2006", "title": "How Perception Adheres Color to Objects and Surfaces: Studies Using Visual Illusions and Transcranial Magnetic Stimulation", "advisor": "Shimojo, Shinsuke", "url": "https://resolver.caltech.edu/CaltechETD:etd-09282005-121349", "creators": [ { "name": { "family": "Wu", "given": "Daw-An" }, "id": "Wu-Daw-An", "orcid": "0000-0003-4296-3369", "display_name": "Wu, Daw-An" } ], "advisors": [ { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "orcid": "0000-0002-1290-5232", "role": "advisor", "display_name": "Shimojo, Shinsuke" } ], "committee": [ { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "chair", "display_name": "Allman, John Morgan" }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "member", "display_name": "Koch, Christof" }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "orcid": "0000-0002-1290-5232", "role": "member", "display_name": "Shimojo, Shinsuke" } ], "option_major": [ "biology" ], "doi": "10.7907/QR9F-YR49", "abstract": "In early visual cortex, visual input is encoded primarily as edges. Information then flows into more specialized regions of the brain, which process visual features such as color, motion, etc. This encoding scheme poses some problems in explaining the experience of seeing. If the cortex processes various visual features separately, how do we see unified objects? What mechanisms bind the features together? If the cortex encodes the visual scene in terms of its edges, then how do we see solid surfaces? What mechanisms fill-in the map of outlines?
\r\n\r\nThis thesis investigates the problems of binding and filling-in using the techniques of visual illusion psychophysics and transcranial magnetic stimulation (TMS). We find that TMS can cause an instant replay effect, whereby recently presented visual stimuli are seen again. When TMS induces an instant replay shortly after the presentation of Cai\u2019s asynchronous binding illusion, some subjects see an image of the actual visual stimulus, undistorted. It appears that TMS can selectively activate a hidden, accurate representation of the stimulus, revealing it without the distortion caused by other processes.
\r\n\r\nWe also find a number of cases in which visual features are decomposed and/or misbound. TMS-induced instant replay can cause the color of one object to be bound to the position and orientation of another. It can also separately replay the color and orientation of a grating. In a non-TMS experiment, we create a stimulus that induces a steady-state misbinding of color and motion\u2014-a vivid, long-lasting misbinding effect ideal for neurophysiological investigation. These experiments confirm the separate encoding of visual features and the existence of an active binding mechanism.
\r\n\r\nFinally, we study filling-in by manipulating an effect distilled from the artwork of Julian Stanczak, in which color is perceived to spread discretely among segregated patches of space. We find that color-filling is dependent on perceptual surfaces, such that overlaid surfaces can support separate filling processes. It appears possible that the neural mechanisms of binding and filling-in might be intimately related, both of them highly integrated with the process of surface segregation.
" }, { "name": "Zollars, Eric Stafford", "degree": "PhD", "year": "2006", "title": "Force Field Development in Protein Design", "advisor": "Mayo, Stephen L.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06052006-155305", "creators": [ { "name": { "family": "Zollars", "given": "Eric Stafford" }, "id": "Zollars-Eric-Stafford", "orcid": "0000-0003-0017-9250", "display_name": "Zollars, Eric Stafford" } ], "advisors": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "advisor", "display_name": "Mayo, Stephen L." } ], "committee": [ { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "chair", "display_name": "Pierce, Niles A." }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." } ], "option_major": [ "biochem" ], "doi": "10.7907/55br-9a21", "abstract": "Protein design requires the rapid evaluation of very large numbers of equations during the course of a calculation. These equations must represent the important contributors to protein stability in simple and accurate terms. Some physical phenomena are relatively easy to model such as van der Waals forces. Electrostatics and solvation in a protein environment are forces that are more difficult to adequately capture. Additionally, the balance of the terms used must be determined in order to design sequences that fold to stable, specific folds.
\r\n\r\nThe electrostatic interactions within the protein and between the protein and solvent are important in both the stability and function of the protein. The effects of the protein-solvent interactions are evaluated using implicit models that consider the solvent as a bulk. These interactions are quantified using the Poisson-Boltzmann equation that must be solved using discrete numerical methods. We sought to avoid this performance hit by scaling a simpler model of electrostatics, Coulomb's law, to reproduce one aspect of the protein-solvent interaction: solvent screening. By dividing the Coulombic dielectric into two parts and scaling to correlate with the Poisson-Boltzmann results we significantly increased the strength of electrostatics in our force field that led to the design of a more stable engrailed homeodomain.
\r\n\r\nThe second part of this work describes attempts to reparameterize our protein design force field. Many protein mutants have been expressed and biophysically characterized in the literature. We sought to use the measured stabilities of protein mutants in the literature to balance the terms in the force field. While we were able to produce a force field that could reproduce experimental energies, this force field led to unsatisfactory designed sequences. To more fully satisfy the unique conditions of a protein design force field we explored other optimization techniques and found that the balance of the terms in the existing force field is nearly optimal.
" }, { "name": "Ambroggio, Xavier Ignacio", "degree": "PhD", "year": "2005", "title": "Structural and Functional Studies of Jamm Domain Proteins and Their Role in the Ubiquitin System", "advisor": "Rees, Douglas C.; Deshaies, Raymond Joseph", "url": "https://resolver.caltech.edu/CaltechETD:etd-09302004-090155", "creators": [ { "name": { "family": "Ambroggio", "given": "Xavier Ignacio" }, "id": "Ambroggio-Xavier-Ignacio", "display_name": "Ambroggio, Xavier Ignacio" } ], "advisors": [ { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "role": "advisor", "display_name": "Rees, Douglas C." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "advisor", "display_name": "Deshaies, Raymond Joseph" } ], "committee": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "role": "chair", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "member", "display_name": "Chan, David C." }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "member", "display_name": "Deshaies, Raymond Joseph" } ], "option_major": [ "biology" ], "doi": "10.7907/EVW4-CC51", "abstract": "The JAMM (JAB1/MPN/Mov34 metalloenzyme) motif is a conserved amino acid sequence, EX(n)HS/THX(7)SXXD, found in proteins from all domains of life. Eukaryotic proteins possesing a JAMM motif are responsible for the selective hydrolysis of iso-peptide linkages involving ubiquitin and ubiquitin-like proteins and often exist as subunits of large complexes. The iso-peptidase activity of JAMM proteins plays a major role in key points of regulation in the ubiquitin system. In particular, the JAMM motif of CSN5 of the COP9 signalosome is responsible for the cleavage of the ubiquitin-like Nedd8 from SCF ubiquitin ligases. A homolog of CSN5 in the lid subcomplex of the 19S proteasome regulatory particle, Rpn11, cleaves ubiquitin from proteasome substrates as they are processed by the proteasome. In order to understand the mechanism underlying iso-peptide bond hydrolysis by the JAMM motif, we have solved the crystal structure of a JAMM domain protein from Archaeoglobus fulgidus, AfJAMM. The JAMM motif forms a thermolysin-like active site on a cytidine deaminase fold. We have demonstrated through biochemical analysis of mutations in the JAMM motif of Csn5 that the mechanism of hydrolysis is similar to that of thermolysin. To achieve an integrated understanding of a JAMM domain protein within its cognate complex, we have purified and crystallized the lid subcomplex of the 19S proteasome regulatory particle for structural studies." }, { "name": "Bak-Maier, Magdalena", "degree": "PhD", "year": "2005", "title": "Commissural Axon Kinetics and the Role of Netrin in Early Brain Circuitry Development", "advisor": "Fraser, Scott E.", "url": "https://resolver.caltech.edu/CaltechETD:etd-10042004-113209", "creators": [ { "name": { "family": "Bak-Maier", "given": "Magdalena" }, "id": "Bak-Maier-Magdalena", "display_name": "Bak-Maier, Magdalena" } ], "advisors": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "advisor", "display_name": "Fraser, Scott E." } ], "committee": [ { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "chair", "display_name": "Zinn, Kai George" }, { "name": { "family": "Collazo", "given": "Andres" }, "id": "Collazo-A", "role": "member", "display_name": "Collazo, Andres" }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "neurobio" ], "doi": "10.7907/1S9H-0C66", "abstract": "As neurons begin to differentiate, they send out processes called axons to initiate the formation of functional nerve connections. A specialized structure at the end of an axon called the growth cone is believed to possess the impressive navigational and target recognition ability crucial for this process. The goal of the research presented in this thesis was to understand the cellular and molecular mechanisms that shape axon growth and guidance in vivo during early brain development using a multifaceted experimental approach.
\r\n\r\nTowards this goal we employed the simple, well-characterized neuronal scaffold of the embryonic zebrafish brain in combination with cell labeling techniques and performed studies in three specific areas: (1) the dynamic behaviors of navigating growth cones to obtain information about their cellular interactions with each other and the local environment, (2) the action of specific proteins (netrin and its receptor DCC) known to be involved in axon guidance in order to determine their function in vivo, and (3) the mobility of GFP in growth cones as a way to gain insight into the dynamics of molecular species in these structures as they actively navigate.
\r\n\r\nCritical to our studies was a stable transgenic gata2::GFP zebrafish line in which we found high level of GFP expression in early forebrain neuronal clusters allowing in vivo timelapse study of the growth cones that pioneer the postoptic commissural (POC) axon tract. Following the development of the POC also allowed us to investigate how early commissural growth cones behave at the midline.
\r\n\r\nTimelapse analysis of POC axon kinetics revealed important insight into growth cone interactions with each other and their environment and showed that these have behavioral consequences. While it was known that commissural axons slow down while crossing the midline, our data showed that this is only true for the leader axons. Follower axons do not slow down unless the leader axon is ablated. Together this analysis revealed that in addition to specific molecular cues, axon-axon interactions are important for establishing early axon tract.
\r\n\r\nThis characterization of POC axon kinetics and growth cone behavior in turn, provided us with an assay for studying the specific role of netrin, primarily a midline attractant for commissural axons in the spinal cord and its receptor, deleted in colorectal cancer (DCC). Loss- and gain-of-function experiments in combination with timelapse imaging uncovered a novel function for netrin as a positional repellent cue for POC axons.
\r\n\r\nFinally, prompted by the observed differences in leader and follower POC growth cones, we developed a new experimental approach to assay GFP mobility as a reporter for the diffusion rates of other molecular species inside growth cones in vivo. We found that diffusion rates in actively pioneering growth cones are significantly decreased compared to follower axons suggesting that diffusion rates might be linked to growth cone pathfinding.
\r\n\r\nCollectively, the findings presented in this thesis constitute a framework that allows for an integrative approach of studying growth cone navigation in vivo. Basic integrative knowledge of this sort is expected to aid the development of medical therapies related to nerve injury and repair.
\r\n" }, { "name": "Barbee, Susannah Dale", "degree": "PhD", "year": "2005", "title": "The Functions of Phosphatidylinositol 3-Kinase in T Lymphocyte Development: Roles in Positive Selection and Thymic Exit", "advisor": "Alberola-Ila, Jose", "url": "https://resolver.caltech.edu/CaltechETD:etd-10062004-073848", "creators": [ { "name": { "family": "Barbee", "given": "Susannah Dale" }, "id": "Barbee-Susannah-Dale", "display_name": "Barbee, Susannah Dale" } ], "advisors": [ { "name": { "family": "Alberola-Ila", "given": "Jose" }, "id": "Alberola-Ila-J", "role": "advisor", "display_name": "Alberola-Ila, Jose" } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "chair", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Alberola-Ila", "given": "Jose" }, "id": "Alberola-Ila-J", "role": "co-chair", "display_name": "Alberola-Ila, Jose" }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "member", "display_name": "Baltimore, David L." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/3bbr-b515", "abstract": "Phosphatidylinositol 3-kinase (PI3K) is an important regulator of cell survival, proliferation, activation, and migration in multiple organisms and cell types. We have sought to determine how PI3K may regulate T lymphocyte development, a process that entails exquisitely coordinated phases of proliferation, differentiation, and intra-organ movement. We have generated transgenic mice that express a PI3K gain-of-function mutant specifically in thymocytes. The p110ABD transgene constitutes the adaptor binding domain of the PI3K catalytic subunit and this fragment associates with adaptor subunits in vivo. p110ABD expression induces constitutive PI3K function, as assessed by the activity of the downstream effector Akt. Furthermore, p110ABD-induced PI3K function potentiates Ca ++ influx induced by sub-optimal crosslinking of the antigen receptor (TCR) on immature thymocytes. Enhancing PI3K activity in developing T cells results in the specific accumulation of late-stage, mature HSA lo CD3hi thymocytes of both lineages. The increased numbers of mature thymocytes can be partly attributed to an improvement in positive selection. This is demonstrated by the ability of p110ABD to promote efficient positive selection of transgenic AND TCR thymocytes in a background that mediates sub-optimal differentiation. The improvement in selection is not biased to the CD4 lineage, since CD4 lineage development is not specifically improved in class I-restricted transgenic TCR animals expressing p110ABD. Furthermore, the effect is specific to positive selection since immature thymocyte survival and negative selection are unaffected by p110ABD expression. The increased mature populations are also partly the result of impaired thymocyte emigration. p110ABD T cells colonize the periphery of neonatal animals and irradiated recipients slower than do non-transgenic T cells. The ability of PI3K to regulate positive selection effect is probably due to enhancement of Itk-mediated Ca++ influx. By contrast, the role of PI3K in emigration appears to be independent of known chemotactic or adhesive factors and may instead reflect the importance of subcellular organization for chemokine receptor signaling." }, { "name": "Bhattacharyya, Sujata", "degree": "PhD", "year": "2005", "title": "Embryonic Origin of the Olfactory Sensory System: Fate Map, Lineage Analysis and Specification of the Avian Olfactory Placode", "advisor": "Bronner, Marianne E.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05282005-205447", "creators": [ { "name": { "family": "Bhattacharyya", "given": "Sujata" }, "id": "Bhattacharyya-Sujata", "display_name": "Bhattacharyya, Sujata" } ], "advisors": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "advisor", "display_name": "Bronner, Marianne E." } ], "committee": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "chair", "display_name": "Fraser, Scott E." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." } ], "option_major": [ "biology" ], "doi": "10.7907/hftc-6c39", "abstract": "Coordinating the generation of myriad cell types within the developing nervous system is an exquisite and intensely studied puzzle. The entire vertebrate peripheral nervous system derives from two multipotential cell types in the embryo: neural crest and placodes. Neurogenic placodes are focal ectodermal thickenings present in stereotypic positions in the head. Their derivatives are responsible for much of our sensory perceptions in the craniofacial region. The olfactory placode which gives rise to the olfactory epithelium mediates our sense of smell. Its derivatives include the regenerating olfactory sensory neurons, gonadotropin releasing hormone neurons, olfactory ensheathing glial cells and the basal and supporting cells. While we have some clues to the molecular mechanisms driving its differentiation into the various cell types mentioned above, little is known about the source and induction of the olfactory placode precursor cells in the early embryo.
\r\n\r\nTo trace definitively the origin of the olfactory placode precursor cells, we generated a fate-map and compared it with patterns of gene expression in the region of the chick olfactory placode. To this end, small populations of cells in Hamburger-Hamilton (HH) stage 6 to stage 10 chick embryos were labeled with DiI and DiO and their derivatives were analyzed two days later. At head-fold stages, olfactory placode precursor cells are spread out over a broad domain and intermingle with lens, epidermal and neural precursors. As the neural folds close, the precursors appear to converge anteriorly within the ectoderm. The lens and nasal precursors sort out from each other around HH stage 8, at which time, Pax-6 is differentially upregulated in the region fated to form the lens while Dlx-5 expression is enhanced in the anterior area where the nasal precursors accumulate. To further study the cell movements that lead to the eventual formation of the olfactory placode, I performed confocal time-lapse analysis. The cell rearrangements that I observed are consistent with two possible outcomes: 1.specified lens and nasal precursors have differential adhesive properties and hence sort out or 2.unspecified placodal precursors differentiate according to the environment in which they are positioned by stochastic movements. To distinguish between these possibilities and to clarify whether the precursors are multipotent before they segregate, I have undertaken single cell lineage analysis. Surprisingly, I find no evidence for a shared olfactory and lens placode lineage from single precursors even at early neurula stages, prior to their sorting out from each other in order to contribute to one or the other placode. This raises an interesting question: does the fate of these cells motivate their migration to a certain region of the embryo?
\r\n\r\nIn the next part of my thesis, I have sought to answer a fundamental question in developmental biology, which is how are organs generated in precise and reproducible locations within the body. I have attempted to answer this question in the context of the olfactory sensory system. To understand how and when the nasal structure is first induced, I decided to delineate the tissue that is competent to form the olfactory placode and to determine the spatiotemporal localization of the inducing signals. In general, either one of these two parameters is strictly delimited such that the induced structure arises only in a distinct position. In order to define the extent of competence within the ectoderm to form the nasal placode, I have grafted quail ectoderm from different axial levels to the chick anterior neural fold at stage 8. Cranial and trunk level ectoderm are capable of responding to the inducing signals; they express PAX6 and subsequently form the olfactory placode. However, hindbrain and trunk level ectoderm lose this competence rapidly; by stage 10 neither tissue can express PAX6. This suggests that either the inducing signals are localized anteriorly at early stages or that later signals further refine the olfactory placode-forming region. The presumptive olfactory placode ectoderm is defined by co-expression of several markers. Therefore, I have also analyzed these grafts for DLX3 expression and find a similar trend in loss of competence as seen with PAX6. A prerequisite for studying the induction of a particular fate in a tissue is determining the time at which it is still unspecified i.e. the tissue does not express markers exclusive to its fate when removed from its original context in the embryo and placed in a neutral environment in vitro. I examined the specification of the presumptive olfactory placode ectoderm to express PAX6 and DLX3 and form neurons by culturing this tissue at various stages in three-dimensional collagen gel matrices. Presumptive olfactory placode ectoderm is specified to express PAX6 and DLX3 between stages 8-10. Neuronal specification as assayed by expression of the post-mitotic neuronal marker, Hu, begins around stage 14. This implies that the ectoderm has seen signals that will direct its fate even before it is morphologically visible as a placode. I have also determined the time at which the presumptive olfactory placode ectoderm is irreversibly committed to its fate by grafting this tissue at different stages to the lateral plate ectoderm at the level of the most recently formed somites in the stage 8/9 chick embryo. This occurs by stage 14 as assayed by expression of PAX6 and DLX3, concomitant with a visible thickening of the placode.
\r\n\r\nThe next step is to determine the molecular nature of the inducing signals. Such embryological manipulations in combination with fate-mapping and lineage studies will hopefully afford us some insight into the basic principles by which sensory systems are assembled during development.
" }, { "name": "Choi, Gloria Bohyun", "degree": "PhD", "year": "2005", "title": "Characterization of the Circuits Mediating Innate Reproductive and Defensive Behaviors from the Amygdala to the Hypothalamus", "advisor": "Anderson, David J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05212005-123104", "creators": [ { "name": { "family": "Choi", "given": "Gloria Bohyun" }, "id": "Choi-Gloria-Bohyun", "orcid": "0000-0003-4050-8338", "display_name": "Choi, Gloria Bohyun" } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "advisor", "display_name": "Anderson, David J." } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Alberola-Ila", "given": "Jose" }, "id": "Alberola-Ila-J", "role": "member", "display_name": "Alberola-Ila, Jose" }, { "name": { "family": "Swanson", "given": "Larry W." }, "id": "Swanson-L-W", "role": "member", "display_name": "Swanson, Larry W." } ], "option_major": [ "biology" ], "doi": "10.7907/w6ht-9m14", "abstract": "All metazoan organisms must reproduce and defend themselves in order to survive as individuals and as a species. These innate behaviors are so crucial that they are \"hard-wired\" into the brain during the animal\u2019s development. They are also released primarily by the olfactory stimuli detected by the AOB, which synapses into the MEA. The MEA in turn projects to the medial hypothalamic behavior control column, which contains a series of nuclei orchestrating either reproductive or defensive behaviors. These amygdalar-hypothalamic projections are topographically organized, and the sub-circuitries controlling reproduction and defense are segregated both functionally and anatomically.
\r\n\r\nThe topographically organized projections suggest that these neural pathways for reproduction and defense are likely genetically determined, but genes that might control their wiring have not yet been identified. Such a parallel circuit organization with very few cross-talks between the two sub-circuits also poses the problem of how rapid decisions between competing reproductive and defensive behaviors are made by organisms faced with conflicting cues.
\r\n\r\nUsing oligonucleotide microarrays and laser-capture microdissection, I identified that several LIM homeodomain transcription factors mark different regions of the MEA involved in either reproductive or defensive behaviors. I have characterized the projections of these neurons to the hypothalamus, using both genetically encoded anterograde and traditional retrograde tracers. I have also carried out behavioral experiments to assess their differential activations by reproductive and defensive stimuli.
\r\n\r\nMy results indicate that Lhx6 delineates a reproductive pathway, which involves neurons in both MEApd and BSTpr, and their projections to the three reproductive nuclei in the hypothalamic medial behavioral control column (MPN, VMHvl and PMv). Further analysis reveals, counter-intuitively, that VMHvl receives inhibitory projections from this reproductive pathway, and a convergent excitatory projection from neurons in MEApv that are activated by a predator odor. The results suggest that this point-of-convergence may serve to \"gate\" the expression of reproductive behavior, under conditions where animals are exposed to threatening stimuli. Thus, my data identifies a potential neural substrate within the hypothalamus for controlling behavioral decisions in the face of conflicting cues and a transcription factor family that may contribute to the development of this substrate.
" }, { "name": "Copeland, Jeffrey Michael", "degree": "PhD", "year": "2005", "title": "Identification of Novel Cell Death Regulators in C. elegans and Drosophila", "advisor": "Hay, Bruce A.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06012005-114127", "creators": [ { "name": { "family": "Copeland", "given": "Jeffrey Michael" }, "id": "Copeland-Jeffrey-Michael", "orcid": "0000-0001-5432-3524", "display_name": "Copeland, Jeffrey Michael" } ], "advisors": [ { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "advisor", "display_name": "Hay, Bruce A." } ], "committee": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "chair", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/7T3M-DY09", "abstract": "Apoptosis is a form of cell death executed by a class of cysteine proteases called caspases. Though caspases are well-conserved, the mechanisms by which caspases are regulated vary remarkably. This dissertation addresses three independent aspects of apoptosis and its regulation.
\r\n\r\nIn the developing Drosophila eye, apoptosis is activated to remove extra cells that are initially present between ommatidia. Mutants for the gene echinus have a disorganized eye structure due to a failure of these cell deaths to occur. We demonstrate that echinus resembles a deubiquitinating enzyme, that it is expressed in the pupal eye during the time of cell death, and that echinus acts genetically upstream or independently of the death-inducing genes head involution defective, reaper, and grim. Based on in vitro assays and the fact that the Echinus enzyme lacks a catalytic cysteine residue, we propose that echinus and its orthologs constitute a novel class of inactive deubiquitinating enzymes, perhaps functioning in a dominant-negative manner to inhibit deubiquitination of specific substrates.
\r\n\r\nIn C. elegans, the model for caspase inhibition is quite different from that in Drosophila and in mammals. To look for genes that directly inhibit the CED-3 caspase, we screened a C. elegans cDNA library for CED-3 suppressors in the yeast S. cerevisiae and found several suppressors. Experiments in yeast suggest that one of these genes, Y39B6A.12, requires the prodomain of CED-3 for suppression, and ectopic expression in the Drosophila eye shows that it can suppress apoptosis induced by the Bcl2 family member Debcl.
\r\n\r\nIn Drosophila, DIAP1 is the focal point in the regulation of apoptosis. To identify novel regulators of DIAP1, deficiency chromosomes spanning the Drosophila genome were screened for dominant modifiers of a diap1 knockdown phenotype. Nine deficiencies were isolated that cover no known regulators, and two modifiers were mapped to small genomic regions. This screen has provided a starting point for identifying some of the many uncharacterized genes that are involved in regulating apoptosis.
" }, { "name": "Croal, Laura Rosemary", "degree": "PhD", "year": "2005", "title": "Fe(II) Oxidation by Anaerobic Phototrophic Bacteria: Molecular Mechanisms and Geological Implications", "advisor": "Newman, Dianne K.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06062005-011632", "creators": [ { "name": { "family": "Croal", "given": "Laura Rosemary" }, "id": "Croal-Laura-Rosemary", "display_name": "Croal, Laura Rosemary" } ], "advisors": [ { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "advisor", "display_name": "Newman, Dianne K." } ], "committee": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "chair", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Newman", "given": "Dianne K." }, "id": "Newman-D-K", "orcid": "0000-0003-1647-1918", "role": "member", "display_name": "Newman, Dianne K." }, { "name": { "family": "Rossman", "given": "George Robert" }, "id": "Rossman-G-R", "orcid": "0000-0002-4571-6884", "role": "member", "display_name": "Rossman, George Robert" }, { "name": { "family": "Kirschvink", "given": "Joseph L." }, "id": "Kirschvink-J-L", "orcid": "0000-0001-9486-6689", "role": "member", "display_name": "Kirschvink, Joseph L." }, { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "member", "display_name": "Simon, Melvin I." } ], "option_major": [ "biology" ], "doi": "10.7907/PW00-W724", "abstract": "In this thesis, the hypothesis that photoautotrophic Fe(II)-oxidizing bacteria catalyzed the deposition of Banded Iron Formations (BIFs), an enigmatic class of ancient sedimentary rocks is explored. Ecophysiological, geochemical, genetic and biochemical approaches are taken to elucidate the molecular mechanism of photoautotrophic Fe(II) oxidation in an effort to identify molecular biosignatures that are unique to this metabolism and capable of being preserved BIFs. In an ecophysiological approach, we show that Fe(II) oxidation by these phototrophs proceeds at appreciable rates in the presence of high concentrations of H2 when CO2 is abundant. These findings substantiate a role for the involvement of these phototrophs in BIF deposition under the presumed geochemical conditions of the Archean. In a geochemical approach, we find that although phylogenetically distinct phototrophs fractionate Fe isotopes in a way that is consistent with Fe isotopic values found in Precambrian BIFs, it is unlikely that this fractionation can be used as a biosignature for this metabolism given its similarity to fractionations produced by abiotic Fe(II) oxidation reactions. In two distinct genetic approaches, we identify genes involved in Fe(II) oxidation in Rhodopseudomonas palustris TIE-1 and Rhodobacter SW2. Genes identified in TIE-1 encode a predicted integral membrane protein that appears to be part of an ABC transport system and a putative CobS, an enzyme involved in cobalamin (vitamin B12) biosynthesis. Candidate genes on a cloned fragment of the Rhodobacter SW2 genome that confer Fe(II) oxidation activity to a non-oxidizing strain include those predicted to encode permeases and a protein with potential redox capability. Finally, in a preliminary biochemical approach, c-type cytochromes and other proteins that are exclusive or more highly expressed under Fe(II) growth conditions in TIE-1 and SW2 are identified in SDS-PAGE gels. The work described here furthers our search for a biosignature unique to photoautotrophic Fe(II) oxidation by providing mechanistic information on this metabolism." }, { "name": "Farivar, Shabnam Sarah", "degree": "PhD", "year": "2005", "title": "Cytoarchitecture of the Locust Olfactory System", "advisor": "Laurent, Gilles J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-04212005-143332", "creators": [ { "name": { "family": "Farivar", "given": "Shabnam Sarah" }, "id": "Farivar-Shabnam-Sarah", "display_name": "Farivar, Shabnam Sarah" } ], "advisors": [ { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "advisor", "display_name": "Laurent, Gilles J." } ], "committee": [ { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "chair", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "member", "display_name": "Koch, Christof" }, { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "member", "display_name": "Schuman, Erin Margaret" }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." } ], "option_major": [ "biology" ], "doi": "10.7907/4Y60-KH68", "abstract": "The insect mushroom body (MB) receives and processes olfactory information. The MB is a highly conserved structure found in all but a few insect species, and has been shown to be a relevant area in learning and memory of olfactory information. The functional properties of the intrinsic cells of the MB -- the Kenyon cells (KCs) -- have been extensively studied, and their integrative properties are starting to be understood, particularly in locust. To help decipher its role in odor processing, this thesis presents an in-depth study of the architecture of the locust MB, using a variety of anatomical techniques and original software. Four divisions in the MB's input area, the calyx, are defined and described, as well as a division in one of its output regions, the beta lobe. KCs are characterized based on their morphologies and extents within the calyx divisions and the beta lobe. MB input cells - the projection neurons - are described in relation to their own input area, the antennal lobe, as well as their output to MB calyx divisions. Two classes of cells downstream from the KCs are also defined anatomically and related to immunochemistry on neurotransmitters. A specific area within the brain - the lateral horn lobe - to which projection neurons and extrinsic cells project, is also defined. Similarities of these structures to other insect orders are discussed." }, { "name": "Hart, Christopher Edward", "degree": "PhD", "year": "2005", "title": "Inferring Genetic Regulatory Network Structure: Integrative Analysis of Genome-Scale Data", "advisor": "Wold, Barbara J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-03152005-110423", "creators": [ { "name": { "family": "Hart", "given": "Christopher Edward" }, "id": "Hart-Christopher-Edward", "display_name": "Hart, Christopher Edward" } ], "advisors": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "advisor", "display_name": "Wold, Barbara J." } ], "committee": [ { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "chair", "display_name": "Simon, Melvin I." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Mjolsness", "given": "Eric D." }, "id": "Mjolsness-E-D", "role": "member", "display_name": "Mjolsness, Eric D." }, { "name": { "family": "Winfree", "given": "Erik" }, "id": "Winfree-E", "role": "member", "display_name": "Winfree, Erik" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/2JXP-3G71", "abstract": "With the aim of uncovering regulatory relationships that underly biological processes, we constructed a framework of computational tools and techniques to relate disparate genome-scale data within and across datasets. Using these tools we focus on the yeast cell cycle and the transcriptional network driving the transition into and out of G1. Through integrative analysis of genome-scale datasets we were able to recover many of the previously known transcriptional regulatory connections within the yeast cell cycle. We also found several novel hypothetical connections yet to be experimentally validated.
\r\n\r\nMuch of the analysis of large-scale gene expression data has relied heavily on the application of clustering algorithms to identify sets of co-expressed genes (clusters). In chapter 2 we introduce several new techniques for comparing and evaluating microarray data, specifically focusing on clustering results. We discuss the need for quantitative methods for evaluating clustering methods, and discuss the application of comparative analysis of clustering results.
\r\n\r\nRemarkably, our analysis shows the results from any clustering algorithm are quite sensitive to slight perturbations to the data. Yet, the underlying structure revealed by most clustering algorithms remains fairly stable. These findings have a pragmatic impact on how clustering results should be interpreted and used. Chapter 3 uses the tools introduced in chapter 2 and performs a systematic comparison of the influence of noise on the stability and reliability of clustering results.
\r\n\r\nIn chapter 4 we demonstrate the use of artificial neural networks (ANNs) to infer regulatory networks by combining expression data and protein:DNA binding data. We then compare these regulatory relationships to the presence of transcription factor binding sites. We also note evolutionary stability in some of the components of this network by comparing results to other species of yeast.
" }, { "name": "Hom, Geoffrey Kai Tong", "degree": "PhD", "year": "2005", "title": "Advances in Computational Protein Design: Development of More Efficient Search Algorithms and their Application to the Full-Sequence Design of Larger Proteins", "advisor": "Mayo, Stephen L.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05302005-223153", "creators": [ { "name": { "family": "Hom", "given": "Geoffrey Kai Tong" }, "id": "Hom-Geoffrey-Kai-Tong", "display_name": "Hom, Geoffrey Kai Tong" } ], "advisors": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "advisor", "display_name": "Mayo, Stephen L." } ], "committee": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "orcid": "0000-0002-3671-9354", "role": "chair", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "member", "display_name": "Pierce, Niles A." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." } ], "option_major": [ "biochem" ], "doi": "10.7907/M4R9-YM51", "abstract": "Protein design is the art of choosing an amino acid sequence that will fold into a desired structure. Computational protein design aims to quantify and automate this process. In computational protein design, various metrics may be used to calculate an energy score for a sequence with respect to a desired protein structure. An ongoing challenge is to find the lowest-energy sequences from amongst the vast multitude of sequence possibilities. A variety of exact and approximate algorithms may be used in this search.
\r\n\r\nThe work in this thesis focuses on the development and testing of four search algorithms. The first algorithm, HERO, is an exact algorithm, meaning that it will always find the lowest-energy sequence if the algorithm converges. We show that HERO is faster than other exact algorithms and converges on some previously intractable designs. The second algorithm, Vegas, is an approximate algorithm, meaning that it may not find the lowest-energy sequence. We show that, under certain conditions, Vegas finds the lowest-energy sequence in less time than HERO. The third algorithm, Monte Carlo, is an approximate algorithm that had been developed previously. We tested whether Monte Carlo was thorough enough to do a challenging computational design: the full-sequence design of a protein. Monte Carlo didn\u2019t find the lowest-energy sequence, although a similar sequence from Vegas folded into the desired structure. Several biophysical methods suggested that the Monte Carlo sequence should also fold into the desired structure. Nevertheless, the Monte Carlo structure as determined by X-ray crystallography was markedly different from the predicted structure. We attribute this discrepancy to the presence of a high concentration of dioxane in the crystallization conditions. The fourth algorithm, FC_FASTER, is an approximate algorithm for designs of fixed amino acid composition. Such designs may accelerate improvements to the physical model. We show that FC_FASTER finds lower-energy sequences and is faster than our current fixed-composition algorithm.
" }, { "name": "Huh, Jun Ryul", "degree": "PhD", "year": "2005", "title": "To Die or to Differentiate: Apoptotic and Non-Apoptotic Roles of Death Molecules in Drosophila melanogaster", "advisor": "Hay, Bruce A.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06032005-145438", "creators": [ { "name": { "family": "Huh", "given": "Jun Ryul" }, "id": "Huh-Jun-Ryul", "display_name": "Huh, Jun Ryul" } ], "advisors": [ { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "advisor", "display_name": "Hay, Bruce A." } ], "committee": [ { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "chair", "display_name": "Zinn, Kai George" }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "role": "member", "display_name": "Dunphy, William G." } ], "option_major": [ "biology" ], "doi": "10.7907/Q00G-B953", "abstract": "Virtually every cell, if not all, are ready to die under stressful conditions or by necessity during animal development. In Drosophila, three pro-apoptotic proteins, Rpr/Hid/Grim, had been found to induce cell death by preventing the function of the cell death inhibitor, DIAP1. However, the mechanistic details of this process were largely unknown. We have found that Rpr/Hid/Grim induce DIAP1 destabilization through ubiquitination and general translational inhibition. Moreover, from the in-vitro and in-vivo studies, we also found that ubiquitination of DIAP1 by Hid is dependant on DIAP1\u2019s own ability to ubiquitinate itself. Once the life-or-death decision is made, cells can efficiently start apoptosis by quickly removing pre-existing death inhibitors using these mechanisms.
\r\n\r\nIn addition to the canonical roles of death machinery, we have also studied their roles in non-apoptotic developmental processes. In the testis, germline stem cells ultimately give rise to 64 individual sperms. Spermatocytes, and later, spermatids, develop within a single membranous structure, or syncytium. Formation of free-swimming sperms requires the encapsulation of each spermatid by an independent plasma membrane and the elimination of most of the sperm cytoplasm. We demonstrated that at least three independent caspase activation pathways are likely to be involved in these processes with different spatial and temporal activation patterns, and that a global inhibition of caspase activity results in male sterility.
\r\n\r\nExternal stresses such as radiation and heat shock were known to induce large amounts of cell death (up to 60% of the total cell population) in proliferative tissues like Drosophila larval imaginal discs. Interestingly, larvae exposed to such stress ultimately develop into normal adult flies. This is facilitated by the compensatory proliferation of cells that neighbor the dying cells. In order to study the mechanistic basis for this process, we uncoupled cell death from death activation by expressing Hid in the presence of P35, a viral inhibitor of effecter caspases. Interestingly, neighboring cells of clones expressing Hid underwent compensatory proliferation, which was no longer observed when we blocked the activation of initiator caspase, Dronc. Our observations indicate that non-apoptotic Dronc activity is required for the generation of a non-autonomous proliferation signal.
" }, { "name": "Leung, Thomas Hin-Chai", "degree": "PhD", "year": "2005", "title": "Specificity of Transcription Activation by NF-kB Subunits", "advisor": "Baltimore, David L.", "url": "https://resolver.caltech.edu/CaltechETD:etd-10042004-090452", "creators": [ { "name": { "family": "Leung", "given": "Thomas Hin-Chai" }, "id": "Leung-Thomas-Hin-Chai", "orcid": "0000-0003-1086-0824", "display_name": "Leung, Thomas Hin-Chai" } ], "advisors": [ { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "advisor", "display_name": "Baltimore, David L." } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "chair", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "member", "display_name": "Baltimore, David L." }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "member", "display_name": "Chan, David C." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/100y-3c61", "abstract": "The transcription factor NF-kappaB is a regulator of a wide variety of processes including inflammation, innate and adaptive immunity, apoptosis, and learning. How can one factor be accurately involved in so many different processes and generate without exception precise and appropriate responses? Four members of the NF-kappaB transcription factor family are involved in gene activation, and they hetero- or homodimerize with each other to bind DNA. This allows for many different potential combinations of NF-kappaB dimers. To test whether NF-kappaB-dependent genes require specific NF-kappaB family members for gene activation, cell lines were generated lacking in individual and multiple NF-kappaB proteins. Using TNFalpha as an inducer, a panel of endogenous NF-kappaB responsive genes showed a wide range of subunit specificities. Given that the NF-kappaB consensus binding site sequence is very broad and that crystal structures of NF-kappaB have not identified enough dimer-specific DNA-binding contacts to rationalize specific NF-kappaB binding sites, kappaB sites were compared from a single gene to another and no direct correlation was found between kappab site sequence and kappaB family member requirements. However when interspecies comparisons were made of the same gene, a remarkable constancy of the kappaB site sequence was found, which suggested that individual sites have important functional characteristics. To test this theory, a novel lentiviral system was created that incorporated regulatory sequences into cellular DNA. Then by simply swapping sites between kappaB-dependent genes, NF-kappaB dimer specificity of the promoters was altered and revealed that two kappaB sites can function together as a module to regulate gene activation. Further, although the sequence of the kappaB site is important for determining kappaB family member specificity, rather than determining the ability of a particular dimer to bind effectively, the sequence affects which co-activators will form productive interactions with the bound kappaB dimer. My findings suggest that a particular DNA-binding site may impart a specific configuration to bound transcription factors that specifies the requirement for particular co-activators. Taken together, I have taken the first steps to dissecting how the promoter code influences individual NF-kappaB family members to function on NF-kappaB responsive genes and to regulate gene expression." }, { "name": "Mah, Angie Siu Yee", "degree": "PhD", "year": "2005", "title": "Regulation of Protein Kinase Dbf2 in Mitotic Exit", "advisor": "Deshaies, Raymond Joseph", "url": "https://resolver.caltech.edu/CaltechETD:etd-05242005-130533", "creators": [ { "name": { "family": "Mah", "given": "Angie Siu Yee" }, "id": "Mah-Angie-Siu-Yee", "display_name": "Mah, Angie Siu Yee" } ], "advisors": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "advisor", "display_name": "Deshaies, Raymond Joseph" } ], "committee": [ { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "role": "chair", "display_name": "Dunphy, William G." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "role": "member", "display_name": "Chan, David C." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "member", "display_name": "Deshaies, Raymond Joseph" } ], "option_major": [ "biochem" ], "doi": "10.7907/NS1Z-3K87", "abstract": "Cyclin-dependent kinases (Cdk) direct cell cycle transitions by associating with various cyclins throughout the cell cycle. For cells to exit mitosis, mitotic Cdk activity must be turned off. In Saccharomyces cerevisiae, the mitotic exit network, or MEN, comprises of a group of proteins that form a signaling pathway required for mitotic exit. The MEN regulates the activity of Cdc14, the protein phosphatase critical for inactivating mitotic Cdk. Components of the MEN include the protein kinases Cdc15 and Dbf2, as well as the Dbf2-associated protein Mob1. We determined how these proteins are organized within the MEN by determining the molecular mechanism of Dbf2 activation. Dbf2 requires Mob1 association in order to be active and Cdc15 phosphorylates and thereby activates the Dbf2-Mob1 protein kinase complex. We also determined that the conserved phosphorylation sites of the NDR protein kinase family are required for Dbf2 kinase activity in vitro as well as for DBF2 function in vivo. It is unknown how Dbf2-Mob1 leads to Cdc14 release or how the protein kinase complex functions in cytokinesis. As a result, we sought to identify physiological substrates of Dbf2-Mob1 which would provide insight to Dbf2-Mob1 function in both of these significant cell cycle processes. There is no known physiological substrate for Dbf2-Mob1 we first identified RXXS as the motif that Dbf2-Mob1 preferentially phosphorylates. We then identified a number of in vitro substrates for Dbf2-Mob1, of which the majority contains the RXXS motif. The mechanism of Dbf2 activity has been shown to be conserved in a number of other NDR kinase family members, which have roles in morphogenesis and cell division, and have been implicated in tumorigenesis. Studies on Dbf2 will provide insight into cell cycle processes in budding yeast as well as in higher eukaryotes." }, { "name": "Peng, Joyce Yaochun", "degree": "PhD", "year": "2005", "title": "Structure and Function Prediction of Human Muscarinic Acetylcholine Receptor 1, Cation-\u03c0 Studies, and Protein Design", "advisor": "Goddard, William A., III; Vaidehi, Nagarajan", "url": "https://resolver.caltech.edu/CaltechETD:etd-05312005-114949", "creators": [ { "name": { "family": "Peng", "given": "Joyce Yaochun" }, "id": "Peng-Joyce-Yaochun", "display_name": "Peng, Joyce Yaochun" } ], "advisors": [ { "name": { "family": "Goddard", "given": "William A., III" }, "id": "Goddard-W-A-III", "orcid": "0000-0003-0097-5716", "role": "advisor", "display_name": "Goddard, William A., III" }, { "name": { "family": "Vaidehi", "given": "Nagarajan" }, "id": "Vaidehi-N", "orcid": "0000-0001-8100-8132", "role": "advisor", "display_name": "Vaidehi, Nagarajan" } ], "committee": [ { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "chair", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "role": "member", "display_name": "Campbell, Judith L." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Goddard", "given": "William A., III" }, "id": "Goddard-W-A-III", "orcid": "0000-0003-0097-5716", "role": "member", "display_name": "Goddard, William A., III" } ], "option_major": [ "biology" ], "doi": "10.7907/XVJR-RN32", "abstract": "(1) Muscarinic acetylcholine receptors, a G protein-coupled receptor, are responsible for a wide range of diseases. We predicted the 3D structure of the human M1 muscarinic receptor using the MembStruk method and validated its binding sites for 10 agonists and antagonists using the HierDock method. The predicted binding sites, the intramolecular contacts that stabilize the receptor conformation, and the in silico mutagenesis results, agree well with mutagenesis data. The calculated relative binding energies correlate well with measured binding affinities. In addition, the predicted binding sites provide a structural basis for the large reduction in ligand binding affinity and signaling efficacy by Trp 157 and Pro 159 mutations, which was not previously explained by homology models. The predicted binding sites illustrate the importance of aromatic residues in ligand binding through extensive cation-pi and aromatic-aromatic interactions, with new mutation candidates suggested. The predicted M1 structure improves our understanding of the muscarinic receptors, offers a basis for structure based drug design, and is a successful step toward applying these procedures in predicting the structures of other muscarinic receptor subtypes.
\r\n\r\n(2) We used high-level quantum mechanics to quantify cation-pi interactions in the crystal structure of carbamylcholine binding to Acetylcholine-binding Protein, a nicotinic receptor homolog. The calculated effects of fluorinated unnatural amino acid substitutions also correlate excellently with experimental EC50 data, suggesting that quantum mechanics can accurately predict cation-pi binding in a protein environment and provides a good model system in developing force fields to better describe cation-pi interactions.
\r\n\r\n(3) Histidines are known to modulate pH responsive binding. We designed a series of histidine derivatives by substituting its imidazole ring with functional groups that are small in size and lack the ability to form hydrogen bonds. Quantum mechanical calculations of the acid dissociation constants (pKa) show that these substitutions shift the histidine pKa upward or downward. We report a list of histidine derivatives and their corresponding pKa values that can be used in designing tumor specific drugs (e.g. HER2-Herceptin antibody), drug delivery through pH sensitive hydrogels, drug recycling, catalysis, and biosensors development. An example of how these unnatural histidines can be used is illustrated with 2-methyl histidine incorporated in a c-Myc-Max heterodimer.
" }, { "name": "Reddy, Leila", "degree": "PhD", "year": "2005", "title": "Attention and the Processing of Natural Stimuli: Psychophysics, fMRI and Single Unit Recordings in the Human Brain", "advisor": "Koch, Christof", "url": "https://resolver.caltech.edu/CaltechETD:etd-05312005-170913", "creators": [ { "name": { "family": "Reddy", "given": "Leila" }, "id": "Reddy-Leila", "orcid": "0000-0002-7078-1055", "display_name": "Reddy, Leila" } ], "advisors": [ { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "advisor", "display_name": "Koch, Christof" } ], "committee": [ { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "chair", "display_name": "Koch, Christof" }, { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "orcid": "0000-0001-8837-678X", "role": "member", "display_name": "Siapas, Athanassios G." }, { "name": { "family": "Fried", "given": "Itzhak" }, "id": "Fried-I", "role": "member", "display_name": "Fried, Itzhak" }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "orcid": "0000-0002-1290-5232", "role": "member", "display_name": "Shimojo, Shinsuke" } ], "option_major": [ "cns" ], "doi": "10.7907/D984-0380", "abstract": "How does the visual system process stimuli it encounters frequently? To address this question, we investigated the attentional requirements associated with processing natural stimuli in isolation and in crowded environments. Using the dual-task paradigm, we tested observers on discriminating the gender of faces presented in isolation, in the near-absence of focal attention. We observed that performance on this task for poorly attended faces suffered only minimally. Furthermore, we also showed that finer discriminations, such as face identification (celebrities and unfamiliar faces), are unimpaired when attention is unavailable. From a computational perspective these results are surprising since observers are unable to categorize computationally simpler stimuli (e.g., a red-green color disk from its mirror image) under identical conditions. The brain mechanisms underlying the processing of face-gender were probed with fMRI. We found that the BOLD signal in the near-absence of attention was not significantly reduced, provided the faces were behaviorally relevant. This finding, that top-down expectations can be sufficient for high levels of the BOLD signal, is in contrast to current views that hold that the signal is reduced in the absence of focused attention. We took a closer look at attentional effects on neuronal activity by recording from individual neurons in the human brain while subjects performed a change detection paradigm (in which they reported whether they noticed changes made to (natural) stimuli presented in a crowded environment). Subjects were epileptic patients, implanted with depth electrodes in the medial temporal lobe (MTL) for identification of the seizure foci for potential surgical resection. We observed that neuronal responses when changes were correctly detected were significantly higher compared to incorrect trials. Under the common assumption that incorrect performance reflects the absence of attention, we show that MTL neuronal activity is reduced when attention is unavailable. For each cell, on a trial-by-trial basis, we were able to predict the occurrence of a change (67%) and the patients\u2019 behavior (58%), significantly above chance. Our results show that the brain can process isolated natural stimuli in the near-absence of attention, while in crowded environments, attention plays a key role, as we have observed at the neuronal level" }, { "name": "Shah, Premal S.", "degree": "PhD", "year": "2005", "title": "Advances in Force Field Development and Sequence Optimization Methods for Computational Protein Design", "advisor": "Mayo, Stephen L.", "url": "https://resolver.caltech.edu/CaltechETD:etd-04042005-142719", "creators": [ { "name": { "family": "Shah", "given": "Premal S." }, "id": "Shah-Premal-S", "display_name": "Shah, Premal S." } ], "advisors": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "advisor", "display_name": "Mayo, Stephen L." } ], "committee": [ { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "chair", "display_name": "Rees, Douglas C." }, { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "member", "display_name": "Arnold, Frances Hamilton" }, { "name": { "family": "Pierce", "given": "Niles A." }, "id": "Pierce-N-A", "orcid": "0000-0003-2367-4406", "role": "member", "display_name": "Pierce, Niles A." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." } ], "option_major": [ "biochem" ], "doi": "10.7907/mg9x-s593", "abstract": "The overall goals of computational protein design range from designing new protein folds and protein-protein interfaces to the de novo design of enzymes. All goals require that two equally challenging components of computational protein design be addressed. First, the physical model that describes a protein\u2019s intermolecular and intramolecular interactions must be accurate. Second, energetically optimal amino acid sequences must be identified from an enormous number of possibilities. This thesis describes work that makes progress in both these arenas. In addition, the effectiveness and applicability of computational protein design is demonstrated by tackling challenging design problems.
\r\n\r\nImprovements to the physical model have been made by developing a more accurate method for calculating rotamer (amino acid side-chain conformation) surface areas for use in our surface area-based hydrophobic solvation term. With this method, surface area errors were decreased dramatically and the experimental stabilities of proteins generated from computationally predicted sequences were improved. Also, our direct surface area calculation approach significantly reduced the compute time required for sequence optimization using dead-end elimination (DEE)-based algorithms.
\r\n\r\nAlthough DEE-based algorithms have been effectively used for many challenging design problems, the daunting task of sequence optimization can cause even the most efficient DEE-based methods to fail. We developed a sequence optimization technique called Vegas that combines elements of non-DEE-based as well as DEE-based algorithms. For design problems that were already tractable using DEE-based methods, Vegas delivered the GMEC in significantly less time. In cases where DEE-based algorithms stalled and failed to deliver the GMEC, Vegas produced an answer that, at the time, was better than any other algorithm. This is illustrated by Vegas\u2019 solution to a challenging problem: the full sequence design of a 51-residue fragment of the Drosophila engrailed homeodomain (ENH). We generated a variant of ENH predicted by Vegas and compared its thermodynamic properties with a protein obtained using a Monte Carlo search. We found that the thermodynamic properties of the two molecules were identical. We also solved the solution structure of the Vegas-based molecule using nuclear magnetic resonance (NMR) spectroscopy and found that it folded accurately into the target fold.
\r\n\r\nObtaining water soluble variants of membrane proteins might alleviate some of the problems encountered when working with them and facilitate our understanding of the different forces contributing to protein stabilities in membranes. We made progress in developing an automated design scheme that can generate water soluble variants of membrane proteins. We analyzed and compared the surfaces of membrane proteins and water soluble proteins, and developed a metric for altering membrane protein surfaces. Using this metric, we can design membrane protein surfaces using the ORBIT suite of protein design algorithms and convert them to those resembling water soluble protein surfaces. We tested this strategy on two proteins and although we have not been completely successful, we have established rules and guidelines that will aid future efforts towards achieving this goal.
" }, { "name": "Shin, Donghun", "degree": "PhD", "year": "2005", "title": "Identification and Characterization of Endothelial Specific Genes", "advisor": "Anderson, David J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05232005-173743", "creators": [ { "name": { "family": "Shin", "given": "Donghun" }, "id": "Shin-Donghun", "orcid": "0000-0002-7975-9014", "display_name": "Shin, Donghun" } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "advisor", "display_name": "Anderson, David J." } ], "committee": [ { "name": { "family": "Alberola-Ila", "given": "Jose" }, "id": "Alberola-Ila-J", "role": "chair", "display_name": "Alberola-Ila, Jose" }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/XV0S-9D71", "abstract": "Cardiovascular development and its proper function are essential for the development and survival of animals, while malformation of vasculature leads to a variety of diseases. The significance of vasculature during development and in adulthood has been delineated by investigating the functions of genes expressed in the vasculature. Endothelial cells lining the lumen of vessel tubes with a single layer, had long been considered inert, homogeneous cells. However, molecular and genetic studies have provided numerous pieces of evidence, which indicate that endothelial cells are active, dynamic, heterogeneous cells. Among these studies, molecular differences between arterial and venous endothelial cells were first revealed by the observation that ephrin-B2 and its cognate receptor EphB4 are restrictively expressed in arterial and venous endothelial cells, respectively. These genes are not only molecular markers of arteries and veins, but they also play essential roles in cardiovascular development.
\r\n\r\nTo investigate whether the molecular difference between arteries and veins persists into adulthood, I analyzed ephrin-B2 expression in adult tissues including pathological settings. These data indicate that the molecular distinction is maintained in adults, and ephrin-B2 further distinguishes arterial smooth muscle cells from venous smooth muscle cells in adults.
\r\n\r\nEphrin-B2 was serendipitously identified as an arterial marker; therefore, I performed a systematic screen to isolate novel arterial- and venous-specific genes, whose identification and characterization might improve current understanding of vascular biology. Through this screen, I isolated several novel arterial-restricted genes, and one of these genes, Depp (decidual protein induced by progesterone), was characterized in detail by generating a knockout of the Depp locus. Although the homozygous mutant mice appear phenotypically normal, the detailed analysis of Depp expression reveals the heterogeneity of arterial endothelial cells from the early stage of vascular development.
\r\n\r\nI identified another novel gene, D1.1, through the screen; however, D1.1 is expressed in both arterial and venous endothelial cells. The fact that D1.1 is specifically expressed in endothelial cells and encodes a predicted transmembrane protein, prompted me to characterize D1.1 in detail using a tau-LacZ knock-in to the D1.1 locus. The data from the expression analysis suggest D1.1 as a novel marker of adult neovasculature. In addition, the data using a soluble D1.1-Fc fusion protein in several different acute assays suggest that D1.1 may play a functional role in angiogenesis that is compensated in vivo by other, structurally distinct proteins.
" }, { "name": "Simion, Claudiu", "degree": "PhD", "year": "2005", "title": "Orienting and Preference: An Enquiry into the Mechanisms Underlying Emotional Decision Making", "advisor": "Bogen, Joseph E.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05262005-211552", "creators": [ { "name": { "family": "Simion", "given": "Claudiu" }, "id": "Simion-Claudiu", "display_name": "Simion, Claudiu" } ], "advisors": [ { "name": { "family": "Bogen", "given": "Joseph E." }, "id": "Bogen-J-E", "role": "advisor", "display_name": "Bogen, Joseph E." } ], "committee": [ { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "chair", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Bogen", "given": "Joseph E." }, "id": "Bogen-J-E", "role": "member", "display_name": "Bogen, Joseph E." }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "member", "display_name": "Perona, Pietro" }, { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-R", "orcid": "0000-0002-8053-9692", "role": "member", "display_name": "Adolphs, Ralph" }, { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "orcid": "0000-0002-1290-5232", "role": "member", "display_name": "Shimojo, Shinsuke" } ], "option_major": [ "biology" ], "doi": "10.7907/PWAN-XK37", "abstract": "The present work is an extensive investigation of the contribution of orienting behavior to preference decision making in humans. The central claim of this thesis is that gaze assists cognition in choosing preference, integrating phenomena previously demonstrated in the literature, the mere exposure effect and preferential looking, in a positive feedback loop leading to the conscious decision. In other words, the more we look at something, the more we like it, but also the more we like it, the more we look at it. This leads to the effect we observed when tracking subjects' eye-movements while they were choosing the preferred stimulus in two-alternative forced-choice tasks: the likelihood of gazing towards choice continually increased as the decision moment was approaching. We called this pattern the \"gaze cascade effect\" to illustrate its reinforcing nature, and we showed that it is an indispensable part of any preference decision, no matter the circumstances, as long as subjects' gaze is natural and unrestricted. We obtained the cascade effect even when the stimuli were no longer present on the screen, and the decision was based entirely on internal reconstruction. Moreover, we influenced observers' preference by manipulating their gaze, an effect that could not be accounted for by mere exposure. These results demonstrate that preference formation starts early in the visual processing stream, sharing fast reciprocal connections with simpler, somatic-based behaviors, such as orienting. As general implications, our work contradicts views of the brain as a collection of sequential modules, starting with sensation, through perceptual integration and cognitive association to emotional valence, decision making, motor preparation and motor response. Instead, it supports more recent views, which assume a multitude of parallel modules heavily interconnected and exercising influences on each other from very early on in information processing." }, { "name": "Yu, Hui", "degree": "PhD", "year": "2005", "title": "C. elegans Male Tail Development", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechETD:etd-11142004-223239", "creators": [ { "name": { "family": "Yu", "given": "Hui" }, "id": "Yu-Hui", "display_name": "Yu, Hui" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/gg2d-aa22", "abstract": "The C. elegans hook sensillum is a male copulatory structure developed from the hook sensillum competence group (HCG), P(9-11).p. Male P(9-11).p each adopt one of three potential fates (1\u00ba, 2\u00ba, or 3\u00ba), forming an invariant spatial pattern of 3\u00ba-2\u00ba-1\u00ba. I examined HCG fate specification in various genetic backgrounds and found that both lin-17 Frizzled-like Wnt receptor and bar-1 [beta]-catenin are predominantly expressed in 1\u00ba P11.p. Activation of the bar-1 canonical Wnt pathway by a pry-1(Axin) mutation changes the competence of the anterior P(3-8).p cells and induces ectopic 1\u00ba HCG fate. I also found that the Hox gene mab-5 acts downstream of the Wnt pathway to determine HCG competence. Ectopic mab-5 expression, in conjunction with activated LIN-12 or EGF signaling, causes ectopic HCG fates in anterior P(1-8).p. Furthermore, epistatic interactions between lin-17 and lin-12 showed that generation of 2\u00ba HCG fate in P10.p by LIN-12/Notch activity depends on LIN-17-mediated Wnt signaling. Together these observations suggest that Wnt signaling is the major player that governs HCG patterning and functions in HCG competence, specification, and execution.
\r\n\r\nPrecise execution of the 2\u00ba HCG lineage leads to generation of all five major components of a functional hook sensillum that mediates vulva location behavior during male mating. Both sensory neurons of the hook sensillum, HOA and HOB, are necessary for efficient vulva location. From a genetic screen for altered expression of an HOB marker ceh-26::gfp or abnormal hook morphology, I isolated five mutants. Further analysis of one of these mutants identified an allele of egl-46, a zinc-finger transcription factor. EGL-46, and to some extent, another transcription factor EGL-44, regulate a group of HOB-specific genes. These targets include the homeodomain protein CEH-26, a neuropeptide-like protein NLP-8, a degenerin homologue T28F4.2, and two C. elegans polycystin homologs LOV-1 and PKD-2. Both egl-46 and egl-44 mutants exhibit defective vulva location behavior, suggesting impaired HOB function. The regulator of a general ciliogenic pathway, DAF-19, indirectly affects expression of HOB-specific genes. Expression of DAF-19, EGL-46, and EGL-44 is independent from one another, indicating that general and cell-specific regulatory factors act in parallel to produce cell specificities crucial for HOB sensory function.
" }, { "name": "Azzam, Ramzi Issam", "degree": "PhD", "year": "2004", "title": "The Role of Net1 Phosphorylation in Regulating CDC14 Release During Mitotic Exit", "advisor": "Deshaies, Raymond Joseph", "url": "https://resolver.caltech.edu/CaltechETD:etd-05142004-140316", "creators": [ { "name": { "family": "Azzam", "given": "Ramzi Issam" }, "id": "Azzam-Ramzi-Issam", "display_name": "Azzam, Ramzi Issam" } ], "advisors": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "advisor", "display_name": "Deshaies, Raymond Joseph" } ], "committee": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "chair", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "role": "member", "display_name": "Dunphy, William G." } ], "option_major": [ "biology" ], "doi": "10.7907/W73K-KK96", "abstract": "Exit from mitosis is as an important phase in the cell cycle. The molecular event that triggers the cell cycle transition from anaphase into the G1 state involves the inactivation of the cyclin-dependent kinase complex (Cdk) through multiple mechanisms that lead to both destruction of the cyclin subunit co-activator and direct Cdk kinase inhibition. These multiple mechanisms indicate the importance of regulating the inactivation of Cdk to ensure proper cell cycle progression and cytokinesis. We set out to examine the regulation of the protein phosphatase Cdc14. Cdc14 is thought to act through reversal of phosphorylation on key Cdk substrates that promote mitotic exit by stimulating the destruction and inactivation of Cdk. In Saccharomyces cerevisiae, activation of Cdc14 is achieved via release from its nucleolar inhibitor Net1/Cfi1. This activation is correlated with multi-site phosphorylation of Net1 in cells where Cdc14 appears to be released from the nucleolus. We set out to identify new components of the nucleolar complex known as RENT (Regulator of Nucleolar Silencing and Teleophase) which holds Cdc14 in an inactive state. This led to the identification of Casein Kinase II (CKII) as a new component of RENT. CKII was verified to co-immunoprecipitate with Net1; and mutants in CKII arrest in anaphase with unreleased Cdc14 and unsegregated rDNA. Interestingly, phospho-peptide mapping experiments from in vivo Net1 samples revealed phosphorylation of a CKII consensus sequence within Net1. In vivo mapping also revealed another subset of sites that matched the consensus sequence established for Cdk phosphorylation. Mutational analysis of these sites unveiled their involvement in Cdc14 release during early anaphase and a role for a network of genetically interacting proteins involved in Fourteen Early Anaphase Release (FEAR) in promoting these phosphorylations.
\r\n\r\nIn summary, the regulation of Cdc14 release via phosphorylation of its nucleolar inhibitor Net1 as demonstrated by this work highlights the importance of nucleolar sequesteration and regulated release as a mechanism of controlling important cell cycle factors and events. It also points to a fascinating role for Cdk in insuring its own destruction at the end of the cell cycle, thus promoting transition back into the G1 state.
\r\n" }, { "name": "Baker, Catherine Craig", "degree": "PhD", "year": "2004", "title": "Genetic and Genomic Studies of Shoot and Flower Growth in Arabidopsis", "advisor": "Meyerowitz, Elliot M.", "url": "https://resolver.caltech.edu/CaltechETD:etd-12172003-173451", "creators": [ { "name": { "family": "Baker", "given": "Catherine Craig" }, "id": "Baker-Catherine-Craig", "display_name": "Baker, Catherine Craig" } ], "advisors": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "advisor", "display_name": "Meyerowitz, Elliot M." } ], "committee": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "chair", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/ZZAF-1E26", "abstract": "This thesis is organized around the theme of modulation of transcriptional states in Arabidopsis thaliana. The two particular mechanisms on which this work focuses are (1) microRNA-mediated negative regulation of protein levels (either by mRNA cleavage or by repression of translation) and (2) transduction of extracellular signals into the cell to affect the transcription program.
\r\n\r\nChapter 2 characterizes the role of the EARLY EXTRA PETALS (EEP1) microRNA in the regulation of organ formation in the flower and shoot. The eep1 loss-of-function mutant has extra petals, and it enhances the shoot phenotype of the pinoid mutant, which has defects in auxin signaling and organ formation. EEP1 is nearly identical to a pair of published miRNAs (MIR164a and b); all three are predicted to target the mRNAs of six genes in the NAC family of transcription factors. Two of these genes, CUPSHAPED COTYLEDONS1 and 2 (CUC1 and 2), are redundantly required in flower development. Phenotypic and molecular analysis of lines overexpressing EEP1 are consistent with (1) negative regulation of CUC1 and CUC2 by EEP1 and (2) cleavage of the CUC2 mRNA promoted by EEP1.
\r\n\r\nChapter 3 describes the investigation, by reverse genetics, of five proteins encoded by genes in the CLV3/ERS (CLE) family. Due to the similarity of these proteins to CLAVATA3 (CLV3), the likely secreted ligand for the CLAVATA1 receptor-like kinase, functional analyses were performed in order to determine whether these proteins might also function as ligands for CLV1 or other receptor-like kinases. The results presented here derive from experiments using overexpression, double-stranded RNA interference (dsRNAi), and promoter-glucuronidase (GUS) reporter expression.
\r\n" }, { "name": "Basch, Martin Leandro", "degree": "PhD", "year": "2004", "title": "Early Neural Crest Specification, Induction and Competence", "advisor": "Bronner, Marianne E.", "url": "https://resolver.caltech.edu/CaltechETD:etd-02102004-172305", "creators": [ { "name": { "family": "Basch", "given": "Martin Leandro" }, "id": "Basch-Martin-Leandro", "orcid": "0000-0002-3156-5376", "display_name": "Basch, Martin Leandro" } ], "advisors": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "advisor", "display_name": "Bronner, Marianne E." } ], "committee": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "chair", "display_name": "Fraser, Scott E." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/9SPT-RS22", "abstract": "The neural crest is a transient population of embryonic cells that originates at the border between the neural plate and the non-neural ectoderm. Near the time of neural tube closure, the neural crest go through an epithelial to mesenchymal transition and start an extensive migration throughout the embryo. During migration or shortly after they reached their final position, neural crest cells differentiate to form a wealth of derivatives. The mechanisms of migration and differentiation of neural crest have been vastly studied. Comparatively, much less is known about the embryological origins of the neural crest, and the nature of the interactions that generate them. To clarify the timing and nature of these inductive interactions, I examined the time of competence of the neural plate to become neural crest as well as the time of neural fold specification in chick embryos. The neural plate is competent to respond to inductive interactions with the non-neural ectoderm for a limited period, losing its responsive ability after stage 10. In contrast, non-neural ectoderm from numerous stages retains the potential to induce neural crest cells from competent neural plate. When I tested the ability of neural folds to produce neural crest, I found that folds derived from all rostrocaudal levels of the open neural plate of stage 10 embryos can generate neural crest when cultured in isolation. To further characterize the time of neural crest specification, I isolated regions of the epiblast from stages 3 and 4 embryos and identified a region that is already specified to adopt neural crest fates at the beginning of gastrulation. I describe the early expression pattern of the paired box transcription factor Pax-7, which correlates from stage 4+ onwards with the prospective neural crest forming region. Therefore, I propose that Pax-7 is the earliest neural crest marker described in chick. Furthermore, using a morpholino-based loss of function approach, I show that Pax-7 expression is required during neural crest development in chicks. Taken together, my results suggest that specification of the neural crest begins very early in development and it requires multiple and sustained signals and tissue intractions." }, { "name": "Bernheim, Kyle Alan", "degree": "PhD", "year": "2004", "title": "Functional and Structural Magnetic Resonance Imaging of Humans and Macaques", "advisor": "Andersen, Richard A.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05192004-153312", "creators": [ { "name": { "family": "Bernheim", "given": "Kyle Alan" }, "id": "Bernheim-Kyle-Alan", "display_name": "Bernheim, Kyle Alan" } ], "advisors": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "role": "advisor", "display_name": "Andersen, Richard A." } ], "committee": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "chair", "display_name": "Fraser, Scott E." }, { "name": { "family": "Fineman", "given": "Igor" }, "id": "Fineman-I", "role": "member", "display_name": "Fineman, Igor" }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "role": "member", "display_name": "Andersen, Richard A." }, { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "role": "member", "display_name": "Shimojo, Shinsuke" } ], "option_major": [ "biology" ], "doi": "10.7907/V79Z-V863", "abstract": "Magnetic resonance imaging (MRI) is a technique which finds use in the neurosciences both as an anatomical and functional localization tool. The traditional uses of MRI for structural analysis, such as are commonly found in medicine, can be adapted to serve in place of histological studies for identifying areas of interest in the cortex. Functional MRI (fMRI) is a rapidly developing tangent of MRI which can be used alone or in tandem with classical electrophysiological experiments to investigate neural activity. Although developed intensely for clinical and scientific studies in human subjects, MRI and fMRI have been used increasingly in the non-human primate. This document contains work exemplifying the use of fMRI in both species and methods for pre- and post-surgical anatomical MRI in the non-human primate. Serving as a solid foundation for learning the principles of block-design fMRI, a classic visual illusion, the motion aftereffect, is studied in the human by means of a hemifield visual stimulus using conventional blood oxygen level dependent (BOLD) fMRI. Primary response and levels of motion aftereffect are analyzed in visual cortex, areas pMT and pMST. A novel use of iron oxide nanoparticles as an intravascular contrast agent in the non-human primate is investigated as a method of boosting fMRI contrast, yielding an ultimate gain in contrast-to-noise at the expense of temporal resolution. While anatomical imaging served as a necessary tool for the localization of functional response in the human, further novel techniques were investigated in the non-human primate. A technique for MRI-guided implantation of multiple electrode arrays is considered, to aid the localization of sites of interest in the cortex. The use of MRI as a replacement for histological preparations for purposes of reconstructing electrode penetration sites is documented. These studies exist to aid in bridging the gap between human and non-human MRI and fMRI. Further application of these principles could be extended to the eventual placement of intracortical recording devices in the human, to benefit a patient population needing devices such as a neural prosthesis." }, { "name": "Bush, Eliot Christen", "degree": "PhD", "year": "2004", "title": "Evolution and Scaling in Mammalian Brains", "advisor": "Allman, John Morgan", "url": "https://resolver.caltech.edu/CaltechETD:etd-03292004-144927", "creators": [ { "name": { "family": "Bush", "given": "Eliot Christen" }, "id": "Bush-Eliot-Christen", "orcid": "0000-0001-5476-8005", "display_name": "Bush, Eliot Christen" } ], "advisors": [ { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "advisor", "display_name": "Allman, John Morgan" } ], "committee": [ { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "chair", "display_name": "Allman, John Morgan" }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "role": "member", "display_name": "Perona, Pietro" }, { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "role": "member", "display_name": "Shimojo, Shinsuke" } ], "option_major": [ "biology" ], "doi": "10.7907/P9FG-7K79", "abstract": "Here I look at three stages in the evolutionary development of mammalian brains. Chapter one addresses how connectivity in neocortex scales with brain size. This is of evolutionary interest because it helps define the basic mammalian condition. Neocortical white matter increases disproportionately in large brains. This might reflect increases in the number of connections per neuron. It might also reflect scaling in axon diameter. I compare these hypotheses by examining white matter-gray matter scaling in cerebellum. Because the white matter of cerebellum lacks cortico-cortical connections, the connectivity theory predicts that cerebellar white matter should not hyperscale relative to gray matter. I have measured white matter and gray matter volume in a large sample of mammals and I find that cerebellar white matter does not hyperscale. This supports the proposition that neocortical hyperscaling reflects an increase in the number of connections per neuron in large brains.
\r\n\r\nIn chapter two I use independent contrasts analysis to examine the scaling of frontal cortex in a large sample of mammals. I find significant differences in scaling between primates and carnivores. Primate frontal cortex hyperscales relative to the rest of neocortex and the rest of the brain, and the primate slope is significantly greater than that for carnivores. This suggests that there are substantial differences in frontal cortex structure and development between the two groups. Combined with with anatomical differences, it suggests that primates have evolved a number of unique adaptations in frontal cortex.
\r\n\r\nChapter three examines the evolution of brain size in anthropoid primates. Living anthropoids have larger brains than strepsirrhines. What about early anthropoid fossils? I measure brain size in the early anthropoid Parapithecus grangeri using computed tomography. I find that relative to the living anthropoids, Parapithecus had a small brain for its body size. Thus large brains did not develop at the same time as a number of other anthropoid adaptations.
" }, { "name": "Giannetti, Anthony Michael", "degree": "PhD", "year": "2004", "title": "Biochemical, Biophysical, and Cellular Investigations of the Interactions of Transferrin Receptor with Transferrin and the Hereditary Hemochromatosis Protein, HFE", "advisor": "Bjorkman, Pamela J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05262004-173612", "creators": [ { "name": { "family": "Giannetti", "given": "Anthony Michael" }, "id": "Giannetti-Anthony-Michael", "display_name": "Giannetti, Anthony Michael" } ], "advisors": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "advisor", "display_name": "Bjorkman, Pamela J." } ], "committee": [ { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "chair", "display_name": "Rees, Douglas C." }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "member", "display_name": "Chan, David C." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Roberts", "given": "Richard W." }, "id": "Roberts-R-W", "role": "member", "display_name": "Roberts, Richard W." } ], "option_major": [ "biochem" ], "doi": "10.7907/06QJ-JW93", "abstract": "Hereditary hemochromatosis (HH) is a prevalent genetic disorder that results in the daily excess absorption of dietary iron. If untreated this disease leads to systemic organ failure and death. HH is caused by mutations to the gene coding for a protein called HFE, a type I transmembrane glycoprotein with a demonstrated role in regulating cellular iron homeostasis. HFE binds to the cell-surface receptor transferrin receptor (TfR), a dimeric type II transmembrane glycoprotein responsible for iron uptake into most mammalian cell types. TfR binds iron-loaded transferrin (Fe-Tf) from the blood and transports it to acidic recycling endosomes where iron is released from Fe-Tf in a TfR-facilitated process. Iron-free transferrin (apo-Tf) remains bound to TfR and is recycled to the cell surface, where apo-Tf rapidly dissociates from TfR upon exposure to the basic pH of blood. HFE and Fe-Tf can bind simultaneously to TfR to form a ternary complex, but HFE binding to TfR lowers the apparent affinity of the Fe-Tf/TfR interaction. This reduction could result from direct competition between HFE and Fe-Tf for receptor binding sites, from negative cooperativity, or both. We sought to understand the mechanism of HFE, Fe-Tf, and apo-Tf binding by TfR to help define HFE's role in iron homeostasis. We determined the binding constants for HFE, Fe-Tf, and apo-Tf to an extensive set of site-directed TfR mutants and discovered that HFE and Tf bind to an overlapping site on TfR, indicating the two proteins compete with each other for receptor binding. The mutagenesis results also identified differences in the contact points between TfR and the two forms of Tf, Fe-Tf and apo-Tf. By combining the mutations that are required for apo-Tf, but not Fe-Tf, binding we find that a highly conserved hydrophobic patch on the TfR surface is required for the receptor-mediated stimulation of iron release from Fe-Tf. From these data we propose a structure-based model for the mechanism of TfR-assisted iron release.
\r\n\r\nTo explore the mechanism of the HFE-induced affinity reduction for Fe-Tf binding by TfR, we engineered a heterodimeric TfR (hdTfR) that contains mutations such that one TfR chain binds only HFE and the other binds only Fe-Tf. Competition binding experiments using hdTfR demonstrate that TfR does not exhibit cooperativity in heterotropic ligand binding, suggesting that some or all of HFE's effects on iron homeostasis result from competition with Fe-Tf for TfR binding. Using transfected cell lines we show that HFE is dependent on its interactions with TfR for transport to endosomal compartments and that competition with extracellular Fe-Tf can alter HFE trafficking patterns. These data suggest that HFE's role in iron homeostasis is as a sensor of body iron status.
" }, { "name": "Gong, Ying", "degree": "PhD", "year": "2004", "title": "Cell Polarity and Morphogenesis: Functions and Mechanisms of Cell Divisions in Vertebrate Gastrulation", "advisor": "Fraser, Scott E.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06052004-132200", "creators": [ { "name": { "family": "Gong", "given": "Ying" }, "id": "Gong-Ying", "display_name": "Gong, Ying" } ], "advisors": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "advisor", "display_name": "Fraser, Scott E." } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biochem" ], "doi": "10.7907/5TZ8-W842", "abstract": "Gastrulation shapes a vertebrate embryo from an egg-shaped aggregate of cells. In many vertebrates, massive cell divisions occur during gastrulation. In this thesis, I investigate the pattern, function, and regulation of mitotic divisions in zebrafish gastrulation. Using in vivo confocal imaging and quantitative analysis, I find that cells in dorsal axial tissues preferentially divide along the direction of tissue elongation, i.e., the anterior-posterior axis of the embryo. Establishment of the spindle polarity requires Silberblick/Wnt11, Dishevelled and Strabismus acting via the non-canonical Wnt/planar cell polarity (Wnt/PCP) pathway. On the subcellular level, oriented cell division is mediated by spindle rotation. The mitotic spindle forms at a random orientation and rotates at metaphase to line up with the anterior-posterior axis. Wnt/PCP signalling is not required for the spindle to rotate but dictates its destination. These data, together with previous work by others, demonstrate that cell polarization underlies the morphogenetic machinery that shapes the head-to-tail axis. In addition, Wnt/PCP signalling is involved in polarizing the cells, whose responses include medial-lateral elongation of the cell body and localization of protrusions, and anterior-posterior positioning of the mitotic spindle. These two types of polarized cell behaviours cooperate to shape the anterior-posterior body axis of the embryo. This work also demonstrates that the Wnt/PCP pathway is evolutionarily conserved as a strategy for cell polarization.\r\n" }, { "name": "Huang, Po-Ssu", "degree": "PhD", "year": "2004", "title": "Computational Design and Experimental Characterization of Protein Oligomers", "advisor": "Mayo, Stephen L.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06012004-214823", "creators": [ { "name": { "family": "Huang", "given": "Po-Ssu" }, "id": "Huang-Po-Ssu", "orcid": "0000-0002-7948-2895", "display_name": "Huang, Po-Ssu" } ], "advisors": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "advisor", "display_name": "Mayo, Stephen L." } ], "committee": [ { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "chair", "display_name": "Rees, Douglas C." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Roberts", "given": "Richard W." }, "id": "Roberts-R-W", "orcid": "0000-0002-8587-5097", "role": "member", "display_name": "Roberts, Richard W." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." } ], "option_major": [ "biochem" ], "doi": "10.7907/3DZW-2R54", "abstract": "Previous efforts in designing protein binding interfaces have focused on altering binding specificities. These methods fall short, however, when applied to the design of novel binding sites due to difficulties in accurately modeling protein backbones. The goal of this project is to create dimers from monomeric proteins. We developed a special docking algorithm that positions the member protein subunits to a plausible configuration with respect to each other using parameters determined from known complex structures. The docking procedure treats the proteins as rigid bodies and uses Fourier correlation theorem and fast Fourier transform to efficiently search for dimers with the highest interfacial surface complementarities. Using the docked structures as scaffolds for design and employing hydrophobic surface residues to drive dimer formation, we have demonstrated two successful designs, one heterodimer and one homodimer, using protein G and engrailed homeodomain respectively as the starting monomeric proteins. The designed dimers were characterized using circular dichroism, nuclear magnetic resonance, analytical ultracentrifugation, and X-ray crystallography methods. This is the first report of computationally designed de novo protein homodimers generated using a combination of protein docking and protein design tools. These results suggest that this strategy can be used to address the protein recognition problem, and is generally applicable to creating novel binding sites with compatible binding partners." }, { "name": "Meulemans, Daniel Keith", "degree": "PhD", "year": "2004", "title": "Genetic Correlates of Neural Crest Evolution", "advisor": "Bronner, Marianne E.", "url": "https://resolver.caltech.edu/CaltechETD:etd-09292003-170037", "creators": [ { "name": { "family": "Meulemans", "given": "Daniel Keith" }, "id": "Meulemans-Daniel-Keith", "orcid": "0000-0002-8182-6028", "display_name": "Meulemans, Daniel Keith" } ], "advisors": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "advisor", "display_name": "Bronner, Marianne E." } ], "committee": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "chair", "display_name": "Fraser, Scott E." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." } ], "option_major": [ "biology" ], "doi": "10.7907/XGSJ-Y873", "abstract": "Neural crest cells are unique to vertebrates and generate most of the adult structures that distinguish them from their closest invertebrate relatives, the cephalochordates. To elucidate the molecular bases of neural crest evolution, I analyzed the expression, function, and cis-regulation of amphioxus genes with vertebrate homologs having established roles in neural crest development. By comparing these amphioxus genes with their agnathan and gnathostome homologs, I have uncovered genetic changes coincident with, and potentially causal to, the origins of neural crest. I demonstrate that three transcriptional regulators involved with neural crest development, AP-2, Id, and SoxE, were recruited to the neural plate border early in vertebrate evolution? implying that genetic cooption of high-order transcription factors was a major driving force in neural crest evolution. I also show that the function of the Snail protein in establishing the neural plate border was not significantly altered during vertebrate evolution, although vertebrate Snail genes may have evolved novel domains necessary for later functions in neural crest cells. Finally, I began characterizing the cis-regulation of vertebrate and amphioxus Slug/Snail orthologs to determine if divergent aspects of Snail gene expression (i.e., expression in neural crest cells) are reflected in structural differences in Snail cis-regulatory DNA. Using this 3-tiered approach I have begun to define the novel genetic regulatory interactions that drove the evolution of neural crest cells in the vertebrate lineage." }, { "name": "Perez-Orive, Javier", "degree": "PhD", "year": "2004", "title": "Neural Oscillations and the Decoding of Sensory Information", "advisor": "Laurent, Gilles J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05242004-174642", "creators": [ { "name": { "family": "Perez-Orive", "given": "Javier" }, "id": "Perez-Orive-Javier", "orcid": "0000-0002-4799-0326", "display_name": "Perez-Orive, Javier" } ], "advisors": [ { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "role": "advisor", "display_name": "Laurent, Gilles J." } ], "committee": [ { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "chair", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Siapas", "given": "Athanassios G." }, "id": "Siapas-A-G", "role": "member", "display_name": "Siapas, Athanassios G." }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "role": "member", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "role": "member", "display_name": "Perona, Pietro" } ], "option_major": [ "cns" ], "doi": "10.7907/8VSB-VD89", "abstract": "An important problem in neuroscience is to understand how the brain encodes information. A hypothesis is that differences in the timing of action potentials, reflecting synchronization changes among neuronal ensembles --often occurring in the context of oscillations-- can be meaningful to downstream neurons detecting coincident input. Several properties, such as active conductances, feedforward inhibition and oscillatory input, could potentially influence whether a neuron acts as a coincidence detector. Although different neural circuits in various animal groups will use different strategies to solve somewhat varying problems, there will also be many powerful solutions to coding problems that will be used repeatedly across diverse processing stages and animal phyla. The insect olfactory system, sharing many design similarities with other systems while having a reduced complexity, provides an excellent model in which to study the functional interactions of all these coding features.
\r\n\r\nThis dissertation focuses on the decoding of olfactory information by the mushroom body (MB), the second relay of the insect olfactory system, which receives oscillating input from the antennal lobe (the first relay, analogous to the vertebrate olfactory bulb). Kenyon cells (KCs), the intrinsic neurons of the MB, are found to respond very specifically to odors. These responses typically consist of one or two reliable action potentials, phase-locked to the global oscillations, over extremely low baseline firing rates. This leads to a dramatic sparsening of the olfactory representation in the MB. Several circuit and intrinsic properties are found to take part in this transformation. Feedforward inhibition contributes to odor specificity and sparseness: blocking inhibitory input to the KCs broadened their odor tuning and abolished their phase-locking, supporting the idea that feedforward inhibition limits the temporal window over which KCs integrate their inputs. Voltage-dependent conductances contribute to a supralinear summation of coincident postsynaptic potentials and a reduction of their half-widths, indicating that KC intrinsic properties further contribute to coincidence detection. Taken together, these results indicate that oscillations serve as a framework on which KCs act as coincidence detectors and sparsen the olfactory representation. Abolishing the input oscillations disrupts KC odor responses, decreasing their specificity and the sparseness in the MB.
\r\n\r\nThe work in this dissertation describes a mechanism for decoding timing information and indicates that not all spikes are equally relevant to downstream neurons, their specific relevance depending on whether they are correlated, within a specific phase of an oscillation cycle, with other input spikes. These general features can also provide useful insights into neural coding in more complex neural systems, where all the mechanisms described here have been separately observed. This work illustrates how these mechanisms can interact to code sensory information and bring about drastic transformations of neural representations, increasing our understanding of how nervous systems can process information.
" }, { "name": "Peters, Robert Jacob", "degree": "PhD", "year": "2004", "title": "Visual, Attention and Object Categorization: From Psychophysics to Computational Models", "advisor": "Koch, Christof", "url": "https://resolver.caltech.edu/CaltechETD:etd-06062004-213811", "creators": [ { "name": { "family": "Peters", "given": "Robert Jacob" }, "id": "Peters-Robert-Jacob", "display_name": "Peters, Robert Jacob" } ], "advisors": [ { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "advisor", "display_name": "Koch, Christof" } ], "committee": [ { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "chair", "display_name": "Koch, Christof" }, { "name": { "family": "Itti", "given": "Laurent" }, "id": "Itti-L", "role": "member", "display_name": "Itti, Laurent" }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "member", "display_name": "Perona, Pietro" }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "member", "display_name": "Andersen, Richard A." }, { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "orcid": "0000-0002-1290-5232", "role": "member", "display_name": "Shimojo, Shinsuke" } ], "option_major": [ "cns" ], "doi": "10.7907/RM8K-2439", "abstract": "This thesis is arranged in two main parts. Each part relies an approach using the methods of psychophysics and computational modeling to bring abstract or high-level theories of vision closer to a concrete neurobiological foundation.\r\n\r\nThe first part addresses the topic of visual object categorization. Previous studies using high-level models categorization have left unresolved issues of neurobiological relevance, including how features are extracted from the image and the role played by memory capacity in categorization performance. We compared the ability of a comprehensive set of models to match the categorization performance of human observers while explicitly accounting for the models' numbers of free parameters. The most successful models did not require a large memory capacity, suggesting that a sparse, abstracted representation of category properties may underlie categorization performance. This type of representation--different from classical prototype abstraction--could also be extracted directly from two-dimensional images via a biologically plausible early vision model, rather than relying on experimenter-imposed features.\r\n\r\nThe second part addresses visual attention in its bottom-up, stimulus-driven form. Previous research showed that a model of bottom-up visual attention can account in part for the spatial positions of locations fixated by humans while free-viewing complex natural and artificial scenes. We used a similar framework to quantify how the predictive ability of such a model may be enhanced by new model components based on several specific mechanisms within the functional architecture of the visual system. These components included richer interactions among orientation-tuned units, both at short-range (for clutter reduction) and at long-range (for contour facilitation). Subjects free-viewed naturalistic and artificial images while their eye movements were recorded. The resulting fixation locations were compared with the models' predicted salience maps. We found that each new model component was important in attaining a strong quantitative correspondence between model and behavior. Finally, we compared the model predictions with the spatial locations obtained from a task that relied on mouse clicking rather than eye tracking. As these models become more accurate in predicting behaviorally-relevant salient locations, they become useful to a range of applications in computer vision and human-machine interface design.\r\n" }, { "name": "Sakamoto, Kathleen Miho", "degree": "PhD", "year": "2004", "title": "Targeting Proteins for Ubiquitination and Degradation in the Treatment of Human Disease", "advisor": "Deshaies, Raymond Joseph", "url": "https://resolver.caltech.edu/CaltechETD:etd-12292003-134700", "creators": [ { "name": { "family": "Sakamoto", "given": "Kathleen Miho" }, "id": "Sakamoto-Kathleen-Miho", "orcid": "0000-0003-0494-8838", "display_name": "Sakamoto, Kathleen Miho" } ], "advisors": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "advisor", "display_name": "Deshaies, Raymond Joseph" } ], "committee": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "chair", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "member", "display_name": "Baltimore, David L." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "role": "member", "display_name": "Dunphy, William G." } ], "option_major": [ "biology" ], "doi": "10.7907/2RN6-TE18", "abstract": "Protein degradation is one of the tactics employed by the cell for irreversibly inactivating proteins. In eukaryotes, ATP-dependent protein degradation in the cytoplasm and nucleus is carried out by the 26S proteasome. Most proteins are targeted to the 26S proteasome by covalent attachment of a multiubiquitin chain. A key component of the enzyme cascade that results in attachment of the multiubiquitin chain to the target or labile protein is the ubiquitin ligase that controls the specificity of the ubiquitination reaction. Defects in ubiquitin-dependent proteolysis have been shown to result in a variety of human diseases, including cancer, neurodegenerative diseases, and metabolic disorders.
\r\n\r\nThe SCF (Skp1-Cullin-F-box-Hrt1) complex is a heteromeric ubiquitin ligase that multiubiquitinates proteins important for signal transduction and cell cycle progression. A technology was developed known as Protac (Proteolysis Targeting Chimeric Molecule), that acts as a bridge, bringing together the SCF ubiquitin ligase with a protein target, resulting in its ubiquitination and degradation. The Protac contains a peptide moiety at one end that is recognized by SCF that is chemically linked to the binding partner or ligand of the target protein. The first demonstration of the efficacy of Protac technology was the successful recruitment, ubiquitination, and degradation of the protein Methionine Aminopeptidase-2 (MetAP-2) through a covalent interaction between MetAP-2 and Protac. Subsequently, we demonstrated that Protacs could effectively ubiquitinate and degrade cancer-promoting proteins (estrogen and androgen receptors) through non-covalent interactions in vitro and in cells. Finally, cell-permeable Protacs can also promote the degradation of proteins in cells. Biologically, this work signifies the amazing versatility and flexibility of the ubiquitin-proteasome system. Technologically, this work represents the development of a novel ?chemical genetics? approach to selectively target proteins for degradation. Practically, this work is ?Proof of Concept? that exploiting the cell?s natural proteolytic machinery is a potential avenue for the treatment of human disease.
" }, { "name": "Smith, W. Bryan", "degree": "PhD", "year": "2004", "title": "Local Control of Synaptic Strength: Neurotrophic and Dopaminergic Modulation of Dendritic Protein Synthesis", "advisor": "Schuman, Erin Margaret", "url": "https://resolver.caltech.edu/CaltechETD:etd-05262004-125208", "creators": [ { "name": { "family": "Smith", "given": "W. Bryan" }, "id": "Smith-W-Bryan", "display_name": "Smith, W. Bryan" } ], "advisors": [ { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "advisor", "display_name": "Schuman, Erin Margaret" } ], "committee": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "chair", "display_name": "Lester, Henry A." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "member", "display_name": "Schuman, Erin Margaret" }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "member", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "member", "display_name": "Kennedy, Mary B." } ], "option_major": [ "appmath" ], "doi": "10.7907/15vm-zk49", "abstract": "Understanding the cell biological mechanisms responsible for the plasticity of central neurons is key to our understanding of brain function. In an effort to better characterize the cell and molecular biology of individual neurons, we have studied the role of local, dendritic protein synthesis in hippocampal synaptic plasticity. Such a localized control of protein synthesis provides a means of achieving input-specificity, the observation that synapses on a given neuron are able to independently scale the strength of their connections. This property of synaptic enhancement is correlated with the encoding capacity of an individual cell: the greater the input specificity, the more information a cell can encode.
\r\n\r\nHere we show two pathways for inducing local protein synthesis, one mediated by the brain-derived neurotrophic factor, and another by the D1/D5 dopaminergic signaling system. Local protein synthesis stimulated by the dopaminergic pathway leads directly to an increase in synaptic strength by increasing production and synaptic localization of AMPA receptors, the glutamate-gated ion channels that mediate fast synaptic transmission at central synapses. We also present a set of software tools for quantitative analysis of 3-D colocalization and 2-D spatial correlation in immunofluorescence microscopy. These tools will greatly assist in exploratory data analysis of the dynamics of protein distributions in neurons and other cells.
" }, { "name": "V\u00e1zquez, Luis Enrique", "degree": "PhD", "year": "2004", "title": "SynGAP Controls Synapse Formation by Regulating Spine Development and Morphology", "advisor": "Kennedy, Mary B.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06012004-144341", "creators": [ { "name": { "family": "V\u00e1zquez", "given": "Luis Enrique" }, "id": "V\u00e1zquez-Luis-Enrique", "orcid": "0000-0003-0197-2058", "display_name": "V\u00e1zquez, Luis Enrique" } ], "advisors": [ { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "display_name": "Kennedy, Mary B." } ], "committee": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "chair", "display_name": "Fraser, Scott E." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "role": "member", "display_name": "Kennedy, Mary B." } ], "option_major": [ "biology" ], "doi": "10.7907/v7bp-jb59", "abstract": "SynGAP is a brain-specific Ras GTPase-activating protein that is an abundant component of the signaling complex associated with the NMDA-type glutamate receptor. We generated mutant mice lacking synGAP to study its physiological role. Homozygous mutant mice die in the first few days after birth; however, neurons from mutant embryos can be maintained in culture. Here we report that spine maturation and synapse formation are accelerated in cultured mutant neurons, and the spines of mature mutant neurons are significantly larger than those of wild type. Clusters of PSD-95, and subunits of AMPA-type and NMDA-type glutamate receptors are larger and brighter, and appear in spines of mutant neurons by day 10 in vitro; whereas in wild-type neurons they are still mostly located in dendritic shafts. The frequency and amplitude of miniature excitatory postsynaptic currents are larger in mutant neurons at day 10 in vitro, confirming that they have more functional synapses, with more AMPA receptors in them. At day 21 in vitro, the spines of mutant neurons remain significantly larger than those of wild type. The mutant phenotype at day 10 in vitro can be rescued by introduction of recombinant wild-type synGAP on day 9. In contrast, introduction of synGAP with a mutated GAP domain or a deletion of the terminal domain that binds to PSD-95 does not rescue the mutant phenotype, indicating that both domains play a role in control of spine maturation. Thus, the GAP activity of synGAP, as well as its association with PSD-95, is important for normal regulation of spine and synapse maturation in hippocampal neurons." }, { "name": "Yang, Lili", "degree": "PhD", "year": "2004", "title": "Towards Engineering Immunity", "advisor": "Baltimore, David L.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06042004-000641", "creators": [ { "name": { "family": "Yang", "given": "Lili" }, "id": "Yang-Lili", "display_name": "Yang, Lili" } ], "advisors": [ { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "advisor", "display_name": "Baltimore, David L." } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "chair", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "member", "display_name": "Baltimore, David L." }, { "name": { "family": "Alberola-Ila", "given": "Jose" }, "id": "Alberola-Ila-J", "role": "member", "display_name": "Alberola-Ila, Jose" }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/XTDP-SC35", "abstract": "The aim of engineering immunity is to harness and engineer the immune system to treat infectious diseases and cancer. Towards this goal, accumulating evidence shows that the immune system can be manipulated to achieve the desired and improved functions. In the context of cancer therapy, many strategies have appeared to utilize the principle of immune defense to safely and effectively target tumor cells for destruction. These strategies fall into two categories: active immunotherapy and passive immunotherapy. Active immunotherapy involves activating the effectors in the host immune system to inhibit cancer cell growth and reject tumors (e.g., cancer vaccination), while passive immunotherapy is a term for directly providing the host with effectors to react against cancer (e.g., adoptive transfer of in vitro expanded antitumor T cells).
\r\n\r\nWe propose a concept of instructive immunotherapy for cancer. This concept is to use a strategy to guide the host in developing in vivo effector cells capable of targeting cancer. This strategy arises from combination of gene therapy, stem cell therapy and immunotherapy to program hematopoietic stem cells (HSCs) to develop into lymphocytes with desired antitumor specificity. Therefore, taking advantage of the longevity and self-renewal of HSCs, life-long supplies of tumor-specific lymphocytes can be generated in vivo, which exceed the current methods of repetitive immunization and adoptive transfer.
\r\n\r\nTo test the feasibility of this approach, I describe in Chapter 2 the procedure of retrovirus-mediated gene transfer of TCR cDNA into RAG1-deficient HSCs. Subsequent transfer of these genetic modified HSCs into RAG1-deficient mice allows the long-term production of functional antigen-specific T cells.
\r\n\r\nChapter 3 describes a method to impart anti-tumor specificity to the wild-type mouse T cell repertoire. To achieve this, genes encoding a CD8 T cell receptor with the desired anti-tumor specificity were delivered into wild-type HSCs via a retroviral vector. When transferred into host mice, these genetically modified HSCs generated a large population of anti-tumor cytotoxic T cells, accounting for more than 20% of peripheral CD8 T cells. These cells displayed a normal response to antigen stimulation and had the ability to generate and maintain long-term memory. Significant tumor rejection was observed in mice containing these T cells, demonstrating feasibility of instructive cancer immunotherapy.
\r\n\r\nIn recognition of the important roles of helper T cells in anti-tumor immunity, Chapter 4 elaborates a two-arm model to augment tumor-specific immune responses. In the experiment, the two arms, both anti-tumor CD4- and CD8 T cells, were generated by HSC gene transfer method. The resultant immune system in mice could not only suppress tumor growth, but could also eradicate large, solid and vascularized tumors. Coupled with results described in Chapter 3, we demonstrated the great potential of instructive cancer immunotherapy and expanded the scope of engineering immunity.
\r\n\r\nSuccessful immunotherapy relies on understanding the molecular mechanisms that control immune responses. For instance, although IL-2 has been approved by FDA to treat renal cancer and melanoma, many results from mice show that the physiological role of IL-2 is complex and unpredictable, hindering the design of better strategies, that would maximize the therapeutic impact of IL-2. I address the role of IL-2 in negative regulatory function and T cell memory in last two chapters, both of which are important for achieve the overall success of immunotherapy and engineering immunity. Chapter 5 describes the role of IL-2 in maintaining regulatory T cell homeostasis and self-tolerance, and correlates this role with the signaling molecule STAT5. The final chapter (Chapter 6) details the role of IL-2 in generation of CD4 T cell memory.
" }, { "name": "Bui, Y\u00ea\u0144 Kim", "degree": "PhD", "year": "2003", "title": "Genetic Analysis of LET-23 Mediated IP\u2083 Signaling in Caenorhabditis elegans", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechETD:etd-09092002-222935", "creators": [ { "name": { "family": "Bui", "given": "Y\u00ea\u0144 Kim" }, "id": "Bui-Y\u00ea\u0144-Kim", "display_name": "Bui, Y\u00ea\u0144 Kim" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "member", "display_name": "Schuman, Erin Margaret" }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "orcid": "0000-0002-3671-9354", "role": "member", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biology" ], "doi": "10.7907/92D0-6V64", "abstract": "Our understanding of signal transduction has increased from the use of genetically tractable organisms combined with biochemical anaylsis in cell culture. An example is LET-23 receptor tyrosine kinase signaling in Caenorhabditis elegans. The epidermal growth factor receptor homolog, LET-23 RTK, mediates multiple functions: development of the male tale, vulva induction, viability, and fertility. One of the ways in which activation of the same receptor can generate a specific response is through distinct signaling pathways downstream. Fertility is mediated by a RAS-independent inositol 1,4,5- triphopshate (IP\u2083) signaling pathway downstream of LET-23 activation.
\r\n\r\nIn this thesis, I take a genetic route to the analysis of IP\u2083 signaling in C. elegans, which mediates ovulation in the fertility pathway. Genetic screens for suppressor or enhancers of mutant phenotypes remain an imporant tool for dissecting signaling pathways. They can uncover new genes or new mutations in existing genes, which upon analysis, will help us understand more about how that particular protein functions. I describe a genetic screen to identify genes that act to suppress the ovulation/sterility defects associated with both a a gain of function in the IP\u2083 receptor and loss of function in the IP\u2083 3-kinase. Initial characterization of four suppressors identified are reported. Disruption of genes identified by genome sequencing has allowed us to determine whether a protein is essential for a given response. The importance of regulating IP\u2083 levels is ilustrated by the complexity of kinases and phosphatases that metabolize IP\u2083. Nomarksi video microscopy analysis shows the C. elegans IP\u2083 3-kinase defective mutant, lfe-2, has no ovulation phenotype. Using reverse genetics, I targeted a deletion in the C. elegans 5-phosphatase, ipp-5, and demonstrate that IPP-5 plays a critical negative regulatory function for distal spermatheca contraction behavior. Evidence for levels of IP\u2083 signaling regulating spermatheca contractions, which affects fertitlity, comes from my analysis of multiple mutants that perturb IP\u2083 signaling. The work presented in this thesis provide the most extensive genetic analysis of IP\u2083 signaling to date. These results imply thresholds are important for achieving an appropriate response. Finally, I present the genetic characterization of a novel phospholipase C, Ce PLC210 and implicate its critical function for regulating spermatheca-uturine valve contraction behavior. A multitude of proteins is involved in generating a precise biological response.
" }, { "name": "Burkemper, Bruce Seymour", "degree": "PhD", "year": "2003", "title": "Biochemical Characterization of Drosophila Receptor Tyrosine Phosphatases", "advisor": "Zinn, Kai George", "url": "https://resolver.caltech.edu/CaltechETD:etd-03212003-142207", "creators": [ { "name": { "family": "Burkemper", "given": "Bruce Seymour" }, "id": "Burkemper-Bruce-Seymour", "orcid": "0000-0003-3689-7481", "display_name": "Burkemper, Bruce Seymour" } ], "advisors": [ { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "advisor", "display_name": "Zinn, Kai George" } ], "committee": [ { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "chair", "display_name": "Zinn, Kai George" }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "role": "member", "display_name": "Kennedy, Mary B." } ], "option_major": [ "biology" ], "doi": "10.7907/P1HY-MF28", "abstract": "Two classes of enzymes are responsible for modulation of intracellular phosphotyrosine levels, namely protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Together these enzymes maintain the appropriate balance of phosphoproteins required for a variety of developmental processes including axon pathfinding. In Drosophila, five receptor-like protein tyrosine phosphatases (RPTPs) regulate axon pathfinding, but little is known about their downstream signaling pathways or the means by which their enzymatic activity is regulated.
\r\n\r\nChapter 2 of this thesis deals with experiments to test whether dimerization regulates the activity of these enzymes. Crystallographic data indicates that some RPTPs form dimers in which each monomer is precluded from binding substrate due to the insertion of a helix-turn-helix segment of the opposing monomer into the active site. I introduced ?tagged? RPTP constructs into Drosophila S2 tissue culture cells and tested for dimer formation using immunoprecipitation and Western blotting. I did not detect stable dimers, however. This may suggest that dimer formation requires other protein components (such as the putative RPTP ligands) that are not expressed in S2 cells.
\r\n\r\nIn Chapter 3 I investigated the possibility that Roundabout (Robo), a receptor mediating axonal repulsion from the embryonic midline, is a substrate for RPTPs DPTP69D and/or DPTP10D. Previous genetic studies implicate these RPTPs in participating in the Robo signaling pathway. Experiments detailed here show that Robo can be phosphorylated on tyrosine residues in S2 cells, characteristic of an RPTP substrate. However, Robo did not co-immunoprecipitate with ?substrate trap? mutants of either of these RPTPs, possibly because their interaction is dependent on co-factors not present in the cell culture system.
\r\n\r\nChapter 4 is a characterization of DPTP69D-associated proteins purified from embryos expressing a substrate trap version of DPTP69D. We identified one of the associated proteins as non-muscle myosin II heavy chain (nmm II hc). Proper regulation of nmm II hc is essential for axon patterning in mushroom bodies (MBs). I found that expression of the DPTP69D trap in MBs results in an axon retraction phenotype similar to that seen when nmm II hc activity is elevated, suggesting that this protein may be a target for DPTP69D activity.
" }, { "name": "Chang, Grace C.", "degree": "PhD", "year": "2003", "title": "Neural Representation of Surface Ordering in Visual Areas V1, V2 and MT", "advisor": "Koch, Christof", "url": "https://resolver.caltech.edu/CaltechETD:etd-05302003-111238", "creators": [ { "name": { "family": "Chang", "given": "Grace C." }, "id": "Chang-Grace-C", "display_name": "Chang, Grace C." } ], "advisors": [ { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "advisor", "display_name": "Koch, Christof" } ], "committee": [ { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "chair", "display_name": "Koch, Christof" }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "member", "display_name": "Perona, Pietro" }, { "name": { "family": "Albright", "given": "Thomas" }, "id": "Albright-T", "role": "member", "display_name": "Albright, Thomas" } ], "option_major": [ "cns" ], "doi": "10.7907/3SS9-A441", "abstract": "Visual cortical areas V1, V2 and MT may participate in the representation of surface ordering, the arrangement of one surface in front of another. This work investigates the role of neurons of V1 and V2 in figure-ground representation in static stimuli, as well as the role of MT in surface ordering in dynamic stimuli.
\r\n\r\nElectrical recordings were made in V1 and V2 to determine whether neurons in these areas encode information about the identities of figure and ground, and also whether they respond to figure-ground cues. We recorded from 3 monkeys, one trained on a fixation task, and the other two on a match-to-sample task that ensured attention to the stimuli. The stimuli consisted of rectangles of differing contrast arranged in an unambiguous or ambiguous figure-ground configuration. The stimuli were positioned such that the cells' receptive fields were located either at the border between rectangles or in the interiors of rectangles. Cells demonstrating selectivity at borders or interiors of unambiguous figure-ground stimuli were considered selective for border ownership or figure vs. ground, respectively. Cells showing selectivity at borders or interiors of ambiguous figure-ground stimuli were considered selective for figure-ground cues.
\r\n\r\nPreliminary experiments on the fixating monkey suggested that a small fraction of cells in V1 and V2 might play a role in figure-ground interpretation. The results from the awake behaving monkeys further support the hypothesis that V1 and V2 play a role in figure-ground perception. In both areas we found cells demonstrating selectivity for border ownership, and in V2 we found cells demonstrating selectivity for figure over ground. However, in V1 and V2 there was also evidence a separate population was responding to the presence of figure-ground cues in the stimulus.
\r\n\r\nThe experiments in MT were performed on two awake behaving monkeys. The stimuli were transparent rotating cylinders comprised of random dots moving along a sinusoidal gradient. The stimuli were bistable?perceived to rotate in one direction or its opposite. The monkeys indicated in which direction they perceived the cylinder?s front surface rotating. Cells were found whose firing correlated with the monkeys? bistable percept, even though the stimuli were identical.
" }, { "name": "Datta, Deepshikha", "degree": "PhD", "year": "2003", "title": "Protein-Ligand Interactions: Docking, Design and Conformation Change", "advisor": "Mayo, Stephen L.", "url": "https://resolver.caltech.edu/CaltechETD:etd-03242003-111426", "creators": [ { "name": { "family": "Datta", "given": "Deepshikha" }, "id": "Datta-Deepshikha", "display_name": "Datta, Deepshikha" } ], "advisors": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "advisor", "display_name": "Mayo, Stephen L." } ], "committee": [ { "name": { "family": "Goddard", "given": "William A., III" }, "id": "Goddard-W-A-III", "orcid": "0000-0003-0097-5716", "role": "chair", "display_name": "Goddard, William A., III" }, { "name": { "family": "Tirrell", "given": "David A." }, "id": "Tirrell-D-A", "orcid": "0000-0003-3175-4596", "role": "member", "display_name": "Tirrell, David A." }, { "name": { "family": "Roberts", "given": "Richard W." }, "id": "Roberts-R-W", "orcid": "0000-0002-8587-5097", "role": "member", "display_name": "Roberts, Richard W." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." } ], "option_major": [ "biochem" ], "doi": "10.7907/36FS-2262", "abstract": "Virtual ligand screening has proven to be a successful strategy in drug design. An in house-developed procedure (HierDock), a coarse grain docking method followed by a fine grain search procedure, was used to determine the binding site for sugars in the outer membrane protein A in E.coli, a key interaction in the pathogenesis of neonatal meningitis. These results are being further extended in suggesting possible peptide antagonists and drugs for therapeutic strategies.
\r\n\r\nPrediction of binding site of ligands in proteins, starting with the apo-protein is one of the challenges in the field of virtual ligand screening. HeirDock was modified for accurately predicting the ligand binding sites in apo-proteins that undergoes significant structural changes on binding to a ligand. The method was evaluated for finding the binding site for methionine in methionyl tRNA synthetase. We followed up on our understanding of binding mechanism in aminoacyl tRNA synthetases by attempting to design these enzymes to bind to non-natural amino acids. Using the computational protein design software (ORBIT), a phenylalanyl-tRNA synthetase variant that allows efficient in vivo incorporation of aryl ketone functionality into proteins was designed.
\r\n\r\nLigand-induced conformation changes are commonly seen in proteins. We have developed a procedure by combining computational protein design with methods from mean-field theory to design protein sequences capable of switching between two completely different protein folds on chelating to metal. This method is potentially useful in characterizing protein sequence-structure relationships.
" }, { "name": "Ewald, Andrew Josef", "degree": "PhD", "year": "2003", "title": "The Molecular Control of Cell Movements during Early Vertebrate Development", "advisor": "Fraser, Scott E.", "url": "https://resolver.caltech.edu/CaltechETD:etd-03312003-125746", "creators": [ { "name": { "family": "Ewald", "given": "Andrew Josef" }, "id": "Ewald-Andrew-Josef", "orcid": "0000-0002-1964-0740", "display_name": "Ewald, Andrew Josef" } ], "advisors": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "advisor", "display_name": "Fraser, Scott E." } ], "committee": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "role": "chair", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Roberts", "given": "Richard W." }, "id": "Roberts-R-W", "role": "member", "display_name": "Roberts, Richard W." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biochem" ], "doi": "10.7907/67Z0-H535", "abstract": "The early development of vertebrate embryos is characterized by massive, coordinated cell movements. These movements shape the embryo, distribute different cell types, shape complex tissues, and bring tissues into their correct spatial relationships. We have examined two early cell movements: the dorsal mesoderm of the frog embryo during gastrulation as a model for the coordinated movement of connected sheets of cells and the neural crest in the chicken embryo, as a model for cell migration.
\r\n\r\nThe dorsal mesoderm in the frog embryo moves as a sheet of cells, due to strong connections among the cells. Cell intercalation within this sheet drives the elongation of the embryo during the process of gastrulation, whereby the round, morphologically symmetric early embryo is converted into a tadpole. We have demonstrated the existence of propagating intercellular waves of calcium within the dorsal mesoderm during gastrulation. These waves appear to be specific to the dorsal mesoderm and directly required for the cell movements of gastrulation. To build an integrated picture of how different signaling pathways interact to control gastrulation, we have developed a novel means of quantitatively imaging whole embryos with subcellular resolution. We have used this digital atlas to carefully examine the major events of gastrulation in normal embryos and embryos overexpressing a mutant form of the Disheveled protein.
\r\n\r\nThe neural crest is a transient population of cells in the vertebrate embryo that arises in the neural tube and migrates to give rise to neurons, glia, bone and other cell types. During migration individual neural crest cells make extensive temporary connections with other cells, but migrate as individuals, rather than as a connected sheet. We have used patterned substrates and optical tweezers to present them with carefully controlled molecular stimuli. We have characterized their normal cellular behaviors and their response to ephrin-B ligands in a spatially and temporally defined manner.
" }, { "name": "Kirouac, Martha", "degree": "PhD", "year": "2003", "title": "cis-Regulatory Control of Three Cell Fate-Specific Genes in Vulval Organogenesis of C. elegans and C. briggsae", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechETD:etd-09252002-111831", "creators": [ { "name": { "family": "Kirouac", "given": "Martha" }, "id": "Kirouac-Martha", "display_name": "Kirouac, Martha" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "member", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biology" ], "doi": "10.7907/3WVY-RM54", "abstract": "The great-grandprogeny of the Caenorhabditis elegans vulval precursor cells (VPCs) adopt one of the final vulA, B1, B2, C, D, E and F cell types in a precise spatial pattern. Formation of the pattern of vulval cell types is likely to depend upon the cis-regulatory regions of the transcriptional targets of these intercellular signals in vulval development. The outcome of such differential activation will result in individual cell types. egl-17, zmp-1, cdh-3 are expressed differentially in the developing vulva cells, providing a potential readout for different signaling pathways. To understand how different signaling pathways interact to specify unique vulval cell types in a precise pattern, I have identified upstream cis-regulatory regions that are sufficient for their ability to confer vulval cell type-specific regulation when fused in cis to the basal pes-10 promoter. In the egl-17 promoter, I have identified a 143 base pair (bp) region that drives vulC and vulD expression, and a 102 bp region that is sufficient to drive the early expression in presumptive vulE and vulF cells. In the zmp-1 promoter, I have identified a 300 bp region that is sufficient to drive expression in vulE, vulA and the anchor cell. In the cdh-3 promoter, I have identified a 689 bp region sufficient to drive expression in the anchor cell and vulE, vulF, vulD and vulC, a 155 bp region sufficient to drive only anchor cell expression, and a separate 563 bp region that was also sufficient to drive expression in these vulval cells. I have identified the C. briggsae homologs of these three genes, and the corresponding control regions, and tested these regions in both C. elegans and C. briggsae. I find that these regions of similarity in C. elegans and C. briggsae upstream of egl-17, zmp-1, and cdh-3 promote expression in vulval cells and the anchor cell. Using the regions defined by the sufficiency analysis and phylogenetic footprinting, I have been able to isolate over-represented sequences that may play important roles in conferring vulval and anchor cell expression." }, { "name": "Qui\u00f1\u00f3nez, Carlo Joseph", "degree": "PhD", "year": "2003", "title": "Theory and Design of Relaxometric Probes", "advisor": "Fraser, Scott E.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05292003-182043", "creators": [ { "name": { "family": "Qui\u00f1\u00f3nez", "given": "Carlo Joseph" }, "id": "Qui\u00f1\u00f3nez-Carlo-Joseph", "display_name": "Qui\u00f1\u00f3nez, Carlo Joseph" } ], "advisors": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "advisor", "display_name": "Fraser, Scott E." } ], "committee": [ { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "member", "display_name": "Koch, Christof" }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Peters", "given": "Jonas C." }, "id": "Peters-J-C", "orcid": "0000-0002-6610-4414", "role": "member", "display_name": "Peters, Jonas C." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biology" ], "doi": "10.7907/X9G0-2M43", "abstract": "In an effort to rationally design an apoptosis-sensitive MRI contrast agent, two novel gadolinium complexes were designed, synthesized and evaluated as components of a relaxometric probe. The first, AEDO3A will prove a useful building block for in the area of the relaxometric probe design. AEDO3A-Gd has a relaxivity of 2.2 mM-1s-1 and 3.4 mM-1s-1 at 60Mhz and 500MHz, respectively If AEDO3A-Gd is the \"on\" state and assuming reasonable relaxivities for the \"off\" state, tissue contrast modeling of the smallest-detectable relaxivity change suggests AEDO3A is suitable for incorporation into relaxometric probes.
\r\n\r\n1-(2-Aspartryl-aminoethyl)-4,7,10-tri(carboxymethyl)-cyclen (Asp-AEDO3A) is evaluated as the second component of an apoptosis-sensitive relaxometric probe system. The synthesis and characterization of the ligand and its Gd and Tb complexes is described. Fluorescence lifetime data of the terbium complex indicate the presence of 0.6 water molecules in the inner coordination sphere. The gadolinium complex has a relaxivity of 1.4 mM-1s-1 and 1.7 mM-1s-1 at 60Mhz and 500MHz, respectively. Toxicity studies demonstrated Xenopus embryos tolerated Asp-AEDO3A-Gd at magnetically useful concentrations as predicted by tissue contrast modeling. X-ray crystallography data are presented for both the ligand and gadolinium complex.
\r\n\r\nUnfortunately, no enzymatic processing of Asp-AEDO3A-Gd was observed under any conditions. This phenomenon was attributed to heretofore-unknown coordination chemistry for gadolinium elucidated from the X-ray crystal structure. This novel N-carboxamido coordination, while problematic for the application of Asp-AEDO3A-Gd as a relaxometric probe, will be potentially useful in other areas of contrast agent design.
\r\n" }, { "name": "Voigt, Christopher Ashby", "degree": "PhD", "year": "2003", "title": "Computationally Optimizing the Directed Evolution of Proteins", "advisor": "Wang, Zhen-Gang; Arnold, Frances Hamilton; Mayo, Stephen L.", "url": "https://resolver.caltech.edu/CaltechETD:etd-08192002-161141", "creators": [ { "name": { "family": "Voigt", "given": "Christopher Ashby" }, "id": "Voigt-Christopher-Ashby", "orcid": "0000-0003-0844-4776", "display_name": "Voigt, Christopher Ashby" } ], "advisors": [ { "name": { "family": "Wang", "given": "Zhen-Gang" }, "id": "Wang-Zhen-Gang", "orcid": "0000-0002-3361-6114", "role": "advisor", "display_name": "Wang, Zhen-Gang" }, { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "co-advisor", "display_name": "Arnold, Frances Hamilton" }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "co-advisor", "display_name": "Mayo, Stephen L." } ], "committee": [ { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "chair", "display_name": "Rees, Douglas C." }, { "name": { "family": "Arnold", "given": "Frances Hamilton" }, "id": "Arnold-F-H", "orcid": "0000-0002-4027-364X", "role": "member", "display_name": "Arnold, Frances Hamilton" }, { "name": { "family": "Roberts", "given": "Richard W." }, "id": "Roberts-R-W", "orcid": "0000-0002-8587-5097", "role": "member", "display_name": "Roberts, Richard W." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Fontana", "given": "Walter" }, "id": "Fontana-Walter", "role": "member", "display_name": "Fontana, Walter" }, { "name": { "family": "Wang", "given": "Zhen-Gang" }, "id": "Wang-Zhen-Gang", "orcid": "0000-0002-3361-6114", "role": "member", "display_name": "Wang, Zhen-Gang" } ], "option_major": [ "biochem" ], "doi": "10.7907/E4GF-EQ41", "abstract": "Directed evolution has proven a successful strategy for protein engineering. To accelerate the discovery process, we have developed several computational methods to optimize the mutant libraries by targeting specific residues for mutagenesis, and subunits for recombination. In achieving this goal, a statistical model was first used to study the dynamics of directed evolution as a search algorithm. These simulations improved our understanding of the relationship between parameters describing the search space (e.g., interactions between amino acids) and experimental search parameters (e.g., mutation rate and library size). Based on these simulations, a more detailed model was used to calculate the structural tolerance of each residue to amino acid substitutions. Further, a computational model was developed to optimize recombination experiments, based on the three-dimensional structure. Together, these computational techniques represent a major step towards information-driven combinatorial protein design. " }, { "name": "Zhou, Qiao", "degree": "PhD", "year": "2003", "title": "Glial Cell Development in the Vertebrate Central Nervous System", "advisor": "Anderson, David J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-04092003-155305", "creators": [ { "name": { "family": "Zhou", "given": "Qiao" }, "id": "Zhou-Qiao", "display_name": "Zhou, Qiao" } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "advisor", "display_name": "Anderson, David J." } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Alberola-Ila", "given": "Jose" }, "id": "Alberola-Ila-J", "role": "member", "display_name": "Alberola-Ila, Jose" }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" } ], "option_major": [ "biology" ], "doi": "10.7907/SV83-BY17", "abstract": "Neurons and glial cells are the two most fundamental cell types of the vertebrate central nervous system (CNS). While neurons are directly responsible for information processing via their electrical activities, glial cells play essential supportive roles. For example, oligodendroglia insulates axons, microglia performs immune functions, and astroglia maintains homeostasis of the entire CNS. Malfunction of glial cells causes numerous debilitating diseases directly (such as glial tumors), or indirectly by disrupting the normal functions of neurons that they support (as in multiple sclerosis).
\r\n\r\nDespite their functional importance, relatively little is known about the development of vertebrate CNS glial cells. Focusing on the possibility that members of the basic helix-loop-helix (bHLH) transcription factors may play important roles in the development of vertebrate glial cells, similar to their functions in neurons, I searched for novel bHLH factors expressed in glial cells. A new family of bHLH factors was found and named Olig. Intriguingly, one member of this family, Olig2, is sequentially expressed first in motoneuron progenitors and later in the oligodendroglia. The sequence and expression pattern of Olig2 is highly conserved among different vertebrate species including fish, birds and mammals.
\r\n\r\nTo understand the role of Olig2 in oligodendroglia development, I ectopically expressed Olig2 singly or in combination with other factors in chick embryos. My result suggests that Olig2 can promote oligodendrocyte formation in the absence of neurogenic bHLH factors, which are negative regulators of glial fate. Other groups of researchers reported that in the presence of neurogenic factors, Olig2 promotes motoneuron development instead. Olig2 gene is therefore sufficient to specify the fate of either a neuronal subtype or a glial subtype, together with neurogenic factors.
\r\n\r\nTo further assess whether Olig genes are required for motoneuron and oligodendroglia development, I knocked out both Olig2 and Olig1 genes in mouse. In double null mutants, spinal motoneurons and oligodendroglia precursors from the entire CNS fail to develop, demonstrating that Olig genes are absolutely necessary for the generation of these cell types. Unexpectedly, in the absence of both Olig1 and Olig2, spinal motoneurons are transformed into V2 interneurons whereas oligodendroglial cells are respecified as astroglial cells. These results suggest that Olig genes are not involved in neuron-glia decision, but rather in specifying subtype identities of neuron and glia. Given that motoneurons and oligodendrocytes likely derive from common precursors, the expression of Olig may serve to couple the subtype identities of both neurons and glial cells sequentially generated from the same stem cells.
\r\n\r\nThe series of studies on Olig genes contributed on two areas of neural development. First, they shed important light on the specification of oligodendrocyte and astrocyte, the two major glial types in the vertebrate CNS. Second, they revealed that cell fate determinations of neuron and glia are not two unrelated events as often believed, on the contrary, they are deeply intertwined.
" }, { "name": "Aakalu, Girish Nanda", "degree": "PhD", "year": "2002", "title": "Building the Molecular Machinery of Memory: Local Protein Synthesis in Hippocampal Neurons", "advisor": "Schuman, Erin Margaret", "url": "https://resolver.caltech.edu/CaltechTHESIS:01232012-132700275", "creators": [ { "name": { "family": "Aakalu", "given": "Girish Nanda" }, "id": "Aakalu-Girish-Nanda", "display_name": "Aakalu, Girish Nanda" } ], "advisors": [ { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "advisor", "display_name": "Schuman, Erin Margaret" } ], "committee": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "chair", "display_name": "Fraser, Scott E." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "orcid": "0000-0002-3671-9354", "role": "member", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "member", "display_name": "Schuman, Erin Margaret" } ], "option_major": [ "biology" ], "doi": "10.7907/CAY9-6643", "abstract": "Synaptic plasticity is the most widely accepted cellular and molecular model for learning and memory. Although the idea that information is encoding through changes in\r\nsynaptic strength is simple, the requirement for new protein synthesis to maintain long-lasting forms of plasticity threatens to make this model untenably complex. This complexity arises from the fact that an individual neuron can have thousands of connections, small groups of which change strength independently of others. Since the\r\nnecessary proteins are likely the effectors of long-term plasticity, non-specific delivery would lead to loss of plasticity-encoded information. Thus it is critical that the effector proteins be faithfully delivered only to the correct sites.
\r\n\r\nOne way to accomplish this task would be to allow the synapses local control over the necessary protein synthesis and delivery. Previous studies have suggested this possibility of dendritic local protein synthesis (LPS). In this thesis we describe the visualization, in real time, of the synthesis of a GFP-based reporter in the dendrites of cultured rat hippocampal neurons. By utilizing a number of physical, optical and molecular manipulations we have insured that the observed synthesis was free of any\r\nsomatic contribution thereby providing the first definitive evidence for the existence of dendritic LPS in mature vertebrate neurons.
\r\n\r\nWe also describe the regulation of dendritic LPS by two forms of plasticity-inducing stimuli. First, we show that dendritic LPS can be stimulated by brain derived neurotrophic factor (BDNF), a molecule capable of causing persistent synaptic enhancement. Second, we show that a chronic blockade of synaptic activity, which results in a form of synaptic enhancement termed \"disuse hypersensitivity\", appears to enhance dendritic LPS.
\r\n\r\nFinally, we discuss a technique that can facilitate the study of the necessity of dendritic LPS for long-lasting plasticity. By using a \"caged\" protein synthesis inhibitor,\r\nwe are able to abolish protein synthesis in a spatially restricted manner. Thus it is now possible to conduct experiments where dendritic LPS is inhibited while somatic synthesis is permitted. If plasticity is not maintained under these conditions, we will have satisfying evidence of the necessity of dendritic LPS for long-lasting plasticity.
" }, { "name": "Arthur, Benjamin Jacob", "degree": "PhD", "year": "2002", "title": "Neural Computations Leading to Space-specific Auditory Responses in the Barn Owl", "advisor": "Konishi, Masakazu", "url": "https://resolver.caltech.edu/CaltechETD:etd-04172002-150548", "creators": [ { "name": { "family": "Arthur", "given": "Benjamin Jacob" }, "id": "Arthur-Benjamin-Jacob", "display_name": "Arthur, Benjamin Jacob" } ], "advisors": [ { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "advisor", "display_name": "Konishi, Masakazu" } ], "committee": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "chair", "display_name": "Lester, Henry A." }, { "name": { "family": "Pine", "given": "Jerome" }, "id": "Pine-J", "role": "member", "display_name": "Pine, Jerome" }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" } ], "option_major": [ "cns" ], "doi": "10.7907/SY24-X458", "abstract": "Sound localization is the ability to pinpoint the direction a sound is coming from based on auditory cues alone. Neurons in the brain which mediate this behavior are active only when sound comes from a particular direction. This thesis uses physiological and anatomical methods to investigate the computations which lead to such space-specific neural responses in the barn owl.
\r\n \r\nChapter 3 studies a behavioral and neural phenomenon called phase ambiguity, which arises from the way in which the auditory nerve and cochlear nuclei encode acoustic information. Phase ambiguity causes errors in sound localization to be made for tonal stimuli, and is resolved through the convergence of information across different frequencies in broadband noise stimuli. Data presented here show that a continuous band of noise is not necessary; a set of tones spaced at the critical bandwidth resolves phase ambiguity just as well as a noise stimulus. This is due to a sub-linear interaction for tones of nearby frequencies.
\r\n \r\nChapter 4 addresses the head-related transfer function (HRTF) model of sound localization. While traditional barn owl models use linear equations to relate interaural time differences (ITD) to azimuth and interaural intensity differences (IID) to elevation, the HRTF model purports that IID is dependent on frequency to such an extent that pattern recognition is used to match the spectral shape of IID in the stimulus to that characteristic of particular directions in space. Data presented here confirm predictions made by the HRTF model that IID tuning changes with frequency in space-mapped neurons, and that two-tone stimuli whose IIDs match these changes elicit better responses than those which do not.
\r\n \r\nChapter 5 investigates the computation of space-specificity in the forebrain. Previous anatomical studies have suggested that the space-specificity seen there is not merely inherited from the space map in the midbrain, but rather arises, at least in part, independently. The data presented here reconfirm that the forebrain pathway branches off from the midbrain pathway before the convergence across frequencies leads to space-specific neurons. All previous computations, however, including the formation of ITD-IID combination sensitivity, seem to be shared.
\r\n \r\nCollectively, these three studies expand our knowledge of the neurophysiology of sound localization in the barn owl by detailing specific mechanisms underlying the computation of space-specific neural responses.
" }, { "name": "Backer, Alejandro (Alex)", "degree": "PhD", "year": "2002", "title": "Pattern Recognition in the Olfactory System of the Locust: Priming, Gain Control and Coding Issues", "advisor": "Laurent, Gilles J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-11212002-185244", "creators": [ { "name": { "family": "Backer", "given": "Alejandro (Alex)" }, "id": "Backer-Alejandro", "display_name": "Backer, Alejandro (Alex)" } ], "advisors": [ { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "advisor", "display_name": "Laurent, Gilles J." } ], "committee": [ { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "chair", "display_name": "Koch, Christof" }, { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "member", "display_name": "Schuman, Erin Margaret" }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "member", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "member", "display_name": "Perona, Pietro" } ], "option_major": [ "biology" ], "doi": "10.7907/3EW4-YW92", "abstract": "Object recognition requires both specificity, to ensure that stimuli with distinct behavioral relevance\r\nare distinguished, and invariance, to ensure that different instances of the same stimulus\r\nare recognized as the same under varied conditions (intensity, pitch, position,...). Psychophysical\r\nstudies show that an odor can be perceived as identical over significant ranges of concentrations.\r\nWhether concentration invariance results, at least in part, from low-level neural phenomena rather\r\nthan cognitive grouping is so far unknown.
\r\n\r\nI explore, firstly, the contribution of projection neurons (PNs) in the antennal lobe of the\r\nlocust, the analog of the vertebrate olfactory bulb, to the recognition of odor identity across concentrations;\r\nand secondly, what role spike timing, neuronal identity, and synchronization among neuronal\r\nassemblies play in the encoding and decoding of odor information by downstream neurons.
\r\n\r\nI show the following:
\r\nA novel computerized odor delivery system capable of delivering binary mixtures in arbitrary ratios and with arbitrary timecourses selected in real-time.
\r\nThe locust can recognize odors, and shows innate olfactory preferences.
PNs solve the task of encoding both odorant concentration and odorant identity, independently\r\nof concentration, in three ways. First, by multiplexing information in different response dimensions\r\nusing a code that involves neuronal identity, spike timing (on a timescale slower than\r\npreviously believed) and synchronization across a neuronal assembly. Second, via a novel phenomenon\r\nof experience-dependent plasticity that contributes to PNs\u2019 invariance to concentration and\r\nsensitizes PNs after exposure to an odor at high concentration, contrary to the adaptation exhibited\r\nby receptors. Third, a phenomenon of gain control, whereby excitatory and inhibitory responses balance\r\nout massive changes in receptor activity as a function of odorant concentration, maintains the\r\noutput of PNs within a small dynamic range.
\r\n\r\nA further mechanism of gain control contributing to keep the activity of early olfactory circuits\r\nrelatively constant across the wide dynamic range of odorant concentrations in the air is the\r\nphysical chemistry of odorant reception confers the olfactory system invariance to odorant volatility,\r\na physical property that has hitherto been believed to play a fundamental role in an odorant\u2019s effectiveness.
\r\n\r\nResponse patterns sometimes exhibit stable representations over large composition ranges\r\nand then abrupt transitions as a function of concentration and mixture composition, suggesting the\r\ndifference between \u201csame\u201d and \u201cdifferent\u201d odors may be delineated by sharp boundaries in odor\r\nspace.
\r\n\r\nFinally, how is the distributed code for odors in PN assemblies decoded? I show that\r\nalthough synchronization among PN assemblies does not augment stimulus information in PN temporal\r\nresponses, it is necessary for the read-out of odor information by downstream neurons.\r\nIn sum, early olfactory circuits appear to employ plasticity, gain control and temporal coding\r\nacross synchronizing neuronal assemblies to solve the odor recognition problem across multiple\r\nconcentrations.
\r\n\r\nAppendices show that the variability of PN responses is correlated across neurons, show\r\nhow to produce non-cyclic Winnerless Competition (WLC) and a learning rule that causes random\r\nnetworks to self-organize into WLC, present an exact hypothesis test for binomial distributions,\r\nimprovements to sliding-window cross-correlation and to the K-nearest neighbor classification algorithm,\r\na combinatorial analysis of the connectivity between the locust antennal lobes and mushroom\r\nbodies, a didactic exposition of Victor and Purpura\u2019s spike cost-based metric and an experiment\r\nshowing heterogeneity along the length of the locust antenna.
\r\n\r\n\r\n" }, { "name": "Bolon, Daniel N.", "degree": "PhD", "year": "2002", "title": "Computational Enzyme Design", "advisor": "Mayo, Stephen L.", "url": "https://resolver.caltech.edu/CaltechETD:etd-01252002-100801", "creators": [ { "name": { "family": "Bolon", "given": "Daniel N." }, "id": "Bolon-Daniel-N", "orcid": "0000-0001-5857-6676", "display_name": "Bolon, Daniel N." } ], "advisors": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "advisor", "display_name": "Mayo, Stephen L." } ], "committee": [ { "name": { "family": "Goddard", "given": "William A., III" }, "id": "Goddard-W-A-III", "orcid": "0000-0003-0097-5716", "role": "chair", "display_name": "Goddard, William A., III" }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." } ], "option_major": [ "biochem" ], "doi": "10.7907/W7F3-DS14", "abstract": "The long-term objective of computational enzyme design is the ability to generate efficient protein catalysts for any chemical reaction. This thesis develops and experimentally validates a general computational approach for the design of enzymes with novel function.
\r\n \r\nIn order to include catalytic mechanism in protein design, a high-energy state (HES) rotamer (side chain representation) was constructed. In this rotamer, substrate atoms are in a HES. In addition, at least one amino acid side chain is positioned to interact favorably with substrate atoms in their HES and facilitate the reaction. Including an amino acid side chain in the HES rotamer automatically positions substrate relative to a protein scaffold and allows protein design algorithms to search for sequences capable of interacting favorably with the substrate. Because chemical similarity exists between the transition state and the high-energy state, optimizing the protein sequence to interact favorably with the HES rotamer should lead to transition state stabilization. In addition, the HES rotamer model focuses the subsequent computational active site design on a relevant phase space where an amino acid is capable of interacting in a catalytically active geometry with substrate.
\r\n\r\nUsing a HES rotamer model of the histidine mediated nucleophilic hydrolysis of p-nitrophenyl acetate, the catalytically inert 108 residue E. coli thioredoxin as a scaffold, and the ORBIT protein design software to compute sequences, an active site scan identified two promising active site designs. Experimentally, both candidate ?protozymes? demonstrated catalytic activity significantly above background. In addition, the rate enhancement of one of these ?protozymes? was the same order of magnitude as the first catalytic antibodies.
\r\n\r\nBecause polar groups are frequently buried at enzyme-substrate interfaces, improved modeling of buried polar interactions may benefit enzyme design. By studying native protein structures, rules have been developed within the scope of protein design that require core polar residues to largely satisfy their hydrogen bonding potential. Using this polar strategy to design the core of thioredoxin resulted in a protein that was thermodynamically stabilized relative to both the wt protein and a protein designed without core polar residues.
\r\n\r\nThe enzyme design procedures presented here may serve as a platform to develop more detailed methods. It is hoped that the development and experimental testing of more detailed methods will continue to improve our understanding of enzyme mechanism and lead to the long-term goal of designing highly efficient enzymes.
" }, { "name": "Chen, Tianxin (Cynthia)", "degree": "PhD", "year": "2002", "title": "Regulatory Mechanisms of the Heat Shock Response", "advisor": "Parker, Carl Stevens", "url": "https://resolver.caltech.edu/CaltechTHESIS:01252012-152553827", "creators": [ { "name": { "family": "Chen", "given": "Tianxin (Cynthia)" }, "id": "Chen-Tianxin-(Cynthia)", "display_name": "Chen, Tianxin (Cynthia)" } ], "advisors": [ { "name": { "family": "Parker", "given": "Carl Stevens" }, "id": "Parker-C-S", "role": "advisor", "display_name": "Parker, Carl Stevens" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biochem" ], "doi": "10.7907/4BC0-RM51", "abstract": "Although Drosophila heat shock transcription factor (dHSF) is abundant in the early embryos, it does not enter the nucleus in response to heat shock. Using the nuclear\r\nlocalization signal (NLS) of dHSF as bait in a yeast two-hybrid system, we identified and cloned a nuclear transport protein, Drosophila Karyopherin \u03b13 (dKap \u03b13). dKap \u03b13 binds specifically to the dHSF's NLS, but not to mutant NLSs that abolish transport in vivo. The early embryo is deficient in dKap \u03b13 protein through cycle 12, resulting in dHSF nuclear exclusion. From cycle 13 onward the transport factor is present and the dHSF is localized within the nucleus, allowing the embryo to respond to heat shock. The functional domain organization of dKap \u03b13 was mapped in detail using yeast two-hybrid analysis and immuno-fluorescence staining.
\r\n\r\nThe function and specificity of the multiple Karyopherin \u03b1s found in higher eukaryotes is not understood. RNA interference was used to knock out each Kap \u03b1 protein individually in Drosophila S2 cells. We found that RNA polymerase II, TATA binding protein and heat shock transcription factor were transported into the nucleus by\r\ndifferent karyopherins, indicating that unique and non-overlapping pathways exist.
\r\n\r\nIn Saccharomyces cerevisiae, the transcriptional activity of HSF is repressed at non-stress temperatures. Certain mutations of Arginine 274 in the DNA-binding domain\r\n(DBD) increase both basal and induced activities of HSF. We demonstrate that the mutations reduce the association between the DBD/oligomerization domain and the\r\ntranscription activation domains. Our studies suggest that the DBD of HSF can interact with activation domains directly, and this interaction is important for the repression of HSF activity.
\r\n\r\nRNA polymerase II is bound to Drosophila Hsp70 promoters in the absence of heat shock, and paused after initiating a short transcript. Heat shock induces RNA polymerase II hyperphosphorylation and stimulates RNA polymerase II into productive elongation. We demonstrate that knocking out the expression of positive transcription elongation factor b (P-TEFb) significantly reduces Hsp70 mRN A transcription. P-TEFb is bound to HSF upon heat shock, and the complex can phosphorylate polymerase II C-terminal domain (CTD) in vitro. CTD kinase activity is inhibited by either RNAi targeting CDK9, or CDK9 inhibitor DRB.
\r\n" }, { "name": "Choe, Wonchae", "degree": "PhD", "year": "2002", "title": "Biochemical and Biological in vivo Functions of Dna2p in Saccharomyces cerevisiae\r ", "advisor": "Campbell, Judith L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04252012-093049065", "creators": [ { "name": { "family": "Choe", "given": "Wonchae" }, "id": "Choe-Wonchae", "display_name": "Choe, Wonchae" } ], "advisors": [ { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "role": "advisor", "display_name": "Campbell, Judith L." } ], "committee": [ { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "role": "chair", "display_name": "Campbell, Judith L." }, { "name": { "family": "Abelson", "given": "John N." }, "id": "Abelson-J-N", "role": "member", "display_name": "Abelson, John N." }, { "name": { "family": "Parker", "given": "Carl Stevens" }, "id": "Parker-C-S", "role": "member", "display_name": "Parker, Carl Stevens" }, { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "role": "member", "display_name": "Dunphy, William G." } ], "option_major": [ "biology" ], "doi": "10.7907/P30H-JP90", "abstract": "We have characterized a temperature-sensitive yeast mutant, dna2ts, which is defective in DNA replication. DNA2 is essential and encodes a 172-kDa protein with a DNA helicase motif at its C-terminal portion. A homology search showed that Dna2p is conserved structurally among species. Even Xenopus laevis Dna2 was able to complement an S. cerevisiae dna2-1 mutant strain for growth at the nonpermissive temperature, suggesting that Dna2p is conserved also functionally. The site of the dna2-1 mutation was mapped using a marker rescue technique and turned out proline 504 to serine, placing the dna2-1 mutation in the N-terminal portion of the protein, suggesting that N-terminal portion of the protein is important for the activity of Dna2p. Recombinant ScDna2p was expressed in insect cells and purified. With the purified protein, we were able to demonstrate that Dna2p was a single-stranded DNA endonuclease/helicase and a single stranded DNA dependent ATPase, suggesting that Dna2p has various biochemical functions.
\r\n\r\nWe also found that Dna2 helicase-nuclease is a component of telomeric chromatin. Both chromatin immunoprecipitation and immunofluorescence showed that Dna2p associates with telomeres but not the bulk of chromosomal DNA in G1 phase. In S phase, there is a dramatic redistribution ofDna2p from the telomeres to sites throughout replicating chromosomes. Dna2p is again localized to telomeres in late S, where it remains through G2 and until the next S phase. Telomeric localization of Dna2p required Sir3p, since the amount ofDna2p found at telomeres by two different assays, one hybrid and ChIP, is severely reduced in strains lacking Sir3p. The Dna2p is also distributed throughout the\r\nnucleus in cells growing in the presence of DSB-inducing agents such as bleomycin.
\r\n\r\nFinally, we show that Dna2p is functionally required for telomerase-dependent de novo telomere synthesis and also participates in telomere lengthening in mutants lacking\r\ntelomerase, and that genetic instability due to dna2 mutations lead to premature aging phenotype.
\r\n" }, { "name": "Du, Fangyong", "degree": "PhD", "year": "2002", "title": "Allosteric Activation of the Ubiquitin ligase UBR 1 by Short Peptides: Molecular Mechanisms and Physiological Functions", "advisor": "Varshavsky, Alexander J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02232012-110128910", "creators": [ { "name": { "family": "Du", "given": "Fangyong" }, "id": "Du-Fangyong", "display_name": "Du, Fangyong" } ], "advisors": [ { "name": { "family": "Varshavsky", "given": "Alexander J." }, "id": "Varshavsky-A-J", "role": "advisor", "display_name": "Varshavsky, Alexander J." } ], "committee": [ { "name": { "family": "Varshavsky", "given": "Alexander J." }, "id": "Varshavsky-A-J", "role": "chair", "display_name": "Varshavsky, Alexander J." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "member", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "role": "member", "display_name": "Dunphy, William G." }, { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "role": "member", "display_name": "Campbell, Judith L." }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" } ], "option_major": [ "biology" ], "doi": "10.7907/S3VT-0641", "abstract": "The N-end rule relates the in vivo half life of a protein to the identity of its\r\nN-terminal residue. UBR1, the E3 of the N-end rule pathway in Sacchnromzyces\r\ncerevisiae, targets proteins that bear destabilizing N-terminal residues for Ub-dependent,\r\nprocessive degradation. UBR1 binds protein substrates or dipetides\r\nthrough two distinct sites: the type 1 site, specific for basic residues, and the type\r\n2 site, specific for bulky hydrophobic residues. UBR1 also recognizes an internal\r\ndegradation signal of the 35 kDa homeodomain protein CUP9, a transcriptional\r\nrepressor of the di- and tripeptide transporter PTR2.
\r\n\r\n\r\nHere I report that the internal degradation signal of CUP9 is recognized\r\nby UBR1 through its third, distinct substrate-binding site. Occupation of the type\r\n1 or type 2 sites of UBR1 by dipeptides allosterically stimulates the\r\nUBR1-dependent multi-ubiquitylation of CUP9 in an in vitro system, which\r\nconsists of purified components of the yeast N-end rule pathway. UBR1 is the\r\nfirst E3 shown to be allosterically regulated by small compounds. This\r\nregulation underlies, in vivo, the accelerated UBR1-dependent degradation of\r\nCUP9 in the presence of dipeptides with destabilizing N-terminal residues. The\r\nresult is a positive feedback circuit that controls the peptide import in\r\nS. cerevisiae. Specifically, the imported dipeptides bind to UBR1 and accelerate\r\nthe UBR1-dependent degradation of CUP9, thereby derepressing the\r\ntranscription of PTR2 and increasing the cell's capacity to import peptides.
\r\n\r\n\r\nI also describe a new, autoinhibition-based molecular mechanism\r\nunderlying the activation of UBR1 by dipeptides. UBR1 is an autoinhibited\r\nprotein, in that the binding of dipeptides to the type 1 and type 2 sites of UBR1\r\nenhances the dissociation of the C-terminal autoinhibitory domain of UBR1 from its substrate-binding N-terminal region. Moreover, this dissociation, which\r\nallows the interaction between UBRl and CUP9, is strongly increased only if\r\nboth type 1 and type 2 sites of UBRl are occupied by dipeptides. An\r\nautoinhibitory mechanism discovered in the S. cerevisiae UBRl is likely to recur\r\nin metazoan homologs of UBRl, and may also be involved in controlling the\r\nactivity of other Db-dependent pathways.
\r\n\r\n" }, { "name": "Dubowitz, David Julian", "degree": "PhD", "year": "2002", "title": "Functional Magnetic Resonance Imaging in Rhesus Macaque Monkeys", "advisor": "Andersen, Richard A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01252012-103337039", "creators": [ { "name": { "family": "Dubowitz", "given": "David Julian" }, "id": "Dubowitz-David-Julian", "display_name": "Dubowitz, David Julian" } ], "advisors": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "role": "advisor", "display_name": "Andersen, Richard A." } ], "committee": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "role": "chair", "display_name": "Andersen, Richard A." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "role": "member", "display_name": "Shimojo, Shinsuke" }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Burdick", "given": "Joel Wakeman" }, "id": "Burdick-J-W", "role": "member", "display_name": "Burdick, Joel Wakeman" } ], "option_major": [ "cns" ], "doi": "10.7907/JBRJ-AS69", "abstract": "This thesis presents a method for functional magnetic resonance imaging in the brain of the rhesus macaque monkey, Macaca muialla. Experiments were performed in an awake-behaving animal at 1.5 T in a conventional clinical magnetic resonance system. Strategies to train the animal within the MR environment and to ensure behavioral compliance are described. Limitations of studying macaque functional neuroanatomy at this magnetic field strength are also discussed. Methods to improve signal-to-noise beyond the scope of conventional BOLD imaging at 1.5 T are presented, including the use of very high magnetic fields for functional imaging (11.7 T), as well as novel methods to improve sensitivity using intravascular iron oxide contrast media. Using the techniques developed for this thesis, a series of studies are presented to examine the visual pathways in the primate brain, allowing direct comparison of functional neuroanatomy between nonhuman primates and human cortex. Although the two species are anatomically different, direct functional homology within the visual cortex is demonstrated.\r\n" }, { "name": "Gerety, Sebastian de la Soudi\u00e8re", "degree": "PhD", "year": "2002", "title": "Eph Signaling in Vascular Development", "advisor": "Anderson, David J.; Fraser, Scott E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04262012-140534350", "creators": [ { "name": { "family": "Gerety", "given": "Sebastian de la Soudi\u00e8re" }, "id": "Gerety-Sebastian-de-la-Soudi\u00e8re", "display_name": "Gerety, Sebastian de la Soudi\u00e8re" } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "advisor", "display_name": "Anderson, David J." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "advisor", "display_name": "Fraser, Scott E." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/SJM2-8882", "abstract": "One of the most striking features of developmental biology is the dramatic morphological changes that an embryo must undergo to achieve its final form. Arguably, the most stunning example of this is found in the embryonic vasculature: not only does the vasculature undergo morphological changes, it must continue to do so adaptively as the size and nutritional needs of the embryo change during gestation. As embryonic blood flow starts long before the end of vessel morphogenesis, the vessels must maintain the integrity of their cell-cell contacts while at the same time remodeling into their final state. Receptor tyrosine kinases and their ligands have been implicated in the regulation of blood vessel growth and remodeling during development. Recently, the Eph receptors and their ephrin ligands were found to be expressed in the developing vasculature. While one Eph receptor, EphB4, is restricted to veins, its specific ligand, ephrinB2, is restricted to arteries. Furthermore, the ephrinB2 knockout mice exhibit defects in blood vessel remodeling, angiogenesis. Although the reciprocal expression of ephrinB2 and EphB4 suggested that Eph signaling from arteries to veins was important for blood vessel development, the presence of additional Eph receptors suggested EphB4 might not be required for this process. Additionally, the widespread expression of ephrinB2 outside the vasculature suggested that vascular-specific expression of this ligand might not be the tissue source necessary for angiogenic remodeling.
\r\n \r\nTo determine which Eph receptor was mediating the ephrinB2 signal, I generated a knockout of the EphB4 gene in mouse. A reporter gene replacement in the EphB4 locus confirmed the vein-biased expression of this receptor. Homozygous EphB4 mutant mice exhibit angiogenesis and cardiac defects, and embryonic lethality indistinguishable from those of ephrinB2 knockout mice. This suggests that EphB4 is the main Eph receptor responsible for transducing the angiogenic ephrinB2 signal. To examine the importance of endothelial specific expression of ephrinB2 in angiogenesis, in contrast to its nonvascular expression, I generated a conditional ephrinB2 mouse. These mice carry a functional ephrinB2 gene, which can be inactivated in a tissue specific manner. Mice with endothelial-specific inactivation of ephrinB2 (and intact non-vascular ephrinB2 expression) exhibit severe angiogenesis and cardiac defects identical to those of the conventional ephrinB2 mutant mice. This suggests that vascular ephrinB2 is essential, and cannot be compensated for by non-vascular ephrinB2 from surrounding vessels.
\r\nThese studies have clarified two important issues. The first is that the ephrinB2 signal is received by EphB4 expressing endothelial cells (of the veins), rather than by perivascular cells that also express Eph receptors. Second, cphrinB2 expression in endothelial cells of the vessels is an essential tissue source of angiogenic ephrin signals. Together, these studies reinforce the original interpretation of the ephrinB2 mutant, that Eph signaling between arteries to veins is essential for angiogenesis in the early embryo.
" }, { "name": "Hamburger, Zsuzsa Andrea", "degree": "PhD", "year": "2002", "title": "Crystallographic Studies of Invasin, a Bacterial Adhesion Molecule from Yersinia pseudotuberculosis", "advisor": "Bjorkman, Pamela J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06112002-135418", "creators": [ { "name": { "family": "Hamburger", "given": "Zsuzsa Andrea" }, "id": "Hamburger-Zsuzsa-Andrea", "display_name": "Hamburger, Zsuzsa Andrea" } ], "advisors": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "advisor", "display_name": "Bjorkman, Pamela J." } ], "committee": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "chair", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." } ], "option_major": [ "biology" ], "doi": "10.7907/NKAY-AB98", "abstract": "Bacterial pathogens, such as Yersinia pseudotuberculosis, must bind and enter normally non-phagocytic cells to establish infection. The protein responsible for mediating uptake of the bacterium is a 986-residue outer membrane protein called invasin. Invasin binds to several members of the beta 1 integrin family, presumably activating a reorganization of the host cytoskeleton to form pseudopods that envelop the bacterium. Integrin binding has been localized to the extracellular region of invasin (Inv497) comprised by the COOH-terminal 497 residues. In order to gain insight into host cell entry by Yersinia pseudotuberculosis, we solved the 2.3 \u0160crystal structure of Inv497. The structure reveals five domains that form a 180 \u0160rod with structural similarities to tandem fibronectin-III domains. The integrin-binding surfaces of invasin and fibronectin include similarly located key residues, but in the context of different folds and surface shapes. The structures of invasin and fibronectin provide an example of convergent evolution, in which invasin presents an optimized surface for integrin binding compared with host substrates. We have also initiated structural analyses of the NH2-terminal ~500 residues of invasin, which are required for outer membrane localization and for presentation of the integrin-binding region of invasin. We expressed this region of invasin as inclusion bodies in E. coli, and refolded the protein in the presence of detergents. We have also obtained microcrystals of this membrane protein. Circular dichroism studies indicate that this region of invasin is composed of mainly beta-structure. As the transmembrane regions of outer membrane proteins of known structure are beta-barrels, this region of invasin is also presumed to fold into such a structure. Proteolysis experiments suggest that the NH2-terminal 70 amino acids of invasin may form a separate domain from the invasin transmembrane region, analogous to that found in another outer membrane protein, the sucrose-specific porin ScrY. Equilibrium sedimentation analytical ultracentrifugation studies indicate the protein is monomeric in solution. Black bilayer experiments suggest that this region of the protein does not contain a pore and thus plays the role of an outer membrane anchor for the presentation of the integrin-binding domain on the cell surface" }, { "name": "Kreiman, Gabriel Alejandro", "degree": "Masters", "year": "2002", "title": "Neural Coding and Feature Extraction of Time-Varying Signals", "advisor": "Koch, Christof", "url": "https://resolver.caltech.edu/CaltechETD:etd-05262002-173148", "creators": [ { "name": { "family": "Kreiman", "given": "Gabriel Alejandro" }, "id": "Kreiman-Gabriel-Alejandro", "orcid": "0000-0003-3505-8475", "display_name": "Kreiman, Gabriel Alejandro" } ], "advisors": [ { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "advisor", "display_name": "Koch, Christof" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "cns" ], "doi": "10.7907/2V9Y-A983", "abstract": "What are the neuronal codes that the brain uses to represent information? This constitutes one of the most fascinating and challenging questions in Neuroscience. Here we report the results of our investigations about the mechanisms of stimulus encoding and feature extraction using the weakly electric fish Eigenmannia as a model. In many circumstances, sensory systems are subject to natural stimuli that are constantly changing. Therefore we decided to study the representation of time varying signals. Eigenmannia constitutes an ideal system to combine neurophysiological and computational techniques to study neural coding. We have characterized the variability of neuronal responses with a new approach by using parameterized distances between spike trains defined by Victor and Purpura. This measure of variability is widely applicable to neuronal responses, irrespective of the type of stimuli used (deterministic versus random) or the reliability of the recorded spike trains. We also quantitatively defined and evaluated the robustness of the neural code to spike time jittering, spike failures and spontaneous spikes. Our data show that the intrinsic variability of single spike trains lies outside of the range where it might degrade the information conveyed, yet still allows for improvement in coding by averaging across multiple afferent fibers. We also built a phenomenological model of P-receptor afferents incorporating both their linear transfer properties and the variability of their spike trains. We then studied the extraction of features from the time varying signal by bursts of spikes of multiple pyramidal cells, the next stage of information processing. To address the question of whether correlated responses of nearby neurons within topographic sensory maps are merely a sign ofredundancy or carry additional information we recorded simultaneously from pairs of electrosensory pyramidal cells with overlapping receptive fields in the hindbrain of weakly electric fish. We found that nearby pyramidal cells exhibit strong stimulus-induced correlations. The detailed stimulus encoding by pairs of pyramidal cells was inferior to that from single primary afferents. However, the detection of coincident bursts of activity could significantly enhance the extraction of upstrokes and downstrokes in the stimulus amplitude. Our investigations reveal mechanisms by which the nervous system can accurately and robustly transduce a time-varying signal into a digital spike train and then extract behaviorally relevant features." }, { "name": "Kreiman, Gabriel Alejandro", "degree": "PhD", "year": "2002", "title": "On the Neuronal Activity in the Human Brain During Visual Recognition, Imagery and Binocular Rivalry", "advisor": "Koch, Christof", "url": "https://resolver.caltech.edu/CaltechETD:etd-05262002-171928", "creators": [ { "name": { "family": "Kreiman", "given": "Gabriel Alejandro" }, "id": "Kreiman-Gabriel-Alejandro", "orcid": "0000-0003-3505-8475", "display_name": "Kreiman, Gabriel Alejandro" } ], "advisors": [ { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "advisor", "display_name": "Koch, Christof" } ], "committee": [ { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "chair", "display_name": "Koch, Christof" }, { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "member", "display_name": "Schuman, Erin Margaret" }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "member", "display_name": "Andersen, Richard A." }, { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "orcid": "0000-0002-1290-5232", "role": "member", "display_name": "Shimojo, Shinsuke" } ], "option_major": [ "biology" ], "doi": "10.7907/E0XZ-QP78", "abstract": "How does the neuronal activity in our brains give rise to our perceptions? We recorded the electrophysiological activity of over one thousand individual neurons in the human brain during object recognition, binocular rivalry, visual imagery and sleep. Subjects were patients with intractable epilepsy implanted with depth electrodes in targets including the amygdala, entorhinal cortex and hippocampus to localize the seizure focus for potential surgical resection. This has allowed us to explore the neuronal responses during visual processing in humans at an unprecedented level of spatial and temporal resolution. We observed a high degree of selectivity in the responses to complex visual stimuli. Some units were selective to categories of pictures including faces, houses, objects, famous people and animals while others responded only to one or a few stimuli, suggesting a sparse representation of visual information in the medial temporal lobe. Most of the selective neurons modulated their responses depending on the subject's percept during flash suppression. To further explore the correlation between perception and neuronal activity we investigated the vivid images that can be voluntarily generated in our minds in the absence of concomitant visual input. Our study revealed neuronal correlates of visual imagery and supports a common substrate for the processing of visual input and recall. Since visual memory is also prominent during dreams, we investigated the neuronal responses during different stages of the sleep-wake cycle. We observed an increase in synchrony during slow wave sleep compared to the wake and rapid-eye-movement sleep states. Our results suggest that neuronal activity in the human medial temporal lobe correlates with perception, shows a strong degree of invariance to changes in the input and could be involved in processing, storing and recalling visual information. \r\n" }, { "name": "Leonardo, Anthony Michael", "degree": "PhD", "year": "2002", "title": "Neural Dynamics Underlying Complex Behavior in a Songbird", "advisor": "Konishi, Masakazu", "url": "https://resolver.caltech.edu/CaltechETD:etd-05092002-165316", "creators": [ { "name": { "family": "Leonardo", "given": "Anthony Michael" }, "id": "Leonardo-Anthony-Michael", "display_name": "Leonardo, Anthony Michael" } ], "advisors": [ { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "advisor", "display_name": "Konishi, Masakazu" } ], "committee": [ { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "chair", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "member", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Pine", "given": "Jerome" }, "id": "Pine-J", "role": "member", "display_name": "Pine, Jerome" }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "member", "display_name": "Perona, Pietro" } ], "option_major": [ "cns" ], "doi": "10.7907/33BC-5948", "abstract": "Zebra finches memorize a tutor song as juveniles, and then faithfully reproduce this song for the remainder of their lives. This thesis investigates how the intricate and concerted activity of ensembles of neurons in the zebra finch brain gives rise to this complex behavior.
\r\n \r\nThe first section of the thesis demonstrates how neurons in RA, a pre-motor song control nucleus, coordinate their activity to generate the instantaneous spectral and temporal structure seen in the song. Similar sounds are produced by entirely different ensembles of RA neurons. Additionally, rapidly changing patterns of RA neural activity are capable of generating constant acoustic outputs. This is a new form of neural coding, previously unobserved in any other system. This unusual neural representation provides a concise account of an unexplained aspect of song learning.
\r\n\r\nThe second part of the thesis develops a new, noninvasive method of altering the auditory feedback heard by singing zebra finches. This type of feedback perturbation causes the normally stable songs of adult birds to deteriorate, indicating that auditory feedback is essential to song control throughout life. Restoration of the normal auditory feedback heard by these birds enables the gradual recovery of their original songs, demonstrating that a memory of the original song persists in the song control system despite the destabilization of the behavior. The song is thus maintained by an active feedback control process.
\r\n \r\nThe final project investigates the role of nucleus LMAN in real-time song control via auditory feedback. Neurons in nucleus LMAN were previously believed to compare auditory feedback to a memorized song template. The result of this comparison was thought to be used as an error-correction signal to update the motor control program. By recording from individual LMAN neurons while simultaneously manipulating the auditory feedback heard by the singing bird, it is shown that during singing, rather than auditory feedback, LMAN processes an efference copy of the motor commands used to generate the bird's song. This suggests a different model of the system, in which the efference copy is the basis of the real-time error-correction signal.
\r\n \r\nTaken together, these three projects significantly expand our knowledge of the neural mechanisms responsible for the generation of birdsong.
\r\n" }, { "name": "Moreno, Tanya Ann Munnecke", "degree": "PhD", "year": "2002", "title": "Noelins in Neural Development", "advisor": "Bronner, Marianne E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04272012-080339328", "creators": [ { "name": { "family": "Moreno", "given": "Tanya Ann Munnecke" }, "id": "Moreno-Tanya-Ann-Munnecke", "display_name": "Moreno, Tanya Ann Munnecke" } ], "advisors": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "advisor", "display_name": "Bronner, Marianne E." } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" } ], "option_major": [ "biology" ], "doi": "10.7907/M8J1-M419", "abstract": "The nervous system arises from embryonic tissues beginning with neural induction, when signals from the mesoderm induce the overlying ectoderm to form neural precursors. During neurogenesis, neurons are selected to differentiate from the induced neural precursors. Here, I describe the isolation and characterization of the\r\nXenopus Noelins, a family of gene isoforms that may play roles in both of these processes. Noelin proteins are alternatively spliced isoforms and are all secreted proteins. Biochemically, Noelin-1 and Noelin-4 interact; moreover, Noelin-4 interacts with BMP-4, a molecule that is implicated in the process of neural induction.
\r\n\r\nNoelin transcripts are detected beginning at late gastrulation stages. Later, transcripts are observed in post-mitotic neural tissues of the central and peripheral\r\nnervous systems, from the neural tube closure stage and continuing through swimming tadpole stage. In avian embryos, Noelins are expressed in the early neural plate and\r\nneural crest, and over-expression of Noelin-1 and-2 leads to excess and prolonged neural crest emigration from the cranial neural tube. The Xenopus Noelin homologs do not\r\nappear to affect neural crest induction or migration; however, Noelin-1 is shown to have a role in promoting neuronal differentiation in neural tissue. Furthermore, the secretion of Noelin-1 is important for its ability to induce certain neural markers to be expressed. Remarkably, Noelin-4 causes neural induction when over-expressed in naive tissue; in whole embryos, it causes expansion and ectopic production of neural tissue and cement gland by conversion of ectoderm.
\r\n\r\nNoelins may also modulate each others' functions: Noelin-1 activity is increased in the presence of Noelin-4, and surprisingly, Noelin-4 is negatively affected by the\r\npresence of Noelin-1. Since Noelin-4 can act as a neural inducer and can interact with BMP-4, a model for this gene's activity is proposed for modulation of BMP signaling.\r\nThe function of Noelin-1 in modulating Noelin-4 activity may be mediated through competition for binding to BMP proteins. I further show that Morpholino antisense\r\noligonucleotides that target all four Noelin isoforms cause a severe neural phenotype: the forebrain, cement gland and cranial ganglia are severely reduced or missing in injected\r\nembryos. These results indicate that Noelin proteins may be essential for normal development of anterior neural tissues. Thus, Noelin isoforms represent novel secreted\r\nfactors involved in nervous system development, from neural induction to neurogenesis.
\r\n" }, { "name": "Rosenbluth, David", "degree": "PhD", "year": "2002", "title": "Eye Position Modulation of Visual Cortex and the Sensory Set Hypothesis", "advisor": "Allman, John Morgan", "url": "https://resolver.caltech.edu/CaltechTHESIS:01312012-161153127", "creators": [ { "name": { "family": "Rosenbluth", "given": "David" }, "id": "Rosenbluth-David", "display_name": "Rosenbluth, David" } ], "advisors": [ { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "advisor", "display_name": "Allman, John Morgan" } ], "committee": [ { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "chair", "display_name": "Perona, Pietro" }, { "name": { "family": "Abu-Mostafa", "given": "Yaser S." }, "id": "Abu-Mostafa-Y-S", "role": "member", "display_name": "Abu-Mostafa, Yaser S." }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "member", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" } ], "option_major": [ "cns" ], "doi": "10.7907/jnhb-e433", "abstract": "What we see depends on where we look. This is obvious as a statement about the nonuniformity of our external visual environment. But it is also true, in a much less obvious sense, as a statement about the internal neurophysiology of the visual system. What we see depends on where we look in the neurophysiological sense that eye position signals have a dramatic effect on the responsiveness of visual cortical neurons. This thesis empirically studies the way in which point of regard (what point in space the eyes are fixating) influences neurons in visual cortical areas V1 and V4 and then presents a theoretical exploration of how these\r\ntwo different ways in which \"What we see depends on where we look\" might be functionally intertwined.
\r\n\r\nThe empirical data presented here adds to the growing body of evidence that eye position signals are ubiquitous in visual cortex, an observation which reopens speculation about the functional role that these signals might play in different visual cortical areas. The presence of eye position signals in visual areas of the ventral visual processing stream raises the possibility that these signals might facilitate object identity. Eye position signals might be exploited by visual cortex as a conditioned stimulus, which can become functionally linked to the responses of visual cortical neurons (unconditional response) through repeated pairing with the unconditioned stimulus, the retinal stimulus, in a classical conditioning paradigm. In this way the visual system would be capable of learning systematic relationships between point of regard and statistical characteristics of the visual environment. The learned response to the conditioned stimulus could then be exploited as a preparatory signal, to speed or otherwise alter visual processing to suit the current context. In exploring this theoretical viewpoint, we discuss the circumstances under which context dependent coding provides\r\nadvantages and how a code switching strategy might be implemented through physiological parcellation mediated by gain control. Eye position signals are here considered to be one among many different types of extra-retinal signals, present in visual cortical areas, whose presence might be similarly exploited. As such, the data and theory presented here should be considered as contributing to the broader literature on the influence of signals from outside the classical receptive field.
\r\n\r\n" }, { "name": "Shuey, David John", "degree": "PhD", "year": "2002", "title": "A Detailed Analysis of the DNA Binding Properties and the Affinity Purification of the Drosophila Heat-Shock Transcription Factor", "advisor": "Parker, Carl Stevens", "url": "https://resolver.caltech.edu/CaltechTHESIS:01312012-161653671", "creators": [ { "name": { "family": "Shuey", "given": "David John" }, "id": "Shuey-David-John", "display_name": "Shuey, David John" } ], "advisors": [ { "name": { "family": "Parker", "given": "Carl Stevens" }, "id": "Parker-C-S", "role": "advisor", "display_name": "Parker, Carl Stevens" } ], "committee": [ { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "chair", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Abelson", "given": "John N." }, "id": "Abelson-J-N", "role": "member", "display_name": "Abelson, John N." }, { "name": { "family": "Parker", "given": "Carl Stevens" }, "id": "Parker-C-S", "role": "member", "display_name": "Parker, Carl Stevens" } ], "option_major": [ "biochem" ], "doi": "10.7907/y91j-s092", "abstract": "The heat shock response presents an extremely attractive model to study the regulation of gene transcription in eucaryotes. I have focused the bulk of my research efforts on investigating the molecular determinants of protein-DNA interaction exhibited by the heat-shock transcription factor (HSTF) of Drosophila. The specific \"contacts\" made by the HSTF upon binding to the heat-shock element (HSE) were exhaustively determined using a variety of chemical and enzymatic probes for a number of HSTF-HSE complexes formed at both hsp70 and hsp83 gene promoters. During the course of these studies it is demonstrated that the HSTF appears to polymerize in a sequence-specific and template-directed manner on each of these promoters, a novelty in this class of regulators. Evidence suggesting that DNA bending may occur during this HSTF-DNA association on the hsp70 promoter is also presented. This observation represented the first report of a eucaryotic transcriptional activator exhibiting this property.
\r\n\r\nSignificantly, during the course of these studies two novel technologies were advanced; namely the gel-based contact point method and sequence-specific DNA affinity purification methodology. These technical advancements and the HSTF-HSE interaction results will be discussed in the body of the thesis in light of their relevance to both heat-shock and global gene expression mechanisms.
" }, { "name": "Strop, Pavel", "degree": "PhD", "year": "2002", "title": "Characterization of the Mechanosensitive Channel of Large Conductance", "advisor": "Mayo, Stephen L.; Rees, Douglas C.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05092002-155216", "creators": [ { "name": { "family": "Strop", "given": "Pavel" }, "id": "Strop-Pavel", "display_name": "Strop, Pavel" } ], "advisors": [ { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "advisor", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "advisor", "display_name": "Rees, Douglas C." } ], "committee": [ { "name": { "family": "Parker", "given": "Carl Stevens" }, "id": "Parker-C-S", "orcid": "0000-0001-9795-4211", "role": "chair", "display_name": "Parker, Carl Stevens" }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." } ], "option_major": [ "biochem" ], "doi": "10.7907/nkqb-gd95", "abstract": "Osmoregulation is an essential process in bacteria and higher organisms regulated by the mechanosensitive ion channels. The mechanosensitive channel of large conductance (MscL) is an integral membrane protein that responds to pressure in an effort to prevent cell lysis during osmotic shock. Conversion of MscL from a membrane bound form to a water soluble form was attempted by three methods: computational design, random mutagenesis and chemical modification. The water soluble form of MscL was achieved with cysteine modification method. The stability, pH dependence, and C-terminal helix of MscL were also investigated.
\r\n \r\nThe structure of the cab beta-class carbonic anhydrase (Cab) has been determined to 2.1 A resolution. Cab exists as a dimer with a fold similar to plant beta-class carbonic anhydrases. The active site zinc is coordinated by Cys32, His87, and Cys90, with the tetrahedral coordination completed by a water molecule. The difference between plant and cab beta-class carbonic anhydrases is in the organization of the hydrophobic pocket. The structure reveals a Hepes molecule near the active site, suggesting a proton transfer pathway to the solvent.
\r\n\r\nThe structure of the nitrogenase iron protein in the all-ferrous [4Fe-4S]0 form has been determined to 2.2 A resolution. The structure demonstrates that major conformational changes are not necessary to accommodate cluster reduction to the [4Fe-4S]0 state. A survey of [4Fe-4S] clusters coordinated by four cysteine ligands reveals that the [4Fe-4S] cluster of the iron protein has the largest accessible surface area, suggesting that solvent exposure may be relevant to the capability of existing in three oxidation states.
\r\n\r\nThe role of surface salt bridges in protein stabilization has been investigated. The NMR structure of a rubredoxin variant (PFRD-XC4) and the thermodynamic analysis of two surface salt bridges is presented here. The analysis shows that the surface sidechain to sidechain salt bridge between does not stabilize PFRD-XC4. The mainchain to sidechain salt bridge, however, stabilizes PFRD-XC4 by 1.5 kcal mol-1. The entropic cost of making a surface salt bridge involving the protein's backbone is reduced, since the backbone has already been immobilized upon protein folding.
\r\n" }, { "name": "Vernooy, Stephanie Yeager", "degree": "PhD", "year": "2002", "title": "Identification of Apoptotic Regulators in Drosophila and their Nonapoptotic Roles in Spermatogenesis: Implications for the Existence of a \"Caspase Cassette\" which Regulates Diverse Biological Processes", "advisor": "Hay, Bruce A.; Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02012012-113408899", "creators": [ { "name": { "family": "Vernooy", "given": "Stephanie Yeager" }, "id": "Vernooy-Stephanie-Yeager", "display_name": "Vernooy, Stephanie Yeager" } ], "advisors": [ { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "advisor", "display_name": "Hay, Bruce A." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "co-advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "chair", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "co-chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "orcid": "0000-0002-3671-9354", "role": "member", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "member", "display_name": "Zinn, Kai George" } ], "option_major": [ "biology" ], "doi": "10.7907/zqpk-k618", "abstract": "Drosophila has long been an attractive, genetically tractable model system in which to study fundamental processes such as apoptosis which are common to higher eukaryotes. Following completion of the Drosophila genome sequence, we carried out comprehensive BLAST searches to annotate it with respect to apoptosis, and found sequence homologues of virtually all mammalian cell death genes with the exception of death receptors. The only Drosophila cell death genes for which mammalian homologues have not been identified are the cell death activators Rpr, Hid, and Grim. However, since proteins with similar activities are present in mammals and since their mechanisms are likely to be conserved even if true sequence homologues are not identified, understanding how Rpr, Hid, and Grim act to bring about death is an important area of research. To better understand their mechanisms of action, we carried out an overexpression screen to identify suppressors of Rpr-, Hid-, and Grim-induced death. We identified the strongest of these suppressors as dBruce, a large protein with an N-terminal baculovirus IAP repeat (BIR), characteristic of inhibitors of apoptosis (IAPs), and a C-terminal ubiquitin conjugation domain (E2). We show that it potently suppresses death induced by Rpr and Grim but not by Hid, and that this activity likely requires its E2 domain. It does not directly promote degradation of Rpr or Grim, but its antiapoptotic action requires that their N-termini, through which they interact with BIR2 of DIAP1, be intact. These data, combined with the inability of dBruce to block death induced by the apical caspase Dronc or the proapoptotic Bcl-2 family member Debcl/Drob-1/dBorg-1/Dbok, suggest that dBruce regulates cell death at a novel point. Interestingly, dBruce mutant males are sterile, but a lack of increased caspase activity in these mutants suggests that dBruce may also play nonapoptotic roles. A closer look at Drosophila male testes revealed the surprising observation that high levels of caspases are present in wild type testes, along with the caspase activator Ark. This provokes speculation that core components of the cell death machinery can function to regulate processes other than apoptosis, such as spermatogenesis." }, { "name": "Zirlinger, Mariela", "degree": "PhD", "year": "2002", "title": "Application of Microarray, Laser Capture and Transgenic Technologies to the Study of Neural Diversity", "advisor": "Anderson, David J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04242012-143624169", "creators": [ { "name": { "family": "Zirlinger", "given": "Mariela" }, "id": "Zirlinger-Mariela", "display_name": "Zirlinger, Mariela" } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "advisor", "display_name": "Anderson, David J." } ], "committee": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "chair", "display_name": "Anderson, David J." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Alberola-Ila", "given": "Jose" }, "id": "Alberola-Ila-J", "role": "member", "display_name": "Alberola-Ila, Jose" }, { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "member", "display_name": "Schuman, Erin Margaret" } ], "option_major": [ "biology" ], "doi": "10.7907/c701-pk41", "abstract": "A major quest in modem neurobiology is to understand how the brain controls behavior. To this end, the convergence of two traditionally separate fields, systems neuroscience and molecular neuroscience, is required. The delineation of brain regions responsible for different behaviors, and in particular, their underlying neural circuits should be accompanied by the appreciation of the molecules that compose such circuits.
\r\n\r\nI have taken two approaches toward unraveling the molecular signatures of specific neural structures.\r\nFirst, I conducted microarray-based RNA expression analyses to search, in a large scale and with no a priori constraints, for differentially expressed gene products in several brain regions, including the amygdala, cerebellum, hippocampus, olfactory bulb and periaqueductal gray. Interestingly, only 0.3% of the genes characterized to date showed restricted expression in distinct brain areas. Further characterization by in situ hybridization was performed for genes enriched in the amygdala, a structure that modulates emotional behavior. Remarkably, this revealed that most region-specific genes possessed expression domains whose limits respected subnuclear boundaries defined by classical cytoarchitectonic criteria.\r\nThese analyses were not only informative about the molecular composition of distinct brain areas, but also\r\nprovided tools to genetically dissect the role of different brain nuclei in specific behaviors.
\r\n\r\nSecond, I have used a genetic strategy to label all cellular derivatives of neural crest precursor cells\r\nexpressing a particular gene, Ngn2. Such lineage tracing study uncovered a segregated cellular subpopulation in the developing peripheral nervous system, which was strongly biased for the generation of sensory rather than autonomic neurons. Despite this fate bias, Ngn2-derived cells in the dorsal root ganglion were equally likely to give rise to neurons or glia. This suggests that some neural crest cells\r\nbecome restricted to sensory or autonomic sub lineages before becoming committed to neuronal or glial\r\nfates. In general, visualization of the behavior of neural progenitors during the formation of the nervous\r\nsystem may further our understanding of the generation of specific neuronal subtypes and, eventually,\r\nneuronal connections that shape the functioning brain.
\r\n\r\nThe combination of strategies here described will enable the characterization of brain regions at the molecular level on a broad, systems-based approach.
\r\n" }, { "name": "Brown, Keith B.", "degree": "PhD", "year": "2001", "title": "Comparative Studies of Vulva Development in C. briggsae", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03062014-111730488", "creators": [ { "name": { "family": "Brown", "given": "Keith B." }, "id": "Brown-Keith-B", "display_name": "Brown, Keith B." } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "orcid": "0000-0002-3671-9354", "role": "member", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." } ], "option_major": [ "biology" ], "doi": "10.7907/n37e-5g80", "abstract": "As evolution progresses, developmental changes occur. Genes lose and gain\r\nmolecular partners, regulatory sequences, and new functions. As a consequence, tissues\r\nevolve alternative methods to develop similar structures, more or less robust. How this\r\noccurs is a major question in biology. One method of addressing this question is by\r\nexamining the developmental and genetic differences between similar species. Several\r\nstudies of nematodes Pristionchus pacificus and Oscheius CEW1 have revealed various\r\ndifferences in vulval development from the well-studied C. elegans (e.g. gonad induction,\r\ncompetence group specification, and gene function.)
\r\n\r\nI approached the question of developmental change in a similar manner by using\r\nCaenorhabditis briggsae, a close relative of C. elegans. C. briggsae allows the use of\r\ntransgenic approaches to determine developmental changes between species. We\r\ndetermined subtle changes in the competence group, in 1\u00b0 cell specification, and vulval\r\nlineage.
\r\n\r\nWe also analyzed the let-60 gene in four nematode species. We found\r\nconservation in the codon identity and exon-intron boundaries, but lack of an extended 3'\r\nuntranslated region in Caenorhabditis briggsae.
" }, { "name": "Chang, Chieh", "degree": "PhD", "year": "2001", "title": "Signal Transduction, Regulation, and Developmental Logic of EGFR Signaling in C. elegans", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03112014-080307279", "creators": [ { "name": { "family": "Chang", "given": "Chieh" }, "id": "Chang-Chieh", "display_name": "Chang, Chieh" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Chan", "given": "David C." }, "id": "Chan-D-C", "orcid": "0000-0002-0191-2154", "role": "member", "display_name": "Chan, David C." }, { "name": { "family": "Alberola-Ila", "given": "Jose" }, "id": "Alberola-Ila-J", "orcid": "0000-0003-2439-553X", "role": "member", "display_name": "Alberola-Ila, Jose" }, { "name": { "family": "Aroian", "given": "Raffi V." }, "id": "Aroian-R-V", "orcid": "0000-0002-9741-3834", "role": "member", "display_name": "Aroian, Raffi V." } ], "option_major": [ "biology" ], "doi": "10.7907/9qcj-az06", "abstract": "RTKs-mediated signaling systems and the pathways with which they interact (e.g., those initiated by G protein-mediated signaling) involve a highly cooperative network that sense a large number of cellular inputs and then integrate, amplify, and process this information to orchestrate an appropriate set of cellular responses. The responses include virtually all aspects of cell function, from the most fundamental (proliferation, differentiation) to the most specialized (movement, metabolism, chemosensation). The basic tenets of RTK signaling system seem rather well established. Yet, new pathways and even new molecular players continue to be discovered. Although we believe that many of the essential modules of RTK signaling system are rather well understood, we have relatively little knowledge of the extent of interaction among these modules and their overall quantitative importance.
\r\n\r\nMy research has encompassed the study of both positive and negative signaling by RTKs in C. elegans. I identified the C. elegans S0S-1 gene and showed that it is necessary for multiple RAS-mediated developmental signals. In addition, I demonstrated that there is a SOS-1-independent signaling during RAS-mediated vulval differentiation. By assessing signal outputs from various triple mutants, I have concluded that this SOS-1-independent signaling is not mediated by PTP-2/SHP-2 or the removal of inhibition by GAP-1/ RasGAP and it is not under regulation by SLI-1/Cb1. I speculate that there is either another exchange factor for RASor an as yet unidentified signaling pathway operating during RAS-mediated vulval induction in C. elegans.
\r\n\r\nIn an attempt to uncover the molecular mechanisms of negative regulation of EGFR signaling by SLI-1/Cb1, I and two other colleagues codiscovered that RING finger domain of SLI-1 is partially dispensable for activity. This structure-function analysis shows that there is an ubiquitin protein ligase-independent activity for SLI-1 in regulating EGFR signaling. Further, we identified an inhibitory tyrosine of LET-23/ EGFR requiring sli-1(+)for its effects: removal of this tyrosine closely mimics loss of sli-1 but not loss of other negative regulator function.
\r\n\r\nBy comparative analysis of two RTK pathways with similar signaling mechanisms, I have found that clr-1, a previously identified negative regulator of egl-15 mediated FGFR signaling, is also involved in let-23 EGFR signaling. The success of this approach promises a similar reciprocal test and could potentially extend to the study of other signaling pathways with similar signaling logic.
\r\n\r\nFinally, by correlating the developmental expression of lin-3 EGF to let-23 EGFR signaling activity, I demonstrated the existence of reciprocal EGF signaling in coordinating the morphogenesis of epithelia. This developmental logic of EGF signaling could provide a basis to understand a universal mechanism for organogenesis.
" }, { "name": "Chi, Yong", "degree": "PhD", "year": "2001", "title": "Negative Regulation of Transcription Factors by Srb10 Cyclin-Dependent Kinase", "advisor": "Dunphy, William G.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03072014-113238525", "creators": [ { "name": { "family": "Chi", "given": "Yong" }, "id": "Chi-Yong", "display_name": "Chi, Yong" } ], "advisors": [ { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "orcid": "0000-0001-7598-8939", "role": "advisor", "display_name": "Dunphy, William G." } ], "committee": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "orcid": "0000-0002-3671-9354", "role": "chair", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "role": "member", "display_name": "Campbell, Judith L." }, { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "orcid": "0000-0001-7598-8939", "role": "member", "display_name": "Dunphy, William G." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." } ], "option_major": [ "biology" ], "doi": "10.7907/tb1q-fz89", "abstract": "The ubiquitin-dependent proteolytic pathway plays an important role in a broad\r\narray of cellular processes, inducting cell cycle control and transcription. Biochemical\r\nanalysis of the ubiquitination of Sic1, the B-type cyclin-dependent kinase (CDK)\r\ninhibitor in budding yeast helped to define a ubiquitin ligase complex named SCFcdc4 (for\r\nSkp1, Cdc53/cullin, F-box protein). We found that besides Sic1, the CDK inhibitor Far1\r\nand the replication initiation protein Cdc6 are also substrates of SCFcdc4 in vitro. A\r\ncommon feature in the ubiquitination of the cell cycle SCFcdc4 substrates is that they must\r\nbe phosphorylated by the major cell cycle CDK, Cdc28. Gcn4, a transcription activator\r\ninvolved in the general control of amino acid biosynthesis, is rapidly degraded in an\r\nSCFcdc4-dependent manner in vivo. We have focused on this substrate to investigate the\r\ngenerality of the SCFcdc4 pathway. Through biochemical fractionations, we found that the\r\nSrb10 CDK phosphorylates Gcn4 and thereby marks it for recognition by SCFcdc4\r\nubiquitin ligase. Srb10 is a physiological regulator of Gcn4 stability because both\r\nphosphorylation and turnover of Gcn4 are diminished in srb10 mutants. Furthermore, we\r\nfound that at least two different CDKs, Pho85 and Srb10, conspire to promote the rapid\r\ndegradation of Gcn4 in vivo. The multistress response transcriptional regulator Msn2 is\r\nalso a substrate for Srb10 and is hyperphosphorylated in an Srb10-dependent manner\r\nupon heat stress-induced translocation into the nucleus. Whereas Msn2 is cytoplasmic in\r\nresting wild type cells, its nuclear exclusion is partially compromised in srb10 mutant\r\ncells. Srb10 has been shown to repress a subset of genes in vivo, and has been proposed\r\nto inhibit transcription via phosphorylation of the C-terminal domain of RNA polymerase\r\nII. Our results suggest a general theme that Srb10 represses the transcription of specific\r\ngenes by directly antagonizing the transcriptional activators." }, { "name": "Gould, Anita", "degree": "PhD", "year": "2001", "title": "Expression of Eph-Family Receptor Tyrosine Kinases and Ephrins in the Tadpole of the Frog Xenopus Laevis, and Possible Roles in the Development of Retinotectal Topography", "advisor": "Dreyer, William J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03102014-111038092", "creators": [ { "name": { "family": "Gould", "given": "Anita" }, "id": "Gould-Anita", "display_name": "Gould, Anita" } ], "advisors": [ { "name": { "family": "Dreyer", "given": "William J." }, "id": "Dreyer-W-J", "role": "advisor", "display_name": "Dreyer, William J." } ], "committee": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "chair", "display_name": "Fraser, Scott E." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Dreyer", "given": "William J." }, "id": "Dreyer-W-J", "role": "member", "display_name": "Dreyer, William J." } ], "option_major": [ "biology" ], "doi": "10.7907/cj8r-nj43", "abstract": "Assembling a nervous system requires exquisite specificity in the construction of\r\nneuronal connectivity. One method by which such specificity is implemented is\r\nthe presence of chemical cues within the tissues, differentiating one region from\r\nanother, and the presence of receptors for those cues on the surface of neurons\r\nand their axons that are navigating within this cellular environment.
\r\n\r\nConnections from one part of the nervous system to another often take the form\r\nof a topographic mapping. One widely studied model system that involves such\r\na mapping is the vertebrate retinotectal projection-the set of connections\r\nbetween the eye and the optic tectum of the midbrain, which is the primary\r\nvisual center in non-mammals and is homologous to the superior colliculus in\r\nmammals. In this projection the two-dimensional surface of the retina is mapped\r\nsmoothly onto the two-dimensional surface of the tectum, such that light from\r\nneighboring points in visual space excites neighboring cells in the brain. This\r\nmapping is implemented at least in part via differential chemical cues in\r\ndifferent regions of the tectum.
\r\n\r\nThe Eph family of receptor tyrosine kinases and their cell-surface ligands, the\r\nephrins, have been implicated in a wide variety of processes, generally involving\r\ncellular movement in response to extracellular cues. In particular, they possess\r\nexpression patterns-i.e., complementary gradients of receptor in retina and\r\nligand in tectum- and in vitro and in vivo activities and phenotypes-i.e.,\r\nrepulsive guidance of axons and defective mapping in mutants,\r\nrespectively-consistent with the long-sought retinotectal chemical mapping\r\ncues.
\r\n\r\nThe tadpole of Xenopus laevis, the South African clawed frog, is advantageous\r\nfor in vivo retinotectal studies because of its transparency and manipulability.\r\nHowever, neither the expression patterns nor the retinotectal roles of these\r\nproteins have been well characterized in this system. We report here\r\ncomprehensive descriptions in swimming stage tadpoles of the messenger RNA\r\nexpression patterns of eleven known Xenopus Eph and ephrin genes, including\r\nxephrin-A3, which is novel, and xEphB2, whose expression pattern has not\r\npreviously been published in detail. We also report the results of in vivo protein\r\ninjection perturbation studies on Xenopus retinotectal topography, which were\r\nnegative, and of in vitro axonal guidance assays, which suggest a previously\r\nunrecognized attractive activity of ephrins at low concentrations on retinal\r\nganglion cell axons. This raises the possibility that these axons find their correct\r\ntargets in part by seeking out a preferred concentration of ligands appropriate to\r\ntheir individual receptor expression levels, rather than by being repelled to\r\ngreater or lesser degrees by the ephrins but attracted by some as-yet-unknown\r\ncue(s).
" }, { "name": "Herman, David Matthew", "degree": "PhD", "year": "2001", "title": "Stereochemically Modified Polyamides for Recognition in the Minor Groove of DNA", "advisor": "Hoffmann, Michael R.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03122014-104807866", "creators": [ { "name": { "family": "Herman", "given": "David Matthew" }, "id": "Herman-David-Matthew", "display_name": "Herman, David Matthew" } ], "advisors": [ { "name": { "family": "Hoffmann", "given": "Michael R." }, "id": "Hoffmann-M-R", "orcid": "0000-0001-6495-1946", "role": "advisor", "display_name": "Hoffmann, Michael R." } ], "committee": [ { "name": { "family": "Parker", "given": "Carl Stevens" }, "id": "Parker-C-S", "orcid": "0000-0001-9795-4211", "role": "chair", "display_name": "Parker, Carl Stevens" }, { "name": { "family": "Dervan", "given": "Peter B." }, "id": "Dervan-P-B", "orcid": "0000-0001-8852-7306", "role": "member", "display_name": "Dervan, Peter B." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Hsieh-Wilson", "given": "Linda C." }, "id": "Hsieh-Wilson-L-C", "orcid": "0000-0001-5661-1714", "role": "member", "display_name": "Hsieh-Wilson, Linda C." }, { "name": { "family": "Hoffmann", "given": "Michael R." }, "id": "Hoffmann-M-R", "orcid": "0000-0001-6495-1946", "role": "member", "display_name": "Hoffmann, Michael R." } ], "option_major": [ "biochem" ], "doi": "10.7907/vsf2-fe75", "abstract": "The design of synthetic molecules that recognize specific sequences of DNA\r\nis an ongoing challenge in molecular medicine. Cell-permeable small molecules\r\ntargeting predetermined DNA sequences offer a potential approach for offsetting\r\nthe abnormal effects of misregulated gene-expression. Over the past twenty years,\r\nProfessor Peter B. Dervan has developed a set of pairing rules for the rational design\r\nof minor groove binding polyamides containing pyrrole (Py), imidazole (Im), and\r\nhydroxypyrrole (Hp). Polyamides have illustrated the capability to permeate cells\r\nand inhibit transcription of specific genes in vivo. This provides impetus to identify\r\nstructural elements that expand the repetoire of polyamide motifs with recognition\r\nproperties comparable to naturally occurring DNA binding proteins. Through the\r\nintroduction of chiral amino acids, we have developed chiral polyamides with\r\nstereochemically regulated binding characteristics. In addition, chiral substituents\r\nhave facilitated the development of new polyamide motifs that broaden binding site\r\nsizes targetable by this class of ligands." }, { "name": "Kamitani, Yukiyasu", "degree": "PhD", "year": "2001", "title": "Psychobiophysics of Transcranial Magnetic Stimulation", "advisor": "Shimojo, Shinsuke", "url": "https://resolver.caltech.edu/CaltechTHESIS:04082013-161544588", "creators": [ { "name": { "family": "Kamitani", "given": "Yukiyasu" }, "id": "Kamitani-Yukiyasu", "orcid": "0000-0002-9300-8268", "display_name": "Kamitani, Yukiyasu" } ], "advisors": [ { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "orcid": "0000-0002-1290-5232", "role": "advisor", "display_name": "Shimojo, Shinsuke" } ], "committee": [ { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "chair", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "orcid": "0000-0002-1290-5232", "role": "member", "display_name": "Shimojo, Shinsuke" }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "member", "display_name": "Koch, Christof" }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "member", "display_name": "Andersen, Richard A." } ], "option_major": [ "cns" ], "doi": "10.7907/zgqd-1976", "abstract": "Transcranial magnetic stimulation (TMS) is a technique that stimulates the brain using a\r\nmagnetic coil placed on the scalp. Since it is applicable to humans non-invasively, directly\r\ninterfering with neural electrical activity, it is potentially a good tool to study the direct relationship\r\nbetween perceptual experience and neural activity. However, it has been difficult\r\nto produce a clear perceptible phenomenon with TMS of sensory areas, especially using a\r\nsingle magnetic pulse. Also, the biophysical mechanisms of magnetic stimulation of single\r\nneurons have been poorly understood.
\r\n\r\nIn the psychophysical part of this thesis, perceptual phenomena induced by TMS of\r\nthe human visual cortex are demonstrated as results of the interactions with visual inputs.\r\nWe first introduce a method to create a hole, or a scotoma, in a flashed, large-field visual\r\npattern using single-pulse TMS. Spatial aspects of the interactions are explored using the\r\ndistortion effect of the scotoma depending on the visual pattern, which can be luminance-defined\r\nor illusory. Its similarity to the distortion of afterimages is also discussed. Temporal\r\ninteractions are demonstrated in the filling-in of the scotoma with temporally adjacent visual\r\nfeatures, as well as in the effective suppression of transient visual features. Also, paired-pulse\r\nTMS is shown to lead to different brightness modulations in transient and sustained\r\nvisual stimuli.
\r\n\r\nIn the biophysical part, we first develop a biophysical theory to simulate the effect of\r\nmagnetic stimulation on arbitrary neuronal structure. Computer simulations are performed\r\non cortical neuron models with realistic structure and channels, combined with the current\r\ninjection that simulates magnetic stimulation. The simulation results account for general\r\nand basic characteristics of the macroscopic effects of TMS including our psychophysical\r\nfindings, such as a long inhibitory effect, dependence on the background activity, and dependence\r\non the direction of the induced electric field.
\r\n\r\nThe perceptual effects and the cortical neuron model presented here provide foundations\r\nfor the study of the relationship between perception and neural activity. Further insights\r\nwould be obtained from extension of our model to neuronal networks and psychophysical\r\nstudies based on predictions of the biophysical model.
\r\n" }, { "name": "Liu, Yang", "degree": "PhD", "year": "2001", "title": "Molecular Mechanism of Sulfated Carbohydrate Recognition: Structural and Biochemical Studies of the Cysteine-Rich Domain of Mannose Receptor", "advisor": "Bjorkman, Pamela J.; Meyerowitz, Elliot M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03102014-140846235", "creators": [ { "name": { "family": "Liu", "given": "Yang" }, "id": "Liu-Yang-Biology", "display_name": "Liu, Yang" } ], "advisors": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "advisor", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "co-advisor", "display_name": "Meyerowitz, Elliot M." } ], "committee": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "chair", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "orcid": "0000-0002-3671-9354", "role": "member", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." } ], "option_major": [ "biology" ], "doi": "10.7907/fj0v-hx76", "abstract": "Mannose receptor (MR) is widely expressed on macrophages, immature dendritic\r\ncells, and a variety of epithelial and endothelial cells. It is a 180 kD type I transmembrane\r\nreceptor whose extracellular region consists of three parts: the amino-terminal cysteine-rich\r\ndomain (Cys-MR); a fibronectin type II-like domain; and a series of eight tandem C-type\r\nlectin carbohydrate recognition domains (CRDs). Two portions of MR have distinct\r\ncarbohydrate recognition properties: Cys-MR recognizes sulfated carbohydrates and the\r\ntandem CRD region binds terminal mannose, fucose, and N-acetyl-glucosamine (GlcNAc).\r\nThe dual carbohydrate binding specificity allows MR to interact with sulfated and nonsulfated\r\npolysaccharide chains, and thereby facilitating the involvement of MR in\r\nimmunological and physiological processes. The immunological functions of MR include\r\nantigen capturing (through binding non-sulfated carbohydrates) and antigen targeting\r\n(through binding sulfated carbohydrates), and the physiological roles include rapid clearance\r\nof circulatory luteinizing hormone (LH), which bears polysaccharide chains terminating with\r\nsulfated and non-sulfated carbohydrates.
\r\n\r\nWe have crystallized and determined the X-ray structures of unliganded Cys-MR (2.0\r\n\u00c5) and Cys-MR complexed with different ligands, including Hepes (1.7 \u00c5), 4SO_4-N-Acetylgalactosamine\r\n(4SO_4-GalNAc; 2.2 \u00c5), 3SO_4-Lewis^x (2.2 \u00c5), 3S04-Lewis^a (1.9 \u00c5),\r\nand 6SO_4-GalNAc (2.5 \u00c5). The overall structure of Cys-MR consists of 12 anti-parallel \u03b2-strands\r\narranged in three lobes with approximate three fold internal symmetry. The structure\r\ncontains three disulfide bonds, formed by the six cysteines in the Cys-MR sequence. The\r\nligand-binding site is located in a neutral pocket within the third lobe, in which the sulfate\r\ngroup of ligand is buried. Our results show that optimal binding is achieved by a\r\ncarbohydrate ligand with a sulfate group that anchors the ligand by forming numerous\r\nhydrogen bonds and a sugar ring that makes ring-stacking interactions with Trpll7 of CysMR.\r\nUsing a fluorescence-based assay, we characterized the binding affinities between CysMR\r\nand its ligands, and rationalized the derived affinities based upon the crystal structures.\r\nThese studies reveal the mechanism of sulfated carbohydrate recognition by Cys-MR and\r\nfacilitate our understanding of the role of Cys-MR in MR recognition of its ligands.
\r\n" }, { "name": "Lyapina, Svetlana Anatol'Evna", "degree": "PhD", "year": "2001", "title": "Characterization of the Human SCF Ubiquitin Ligases - Structure, Function, and Regulation", "advisor": "Dunphy, William G.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03072014-090536401", "creators": [ { "name": { "family": "Lyapina", "given": "Svetlana Anatol'Evna" }, "id": "Lyapina-Svetlana-Anatol'Evna", "display_name": "Lyapina, Svetlana Anatol'Evna" } ], "advisors": [ { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "orcid": "0000-0001-7598-8939", "role": "advisor", "display_name": "Dunphy, William G." } ], "committee": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "orcid": "0000-0002-3671-9354", "role": "chair", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "role": "member", "display_name": "Campbell, Judith L." }, { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "orcid": "0000-0001-7598-8939", "role": "member", "display_name": "Dunphy, William G." }, { "name": { "family": "Varshavsky", "given": "Alexander J." }, "id": "Varshavsky-A-J", "orcid": "0000-0002-4011-258X", "role": "member", "display_name": "Varshavsky, Alexander J." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." } ], "option_major": [ "biology" ], "doi": "10.7907/apdy-3d30", "abstract": "The SCF ubiquitin ligase complex of budding yeast triggers DNA replication by\r\ncata lyzi ng ubiquitination of the S phase CDK inhibitor SIC1. SCF is composed of several\r\nevolutionarily conserved proteins, including ySKP1, CDC53 (Cullin), and the F-box protein\r\nCDC4. We isolated hSKP1 in a two-hybrid screen with hCUL1, the human homologue of\r\nCDC53. We showed that hCUL1 associates with hSKP1 in vivo and directly interacts with\r\nhSKP1 and the human F-box protein SKP2 in vitro, forming an SCF-Iike particle. Moreover,\r\nhCUL1 complements the growth defect of yeast CDC53^(ts) mutants, associates with ubiquitination-promoting activity in human cell extracts, and can assemble into functional, chimeric ubiquitin\r\nligase complexes with yeast SCF components. These data demonstrated that hCUL1 functions as\r\npart of an SCF ubiquitin ligase complex in human cells. However, purified human SCF\r\ncomplexes consisting of CUL1, SKP1, and SKP2 are inactive in vitro, suggesting that additional\r\nfactors are required.
\r\n\r\nSubsequently, mammalian SCF ubiquitin ligases were shown to regulate various\r\nphysiological processes by targeting important cellular regulators, like l\u0138B\u03b1, \u03b2-catenin, and p27,\r\nfor ubiquitin-dependent proteolysis by the 26S proteasome. Little, however, is known about the\r\nregulation of various SCF complexes. By using sequential immunoaffinity purification and mass\r\nspectrometry, we identified proteins that interact with human SCF components SKP2 and CUL1\r\nin vivo. Among them we identified two additional SCF subunits: HRT1, present in all SCF\r\ncomplexes, and CKS1, that binds to SKP2 and is likely to be a subunit of SCF5^(SKP2) complexes.\r\nSubsequent work by others demonstrated that these proteins are essential for SCF activity. We\r\nalso discovered that COP9 Signalosome (CSN), previously described in plants as a suppressor of\r\nphotomorphogenesis, associates with CUL1 and other SCF subunits in vivo. This interaction is\r\nevolutionarily conserved and is also observed with other Cullins, suggesting that all Cullin based\r\nubiquitin ligases are regulated by CSN. CSN regulates Cullin Neddylation presumably through CSNS/JAB1, a stochiometric Signalosome subunit and a putative deneddylating enzyme. This\r\nwork sheds light onto an intricate connection that exists between signal transduction pathways\r\nand protein degradation machinery inside the cell and sets stage for gaining further insights into\r\nregulation of protein degradation.
\r\n" }, { "name": "Martin, Warham Lance", "degree": "PhD", "year": "2001", "title": "Protein-Protein Recognition: The Neonatal Fc Receptor and Immunoglobulin G", "advisor": "Bjorkman, Pamela J.; Meyerowitz, Elliot M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03072014-143524462", "creators": [ { "name": { "family": "Martin", "given": "Warham Lance" }, "id": "Martin-Warham-Lance", "display_name": "Martin, Warham Lance" } ], "advisors": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "advisor", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "co-advisor", "display_name": "Meyerowitz, Elliot M." } ], "committee": [ { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "chair", "display_name": "Rees, Douglas C." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." } ], "option_major": [ "biochem" ], "doi": "10.7907/9ek6-6833", "abstract": "The neonatal Fc receptor (FcRn) binds the Fc portion of immunoglobulin G (IgG)\r\nat the acidic pH of endosomes or the gut and releases IgG at the alkaline pH of blood.\r\nFcRn is responsible for the maternofetal transfer of IgG and for rescuing endocytosed\r\nIgG from a default degradative pathway. We investigated how FcRn interacts with IgG\r\nby constructing a heterodimeric form of the Fc (hdFc) that contains one FcRn binding\r\nsite. This molecule was used to characterize the interaction between one FcRn molecule\r\nand one Fc and to determine under what conditions FcRn forms a dimer. The hdFc binds\r\none FcRn molecule at pH 6.0 with a Kd of 80 nM. In solution and with FcRn anchored to\r\nsolid supports, the heterodimeric Fc does not induce a dimer of FcRn molecules. FcRnhdFc\r\ncomplex crystals were obtained and the complex structure was solved to 2.8 \u00c5\r\nresolution. Analysis of this structure refined the understanding of the mechanism of the\r\npH-dependent binding, shed light on the role played by carbohydrates in the Fc binding,\r\nand provided insights on how to design therapeutic IgG antibodies with longer serum\r\nhalf-lives. The FcRn-hdFc complex in the crystal did not contain the FcRn dimer. To\r\ncharacterize the tendency of FcRn to form a dimer in a membrane we analyzed the\r\ntendency of the hdFc to induce cross-phosphorylation of FcRn-tyrosine kinase chimeras.\r\nWe also constructed FcRn-cyan and FcRn-yellow fluorescent proteins and have analyzed\r\nthe tendency of these molecules to exhibit fluorescence resonance energy transfer. As of\r\nnow, neither of these analyses have lead to conclusive results. In the process of acquiring\r\nthe context to appreciate the structure of the FcRn-hdFc interface, we developed a study\r\nof 171 other nonobligate protein-protein interfaces that includes an original principal\r\ncomponent analysis of the quantifiable aspects of these interfaces." }, { "name": "Shou, Wenying", "degree": "PhD", "year": "2001", "title": "Diverse Mechanisms of Regulating the Mitotic Cell Cycle", "advisor": "Dunphy, William G.", "url": "https://resolver.caltech.edu/CaltechETD:etd-02032005-110205", "creators": [ { "name": { "family": "Shou", "given": "Wenying" }, "id": "Shou-Wenying", "orcid": "0000-0001-5693-381X", "display_name": "Shou, Wenying" } ], "advisors": [ { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "orcid": "0000-0001-7598-8939", "role": "advisor", "display_name": "Dunphy, William G." } ], "committee": [ { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "orcid": "0000-0002-3671-9354", "role": "chair", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "orcid": "0000-0001-7598-8939", "role": "member", "display_name": "Dunphy, William G." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." } ], "option_major": [ "biology" ], "doi": "10.7907/TP2C-Q082", "abstract": "The mitotic cell cycle - a process in which one cell divides into two - is carefully regulated in response to signals. Organisms as diverse as yeast and human all drive their cell cycles by turning cyclin-dependent kinases (Cdks) on and off. We have examined Cdk regulation in two systems. First, we isolated a relatively general Cdk inhibitor (CKI) p28Kix1 in Xenopus laevis. p28Kix1 binds to and directly inhibits multiple Cdks, and retards DNA replication and mitosis when added to Xenopus egg extracts. Remarkably, the protein level of p28Kix1 is dramatically upregulated around late gastrulation, suggesting that it is induced in response to a developmental signal and in turn functions to establish a somatic type of cell cycle. Second, we examined how Cdk inactivation is achieved during mitotic exit in the yeast S. cerevisiae. We found that mutants in at least six linkage groups bypassed the mitotic arrest in cdc15\u0394 cells. The net1-1 mutant was studied further. Net1 inhibits mitotic exit by inhibiting Cdc 14, an essential protein phosphatase, using two parallel mechanisms: by binding and inactivating Cdc14, and by tethering Cdc14 to the nucleolus. Correct orientation of the mitotic spindle activates Cdc15, which evicts Cdc14 from the nucleolus into the entire cell. Cdc14 subsequently inactivates Cdks by promoting cyclin degradation and CKI induction, and mitotic exit ensues. Unexpectedly, Netl also regulates the structure and function of the nucleolus: it tethers the transcriptional silencing protein Sir2 to the nucleolus, regulates proper localization of multiple nucleolar antigens and rDNA morphology, and directly binds to and stimulates the activity of RNA Pol I. In summary, we observe that different signals regulate cell cycle progression by controlling Cdk activity, that cell cycle regulators may play important roles in other biological processes, and that localization of a protein to a subcellular structure could imply that it is sequestered instead of employed there." }, { "name": "Vanier, Michael Christopher", "degree": "PhD", "year": "2001", "title": "Realistic Computer Modeling of the Mammalian Olfactory Cortex", "advisor": "Bower, James M.", "url": "https://resolver.caltech.edu/CaltechETD:etd-08162006-130008", "creators": [ { "name": { "family": "Vanier", "given": "Michael Christopher" }, "id": "Vanier-Michael-Christopher", "display_name": "Vanier, Michael Christopher" } ], "advisors": [ { "name": { "family": "Bower", "given": "James M." }, "id": "Bower-J-M", "role": "advisor", "display_name": "Bower, James M." } ], "committee": [ { "name": { "family": "Pine", "given": "Jerome" }, "id": "Pine-J", "role": "chair", "display_name": "Pine, Jerome" }, { "name": { "family": "Bower", "given": "James M." }, "id": "Bower-J-M", "role": "member", "display_name": "Bower, James M." }, { "name": { "family": "Barr", "given": "Alan H." }, "id": "Barr-A-H", "role": "co-chair", "display_name": "Barr, Alan H." }, { "name": { "family": "Goodman", "given": "Rodney M." }, "id": "Goodman-R-M", "role": "co-chair", "display_name": "Goodman, Rodney M." }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "member", "display_name": "Koch, Christof" } ], "option_major": [ "cns" ], "doi": "10.7907/QM8V-8404", "abstract": "A combination of experimental and computer modeling techniques were used to investigate the dynamics and computational functions of the rat olfactory (piriform) cortex. Experimental characterization of synaptic response to afferent and associational fiber voltage shocks were performed, in the presence and absence of the neuromodulator norepinephrine. This data was used to generate computer models of synaptic transmission in piriform cortex. Models of pyramidal neurons and feedback inhibitory interneurons were constructed which accurately match intracellular experimental data in the presence and absence of norepinephrine. In order to achieve this, parameter search tools for automatically matching computer models of neurons to data were developed. Models of feedforward inhibitory interneurons were also constructed. An abstract spike generating model of the olfactory bulb was built. These components were combined to create a realistic computer model of the piriform cortex. This model can accurately replicate the response of the real system to a strong shock stimulus, as reflected in current source density plots. Two versions of the model were created to model the oscillatory response of the system to week shots. The first model replicates the surface field potential with considerable accuracy, but fails to replicate the current source density data. The second model replicates the current source density data and suggests a new organizing principle for the olfactory system based on non-overlapping neuronal groups. This hypothesis is experimentally testable." }, { "name": "Chapman, Tara Lynn", "degree": "PhD", "year": "2000", "title": "Biochemical Characterization of Two Cytomegalovirus MHC Class I Homologs", "advisor": "Bjorkman, Pamela J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08202025-165630518", "creators": [ { "name": { "family": "Chapman", "given": "Tara Lynn" }, "id": "Chapman-Tara-Lynn", "display_name": "Chapman, Tara Lynn" } ], "advisors": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "advisor", "display_name": "Bjorkman, Pamela J." } ], "committee": [ { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "chair", "display_name": "Rees, Douglas C." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "member", "display_name": "Strauss, James H." } ], "option_major": [ "bioch" ], "doi": "10.7907/1jbr-8b50", "abstract": "Cytomegaloviruses are ubiquitous host-specific pathogens that are capable of causing\r\nlife-long persistent infections in immunocompetent hosts. To maintain this persistence in\r\nthe presence of a functional immune system, both human and murine cytomegaloviruses\r\n(HCMV and MCMV, respectively) encode genes that modulate the host immune\r\nresponse. These genes include the MHC class I homologs UL18 from HCMV and m144\r\nfrom MCMV. The host receptor for UL18 has been identified as LIR-1, a B cell,\r\nmonocyte and dendritic cell inhibitory receptor related to natural killer cell inhibitory\r\nreceptors, whereas the receptor for m144 remains unknown. In order to facilitate\r\nunderstanding of the functions of UL18 and m144 in viral pathogenesis and immune\r\nevasion, we have initiated structure/function analyses of ml 44, UL18 and LIR-1. We\r\nshow that soluble m144 associates with the MHC class I light chain, B2-microglobulin,\r\nbut unlike UL18 and class I MHC proteins, m144 does not associate with endogenous\r\npeptides, presumably due to a large deletion in the peptide binding platform. Using\r\nsoluble versions of UL18, class I MHC molecules and LIR-1, we find that LIR-1 interacts\r\nwith the relatively non-polymorphic a3 domain of class I proteins and the analogous\r\nregion of ULI 8 using its N-terminal immunoglobulin-like domain. Recognition of the a3\r\ndomain, which is relatively non-polymorphic in class I MHC molecules, predicts that\r\nLIR-1 can interact with most or all class I MHC molecules, consistent with previous\r\nobservations that LIR-1 binds a wide range of class I proteins. We also find that LIR-1\r\nbinds UL18 with a > 1000-fold higher affinity than it binds classical and non-classical\r\nclass I MHC proteins, illustrating how a viral protein can effectively compete with host\r\nproteins to subvert the host immune response." }, { "name": "Chee-Ruiter, Christine Wai Jun", "degree": "PhD", "year": "2000", "title": "The Biological Sense of Smell: Olfactory Search Behavior and a Metabolic View for Olfactory Perception", "advisor": "Bower, James M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04092013-110459537", "creators": [ { "name": { "family": "Chee-Ruiter", "given": "Christine Wai Jun" }, "id": "Chee-Ruiter-Christine-Wai-Jun", "display_name": "Chee-Ruiter, Christine Wai Jun" } ], "advisors": [ { "name": { "family": "Bower", "given": "James M." }, "id": "Bower-J-M", "role": "advisor", "display_name": "Bower, James M." } ], "committee": [ { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "chair", "display_name": "Koch, Christof" }, { "name": { "family": "Bower", "given": "James M." }, "id": "Bower-J-M", "role": "member", "display_name": "Bower, James M." }, { "name": { "family": "Lewis", "given": "Nathan S." }, "id": "Lewis-N-S", "orcid": "0000-0001-5245-0538", "role": "member", "display_name": "Lewis, Nathan S." }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "orcid": "0000-0002-1290-5232", "role": "member", "display_name": "Shimojo, Shinsuke" } ], "option_major": [ "cns" ], "doi": "10.7907/wdn1-5v55", "abstract": "Part I of the thesis describes the olfactory searching and scanning behaviors of\r\nrats in a wind tunnel, and a detailed movement analysis of terrestrial arthropod olfactory\r\nscanning behavior. Olfactory scanning behaviors in rats may be a behavioral correlate to\r\nhippocampal place cell activity.
\r\n\r\nPart II focuses on the organization of olfactory perception, what it suggests about\r\na natural order for chemicals in the environment, and what this in tum suggests about the\r\norganization of the olfactory system. A model of odor quality space (analogous to the\r\n\"color wheel\") is presented. This model defines relationships between odor qualities\r\nperceived by human subjects based on a quantitative similarity measure. Compounds\r\ncontaining Carbon, Nitrogen, or Sulfur elicit odors that are contiguous in this odor\r\nrepresentation, which thus allows one to predict the broad class of odor qualities a\r\ncompound is likely to elicit. Based on these findings, a natural organization for olfactory\r\nstimuli is hypothesized: the order provided by the metabolic process. This hypothesis is\r\ntested by comparing compounds that are structurally similar, perceptually similar, and\r\nmetabolically similar in a psychophysical cross-adaptation paradigm. Metabolically\r\nsimilar compounds consistently evoked shifts in odor quality and intensity under cross-adaptation,\r\nwhile compounds that were structurally similar or perceptually similar did not. This suggests that the olfactory system may process metabolically similar compounds using the same neural pathways, and that metabolic similarity may be the fundamental metric about which olfactory processing is organized. In other words, the olfactory system may be organized around a biological basis.
\r\n\r\nThe idea of a biological basis for olfactory perception represents a shift in how\r\nolfaction is understood. The biological view has predictive power while the current\r\nchemical view does not, and the biological view provides explanations for some of the\r\nmost basic questions in olfaction, that are unanswered in the chemical view. Existing\r\ndata do not disprove a biological view, and are consistent with basic hypotheses that arise\r\nfrom this viewpoint.
\r\n" }, { "name": "Chen, Wen J.", "degree": "PhD", "year": "2000", "title": "Extragenic Suppressors of Heat Shock Activated GO\u03b1. Topic I: Cyclin in Heat Shock Response. Topic II: Signaling by Go and Gq in C. elegans", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08112020-100312577", "creators": [ { "name": { "family": "Chen", "given": "Wen J." }, "id": "Chen-Wen-J", "display_name": "Chen, Wen J." } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "role": "member", "display_name": "Dunphy, William G." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "member", "display_name": "Simon, Melvin I." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "member", "display_name": "Deshaies, Raymond Joseph" } ], "option_major": [ "biology" ], "doi": "10.7907/2sv5-sd03", "abstract": "In the nematode C. elegans, heterotrimeric G proteins have been shown to regulate the behavior of locomotion, feeding and egg-laying. Go belongs to Gi family and only exists in organisms with a nervous system. The signaling downstream of Go has been a mystery since its discovery, and it is what we are determined to find out.
\r\n\r\nLoss of Go\u03b1 causes animals to be hyperactive and lay eggs constitutively. Overexpressing Go\u03b1 causes opposite phenotypes. Another \u03b1 protein a subunit, Gq, causes phenotypes opposite to Go. To identify G protein effectors in C. elegans, we performed a forward genetic screen for suppressors of activated Go\u03b1 under the control of the heat-shock promoter hsp16-2. Because of the nature of the screen design, we identified two categories of genes. One category acts on heat shock response and the other category acts on G protein pathways. We characterized and positional cloned genes from both categories.
\r\n\r\nThe second chapter of the thesis described sag-4, a cyclin L homologue that specifically affects heat shock promoters and decreases heat-shock induced protein expression. We propose that cyclin Lis likely to be involved in heat shock induced transcription. Other genes in this category, sag-3, sag-5 and sag-8, may also function in similar mechanisms.
\r\n\r\nThe third chapter of the thesis focused on G protein signaling. eat-16 was identified in the screen for suppressors of activated Go\u03b1. We positional cloned it and found it encodes a RGS7 homologue. RGS proteins have been studied as GTPase Activating Protein for the \u03b1 subunits of heterotrimeric G proteins. Although eat-16 was identified in a suppressor screen for activated Go (goa-1), both genetic and biochemical evidence showed that eat-16 is a GAP for Gq (egl-30). We propose that Go and Gq antagonize each other, thereby regulating behaviors. Go might negatively regulate Gq signaling, possibly through eat-16 or other unrevealed genes.
\r\n\r\nChapter four describes our reconstituted system in mammalian cell culture. EAT-16 decreases Gq/G11 mediated PLC activity. GOA-1 and GPB-2 (C. elegans G\u03b25 homologue) also decrease PLC activity induced by Gq/G11. These results are consistent with the hypothesis that Go negatively regulate Gq signaling, and the interaction between G\u03b25 and RGS7 can be one of the steps between Go and Gq.
" }, { "name": "Csete, Marie Elizabeth", "degree": "PhD", "year": "2000", "title": "Less is More: Oxygen and Stem Cell Regeneration", "advisor": "Wold, Barbara J.; Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechETD:etd-12032003-091634", "creators": [ { "name": { "family": "Csete", "given": "Marie Elizabeth" }, "id": "Csete-Marie-Elizabeth", "orcid": "0000-0003-4591-0510", "display_name": "Csete, Marie Elizabeth" } ], "advisors": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "advisor", "display_name": "Wold, Barbara J." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "co-advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "chair", "display_name": "Wold, Barbara J." }, { "name": { "family": "Baltimore", "given": "David L." }, "id": "Baltimore-D-L", "role": "member", "display_name": "Baltimore, David L." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "chair", "display_name": "Fraser, Scott E." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "member", "display_name": "Strauss, James H." } ], "option_major": [ "biology" ], "doi": "10.7907/MP6F-KN97", "abstract": "Recent years have witnessed an explosion in the identification and understanding of stem cells, affording new cellular reagents for the study of regeneration in vitro. Traditionally, regeneration is studied in tissue culture in which the gaseous environment surrounding the cells contains about 20% oxygen. Cells in our bodies are never exposed to such high levels of oxygen, well out of normal physiologic range. In this work, stem cell regeneration in several systems was studied in traditional 20% oxygen culture and in oxygen levels more reflective of normal physiology. These lower oxygen-cultured progenitors behaved differently than those cultured in traditional environments. In several stem cell systems low oxygen significantly increased proliferation of progenitor populations, and in central nervous system stem cells, also decreased apoptotic death. More physiologic levels of oxygen in culture also led to regeneration of different daughter progeny populations with a distribution of phenotypes distinct from that generated in 20% oxygen. For example in CNS stem cells, a significantly greater yield of dopaminergic and serotonergic neurons was generated in low oxygen compared to 20% oxygen. Skeletal muscle satellite stem cells in high oxygen were significantly more likely to assume an adipocyte phenotype than those cultured in low oxygen. Furthermore, genes expressed during regeneration in physiologic vs. 20% oxygen were different from each other in timing and in abundance. These data suggest that oxygen manipulations will be useful to increase the survival and expansion of progenitor populations for research and possible transplantation, as well as for the survival and expansion of selected regenerated progeny. Furthermore, oxygen levels are a useful manipulation to help isolate and identify pathways used during regeneration and differentiation.
" }, { "name": "Egnor, Susan Elizabeth Roian", "degree": "PhD", "year": "2000", "title": "The Role of Spectral Cues in Sound Localization by the Barn Owl", "advisor": "Konishi, Masakazu", "url": "https://resolver.caltech.edu/CaltechTHESIS:08152025-184139088", "creators": [ { "name": { "family": "Egnor", "given": "Susan Elizabeth Roian" }, "id": "Egnor-Susan-Elizabeth-Roian", "orcid": "0000-0003-0992-6240", "display_name": "Egnor, Susan Elizabeth Roian" } ], "advisors": [ { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "advisor", "display_name": "Konishi, Masakazu" } ], "committee": [ { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "chair", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "member", "display_name": "Andersen, Richard A." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "abstract": "Toe barn owl (Tyto alba) is a nocturnal predator with excellent spatial hearing.\r\nEvolutionary pressure on the auditory system of the barn owl has produced\r\nnumerous adaptations for processing spatial information; my dissertation\r\naddresses two new aspects of sound localization in this auditory specialist.
\r\n\r\nI. Barn owls have been shown to use interaural time differences (ITDs) and\r\ninteraural intensity differences (IIDs) to localize the sources of sounds. Such\r\nbinaural difference cues are also used by a variety of mammals, including\r\nhumans. Mammals also exploit a monaural sound localization cue. When\r\nsound arrives at the eardrum it has been filtered by the external ear in a\r\nlocation- and frequency-dependent manner; this spectral cue underlies our ability\r\nto localize in the vertical plane. Recent measurements in barn owls have shown\r\nthat spectral features similar to those thought to encode vertical position in\r\nhumans also exist in barn owls. I show that radical variations in the monaural\r\nspectra which do not produce concomitant changes in the binaural difference\r\nspectrum have no effect on barn owl sound localization behavior. In contrast to\r\nhumans, barn owls do make some use of the frequency-specific IID information\r\nin the binaural difference spectrum.
\r\n\r\nII. In order to use a binaural difference cue, the sound from a source that enters\r\nthe left ear must be compared with sound from the same source that enters the\r\nright ear. This results in the perception of a single auditory image; this process\r\nis known as binaural fusion. One feature of sound which is important to fusion\r\nis the degree to which the sounds arriving at the two ears are correlated. The\r\nability of both barn owls and humans to localize based on ITD is eliminated if\r\nbinaural correlation is reduced to zero. However, I show that barn owls can\r\nextract the IID of a binaurally uncorrelated signal, and use that IID to control an\r\nauditory saccade. In addition I show that neurons in the thalamic auditory\r\npathway, in contrast to the more well understood collicular auditory pathway,\r\nencode the IID of binaurally unc01Telated sounds.
" }, { "name": "Gonzalez-Serricchio, Aidyl Sofia", "degree": "PhD", "year": "2000", "title": "Negative Regulation of Cell Fate Specification by the lin-15 Locus During Vulval Induction in Caenorhabditis elegans", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:07022021-160304976", "creators": [ { "name": { "family": "Gonzalez-Serricchio", "given": "Aidyl Sofia" }, "id": "Gonzalez-Serricchio-Aidyl-Sofia", "display_name": "Gonzalez-Serricchio, Aidyl Sofia" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." } ], "option_major": [ "biology" ], "doi": "10.7907/ppq9-ps50", "abstract": "We have visualized extrachromosomal arrays by targeting the green fluorescent protein (GFP) to a specific DNA sequence (lac operator) incorporated into Caenorhabditis elegans' transgenes. This system can be used to determine polyploidy and to investigate chromosome segregation. This technique also allows rapid, accurate determination of spontaneous loss of an array, thereby allowing high-resolution mosaic analysis. We carried out genetic mosaic analysis on lin-3 (epidermal growth factor) using the GFP-Lacl + lacO method. This methodology confirmed lin-3's site of action for vulval induction is at the anchor cell. This result also proved this technique works.
\r\n\r\nWe used both the GFP-Lacl + lacO256 system as well as the ncl-1 gene as genetic mosaic markers to determine the site of action of lin-15A and lin-15B. Both markers indicate that lin-15A gene function is required within the vulval precursor cells (VPCs) to prevent an excessive number of VPCs from generating vulval progeny. The mosaic expression pattern for lin-15B is broad therefore, proven difficult to pinpoint a site of action.
\r\n\r\nThe products of the lin-15 gene were first defined genetically as negative regulators of the vulval induction pathway. It encodes two novel hydrophilic proteins, LIN-15A and LIN-15B. According to antibody stainings and GFP expression patterns, both proteins are nuclear and present in almost all the cells. lin-15 is part of the synthetic multivulva (synMuv) set of genes which are comprised of two classes, A and B. Mutation of both an A and a B gene is required to obtain a multivulva (Muv) phenotype. Further characterization of the lin-15 locus reveals an effect on fertility.
" }, { "name": "Greenwood, Amy L.", "degree": "PhD", "year": "2000", "title": "The Generation of Peripheral Neuron Diversity from Mammalian Progenitor Cells In Vitro", "advisor": "Anderson, David J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08062025-230309605", "creators": [ { "name": { "family": "Greenwood", "given": "Amy L." }, "id": "Greenwood-Amy-L.", "display_name": "Greenwood, Amy L." } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "advisor", "display_name": "Anderson, David J." } ], "committee": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "chair", "display_name": "Anderson, David J." }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biology" ], "doi": "10.7907/6pn4-h629", "abstract": "The vertebrate peripheral nervous system is comprised of many different kinds of\r\nneurons that develop from a common progenitor pool, the neural crest. Factors that\r\ncontrol the specification of most neuronal subtypes are not well understood. The studies\r\npresented in this thesis describe how and when sensory precursors become different from\r\nthose of the autonomic lineage, and how subtype diversity is generated within the sensory\r\nneuron class.
\r\n\r\nThe development of peripheral neurons was assayed in vitro using rat neural tube\r\nexplant cultures as a source of neural crest, and antibody staining for lineage-specific\r\ntranscription factors to identify neuronal subtypes. A chemically defined medium\r\nsupported the differentiation of sensory but not autonomic neurons in these explants.\r\nWhen cultures were challenged with the extrinsic factor BMP2 to induce autonomic\r\nneurons, the division and differentiation of sensory precursors was unperturbed.\r\nTherefore, the neural crest contains a population of di vi ding precursors that are\r\noperationally determined to the sensory neuron fate.
\r\n\r\nThe majority of sensory neurons that differentiated in defined medium displayed\r\ncharacteristics of the muscle afferent subtype including dependence on the neurotrophins\r\nBDNF and NT-3, expression of the marker ER81, and lack of expression of trkA. In\r\ncontrast, the addition of serum or BMP2 induced the development of many neurons that\r\nexpressed the cutaneous afferent marker, trkA. Neither serum nor BMP2 is sufficient to\r\ninduce trkA in post-migratory, differentiated neurons; instead, these factors induce the\r\nneural tube to produce additional sensory precursors, some of which differentiate into\r\ncutaneous afferents. We also observed that the neurotrophins BDNF and NT-3, which\r\ncould directly regulate ER81 expression in differentiated neurons, did not prevent the\r\ndevelopment of trkA expressing neurons. This implies that serum/BMP2 have early\r\naffects on the specification of sensory subtypes while neurotrophins regulate acquisition\r\nof particular subtype characteristics.
\r\n\r\nIn addition to their ability to promote the development of the cutaneous afferent\r\nsensory subtype, serum!BMP2 induced patterning in neural tube explants. In the\r\npresence of these factors, the location of sensory and autonomic neurons in the neural\r\ncrest outgrowth of the explant resembled the arrangement of these neuronal subtypes\r\nalong the dorsoventral axis in vivo.
" }, { "name": "Itti, Laurent", "degree": "PhD", "year": "2000", "title": "Models of Bottom- Up and Top-Down Visual Attention", "advisor": "Koch, Christof", "url": "https://resolver.caltech.edu/CaltechETD:etd-12022005-103530", "creators": [ { "name": { "family": "Itti", "given": "Laurent" }, "id": "Itti-Laurent", "orcid": "0000-0002-0168-2977", "display_name": "Itti, Laurent" } ], "advisors": [ { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "advisor", "display_name": "Koch, Christof" } ], "committee": [ { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "chair", "display_name": "Koch, Christof" }, { "name": { "family": "Psaltis", "given": "Demetri" }, "id": "Psaltis-D", "orcid": "0000-0003-4684-8800", "role": "member", "display_name": "Psaltis, Demetri" }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "member", "display_name": "Perona, Pietro" }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "member", "display_name": "Andersen, Richard A." }, { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "orcid": "0000-0002-1290-5232", "role": "member", "display_name": "Shimojo, Shinsuke" } ], "option_major": [ "cns" ], "doi": "10.7907/MD7V-NE41", "abstract": "When we observe our visual environment, we do not perceive all its components as being equally interesting. Some objects automatically and effortlessly \"pop out\" from their surroundings, that is, they draw our visual attention, in a \"bottom up\" manner, towards them. In a first approximation, focal visual attention acts as a rapidly shiftable \"spotlight,\" which allows only the selected information to reach higher levels of processing and representation. Most models of the bottom-up control of attention are based on the concept of a saliency map, that is, an explicit two-dimensional map that encodes the conspicuity of objects in the visual environment. Competition among neurons in this map gives rise to a single winning location that corresponds to the next attended target. Inhibiting this location automatically allows the system to attend to the next most salient location. A first body of work in this thesis describes a detailed computer implementation of such a scheme, focusing on the problem of combining information across modalities, here orientation, intensity and color information, in a purely stimulus-driven manner. The model is applied to common psychophysical stimuli as well as to very demanding visual search tasks. Its successful performance is used to address the extent to which the primate visual system carries out visual search via one or more such saliency maps and how this can be tested.
\r\n\r\nWe next address the question of what happens once our attention is focused onto a restricted part of our visual field. There is mounting experimental evidence that attention is far more sophisticated than a simple feed-forward spatially-selective filtering process. Indeed, visual processing appears to be significantly different inside the attentional spotlight than outside. That is, in addition to its properties as a feed-forward information processing and transmission bottleneck, focal visual attention feeds back and locally modulates, in a \"top down\" manner, the visual processing and representation of selected objects. The second body of work presented in this thesis is concerned with a detailed computational model of basic pattern vision in humans and its modulation by top-down attention. We start by acquiring a complete dataset of five different simple psychophysical experiments, including discriminations of contrast, orientation and spatial frequency of simple pattern stimuli by human observers. This experimental dataset places strict constraints on our model of early pattern vision. The model, however, is eventually able to reproduce the entire dataset while assuming plausible neurobiological components. The model is further applied to existing psychophysical data which demonstrates how top-down attention alters performance in these simple psychophysical discrimination experiments. Our model is able to quantitatively account for all observations by assuming that attention strengthens the non-linear cortical interactions among visual neurons.
\r\n\r\nTogether, the two aspects of attention studied in this thesis lead us to consider the essential role of non-linear computations in visual processing. We suggest that visual processing, even at its earliest levels, is best characterized not by linear response functions and spatial convolutions, but rather by non-linearly interacting computational devices.
" }, { "name": "Iverson, Tina Michelle", "degree": "PhD", "year": "2000", "title": "Crystallographic Investigations of Respiratory Proteins", "advisor": "Rees, Douglas C.", "url": "https://resolver.caltech.edu/CaltechTHESIS:11252025-213630272", "creators": [ { "name": { "family": "Iverson", "given": "Tina Michelle" }, "id": "Iverson-Tina-Michelle", "orcid": "0000-0001-8816-6352", "display_name": "Iverson, Tina Michelle" } ], "advisors": [ { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "advisor", "display_name": "Rees, Douglas C." } ], "committee": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "chair", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Gray", "given": "Harry B." }, "id": "Gray-H-B", "orcid": "0000-0002-7937-7876", "role": "member", "display_name": "Gray, Harry B." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." } ], "option_major": [ "bioch" ], "doi": "10.7907/ftcb-ry72", "abstract": "All organisms require a respiratory process to produce energy. In eukaryotes,\r\nthis process occurs in the mitochondria, and requires a respiratory chain of four integral\r\nmembrane proteins as well as a membrane-soluble quinone pool and cytochrome c. The\r\nrespiratory proteins transfer electrons to oxygen as the terminal electron acceptor with\r\nthe electron transfer coupled to the translocation of protons across the mitochondrial\r\nmembrane. Not all organisms use oxygen as the terminal electron acceptor of their\r\nelectron transport chain. One of the more common alternative electron acceptors is\r\nfumarate, but other common electron acceptors include nitrogen-containing compounds,\r\nthe transformation of which represents an important step in the biological nitrogen cycle.\r\nThis thesis discusses the structural investigations of proteins involved in diverse\r\nrespiratory processes. The crystal structure of the Escherichia coli fumarate reductase,\r\nan integral-membrane enzyme complex involved in anaerobic respiration with fumarate\r\nas the terminal electron acceptor, has been solved. This structure both suggests the\r\nmechanism of the terminal step of anaerobic fumarate respiration and gives a model for\r\nthe function of the homologous protein succinate dehydrogenase from mitochondrial\r\nrespiration. The crystal structure of cytochrome c554 from the chemoautotrophic\r\nnitrifer Nitrosomonas europaea shows a heme-packing motif that may be important in\r\nrespiratory pathways that require the simultaneous transfer of multiple electrons." }, { "name": "Kielkopf, Clara Louise", "degree": "PhD", "year": "2000", "title": "Structural Basis of DNA Recognition by Synthetic Ligands", "advisor": "Rees, Douglas C.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03302018-093456214", "creators": [ { "name": { "family": "Kielkopf", "given": "Clara Louise" }, "id": "Kielkopf-Clara-Louise", "display_name": "Kielkopf, Clara Louise" } ], "advisors": [ { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "role": "advisor", "display_name": "Rees, Douglas C." } ], "committee": [ { "name": { "family": "Dervan", "given": "Peter B." }, "id": "Dervan-P-B", "role": "chair", "display_name": "Dervan, Peter B." }, { "name": { "family": "Barton", "given": "Jacqueline K." }, "id": "Barton-J-K", "role": "member", "display_name": "Barton, Jacqueline K." }, { "name": { "family": "Roberts", "given": "Richard W." }, "id": "Roberts-R-W", "role": "member", "display_name": "Roberts, Richard W." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "role": "member", "display_name": "Bjorkman, Pamela J." } ], "option_major": [ "biology" ], "doi": "10.7907/73K1-K290", "abstract": "The DNA double helix presents functional groups in the major and minor grooves that can be used by ligands for readout of the base pair sequence. The overall flexibility and shape also distinguishes various sequences. Although the information displayed in the major groove is more diverse, small DNA-binding polyamides predictably distinguish all four base pairs in the minor groove. In this work, the structural basis of this recognition has been studied using x-ray crystallographic techniques, and is described for G\u2022C (Chapter 2) and T\u2022A base pairs (Chapter 3-4). The polyamides directly read the DNA sequence using a combination of specific hydrogen bonds and shape selection. A second class of ligands called intercalators bind to DNA via the major groove by slipping a planar aromatic ligand between the bases. Although this shape-selective DNA binding is relatively nonspecific, the substitution of the ancillary ligands on octahedral metallointercalators allows specific sequences to be recognized in the major groove. The high resolution crystal structure of a designed metal complex bound to its target site reveals the nature of these specific contacts, as well as the precise stacking of the ligand between the bases. A detailed understanding of specific DNA recognition by small molecules is important for their further development as tools for molecular biology and medicine. These structures reveal how synthetic ligands can distinguish all four base pairs in both the major and the minor grooves of DNA." }, { "name": "Paquette, Alice Jean", "degree": "PhD", "year": "2000", "title": "The Role of the Neuron-Restrictive Silencer Factor During Vertebrate Embryogenesis", "advisor": "Andersen, Richard A.; Meyerowitz, Elliot M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08262025-205736197", "creators": [ { "name": { "family": "Paquette", "given": "Alice Jean" }, "id": "Paquette-Alice-Jean", "display_name": "Paquette, Alice Jean" } ], "advisors": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "advisor", "display_name": "Andersen, Richard A." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "co-advisor", "display_name": "Meyerowitz, Elliot M." } ], "committee": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "chair", "display_name": "Anderson, David J." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "member", "display_name": "Andersen, Richard A." } ], "option_major": [ "biology" ], "doi": "10.7907/vyb0-eg86", "abstract": "The gene-expression profile of a cell in large part determines what functions that cell can\r\nperform. Cell-type specific gene expression is set up over the course of development as\r\ndifferent tissues and cell types arise. By studying the mechanisms of cell-type specific\r\ngene expression, one may uncover processes involved in the generation of different cell\r\ntypes.
\r\n\r\nThe neuron-restrictive silencer factor (NRSF) was isolated in an effort to discover\r\nmechanisms involved in the generation of neurons during vertebrate embryogenesis.\r\nNRSF is a zinc-finger transcriptional repressor that is known to have many, primarily\r\nneuron-specific genes as putative direct targets. It is expressed widely outside of the\r\nnervous system, and in the nervous system it is expressed in neural progenitors. NRSF is\r\ndownregulated in differentiated neurons. Given its action as a repressor, its expression\r\npattern, and its list of potential target genes, NRSF was likely to play an important role\r\nin neural development.
\r\n\r\nWe chose to test this idea by both inhibiting NRSF function and overexpressing\r\nNRSF in chicken embryos. In order to inhibit NRSF function in vivo we infected embryos\r\nwith a retrovirus encoding a dominant-negative form of NRSF. Ectopic expression of\r\nthree neuronal target genes was observed in non-neural tissue. Within the nervous\r\nsystem, two of these genes were derepressed in neural progenitors. Premature\r\nneurogenesis, however, was not seen, and the low-level expression of neuron-specific\r\ngenes was insufficient to convert neural progenitors into ectopic neurons.
\r\n\r\nOverexpression of NRSF was performed by electroporating expression constructs\r\nfor NRSF into one side of the neural tube of developing chicken embryos. One target\r\ngene that is normally expressed at low levels in neural progenitors was repressed in this\r\ncell type. Another target gene was repressed in the differentiated neurons. This gene,\r\nNg-CAM, is important for axon pathfinding of spinal-cord commissural neurons. Some\r\naxons of NRSF-overexpressing commissural neurons showed pathfinding errors. The\r\nextent of neurogenesis and the expression of some NRSF target genes, however, was\r\napparently unaffected by this manipulation. NRSF, therefore, is important for proper\r\ngene regulation during neural development, but is not a direct regulator of the process\r\nof neurogenesis itself.
" }, { "name": "Pezaris, John Stylianos", "degree": "PhD", "year": "2000", "title": "Response of Multiple Simultaneously Recorded Macaque Area LIP Neurons in a Memory Saccade Task", "advisor": "Andersen, Richard A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04102013-085014457", "creators": [ { "name": { "family": "Pezaris", "given": "John Stylianos" }, "id": "Pezaris-John-Stylianos", "display_name": "Pezaris, John Stylianos" } ], "advisors": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "advisor", "display_name": "Andersen, Richard A." } ], "committee": [ { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "chair", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "member", "display_name": "Andersen, Richard A." }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "member", "display_name": "Koch, Christof" }, { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "member", "display_name": "Schuman, Erin Margaret" }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "member", "display_name": "Perona, Pietro" } ], "option_major": [ "cns" ], "doi": "10.7907/x9jm-8y88", "abstract": "Cells in the lateral intraparietal cortex (LIP) of rhesus macaques respond vigorously and in spatially-tuned fashion to briefly memorized visual stimuli. Responses to stimulus presentation, memory maintenance, and task completion are seen, in varying combination from neuron to neuron. To help elucidate this functional segmentation a new system for simultaneous recording from multiple neighboring neurons was developed. The two parts of this dissertation discuss the technical achievements and scientific discoveries, respectively.
\r\n\r\nTechnology. Simultanous recordings from multiple neighboring neurons were made with four-wire bundle electrodes, or tetrodes, which were adapted to the awake behaving primate preparation. Signals from these electrodes were partitionable into a background process with a 1/f-like spectrum and foreground spiking activity spanning 300-6000 Hz. Continuous voltage recordings were sorted into spike trains using a state-of-the-art clustering algorithm, producing a mean of 3 cells per site. The algorithm classified 96% of spikes correctly when tetrode recordings were confirmed with simultaneous intracellular signals. Recording locations were verified with a new technique that creates electrolytic lesions visible in magnetic resonance imaging, eliminating the need for histological processing. In anticipation of future multi-tetrode work, the chronic chamber microdrive, a device for long-term tetrode delivery, was developed.
\r\n\r\nScience. Simultaneously recorded neighboring LIP neurons were found to have similar preferred targets in the memory saccade paradigm, but dissimilar peristimulus time histograms, PSTH). A majority of neighboring cell pairs had a difference in preferred directions of under 45\u00b0 while the trial time of maximal response showed a broader distribution, suggesting homogeneity of tuning with heterogeneity of function. A continuum of response characteristics was present, rather than a set of specific response types; however, a mapping experiment suggests this may be because a given cell's PSTH changes shape as well as amplitude through the response field. Spike train autocovariance was tuned over target and changed through trial epoch, suggesting different mechanisms during memory versus background periods. Mean frequency-domain spike-to-spike coherence was concentrated below 50 Hz with a significant maximum of 0.08; mean time-domain coherence had a narrow peak in the range \u00b110 ms with a significant maximum of 0.03. Time-domain coherence was found to be untuned for short lags (10 ms), but significantly tuned at larger lags (50 ms).
" }, { "name": "Siegel, Micah Seth", "degree": "PhD", "year": "2000", "title": "Genetically Engineered Sensors of Cell Signaling", "advisor": "Mead, Carver", "url": "https://resolver.caltech.edu/CaltechTHESIS:04082013-160433360", "creators": [ { "name": { "family": "Siegel", "given": "Micah Seth" }, "id": "Siegel-Micah-Seth", "display_name": "Siegel, Micah Seth" } ], "advisors": [ { "name": { "family": "Mead", "given": "Carver" }, "id": "Mead-C-A", "orcid": "0000-0003-4051-0462", "role": "advisor", "display_name": "Mead, Carver" } ], "committee": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "chair", "display_name": "Fraser, Scott E." }, { "name": { "family": "Isacoff", "given": "Ehud Y." }, "id": "Isacoff-Ehud-Y", "orcid": "0000-0003-4775-9359", "role": "member", "display_name": "Isacoff, Ehud Y." }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "member", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Mead", "given": "Carver" }, "id": "Mead-C-A", "orcid": "0000-0003-4051-0462", "role": "member", "display_name": "Mead, Carver" } ], "option_major": [ "cns" ], "doi": "10.7907/92c5-r851", "abstract": "Measuring electrical activity in large numbers of cells with high spatial and temporal resolution is\r\na fundamental problem for the study of neural development and information processing. To address this\r\nproblem, we have constructed FlaSh: a novel, genetically-encoded probe that can be used to measure trans-membrane\r\nvoltage in single cells. We fused a modified green fluorescent protein (GFP) into a voltage-sensitive\r\npotassium channel so that voltage dependent rearrangements in the potassium channel induce\r\nchanges in the fluorescence of GFP. A voltage sensor encoded into DNA has the advantage that it may be\r\nintroduced into an organism non-invasively and targeted to specific developmental stages, brain regions,\r\ncell types, and sub-cellular compartments.
\r\n\r\nWe also describe modifications to FlaSh that shift its color, kinetics, and dynamic range. We used\r\nmultiple green fluorescent proteins to produce variants of the FlaSh sensor that generate ratiometric signal\r\noutput via fluorescence resonance energy transfer (FRET). Finally, we describe initial work toward FlaSh\r\nvariants that are sensitive to G-protein coupled receptor (GPCR) activation. These sensors can be used to\r\ndesign functional assays for receptor activation in living cells.
" }, { "name": "Sun, Qi", "degree": "PhD", "year": "2000", "title": "Molecular genetics of axon guidance in Drosophila melanogaster", "advisor": "Zinn, Kai George", "url": "https://resolver.caltech.edu/CaltechETD:etd-03242005-130557", "creators": [ { "name": { "family": "Sun", "given": "Qi" }, "id": "Sun-Q", "display_name": "Sun, Qi" } ], "advisors": [ { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "advisor", "display_name": "Zinn, Kai George" } ], "committee": [ { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "chair", "display_name": "Zinn, Kai George" }, { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "role": "member", "display_name": "Hay, Bruce A." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/4PTH-AZ39", "abstract": "Understanding the cellular and molecular mechanisms governing axon guidance and synaptogenesis is a central issue in developmental neurobiology. The Drosophila embryonic central nervous system, with its simplicity and genetic accessibility, is an ideal model system to examine these problems.\n\nOne signaling mechanism by which growth cones respond to guidance factors is the control of tyrosine phosphorylation. Genetic studies in Drosophila have shown that neural receptor protein tyrosine phosphatases (RPTPs) are important regulators of motor axon guidance decisions. Chapters 2 and 3 of this thesis describe genetic studies of the RPTP gene DPTP10D. Removing DPTP1OD and DPTP69D causes axon pathfinding errors at the midline and within the longitudinal tracts. These RPTPs genetically interact with genes involved in repulsion from the midline, including Slit, Roundabout and Commissureless. Slit is a midline repulsive signal, while Roundabout is the receptor for Slit. The phosphatases are likely to be components of signaling pathways downstream of Roundabout. DPTP10D is also involved in growth cone guidance decisions in the embryonic neuromuscular system. Phenotypic analyses of RPTP mutant combinations show that DPTPlOD works together with other RPTPs to promote bifurcation of the SNa nerve and allow the ISNb nerve to separate from the common ISN pathway. DPTPlOD, however, has a competitive relationship with the other RPTPs in controlling growth of the ISM These results show that the functional relationships among the four neural RPTPs are complex. At individual choice points, RPTPs can have cooperative, collaborative, or antagonistic functions in controlling guidance.\n\nIn Chapter 4, I describe an overexpression/misexpression P element screen for new genes involved in axon guidance. This screen allows identification and rapid cloning of genes that cause axon guidance defects when they are overexpressed in all neurons or all muscle fibers. One known axon guidance gene and several novel genes have already been identified in this screen, indicating that it may provide a powerful method to identify new genes that regulate axon guidance and synaptogenesis.\n\nThere are also three Appendices in the thesis. Two of these are reviews that I coauthored. The third Appendix describes a genetic analysis of the RPTP substrate protein gp150." }, { "name": "Turner, Glenn Cameron", "degree": "PhD", "year": "2000", "title": "Functions of the ubiquitin-proteasome system in Saccharomyces cerevisiae : cotranslational protein degradation and regulation of the UBR1 pathway", "advisor": "Varshavsky, Alexander J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-09222005-131809", "creators": [ { "name": { "family": "Turner", "given": "Glenn Cameron" }, "id": "Turner-G-C", "display_name": "Turner, Glenn Cameron" } ], "advisors": [ { "name": { "family": "Varshavsky", "given": "Alexander J." }, "id": "Varshavsky-A-J", "role": "advisor", "display_name": "Varshavsky, Alexander J." } ], "committee": [ { "name": { "family": "Varshavsky", "given": "Alexander J." }, "id": "Varshavsky-A-J", "role": "chair", "display_name": "Varshavsky, Alexander J." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "member", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "role": "member", "display_name": "Dunphy, William G." } ], "option_major": [ "biology" ], "doi": "10.7907/X5T1-TS17", "abstract": "The ubiquitin-proteasome system is the major pathway for protein degradation in the cytoplasm of eukaryotic cells. This pathway serves two main functions: protein quality control - removing damaged or misfolded proteins, and concentration control - regulating levels of the protein components of biochemical switches and oscillators.\n\nMisfolded proteins expose hydrophobic patches that act as degradation signals recognized by the ubiquitin-proteasome system. Nascent proteins being synthesized by the ribosome expose similar patches that might also serve as degradation signals. I show here that nascent polypeptides carrying a strong degradation signal of the Ub-proteasome system experience a kinetic competition between degradation and biogenesis. These results suggest that there may be a proofreading pathway for protein folding that recognizes and degrades proteins that fail to fold correctly.\n\nLevels of regulatory proteins must be adjusted in response to many different signals, both environmental and cell-intrinsic. I show here that the activity of a specific ubiquitin-protein ligase (E3), Ubr1, is allosterically regulated. UbrI regulates dipeptide uptake in saccharomyces cerevisiae by controlling the degradation of Cup9, a homeodomain-containing repressor of the dipeptide transporter Ptr2. UbrI is allosterically activated by dipeptides bearing destabilizing residues according to the N-end rule. The import of these dipeptides stimulates Ubri, increasing Cup9 degradation, thereby de-repressing ptr2 expression. Thus, the expression of the machinery required for dipeptide uptake is coupled to the availability of dipeptides.\n\nI also outline a novel pathway governing Ubrl activity. Free amino acids induce Ptr2 expression via a signal transduction cascade containing Ssy1, a putative transmembrane amino acid receptor, and Ptr3, a novel downstream signaling component. One of the targets of this signal transduction pathway is Ubr1. Ubrl is activated in the presence of amino acids, accelerating Cup9 degradation, thus inducing Ptr2.\n" }, { "name": "Wang, Minqin", "degree": "PhD", "year": "2000", "title": "Pattern formation during Caenorhabditis Elegans vulval development", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10082013-155308536", "creators": [ { "name": { "family": "Wang", "given": "Minqin" }, "id": "Wang-M", "display_name": "Wang, Minqin" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "role": "member", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "member", "display_name": "Lewis, Edward B." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "member", "display_name": "Meyerowitz, Elliot M." } ], "option_major": [ "biology" ], "doi": "10.7907/j9v8-2z18", "abstract": "Pattern formation during animal development involves at least three\r\nprocesses: establishment of the competence of precursor cells to respond to\r\nintercellular signals, formation of a pattern of different cell fates adopted by\r\nprecursor cells, and execution of the cell fate by generating a pattern of\r\ndistinct descendants from precursor cells. I have analyzed the fundamental\r\nmechanisms of pattern formation by studying the development of\r\nCaenorhabditis elegans vulva.
\r\n\r\nIn C. elegans, six multipotential vulval precursor cells (VPCs) are\r\ncompetent to respond to an inductive signal LIN-3 (EGF) mediated by LET-\r\n23 (RTK) and a lateral signal via LIN-12 (Notch) to form a fixed pattern of 3\u00b0-3\u00b0-2\u00b0-1\u00b0-2\u00b0-3\u00b0. Results from expressing LIN-3 as a function of time in\r\nanimals lacking endogenous LIN-3 indicate that both VPCs and VPC\r\ndaughters are competent to respond to LIN-3. Although the daughters of\r\nVPCs specified to be 2\u00b0 or 3\u00b0 can be redirected to adopt the 1\u00b0fate, the\r\ndecision to adopt the 1\u00b0 fate is irreversible. Coupling of VPC competence to\r\ncell cycle progression reveals that VPC competence may be periodic during\r\neach cell cycle and involve LIN-39 (HOM-C). These mechanisms are\r\nessential to ensure a bias towards the 1\u00b0 fate, while preventing an excessive\r\nresponse.
\r\n\r\nAfter adopting the 1\u00b0 fate, the VPC executes its fate by dividing three\r\nrounds to form a fixed pattern of four inner vulF and four outer vulE descendants. \r\nThese two types of descendants can be distinguished by a\r\nmolecular marker zmp-1::GFP. A short-range signal from the anchor cell\r\n(AC), along with signaling between the inner and outer 1\u00b0 VPC descendants\r\nand intrinsic polarity of 1\u00b0 VPC daughters, patterns the 1\u00b0 lineage. The Ras\r\nand the Wnt signaling pathways may be involved in these mechanisms.
\r\n\r\nThe temporal expression pattern of egl-17::GFP, another marker ofthe\r\n1\u00b0 fate, correlates with three different steps of 1\u00b0 fate execution: the\r\ncommitment to the 1\u00b0 fate, as well as later steps before and after\r\nestablishment of the uterine-vulval connection. Six transcription factors,\r\nincluding LIN-1(ETS), LIN-39 (HOM-C), LIN-11(LIM), LIN-29 (zinc finger),\r\nCOG-1 (homeobox) and EGL-38 (PAX2/5/8), are involved in different steps\r\nduring 1\u00b0 fate execution.
\r\n" }, { "name": "Wang, Susan Leishua", "degree": "PhD", "year": "2000", "title": "Turning on Death in the Fly: Regulation of Apoptosis in Drosophila melanogaster", "advisor": "Wold, Barbara J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08182025-182257078", "creators": [ { "name": { "family": "Wang", "given": "Susan Leishua" }, "id": "Wang-Susan-Leishua", "display_name": "Wang, Susan Leishua" } ], "advisors": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "advisor", "display_name": "Wold, Barbara J." } ], "committee": [ { "name": { "family": "Hay", "given": "Bruce A." }, "id": "Hay-B-A", "orcid": "0000-0002-5486-0482", "role": "chair", "display_name": "Hay, Bruce A." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Deshaies", "given": "Raymond Joseph" }, "id": "Deshaies-R-J", "orcid": "0000-0002-3671-9354", "role": "member", "display_name": "Deshaies, Raymond Joseph" }, { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "orcid": "0000-0001-7598-8939", "role": "member", "display_name": "Dunphy, William G." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." } ], "option_major": [ "biology" ], "abstract": "Apoptosis, an evolutionarily conserved form of cell suicide, is implemented by a\r\nfamily of cysteine proteases termed caspases. Caspases are constitutively expressed in\r\nmost cells and function in a proteolytic cascade activated by diverse extracellular and\r\nintracellular death stimuli. What molecules govern caspase activity so that it is limited to\r\ndoomed cells? What mechanisms do these regulators employ? Caspases and their\r\nregulators have been identified in a wide variety of species. This dissertation describes the\r\nanalysis of caspase regulators in Drosophila melanogaster, which has led to the elucidation\r\nof some mechanisms that control caspase activity.
\r\n\r\nIn Drosophila, three genes, reaper (rpr), head involution defective (hid), and grim,\r\nare essential for most normally occurring cell death, and are sufficient to initiate caspase-dependent\r\ndeath when expressed in cells that typically live. The Drosophila IAP\r\nhomologue DIAP1 is a dosage-dependent suppressor of normally occurring, as well as rpr-and\r\nhid-dependent cell death. We show that DIAP1 physically interacts with RPR, HID,\r\nand GRIM, and binds to and inhibits the activity of Drosophila caspases. Using a yeast-based\r\ncaspase activity reporter system and an in vitro reconstitution assay, we find that\r\nHID blocks DIAP1's ability to inhibit caspase activity and provide evidence that RPR and\r\nGRIM can behave similarly to activate caspases. These observations define a novel point at\r\nwhich caspase activity can be regulated and suggest that one mechanism by which RPR,\r\nHID, and GRIM can promote apoptosis is by disrupting productive IAP-caspase\r\ninteractions.
\r\n\r\nSupporting this hypothesis we show that DIAP1 is required to block apoptosis-inducing\r\ncaspase activity during Drosophila embryonic development. Elimination of\r\nDIAPl function results in global early embryonic cell death and a large increase in DIAP1-inhibitable\r\ncaspase activity. DIAP1 is still required for cell survival when expression of\r\nrpr, hid, and grim is eliminated. Since the death program is constitutively expressed in\r\nmost cells, the mechanism of cell death activation defined by RPR, HID, and GRIM may\r\nbe quite general.
" }, { "name": "Bashirullah, Arash", "degree": "PhD", "year": "1999", "title": "Temporal and spacial control of RNA stability in the early embryo of Drosophila melanogaster", "advisor": "Lipshitz, Howard D.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03052014-104605861", "creators": [ { "name": { "family": "Bashirullah", "given": "Arash" }, "id": "Bashirullah-A", "display_name": "Bashirullah, Arash" } ], "advisors": [ { "name": { "family": "Lipshitz", "given": "Howard D." }, "id": "Lipshitz-H-D", "role": "advisor", "display_name": "Lipshitz, Howard D." } ], "committee": [ { "name": { "family": "Lipshitz", "given": "Howard D." }, "id": "Lipshitz-H-D", "role": "chair", "display_name": "Lipshitz, Howard D." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" } ], "option_major": [ "biology" ], "doi": "10.7907/HWD3-HB90", "abstract": "Early embryogenesis in metazoa is controlled by maternally synthesized\r\nproducts. Among these products, the mature egg is loaded with transcripts\r\nrepresenting approximately two thirds of the genome. A subset of this maternal\r\nRNA pool is degraded prior to the transition to zygotic control of development.\r\nThis transfer of control of development from maternal to zygotic products is\r\nreferred to as the midblastula transition (or MBT). It is believed that the\r\ndegradation of maternal transcripts is required to terminate maternal control of\r\ndevelopment and to allow zygotic control of development to begin.\r\nUntil now this process of maternal transcript degradation and the subsequent\r\ntiming of the MBT has been poorly understood. I have demonstrated that in the\r\nearly embryo there are two independent RNA degradation pathways, either of\r\nwhich is sufficient for transcript elimination. However, only the concerted action\r\nof both pathways leads to elimination of transcripts with the correct timing, at\r\nthe MBT. The first pathway is maternally encoded, is triggered by egg\r\nactivation, and is targeted to specific classes of mRNAs through cis-acting\r\nelements in the 3' untranslated region (UTR}. The second pathway is activated\r\n2 hr after fertilization and functions together with the maternal pathway to\r\nensure that transcripts are degraded by the MBT. In addition, some transcripts\r\nfail to degrade at select subcellular locations adding an element of spatial\r\ncontrol to RNA degradation. The spatial control of RNA degradation is achieved\r\nby protecting, or masking, transcripts from the degradation machinery. The\r\nRNA degradation and protection events are regulated by distinct cis-elements\r\nin the 3' untranslated region (UTR). These results provide the first systematic\r\ndissection of this highly conserved process in development and demonstrate\r\nthat RNA degradation is a novel mechanism used for both temporal and spatial\r\ncontrol of development." }, { "name": "Batista, Aaron Paul", "degree": "PhD", "year": "1999", "title": "Contributions of Parietal Cortex to Reach Planning", "advisor": "Andersen, Richard A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04092013-091722066", "creators": [ { "name": { "family": "Batista", "given": "Aaron Paul" }, "id": "Batista-Aaron-Paul", "display_name": "Batista, Aaron Paul" } ], "advisors": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "advisor", "display_name": "Andersen, Richard A." } ], "committee": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "chair", "display_name": "Andersen, Richard A." }, { "name": { "family": "Shimojo", "given": "Shinsuke" }, "id": "Shimojo-S", "orcid": "0000-0002-1290-5232", "role": "member", "display_name": "Shimojo, Shinsuke" }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "member", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "member", "display_name": "Perona, Pietro" }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "member", "display_name": "Koch, Christof" } ], "option_major": [ "cns" ], "doi": "10.7907/p8wm-2z13", "abstract": "Sensory-motor circuits course through the parietal cortex of the human and monkey brain. How\r\nparietal cortex manipulates these signals has been an important question in behavioral neuroscience.\r\nThis thesis presents experiments that explore the contributions of monkey parietal cortex to sensory-motor\r\nprocessing, with an emphasis on the area's contributions to reaching. First, it is shown that\r\nparietal cortex is organized into subregions devoted to specific movements. Area LIP encodes plans\r\nto make saccadic eye movements. A nearby area, the parietal reach region (PRR), plans reaches. A\r\nseries of experiments are then described which explore the contributions of PRR to reach planning.\r\nReach plans are represented in an eye-centered reference frame in PRR. This representation is shown\r\nto be stable across eye movements. When a sequence of reaches is planned, only the impending\r\nmovement is represented in PRR, showing that the area is more related to movement planning than\r\nto storing the memory of reach targets. PRR resembles area LIP in each of these properties: the two\r\nareas may provide a substrate for hand-eye coordination. These findings yield new perspectives on\r\nthe functions of the parietal cortex and on the organization of sensory-motor processing in primate\r\nbrains." }, { "name": "Dvorak-Carbone, Hannah", "degree": "PhD", "year": "1999", "title": "Contribution of the Temporoammonic Pathway to Hippocampal Processing", "advisor": "Schuman, Erin Margaret", "url": "https://resolver.caltech.edu/CaltechTHESIS:05202015-142019062", "creators": [ { "name": { "family": "Dvorak-Carbone", "given": "Hannah" }, "id": "Dvorak-Carbone-Hannah", "display_name": "Dvorak-Carbone, Hannah" } ], "advisors": [ { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "advisor", "display_name": "Schuman, Erin Margaret" } ], "committee": [ { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "chair", "display_name": "Schuman, Erin Margaret" }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "member", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Pine", "given": "Jerome" }, "id": "Pine-J", "role": "member", "display_name": "Pine, Jerome" } ], "option_major": [ "biology" ], "doi": "10.7907/AYRT-5Y12", "abstract": "The temporoammonic (TA) pathway is the direct, monosynaptic projection from layer III of entorhinal cortex to the distal dendritic region of area CA1 of the hippo\u00ad campus. Although this pathway has been implicated in various functions, such as memory encoding and retrieval, spatial navigation, generation of oscillatory activity, and control of hippocampal excitability, the details of its physiology are not well understood. In this thesis, I examine the contribution of the TA pathway to hippocampal processing. I find that, as has been previously reported, the TA pathway includes both excitatory, glutamatergic components and inhibitory, GABAergic components. Several new discoveries are reported in this thesis. I show that the TA pathway is subject to forms of short-term activity-dependent regulation, including paired-pulse and frequency\u00ad dependent plasticity, similar to other hippocampal pathways such as the Schaffer collateral (SC) input from CA3 to CA1. The TA pathway provides a strongly excitatory input to stratum radiatum giant cells of CA1. The excitatory component of the TA pathway undergoes a long-lasting decrease in synaptic strength following low-frequency stimulation in a manner partially dependent on the activation of NMDA receptors. High\u00ad frequency activation of the TA pathway recruits a feedforward inhibition that can prevent CA1 pyramidal cells from spiking in response to SC input; this spike-blocking effect shows that the TA pathway can act to regulate information flow through the hippocampal trisynaptic pathway." }, { "name": "Jankowsky, Joanna L.", "degree": "PhD", "year": "1999", "title": "The Regulations and Role of Cytokines in Models of Synaptic Activity and Plasticity", "advisor": "Patterson, Paul H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08062025-221529508", "creators": [ { "name": { "family": "Jankowsky", "given": "Joanna L." }, "id": "Jankowsky-Joanna-L.", "orcid": "0000-0002-5593-2310", "display_name": "Jankowsky, Joanna L." } ], "advisors": [ { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "advisor", "display_name": "Patterson, Paul H." } ], "committee": [ { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "chair", "display_name": "Patterson, Paul H." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "orcid": "0000-0003-4274-1862", "role": "member", "display_name": "Bronner, Marianne E." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "member", "display_name": "Schuman, Erin Margaret" } ], "option_major": [ "neurobio" ], "doi": "10.7907/2r9p-rn53", "abstract": "While many functions have been proposed for cytokines in the CNS, most research\r\nhas focused on injury and infection. Initially described as protein signaling molecules\r\nbetween cells of the immune system, many cytokines and their receptors have since been\r\nlocalized to both the central and peripheral nervous systems. Our experiments explore the\r\ncontribution of cytokines to the fundamental brain functions, neuronal activity and synaptic\r\nplasticity.
\r\n\r\nSince cytokines are up-regulated by brain trauma, we first asked if cytokine mRNA\r\nexpression is affected by the procedures used to make hippocampal slices and to study long\r\nterm potentiation (LTP) in vivo. Indeed, expression of many cytokines is altered in the\r\npreparation of hippocampal slices, and we find even more dramatic cytokine changes with\r\nelectrode penetration in vivo. We therefore turned to a chronic in vivo preparation in which\r\nthe effects of injury are eliminated by a three week recovery period between electrode\r\nplacement and electrophysiological stimulation. In this preparation, we find that brain\r\nderived neurotrophic factor expression is decreased by low-frequency test stimuli. More\r\ndramatically, interleukin-6 (IL-6) is up-regulated specifically by LTP induction. Since IL-6\r\ncan regulate neuronal activity, this finding suggests a role for IL-6 in the control of LTP.
\r\n\r\nWe also studied the regulation of neuropoietic cytokines by neuronal activity in the\r\nhippocampus using pilocarpine-induced seizure. Using a semi-quantitative RNase\r\nprotection assay, we find that leukemia inhibitory factor (LIF) and oncostatin M are\r\nstrongly up-regulated by seizure activity. Each cytokine is, however, induced in discrete\r\ncell populations with a distinct time course, suggesting particular roles in both the seizure\r\nand in its pathological consequences.
\r\n\r\nThe relatively rapid induction of LIF in hippocampal astrocytes suggested that this\r\ncytokine could control astrocyte activation in response to seizure. Using the\r\nelectroconvulsive shock (ECS) model of seizure in LIF knockout and wild type mice, and\r\nglial fibrillary acidic protein (GFAP) induction as the marker of astrocyte activation, we\r\nfind that LIF is required for GFAP up-regulation.
\r\n\r\nIn sum, certain cytokines are regulated by particular patterns of neuronal activity,\r\nincluding those thought to be involved in learning and memory. Moreover, one cytokine,\r\nLIF, is required for astrocytic activation, a key process in subsequent hippocampal\r\npathology.
" }, { "name": "Linden, Jennifer Fran", "degree": "PhD", "year": "1999", "title": "Responses to Auditory Stimuli in Macaque Lateral Intraparietal Area", "advisor": "Andersen, Richard A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04092013-145216206", "creators": [ { "name": { "family": "Linden", "given": "Jennifer Fran" }, "id": "Linden-Jennifer-Fran", "display_name": "Linden, Jennifer Fran" } ], "advisors": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "advisor", "display_name": "Andersen, Richard A." } ], "committee": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "chair", "display_name": "Andersen, Richard A." }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "member", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "member", "display_name": "Perona, Pietro" }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "member", "display_name": "Koch, Christof" }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "cns" ], "doi": "10.7907/t6zm-7042", "abstract": "The lateral intraparietal area (LIP) of macaque posterior parietal cortex participates\r\nin the sensorimotor transformations underlying visually guided eye movements. Area\r\nLIP has long been considered unresponsive to auditory stimulation. However, recent\r\nstudies have shown that neurons in LIP respond to auditory stimuli during\r\nan auditory-saccade task, suggesting possible involvement of this area in auditory-to-oculomotor as well as visual-to-oculomotor processing. This dissertation describes\r\ninvestigations which clarify the role of area LIP in auditory-to-oculomotor processing.
\r\n\r\nExtracellular recordings were obtained from a total of 332 LIP neurons in two\r\nmacaque monkeys, while the animals performed fixation and saccade tasks involving\r\nauditory and visual stimuli. No auditory activity was observed in area LIP before\r\nanimals were trained to make saccades to auditory stimuli, but responses to auditory\r\nstimuli did emerge after auditory-saccade training. Auditory responses in area\r\nLIP after auditory-saccade training were significantly stronger in the context of an\r\nauditory-saccade task than in the context of a fixation task. Compared to visual\r\nresponses, auditory responses were also significantly more predictive of movement-related\r\nactivity in the saccade task. Moreover, while visual responses often had a fast\r\ntransient component, responses to auditory stimuli in area LIP tended to be gradual\r\nin onset and relatively prolonged in duration.
\r\n\r\nOverall, the analyses demonstrate that responses to auditory stimuli in area LIP\r\nare dependent on auditory-saccade training, modulated by behavioral context, and\r\ncharacterized by slow-onset, sustained response profiles. These findings suggest that\r\nresponses to auditory stimuli are best interpreted as supramodal (cognitive or motor)\r\nresponses, rather than as modality-specific sensory responses. Auditory responses in\r\narea LIP seem to reflect the significance of auditory stimuli as potential targets for\r\neye movements, and may differ from most visual responses in the extent to which\r\nthey arc abstracted from the sensory parameters of the stimulus.
\r\n" }, { "name": "MacLeod, Katrina Marie", "degree": "PhD", "year": "1999", "title": "Mechanisms and Function of Neural Synchronization in an Insect Olfactory System", "advisor": "Laurent, Gilles J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04202017-091931686", "creators": [ { "name": { "family": "MacLeod", "given": "Katrina Marie" }, "id": "MacLeod-Katrina-Marie", "display_name": "MacLeod, Katrina Marie" } ], "advisors": [ { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "advisor", "display_name": "Laurent, Gilles J." } ], "committee": [ { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "chair", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Bower", "given": "James M." }, "id": "Bower-J-M", "role": "member", "display_name": "Bower, James M." }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "member", "display_name": "Koch, Christof" }, { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "member", "display_name": "Schuman, Erin Margaret" } ], "option_major": [ "biology" ], "doi": "10.7907/GYVD-3755", "abstract": "One of the fundamental questions in modem integrative neurobiology relates to the\r\nencoding of sensory information by populations of neurons, and to the significance of this\r\nactivity for perception, learning, memory and behavior. Synchronization of activity across\r\na population of neurons has been observed many times over, but has never been\r\ndemonstrated to be a necessary component of this coding process. Neural synchronization\r\nhas been found in many brain areas in animals across several phyla, from molluscs to\r\nmammals. Studies in mammals have correlated the degree of neural synchronization with\r\nspecific behavioral or cognitive states, such as sensorimotor tasks, segmentation and\r\nbinocular rivalry suggesting a functional link. In the locust olfactory system, oscillatory\r\nsynchronization is a prominent feature of the odor-evoked neural activity. Stimulation of\r\nthe antenna by odors evokes synchronized firing in dynamic and odor-specific ensembles\r\nof the projection neurons of the antennal lobe, the principal neurons of the first-order\r\nolfactory relay in insects. The coherent activity of these projection neurons underlies an\r\nodor-evoked oscillatory field potential which can be recorded in the mushroom body, the\r\nsecond-order olfactory relay to which they project.
\r\n\r\n\r\nIn this dissertation, we investigated two important questions raised by these\r\nfindings: how are such stimulus-evoked synchronous ensembles generated, and what is\r\ntheir functional significance? To address these questions, we performed\r\nelectrophysiological experiments and recorded odor responses from neurons of the\r\nantennal lobes and mushroom bodies of locusts, in vivo and using natural odor stimulation\r\nin an unanesthetized, semi-intact preparation.
\r\n\r\n\r\nWe demonstrated the critical mechanism involved in neural synchronization of the\r\nantennal lobe neurons. The synchronization of the projection neurons relies critically on\r\nfast GABA (\u03b3-aminobutyric acid) -mediated inhibition from the local interneurons.\r\nProjection neuron synchronization could be selectively blocked by local injection of the\r\nGABA receptor antagonist, picrotoxin. Picrotoxin spared the odor-specific, slow\r\nmodulation of individual projection neuron responses, but desynchronized the firing of the\r\nodor-activated projection neuron assemblies. The oscillatory activity of the local\r\nintemeurons was also blocked by picrotoxin, which indicates that such activity depends on\r\nnetwork synaptic dynamics. We also showed that the mushroom body networks are\r\ncapable of generating oscillatory behavior of a similar frequency as that of its projection\r\nneuron inputs, and that they may thus be \"tuned\" to accept synchronized, oscillatory inputs\r\nof that frequency range.
\r\n\r\n\r\nOur understanding of this mechanism, in tum, made possible the functional\r\ninvestigation of neural synchronization by selective disruption of projection neuron\r\nsynchronization. We studied a population of neurons downstream from the antennal lobe\r\nprojection neurons, the extrinsic neurons of the \u03b2-lobe of the mushroom body (\u03b2LNs).\r\nThese \u03b2LNs were chosen for investigation because they were found to be odor-responsive\r\nand because their position in the olfactory pathway makes them a suitable \"read-out\" of\r\npopulation activity in the antennal lobe. We characterized \u03b2LN odor responses before and\r\nafter selective disruption of the synchronization of the projection neuron ensembles with\r\nlocal picrotoxin injection into the antennal lobe. We showed that the tuning of these \u03b2LN\r\nresponses was altered by PN desynchronization by changing existing responses and\r\ninducing new responses. This alteration in tuning resulted in a significant loss of odor\r\nspecificity in individual \u03b2LN responses, an effect that never occurred in the responses of\r\nindividual, desynchronized projection neurons. We thus propose that neural\r\nsynchronization is indeed important for information processing in the brain: it serves, at\r\nleast in part, as a temporal substrate for the transmission of information that is contained\r\nacross co-activated neurons (relational code) early in the pathway.
" }, { "name": "Sahani, Maneesh", "degree": "PhD", "year": "1999", "title": "Latent variable models for neural data analysis", "advisor": "Andersen, Richard A.; Hopfield, John J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04092013-142650761", "creators": [ { "name": { "family": "Sahani", "given": "Maneesh" }, "id": "Sahani-M", "display_name": "Sahani, Maneesh" } ], "advisors": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "role": "advisor", "display_name": "Andersen, Richard A." }, { "name": { "family": "Hopfield", "given": "John J." }, "id": "Hopfield-J-J", "role": "advisor", "display_name": "Hopfield, John J." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "cns" ], "doi": "10.7907/hwy6-ar88", "abstract": "The brain is perhaps the most complex system to have ever been subjected to rigorous scientific\r\ninvestigation. The scale is staggering: over 10^11 neurons, each making an average of 10^3 synapses,\r\nwith computation occurring on scales ranging from a single dendritic spine, to an entire cortical\r\narea. Slowly, we are beginning to acquire experimental tools that can gather the massive amounts\r\nof data needed to characterize this system. However, to understand and interpret these data will\r\nalso require substantial strides in inferential and statistical techniques. This dissertation attempts\r\nto meet this need, extending and applying the modern tools of latent variable modeling to problems\r\nin neural data analysis.
\r\n\r\nIt is divided into two parts. The first begins with an exposition of the general techniques of\r\nlatent variable modeling. A new, extremely general, optimization algorithm is proposed - called\r\nRelaxation Expectation Maximization (REM) - that may be used to learn the optimal parameter\r\nvalues of arbitrary latent variable models. This algorithm appears to alleviate the common problem\r\nof convergence to local, sub-optimal, likelihood maxima. REM leads to a natural framework for\r\nmodel size selection; in combination with standard model selection techniques the quality of fits may\r\nbe further improved, while the appropriate model size is automatically and efficiently determined.\r\nNext, a new latent variable model, the mixture of sparse hidden Markov models, is introduced, and\r\napproximate inference and learning algorithms are derived for it. This model is applied in the second\r\npart of the thesis.
\r\n\r\nThe second part brings the technology of part I to bear on two important problems in experimental\r\nneuroscience. The first is known as spike sorting; this is the problem of separating the spikes\r\nfrom different neurons embedded within an extracellular recording. The dissertation offers the first\r\nthorough statistical analysis of this problem, which then yields the first powerful probabilistic solution.\r\nThe second problem addressed is that of characterizing the distribution of spike trains recorded\r\nfrom the same neuron under identical experimental conditions. A latent variable model is proposed.\r\nInference and learning in this model leads to new principled algorithms for smoothing and clustering\r\nof spike data.
\r\n" }, { "name": "Tang, Lixin", "degree": "PhD", "year": "1999", "title": "Role for the Cadherin Family of Cell Adhesion Molecules in Synaptic Function in the Adult Hippocampus", "advisor": "Schuman, Erin Margaret", "url": "https://resolver.caltech.edu/CaltechTHESIS:08282025-214605106", "creators": [ { "name": { "family": "Tang", "given": "Lixin" }, "id": "Tang-Lixin", "display_name": "Tang, Lixin" } ], "advisors": [ { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "advisor", "display_name": "Schuman, Erin Margaret" } ], "committee": [ { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "chair", "display_name": "Schuman, Erin Margaret" }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." }, { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "member", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." } ], "option_major": [ "biology" ], "doi": "10.7907/qh82-x455", "abstract": "The cadherins are a family of type I single-pass integral membrane glycoproteins that\r\nspan intercellular junctions and mediate Ca2+-dependent homophilic intercellular interactions.\r\nThe highly conserved cytoplasmic C termini of cadherins interact with the catenins\r\nand the cytoskeleton. The extracellular domain is composed of five repeats with the most\r\ndistal repeat, especially a region containing the highly conserved His-Ala-Val (HA V)\r\nsequence, being critical for homophilic binding.
\r\n\r\nI first examined the expression of cadherins, especially the neural- (N-) and epithelial-\r\n(E-) subtypes, in the adult hippocampus. In situ hybridization experiments indicated\r\nthe presence of mRNAs for both N- and E- cadherins in the adult hippocampus. Immunoblot\r\nanalysis revealed the expression of cadherin proteins in the hippocampal synaptosome\r\nfraction. Immunofluorescent staining indicated that cadherins and catenins are\r\nexpressed at synaptic sites.
\r\n\r\nI investigated the possible role of cadherins in synaptic plasticity at the CAI synapses\r\nin the adult hippocampus. Preincubation of hippocampal slices with function-blocking\r\ncadherin antibodies or HA V-containing antagonistic peptides greatly reduced long-term\r\npotentiation (LTP) whereas basal synaptic properties including input-output relations, and\r\npaired-pulse facilitation were normal. The HAV peptides inhibited LTP in a concentration-\r\ndependent and LTP induction protocol-independent manner.
\r\n\r\nA decrease in the extracellular Ca2+ associated with LTP induction may increase the\r\nvulnerability of Ca2+-sensitive cadherin bonds to cadherin inhibitory reagents. In support\r\nof this hypothesis, I found that doubling of the extracellular Ca2+ abolished the inhibition\r\nof LTP by HAV peptides. Moreover, HAV peptides delivered in a lower Ca2+ solution\r\nreduced previously potentiated responses, suggesting a role for cadherins in both the\r\ninduction and expression of LTP.
\r\n\r\nA recombinant adenovirus containing a dominant-inhibitory cadherin cDNA was\r\nconstructed. I found that hippocampal slices infected with this virus exhibited normal\r\nsynaptic properties but less LTP than adjacent slices infected with an adenovirus containing\r\na reporter gene.
\r\n\r\nI also examined the effect of HAV peptides on presynaptic vesicle exocytosis in\r\nhippocampal cultures using the fluorescent membrane dye FM 1-43. HAV peptides do\r\nnot affect the dye release following stimulation, suggesting cadherin function is not\r\nrequired for normal exocytosis.
\r\n\r\nTaken together, these data suggest cadherins make important contributions to synaptic\r\nplasticity in the adult hippocampus.
" }, { "name": "Trotta, Christopher Robert", "degree": "PhD", "year": "1999", "title": "The Composition, Function and Evolution of the tRNA Splicing Endonuclease", "advisor": "Abelson, John N.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08192025-203726793", "creators": [ { "name": { "family": "Trotta", "given": "Christopher Robert" }, "id": "Trotta-Christopher-Robert", "display_name": "Trotta, Christopher Robert" } ], "advisors": [ { "name": { "family": "Abelson", "given": "John N." }, "id": "Abelson-J-N", "role": "advisor", "display_name": "Abelson, John N." } ], "committee": [ { "name": { "family": "Abelson", "given": "John N." }, "id": "Abelson-J-N", "role": "chair", "display_name": "Abelson, John N." }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "orcid": "0000-0001-8291-5551", "role": "member", "display_name": "Campbell, Judith L." }, { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "orcid": "0000-0001-7598-8939", "role": "member", "display_name": "Dunphy, William G." } ], "option_major": [ "molbiochem" ], "doi": "10.7907/n4y9-jj84", "abstract": "The splicing of tRNA precursors is essential for the production of mature tRNA in\r\norganisms from all major phyla. In Bacteria, intron removal is through autocatalysis of\r\ngroup I introns. In Archaea and Eukaryotes, intron removal is dependent on protein-based\r\nenzymes. Recognition and cleavage of the splice sites is accomplished by the tRNA\r\nsplicing endonuclease. In order to understand how the eukaryotic endonuclease\r\naccomplishes this task, the genes encoding all four subunits of the S. cerevisiae enzyme\r\nhave been cloned. All four genes are essential. Two subunits, Sen2 and Sen34, contain a\r\nhomologous domain of approximately 130 amino acids. Surprisingly this domain is found\r\nin the gene encoding the archaeal tRNA splicing endonuclease of H. volcanii and in other\r\nArchaea. Utilizing the alignment as a guide, a mutation was made in the yeast Sen34\r\nsubunit which demonstrated that the eukaryotic endonuclease contains two functionally\r\nindependent active sites for cleavage of the 5' and 3' splice sites, encoded by the SEN2 and\r\nSEN34 genes, respectively. The homology to the archaeal enzymes suggests an ancient\r\norigin for the tRNA splicing reaction. Exploiting the smaller and simpler archaeal version\r\nof the endonuclease, a crystal structure of the Methanoccocus jannaschii enzyme was\r\ndetermined to a resolution of 2.3 angstroms. The structure indicates that the cleavage\r\nreaction is similar to that of ribonucleaseA. Insight gained from the architecture of this\r\nhomotetrameric enzyme has allowed for a clearer understanding of the heterotetrameric\r\nsplicing endonuclease of yeast. In particular, the eukaryotic enzyme has preserved the\r\nimportant structural features found in the archaeal enzyme which allow for precise spatial\r\npositioning of the two active sites for cleavage." }, { "name": "Wang, Hai", "degree": "PhD", "year": "1999", "title": "Transmembrane Ephrin Ligands in Neural and Vascular Development", "advisor": "Anderson, David J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08212025-015916492", "creators": [ { "name": { "family": "Wang", "given": "Hai" }, "id": "Wang-Hai", "display_name": "Wang, Hai" } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "advisor", "display_name": "Anderson, David J." } ], "committee": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "chair", "display_name": "Anderson, David J." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "member", "display_name": "Zinn, Kai George" } ], "option_major": [ "biology" ], "doi": "10.7907/kga9-kk35", "abstract": "Receptor tyrosine kinases and their ligands play important roles in development. Using an expression cloning approach, I identified two transmembrane ligands - ephrinB1 and ephrinB2 for the receptor tyrosine kinase EphB2. Trunk neural crest cell migration and early motor axon outgrowth are restricted to rostral somite halves. Transmembrane ephrin ligands were localized to the caudal somite halves in both mouse and chick. In vitro functional assays revealed that the ephrin ligands can repulsively guide migrating crest cells and early motor axons that express the receptor EphB2. Targeted gene deletion was used to dissect mouse ephrinB2's role in vivo. By examining multiple markers for hindbrain, somite, migrating crest, and motor axons, I failed to show any obvious phenotypic alterations in the mutant embryos. Related ephrins or unrelated regulators may play redundant roles in the nervous system. Unexpectedly, ephrinB2 mutants die around El0.5 due to severe vascular defects. EphrinB2's unique expression in the blood vessels is restricted to the arteries and not veins. Moreover,\r\nEphB4, another receptor for ephrinB2, was found restricted to veins and not arteries. Thus, ephrinB2 and its receptor EphB4 revealed a molecular distinction between arteries and veins -- two types of vessels defined by the directions of blood flow. Deletion of ephrinB2 in the arteries resulted in defective arterial angiogenesis in the capillary beds. Embryonic veins also suffered from defective remodeling in the absence of a normal artery development. These results indicated that ephrinB2-EphB4 mediated interactions between arteries and veins is essential for the angiogenesis of both types of vessels. The nature of ligand-receptor signaling between arterial and venous endothelial cells in the\r\ncapillaries remain unexplored. There are several implications based on our finding. First, more genes are likely expressed differentially in arteries and veins, consistent with the pathological differences between the two types of vessels. Second, the specification of arterial and venous endothelial cells suggests novel embryonic patterning events. Third, the ephrinB2-EphB4 signaling system between arteries and veins is a potential antiangiogenic target for controlling abnormal vascular development in tumor angiogenesis." }, { "name": "Wehr, Michael Stephen", "degree": "PhD", "year": "1999", "title": "Oscillatory Sequences of Firing in the Locust Olfactory System: Mechanisms and Functional Significance", "advisor": "Laurent, Gilles J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05212002-120039", "creators": [ { "name": { "family": "Wehr", "given": "Michael Stephen" }, "id": "Wehr-Michael-Stephen", "display_name": "Wehr, Michael Stephen" } ], "advisors": [ { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "advisor", "display_name": "Laurent, Gilles J." } ], "committee": [ { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "chair", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "member", "display_name": "Koch, Christof" }, { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "member", "display_name": "Schuman, Erin Margaret" }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "orcid": "0000-0002-7947-0472", "role": "member", "display_name": "Andersen, Richard A." } ], "option_major": [ "cns" ], "doi": "10.7907/QZE9-SZ72", "abstract": "What neural codes does the brain use to represent and process sensory information? Stimulus-evoked oscillatory synchronization of neuronal activity has been observed in many systems, yet the possible functions of such rhythmic synchronization in neural coding remain largely speculative. In the locust, odors appear to be represented by dynamic ensembles of transiently synchronized neurons. The experiments described here explored the design and function of the locust olfactory system, focusing on projection neurons in the antennal lobe. The first goal was to characterize, by means of intracellular and multiple extracellular recordings, the oscillatory synchronization and slow temporal patterns in PN odor responses in vivo. After the system had been characterized, specific coding hypotheses were tested. The results demonstrated that the cycle-by-cycle firing patterns across ensembles of PNs encode odor identity information, but that other response features (such as phase or frequency) do not. Finally the mechanisms for the generation of these dynamics were addressed. Odors do not evoke oscillatory synchronization in the population activity of olfactory receptor afferents, and non-specific, temporally unpatterned electrical stimulation of receptor axons can evoke both oscillatory synchronization and slow temporal patterns in PNs, similar to those evoked by natural stimulation with odors. Oscillatory synchronization of olfactory neurons therefore originates in the antennal lobe, and slow temporal patterns in projection neurons can arise in the absence of temporal patterning of the afferent input." }, { "name": "Cornelison, Dawn D. W.", "degree": "PhD", "year": "1998", "title": "Gene expression in wild-type and MyoD-null satellite cells: regulation of activation, proliferation, and myogenesis", "advisor": "Wold, Barbara J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04072011-104210966", "creators": [ { "name": { "family": "Cornelison", "given": "Dawn D. W." }, "id": "Cornelison-D-D-W", "display_name": "Cornelison, Dawn D. W." } ], "advisors": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "advisor", "display_name": "Wold, Barbara J." } ], "committee": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "chair", "display_name": "Wold, Barbara J." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Bronner", "given": "Marianne E." }, "id": "Bronner-M-E", "role": "member", "display_name": "Bronner, Marianne E." } ], "option_major": [ "biology" ], "doi": "10.7907/09DA-FF55", "abstract": "Regeneration is the process of renewal or repair of damaged cells and tissue. In skeletal muscle, regeneration is accomplished by satellite cells, which are rare,\r\nmononucleate, mitotically quiescent myogenic precursor cells normally present in undamaged muscle tissue. When stimulated by injury, overuse, or disease, satellite cells\r\nwill become activated to proliferate and form a pool of replacement myoblasts which will differentiate to replace necrotic muscle fibers. These cells may also have the quality of self-renewal associated with stem cells. Due mainly to technical difficulties caused by their rarity, difficulty of isolation, and lack of identifying markers, satellite cells have not been as well studied as other myogenic cells. Here I present work in which I establish a\r\nreliable means of isolating and culturing mouse satellite cells resident on single explanted myofibers; a molecular marker for satellite cells which also yields information about their mechanism of activation, and a method of multiplex single-cell RT-PCR which allows\r\nsimultaneous detection of six genes from a single satellite cell. Using these techniques, I have determined the temporal coexpression pattern of the four myogenic regulatory factors (MRFs) in single activated satellite cells over the first four days of a regeneration\r\nresponse in vitro. I have also assayed satellite cell cDNA pools for expression of genes important in regulating myogenesis, cell cycling, and cell fate decisions in other\r\nmyogenic lineages. Finally, I have performed these analyses on MyoD-null satellite cells, which are differentiation-deficient in vivo, and present possible mechanisms for this based on gene expression; this analysis also suggested a potential marker for activated satellite cells which will return to the reserve satellite cell population and may act as myogenic stem cells." }, { "name": "Holt, Gary R.", "degree": "PhD", "year": "1998", "title": "A critical reexamination of some assumptions and implications of cable theory in neurobiology", "advisor": "Koch, Christof", "url": "https://resolver.caltech.edu/CaltechETD:etd-09122006-135415", "creators": [ { "name": { "family": "Holt", "given": "Gary R." }, "id": "Holt-G-R", "display_name": "Holt, Gary R." } ], "advisors": [ { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "role": "advisor", "display_name": "Koch, Christof" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "cns" ], "doi": "10.7907/HPPC-S237", "abstract": "Linear cable theory lies at the core of our understanding of how an individual neuron works. Cable theory usually assumes that neurons do not interact significantly except at specific, anatomically specialized locations (synapses and gap junctions). An analysis of the extracellular electrical fields shows that spikes in one neuron could cause a depolarization of several mV in a dendrite or axon passing by its initial segment. This is somewhat larger than typical chemical synapses; such ephaptic interactions could possibly play a role in controlling action potential failure at branch points.\n\nApplying conclusions of linear cable theory to nonlinear spiking neurons has led to incorrect ideas about neural function. For example, in linear cable theory changing the membrane conductance can be used to scale the amplitude of EPSPs. Shunting inhibition has therefore been repeatedly proposed as a mechanism for division or normalization. This mechanism does not work if the neuron is spiking, i.e., when the output is firing rate rather than EPSP amplitude. When a neuron spikes, its time-averaged voltage does not increase much even if the firing rate goes up; therefore current through a shunt resistance is independent of firing rate, and shunting inhibition acts subtractively rather than divisively.\n\nCable theory also predicts that EPSPs are low-pass filtered by the membrane resistance and capacitance, and investigators have therefore assumed that the membrane time constant determines how fast a neuron can respond. Again, because of the spiking mechanism, the membrane potential never reaches steady state, so the time constant is not obviously relevant. The dynamics of firing rates may be better described by currents than voltages.\n\nApplying this principle to the dynamics of simple feedback networks shows that a key factor in the response time of a network is the adaptation current. Without adaptation, the network time constant can belong because it is the gain of the network multiplied by the synaptic time constants. Adaptation can cancel out the long tails of synaptic current, significantly speeding up response times. Recurrent inhibition has a similar effect.\n\nAnother key factor determining input current is synaptic depression and facilitation. Recurrent networks are especially sensitive to synaptic depression because of the feedback; within a very short period of time the network behaves like a feedforward network because the recurrent synapses have been depressed away. However, facilitation and depression can act together to provide a log-exponential transform, allowing subtractive inhibition at one stage to have a divisive effect at another.\n" }, { "name": "Kiser, Cynthia N", "degree": "PhD", "year": "1998", "title": "Biological electron transfer in copper proteins", "advisor": "Richards, John H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04212014-115617853", "creators": [ { "name": { "family": "Kiser", "given": "Cynthia N" }, "id": "Kiser-C-N", "display_name": "Kiser, Cynthia N" } ], "advisors": [ { "name": { "family": "Richards", "given": "John H." }, "id": "Richards-J-H", "role": "advisor", "display_name": "Richards, John H." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/PWWP-AS34", "abstract": "Nature has used a variety of protein systems to mediate electron transfer. In this thesis I examine aspects of the control of biological electron transfer by two copper proteins that act as natural electron carriers.
\r\n\r\nIn the first study, I have made a mutation to one of the ligand residues in the azurin blue copper center, methionine 121 changed to a glutamic acid. Studies of intramolecular electron transfer rates from that mutated center to covalently attached ruthenium complexes indicate that the weak axial methionine ligand is important not only for tuning the reduction potential of the blue copper site but also for maintaining the low reorganization energy that is important for fast electron transfer at long distances.
\r\n\r\nIn the second study, I begin to examine the reorganization energy of the purple copper center in the CuA domain of subunit II of cytochrome c oxidase. In\r\nthis copper center, the unpaired electron is delocalized over the entire binuclear site. Because long-range electron transfer into and out of this center occurs over\r\nlong distances with very small driving forces, the reorganization energy of the CuA center has been predicted to be extremely low. I describe a strategy for measuring this reorganization energy starting with the construction of a series of mutations introducing surface histidines. These histidines can then be labeled with a series of ruthenium compounds that differ primarily in their reduction\r\npotentials. The electron transfer rates to these ruthenium compounds can then be used to determine the reorganization energy of the CuA site.
\r\n" }, { "name": "Lesa, Giovanni M.", "degree": "PhD", "year": "1998", "title": "Signaling by LET-23, a Caenorhabditis elegans Epidermal Growth Factor Receptor Homolog", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:07292025-220045090", "creators": [ { "name": { "family": "Lesa", "given": "Giovanni M." }, "id": "Lesa-Giovanni-M.", "display_name": "Lesa, Giovanni M." } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "chair", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Lipshitz", "given": "Howard D." }, "id": "Lipshitz-H-D", "orcid": "0000-0002-7372-4419", "role": "member", "display_name": "Lipshitz, Howard D." }, { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "member", "display_name": "Simon, Melvin I." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "member", "display_name": "Zinn, Kai George" } ], "option_major": [ "molbiochem" ], "doi": "10.7907/ywxy-2h20", "abstract": "The Caenorhabditis elegans gene let-23 encodes a tyrosine kinase of the\r\nEpidermal Growth Factor Receptor (EGFR) family. Ligand-independent\r\nactivation of EGFR tyrosine kinases is involved in many types of cancer. A\r\nligand-independent activating mutation of let-23, sa62, has been\r\ncharacterized. sa62 maps to the extracellular domain of the LET-23 protein\r\nand results in excessive proliferation of the cells which forms the\r\nhermaphrodite vulva. Analysis of sa62 has suggested that the region to\r\nwhich it maps is important for transmembrane activation of LET-23\r\nsignaling.
\r\n\r\nlet-23 mediates multiple functions in C. elegans including viability,\r\nvulval differentiation, and fertility. Analysis of tissue specific and loss-of-function\r\nmutations of let-23 suggested that the C-terminus of LET-23 can be\r\ndivided into at least three domains, each mediating a subset of let-23\r\nfunctions. The EGFR family of tyrosine kinases contain potential C-terminal\r\nphosphotyrosines whose role is still unclear. Using in vitro\r\nmutagenesis and transgenic technology, this thesis shows that in vivo the\r\nLET-23 C-terminal tyrosines are required for wild-type activity and that they\r\nare differently used to mediate cell specific, positive, and negative\r\nregulation. One tyrosine is necessary and sufficient for wild-type fertility.\r\nThree other tyrosines are involved in viability and vulval differentiation.\r\nAnother tyrosine appears to mediate tissue specific negative regulation.\r\nTwo mechanisms are proposed for receptor tyrosine kinase signaling: a\r\npositive mechanism, which promote, and a negative mechanism, which\r\ninhibits receptor tyrosine kinase activity. It is also shown that LET-23\r\nactivates at least two pathways: the Ras pathway to mediate viability and\r\nvulval differentiation, and another pathway to mediate fertility. A genetic\r\nscreen to identify genes acting positively in the let-23-mediated fertility\r\npathway is described. In addition, the initial characterization of a gene\r\nacting in this pathway is reported.
" }, { "name": "Protopapas, Alexander D.", "degree": "PhD", "year": "1998", "title": "Pyramidal Cell Responses to Temporally Structured Stimuli: Experiments and Computer Simulations", "advisor": "Bower, James M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:07252025-202233992", "creators": [ { "name": { "family": "Protopapas", "given": "Alexander D." }, "id": "Protopapas-Alexander-D", "display_name": "Protopapas, Alexander D." } ], "advisors": [ { "name": { "family": "Bower", "given": "James M." }, "id": "Bower-J-M", "role": "advisor", "display_name": "Bower, James M." } ], "committee": [ { "name": { "family": "Bower", "given": "James M." }, "id": "Bower-J-M", "role": "chair", "display_name": "Bower, James M." }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" } ], "option_major": [ "biology" ], "doi": "10.7907/zv26-tp58", "abstract": "Oscillations in the field potential recorded from piriform cortex can be broadly categorized\r\ninto slow and fast frequency ranges. The slow wave is correlated with respiration\r\nand sniffing. During respiration it is typically in the 1-4 Hz range but during sniffing\r\nit increases in frequency and is often referred to as the theta rhythm (4-12 Hz). The\r\nfaster oscillations (30-50 Hz, also called gamma) appear in response to odor stimuli\r\nand are always modulated by the slower rhythm. Oscillations in the field potential\r\nare believed to reflect synchronized synaptic input to the dendrites of pyramidal neurons\r\nin the piriform cortex. In this thesis I use a combination of experimental and\r\ncomputer simulation techniques to study the consequences of pyramidal cell input\r\nmeant to approximate the temporal characteristics of cortical oscillations.
\r\n\r\nBecause the precise spatial and temporal control of synaptic inputs is not possible\r\nin an experimental preparation, I constructed a detailed biophysical simulation of a\r\nlayer II pyramidal cell from piriform cortex where such control would be possible. The\r\npassive and active properties of this model were tuned to experimental measurements\r\nthat I made from pyramidal cells in vitro. The model was able to match a wide\r\nrange of physiological behavior including subthreshold oscillations and responses to\r\nmultiple levels of current injection. Spatio-temporal patterns of synaptic input that\r\nhave been suggested to underlie gamma oscillations in piriform cortex were then used\r\nas input to the model. The effects of a single such pattern of input were longer\r\nlasting than the duration of a single gamma oscillation suggesting that a pyramidal\r\ncell integrates input over multiple gamma oscillations during the course of bursts of\r\ngamma oscillations modulated by the respiratory / theta rhythm. When bursts of\r\ngamma activity in the model were separated by 650 msec or more, the first burst\r\nhad no effect on the second, implying that these neurons might be able to isolate the\r\neffects of sufficiently spaced sniffs or bouts of sniffing.
\r\n\r\nTo determine how well current injections with the temporal characteristics of cortical \r\noscillations might be represented in the spike trains of pyramidal cells, I used\r\na reconstruction algorithm to estimate the structure of the stimulus from spike train\r\ndata. By comparing the estimate to the actual stimulus I was able to quantify the\r\namount of stimulus information contained in the spike train. I found that stimuli\r\nfiltered at frequencies of 0-10 Hz and 4-12 Hz were much better represented in the\r\npyramidal cell spike trains than 0-40 Hz stimuli designed to include the entire frequency\r\nrange of cortical oscillations. The effects of norepinephrine (a neuromodulator\r\nreleased during arousal) on spike coding were also studied. I found that while norepinephrine\r\nincreased the amount of stimulus information in the spike train, a change\r\nin decoding strategy to extract this information from the spike train was not required.
" }, { "name": "Sullivan, Brian M.", "degree": "PhD", "year": "1998", "title": "Studies of Nitric Oxide Signal Transduction", "advisor": "Schuman, Erin Margaret", "url": "https://resolver.caltech.edu/CaltechTHESIS:07312025-023246266", "creators": [ { "name": { "family": "Sullivan", "given": "Brian M." }, "id": "Sullivan-Brian-M", "display_name": "Sullivan, Brian M." } ], "advisors": [ { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "advisor", "display_name": "Schuman, Erin Margaret" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/q269-0e91", "abstract": "Nitric oxide (NO) is released by many cells of the body, acting both as a signaling molecule or as a cytotoxic agent. In order to further our understanding of the mechanisms\r\nby which NO may function in the nervous system to regulate synaptic function, the following topics have been studied: 1) The NO-stimulated ADP ribosylation of synaptosomal proteins. 2) The subcellular localization of the neuronal isoform of nitric oxide synthase (nNOS) when the enzyme was overexpressed in embryonic hippocampal neurons by gene transfer with a recombinant adenovirus. 3) The release of NO and its primary metabolite, nitrite, as assayed by an electrochemical NO meter, from cultured cells infected with recombinant adenovirus expressing either nNOS or eNOS.\r\nFurthermore, the effects of inhibiting membrane attachment of eNOS were examined. From these studies, in Chapter 4, we conclude that the known reaction rates of NO with\r\nO2 do not account for the observed large ratio of nitrite to NO." }, { "name": "Vaughn, Daniel E.", "degree": "PhD", "year": "1998", "title": "Molecular Mechanism of pH Dependent Antibody Binding: Structure/Function Studies on the Neonatal Fc Receptor", "advisor": "Bjorkman, Pamela J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08062012-111300229", "creators": [ { "name": { "family": "Vaughn", "given": "Daniel E." }, "id": "Vaughn-Daniel-E", "display_name": "Vaughn, Daniel E." } ], "advisors": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "advisor", "display_name": "Bjorkman, Pamela J." } ], "committee": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "chair", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." } ], "option_major": [ "biology" ], "doi": "10.7907/bzyh-hn75", "abstract": "The work described here is an investigation of the molecular mechanism of pH\r\ndependent immunoglobulin G (IgG) binding by the neonatal Fc receptor (FcRn). FcRn\r\nbinds IgG at acidic, but not alkaline pHs, in two important physiological processes. These\r\nprocesses are the acquisition of passive immunity by the fetus or newborn and protecting\r\nIgG from a default degradative pathway.
\r\n\r\nA biosensor assay is used to characterize the interaction of a soluble form of FcRn\r\nwith IgG. Immobilization of FcRn on the biosensor surface reproduces the high affinity\r\nIgG binding observed for membrane bound FcRn, whereas immobilization of IgG results\r\nin lower affinity binding similar to that of the FcRn/IgG interaction in solution. The\r\nstatistical method of cross-validation is used to show that there are two classes of noninteracting binding sites. The IgG binding interaction is characterized for several mutant FcRns with designed amino acid substitutions. These mutations map the functional IgG\r\nbinding site on FcRn.
\r\n\r\nThe structure of FcRn at an alkaline pH is described. This structure determination\r\nreveals an extensive carbohydrate mediated interaction between the dimer related FeRn\r\nmolecules. The physiological relevance of this interaction is discussed in the context of the FcRn dimerization literature. A further refined structure of FcRn at an acidic pH is\r\ndescribed that includes additional carbohydrate structure. These structures are compared\r\nwith specific attention to the pH dependence of FcRn stability and IgG affinity. Finally, a\r\nmechanism for pH dependent antibody binding to FcRn is proposed based on these structures and the body of structure/function literature concerning this interaction.
" }, { "name": "Kang, Hyejin", "degree": "PhD", "year": "1997", "title": "Modulation of Synaptic Function by Neurotrophic Factors in the Adult Hippocampus", "advisor": "Schuman, Erin Margaret", "url": "https://resolver.caltech.edu/CaltechTHESIS:10282025-173845996", "creators": [ { "name": { "family": "Kang", "given": "Hyejin" }, "id": "Kang-Hyejin", "display_name": "Kang, Hyejin" } ], "advisors": [ { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "role": "advisor", "display_name": "Schuman, Erin Margaret" } ], "committee": [ { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "chair", "display_name": "Schuman, Erin Margaret" }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "member", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "member", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." } ], "option_major": [ "neurobio" ], "doi": "10.7907/08g9-gb22", "abstract": "The neurotrophins including nerve growth factor (NGF), brain-derived neurotrophic\r\nfactor (BDNF), and neurotrophin-3 (NT-3), are a group of signaling factors that are\r\ncrucial for neuronal survival and differentiation during development. Previous studies\r\nhave shown that the hippocampus is a prominent site of expression of the neurotrophins\r\nand their Trk receptors in the adult brain. Interestingly, the expression of BDNF, NT-3,\r\nand their receptors can be regulated by a variety of neuronal activity, which suggests that\r\nthe neurotrophins may also participate in adult synaptic plasticity.
\r\n\r\nThe possibility that the neurotrophins directly modulate synaptic strength in the\r\nmature brain was investigated at the Schaffer collateral-CAI synapses in the adult rat\r\nhippocampus. Transient application of BDNF or NT-3 but not NGF produced a dramatic\r\nand sustained (3 to 4 hours) enhancement of synaptic transmission. Both\r\nelectrophysiological and immunocytochemical evidence indicated that the penetration\r\nand the resulting synaptic potentiation by neurotrophins are influenced by the perfusion\r\nrate at which the neurotrophin is applied to hippocampal synapses.
\r\n\r\nThe potentiating effects ofBDNF and NT-3 could be completely blocked by\r\ninhibiting the function of Trk receptors using either a pharmacological inhibitor of\r\ntyrosine kinases or a function-blocking Trk antibody. In addition, blockade ofL-type\r\ncalcium channels or intracellular calcium stores significantly reduced the potentiation\r\ninduced by either neurotrophin. These data suggest that both the activation of Trk\r\nreceptors and the subsequent increase in intracellular calcium concentration are essential\r\nfor the initiation of neurotrophin-induced synaptic enhancement.
\r\n\r\nThe neurotrophin-induced plasticity exhibits an immediate requirement for protein\r\nsynthesis, which is not somatic in origin. The neurotrophins still produced synaptic\r\npotentiation at synapses isolated from their cell bodies; this plasticity displayed a\r\ndependence on protein synthesis, raising the possibility that these factors may stimulate\r\nlocal protein synthesis within dendrites and promote site-specific modification of\r\nsynaptic function.
\r\n\r\nFinally, I report that the neurotrophins play a functional role in certain forms of\r\nhippocampal long-term potentiation (LTP). The maintenance of LTP induced by thetaburst\r\nor pairing, but not strong tetanic stimulation, relied on intact neurotrophin signaling.\r\nIn long-term LTP, the neurotrophins were primarily involved in maintaining the late\r\nphases of synaptic enhancement, without significantly affecting an early phase of\r\npotentiation. Thus, during adult plasticity, synaptic activity-induced increases in\r\nneurotrophin synthesis and release contribute to the late phase of LTP and may eventually\r\nlead to structural changes at the synapse.
" }, { "name": "Lane, Robert P.", "degree": "PhD", "year": "1997", "title": "Evolution of the Neural Immunoglobulin Supergene Family and Functional Studies of One of its Members", "advisor": "Dreyer, William J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:07162025-220110244", "creators": [ { "name": { "family": "Lane", "given": "Robert P." }, "id": "Lane-Robert-P", "display_name": "Lane, Robert P." } ], "advisors": [ { "name": { "family": "Dreyer", "given": "William J." }, "id": "Dreyer-W-J", "role": "advisor", "display_name": "Dreyer, William J." } ], "committee": [ { "name": { "family": "Dreyer", "given": "William J." }, "id": "Dreyer-W-J", "role": "chair", "display_name": "Dreyer, William J." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "member", "display_name": "Zinn, Kai George" } ], "option_major": [ "molbiochem" ], "doi": "10.7907/rgww-db14", "abstract": "The immunoglobulin supergene family is a diverse set of\r\nmolecules that share in common the immunoglobulin (Ig) domain. In\r\nthe nervous system, a large subfamily of these proteins has been\r\ncharacterized that contain, in addition to N-terminal Ig-like domains,\r\nnumerous fibronectin (Fn) type III repeats. One group of these neural\r\nhomologs has been well characterized and includes Neuroglian,\r\nBravo/Nr-CAM, Neurofascin, L1, Ng-CAM, Contactin/ F11, Axonin-1/\r\nTag-1, Big-1/Tag-like, and Big-2. Each of these proteins have six Ig-like\r\ndomains and either four or five fibronectin type III repeats, and\r\nvarious developmental functions have been attributed to this group,\r\nincluding neurite outgrowth, fasciculation, cell adhesion and axon\r\nguidance.
\r\n\r\nUsing structural modeling and cladistic analyses, the evolutionary\r\nrelationships among these homologous neural Ig superfamily proteins\r\nwere investigated. This study reinforces the idea that individual Ig-like\r\nand Fn domains are probably not distinct functional modules that can\r\nbe shuffled in evolution, but rather that they may act in tandem.\r\nPatterns of conservation and divergence of specific residues along the\r\nvarious phylogenetic branches of the evolutionary tree suggest a model\r\nwhereby important interactions may predominantly map between\r\ndomains, with the \"top\" loops of one domain, the \"bottom\" loops of\r\nthe adjacent domain, and the interdomain residues forming part of a\r\nligand \"pocket\". The evolutionary analyses also permits an evaluation\r\nof the controversial identification of Ng-CAM and L1 as species\r\northologs, and in light of avian-mammalian speciation events, it\r\nappears these proteins are orthologous but perhaps not functionally\r\nidentical.
\r\n\r\nA new member of this neural Ig subfamily has been cloned and\r\nidentified as the human ortholog to the chicken Bravo/Nr-CAM\r\nprotein. The complete coding sequence was determined, and like its\r\nchicken homolog, it is composed of six V-like Ig-like domains, five\r\nfibronectin type III repeats, as well as a transmembrane and\r\nintracellular domain. Overall, the human protein is 82% identical to\r\nthe chicken homolog, although the trans-membrane and intracellular\r\ndomains are 100% conserved at the amino acid level. Independent\r\ncDNA's encoding four distinct isoforms were identified, all of which\r\ncontain alternatively spliced variants around the fifth fibronectin\r\nrepeat, including one isoform previously identified in chicken in\r\nwhich- the entire 93 amino acid domain is spliced. Northern blot\r\nanalysis reveals one mRNA species of approximately 7.0 kb in adult\r\nbrain. Fluorescence in situ hybridization maps the human Bravo/NrCAM\r\ngene to human chromosome 7q31.1-31.2, a locus previously\r\nidentified to contain a tumor suppressor gene.
\r\n\r\nAlthough the cell adhesion (CAM) nomenclature implies that\r\nBravo/ Nr-CAM and its family members function merely as a sort of\r\nindiscriminate cell-cell \"glue\", evidence has mounted that these\r\nproteins participate in receptor-like intracellular signalling functions\r\nwith cell behavioral consequences. Of particular interest with regard to\r\nthe Bravo/Nr-CAM protein is the conserved alternative splicing of the\r\nmembrane-near fibronectin domain, as well as the striking sequence\r\nconservation C-terminal to this alternative exon that extends through\r\nthe membrane and inside the cell. To explore the function of these\r\nsequences, both the 93 amino acid alternative Fn5 exon and the 100%\r\nconserved intracellular domains of Bravo/Nr-CAM were separately\r\nproduced in heterologous expression systems and purified by various\r\nbiochemical techniques. Affinity chromatography and expression\r\nlibrary screening were used in an attempt to identify putative ligands to\r\nthese presumably important protein regions.
\r\n\r\nThe significance of the fifth fibronectin alternative exon usage was\r\nalso investigated by using the expressed domain to raise domain-specific\r\nmonoclonal antibodies, and using the antibodies in a\r\nhistological study of spatial and temporal regulation of these splicing\r\nevents. Using double labeling and confocol microscopy, as well as PCR\r\nanalysis, in all tissues and across all stages of development, both the\r\ndomain-containing and domain-lacking isoforms appear to be\r\nuniformly expressed in the same cells. Therefore, the developmental\r\nfunction of the complex array of alternatively spliced variants around\r\nthe fifth fibronectin domain is subtle. A model is discussed whereby\r\nisoform diversity may provide a means to integrate multiple ligandbinding\r\nevents involving the same protein on the same cell that\r\ninteract with distinct ligands and co-receptors.
" }, { "name": "Liu, Shih-Chii", "degree": "PhD", "year": "1997", "title": "Neuromorphic models of visual and motion processing in the fly visual system", "advisor": "Mead, Carver", "url": "https://resolver.caltech.edu/CaltechETD:etd-01152008-133452", "creators": [ { "name": { "family": "Liu", "given": "Shih-Chii" }, "id": "Liu-Shih-Chii", "display_name": "Liu, Shih-Chii" } ], "advisors": [ { "name": { "family": "Mead", "given": "Carver" }, "id": "Mead-C-A", "orcid": "0000-0003-4051-0462", "role": "advisor", "display_name": "Mead, Carver" } ], "committee": [ { "name": { "family": "Mead", "given": "Carver" }, "id": "Mead-C-A", "orcid": "0000-0003-4051-0462", "role": "chair", "display_name": "Mead, Carver" }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "de Ruyter van Steveninck", "given": "Robert" }, "id": "de-Ruyter-van-Steveninck-Robert", "role": "member", "display_name": "de Ruyter van Steveninck, Robert" }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "member", "display_name": "Koch, Christof" }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "member", "display_name": "Perona, Pietro" } ], "option_major": [ "cns" ], "doi": "10.7907/kxfr-s226", "abstract": "Since the first neuromorphic retina was introduced 10 years ago, we have seen neuromorphic modeling extended to motion processing, saccadic systems, and auditory processing, to name a few. This dissertation extends neuromorphic modeling to the fly visual system. The retinotopic and regular arrangement of the layers in this system makes viable the mapping of the structure of the layers to silicon. The ability of the fly to compute motion reliably with only 24,000 receptors, while consuming only microwatts of power, also makes this system attractive for neuromorphic modeling. I start this dissertation by comparing the filter bandwidth properties and offsets of the fly receptors with those of silicon receptors. The filtering properties and the offsets of the receptors are critical because they determine the limits of subsequent processing circuitry. This work is the first characterization of biological and artificial offsets. Next, I describe an analog circuit that captures some of the adaptation and temporal filtering properties of the cells in the initial layers of the visual system. The adaptation time constant of the circuit is controllable via an external bias. The temporal filtering of the circuit changes with the S/N ratio of signals. This prefiltering preceding the motion areas is important to ensure that the motion computation is robust under different S/N rations. The filtering should be adaptive to match the S/N ratio so as to maximise the information transfer to subsequent processing. Adaptation is also a big component of the motion computation since the visual system has to extract information from a 2 to 3 decade range of speeds of objects under a six decade range of illumination. In this dissertation, I show the first adaptive motion model that matches its time scale to that of the moving image. The model explains experimental data showing motion adaptation in the direction-selective cells of the fly. Finally, I describe the responses of the direction-selective cells to motion. This silicon model is critical in showing that local direction selectivity can be computed from the correlation of continuous-time, graded inputs. The computation integrates the visual information over time in the decision making process and binarized features are not needed for the correlation. This model differs from previous silicon implementations of the Reichardt model that used token-based information for correlation. This model is a closer analogue of the motion computation in flies than previous silicon models." }, { "name": "Miskevich, Frank", "degree": "PhD", "year": "1997", "title": "Expression, Characterization, and Ligand Studies Involving Domains of the Chick Cell Surface Protein Neogenin", "advisor": "Dreyer, William J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08082025-212351994", "creators": [ { "name": { "family": "Miskevich", "given": "Frank" }, "id": "Miskevich-Frank", "display_name": "Miskevich, Frank" } ], "advisors": [ { "name": { "family": "Dreyer", "given": "William J." }, "id": "Dreyer-W-J", "role": "advisor", "display_name": "Dreyer, William J." } ], "committee": [ { "name": { "family": "Dreyer", "given": "William J." }, "id": "Dreyer-W-J", "role": "chair", "display_name": "Dreyer, William J." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Mayo", "given": "Stephen L." }, "id": "Mayo-S-L", "orcid": "0000-0002-9785-5018", "role": "member", "display_name": "Mayo, Stephen L." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "molbiochem" ], "doi": "10.7907/z9xk-ab42", "abstract": "The vertebrate retinotectal projection provides an excellent developmental system\r\nto study mechanisms and molecules involved in precisely patterning the nervous system.\r\nThe retina sends out a single topographic projection which maps in a one to one\r\ncorrespondence in the tectum. This correspondence is brought about by the interplay of\r\nnumerous factors, including electrical activity, extracellular signals, and the interaction of\r\nvarious signal cascades within the retinal ganglion cell.
\r\n\r\nNeogenin is an alternatively spliced transmembrane protein homologous to a\r\nnumber of genes involved in neurite outgrowth and pathfinding. Its developmental\r\nexpression in the retina suggested that it could be involved in differentiation or signaling\r\nevents during the period when optic fibers are making initial connections in the tectum.\r\nThe aim of this study was to identify extracellular and cytoplasmic signals carried by\r\nneogenin.
\r\n\r\nThe immunoglobulin domains of neogenin were heterologously expressed in the\r\nyeast Pichia pastoris and biochemically characterized. This protein was then used to\r\ngenerate monoclonal antibodies against various epitopes in these domains hopefully to\r\nidentify function blocking or cross-species reactive antibodies.
\r\n\r\nThe intracellular isoforms of neogenin were expressed and characterized in E. coli\r\nto identify proteins which interact with neogenin. Proteins of 200, 140, 110, and 55 kD\r\nwere specifically labeled in brain lysates. Both neogenin isoforms react with the proteins\r\nin a calcium dependent fashion. Affinity chromatography, antibody co-precipitation, and\r\nexpression library screening were then attempted to identify these labeled proteins.
" }, { "name": "Shah, Nirao Mahesh", "degree": "PhD", "year": "1997", "title": "Mechanisms of Cell Fate Determination and Differentiation in the Mammalian Neural Crest", "advisor": "Anderson, David J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08052025-215831325", "creators": [ { "name": { "family": "Shah", "given": "Nirao Mahesh" }, "id": "Shah-Nirao-Mahesh", "orcid": "0000-0002-0152-467X", "display_name": "Shah, Nirao Mahesh" } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "advisor", "display_name": "Anderson, David J." } ], "committee": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "chair", "display_name": "Anderson, David J." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "member", "display_name": "Zinn, Kai George" } ], "option_major": [ "biology" ], "doi": "10.7907/ztxp-sd35", "abstract": "The neural crest provides a model system to study the generation of cellular diversity\r\nduring development. Crest cells derive from the vertebrate neural tube and migrate to\r\nmany locations in the embryo where they give rise to different lineages. Cell types\r\ndifferentiating from the neural crest include neurons and glia of the peripheral nervous\r\nsystem, neuroendocrine cells, melanocytes of the skin and ectomesenchymal lineages\r\nsuch as smooth muscle and bone. Multipotential crest cells have been demonstrated to\r\nexist in vivo and in vitro. How do multipotential crest cells make lineage decisions? In\r\none scenario, neural crest cells may differentiate using only cell autonomous\r\nmechanisms. Another scenario is that differentiative signals present in the environment\r\ndirect the lineage choices of multipotential crest cells. These two models represent the\r\nextremes of possible mechanisms of cell fate specification.
\r\n\r\nI have tried to determine the mechanisms involved in lineage specification of rat\r\nneural crest stem cells (NCSCs). NCSCs form clones containing neurons, glia and\r\nsmooth muscle in vitro. I have identified three growth factors that can direct\r\ndifferentiation of NCSCs to each of these fates in culture. Neuregulin-2 (GGF2),\r\nBMP2 and TGF\u03b21 promote predominantly glial, autonomic neuronal and smooth\r\nmuscle differentiation from these cells, respectively. My experiments suggest that these\r\npolypeptides may exert an instructive rather than a selective influence on NCSC lineage\r\nspecification. Furthermore, each of these growth factors is present at a time and place in\r\nthe embryo consistent with it being able to influence crest differentiation. Although\r\nNCSCs possess functional receptors for all three polypeptides, glial differentiation in\r\nGGF2 in vitro may not occur as fast as neuronal and smooth muscle differentiation in\r\nBMP2 and TGF\u03b21, respectively. NCSCs also exhibit differential dosage sensitivity in\r\ntheir differentiation response to these growth factors. Such differences in the response\r\nto environmental signals can affect the outcome of situations in which NCSCs encounter\r\ncompeting instructive cues. Taken together, my results suggest that instructive\r\nenvironmental signals in conjunction with cell intrinsic mechanisms may play an\r\nimportant role in neural crest cell fate specification.
" }, { "name": "Stemmler, Martin Bernard", "degree": "PhD", "year": "1997", "title": "Information maximization and stochastic resonance in single neurons", "advisor": "Koch, Christof", "url": "https://resolver.caltech.edu/CaltechETD:etd-12182007-104908", "creators": [ { "name": { "family": "Stemmler", "given": "Martin Bernard" }, "id": "Stemmler-M-B", "display_name": "Stemmler, Martin Bernard" } ], "advisors": [ { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "role": "advisor", "display_name": "Koch, Christof" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "cns" ], "doi": "10.7907/hg8a-9t13", "abstract": "Does the nervous system \"tune\" itself to perform at peak efficiency? Optimal transmission of information in a single nerve cell occurs when the response is matched to the statistics of naturally occurring stimuli, such that all firing rates are used with equal probability and that redundant temporal correlations in the input are removed. Non-Hebbian, local learning rules are developed to adapt the voltage-dependent ionic conductances in Hodgkin-Huxley models of neurons with the goal of matching the neuron's response to the statistics of natural stimuli. These learning rules allow a nerve cell to maximize the amount of information transmitted about arriving stimuli. At a more detailed level, information transmission in neurons is limited by the noise in the input, defined as the root mean square of the fluctuations in the input. Three different performance measures are shown to scale identically as a function of the noise in simple models of neurons that have both a voltage and current threshold. These performance measures are: the probability of correctly detecting a constant input in a limited time, the signal-to-noise ratio in response to sinusoidal input, and the mutual information between an arbitrarily varying input and the output spike train of the model neuron. Of these, detecting a constant signal is the simplest and most fundamental quantity. For subthreshold signals, the model exhibits stochastic resonance, a non-zero noise amplitude that optimally enhances signal detection. In this case, noise paradoxically does not limit, but instead improves performance. Even though the noise amplitude can dwarf the signal, detection of a weak constant signal using stochastic resonance is still possible when the signal elicits on average only one additional spike. Stochastic resonance could thus play a role in neurobiological sensory systems, where speed is of the utmost importance and averaging over many individual spikes is not possible.\n" }, { "name": "Tocchini-Valentini, Giuseppe D.", "degree": "PhD", "year": "1997", "title": "E. coli tRNA Leucine Identity and Recognition Sets", "advisor": "Abelson, John N.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03012017-091934429", "creators": [ { "name": { "family": "Tocchini-Valentini", "given": "Giuseppe D." }, "id": "Tocchini-Valentini-Giuseppe-D", "display_name": "Tocchini-Valentini, Giuseppe D." } ], "advisors": [ { "name": { "family": "Abelson", "given": "John N." }, "id": "Abelson-J-N", "role": "advisor", "display_name": "Abelson, John N." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/323e-0690", "abstract": "E. coli contains five different tRNAs which recognize the six leucine codons. These tRNAs are all recognized by the single leucyl-tRNA synthetase (LeuRS). We have used in vitro and in vivo methods to determine the set of identity elements which distinguish the set of leucine tRNAs from all other tRNAs allowing the faithful translation of the leucine codons.
\r\n\r\nAn identity swap experiment has been used to determine which of the nucleotides conserved in all leucine tRNAs are identity elements. In this experiment the identity of an amber suppressor tRNASer was changed completely to leucine. This experiment was effective because the anticodons in tRNASer and tRNALeu are not recognized by their respective synthetases and consequently in both cases tRNAs containing the CUA anticodon required in amber suppressors are fully active.
\r\n\r\nIn its minimal form the Ser-Leu swap required six changes, five of which altered the tertiary structure of the tRNA: the G15-C48 tertiary \"Levitt base-pair\" in tRNASer was changed to Al5-U48 found in all leucine tRNAs; it was necessary to insert one nucleotide and to delete one nucleotide so as to position the conserved D-loop Gl8, Gl9 nucleotides as they are in all leucine tRNAs; a base was inserted at position 47n between the base-paired extra stem and the T-stem to achieve a configuration found in all leucine tRNAs; in addition it was necessary to change the G73 \"discriminator\" base in tRNASer to A73, found in all leucine tRNAs. This minimally altered tRNASer inserted exclusively leucine as an amber suppressor and it was an excellent in vitro substrate for LeuRS.
\r\n\r\nBoth tRNASer and tRNALeu are type II tRNAs containing large base-paired extra stem loops. In the case of tRNASer the extra stem loop is a crucial identity element but for tRNALeu earlier in vitro and in vivo experiments had indicated that it is not an identity element. To investigate the role of tRNA tertiary structure in leucine identity we carried out a parallel swap experiment in which the glutamine identity of the amber suppressor tRNASer\u0394 (in which the type II extra stem loop had been replaced by a consensus type I loop) was converted to leucine. This \"type I\" swap experiment was also successful both in vivo and in vitro. Interesting differences in the role of conserved leucine base-pairs in the acceptor stems of leucine tRNAs were observed in the two experiments. In the type II swap the conserved acceptor stem bases were not important. In the type I swap their absence had a large effect both in vivo and in vitro. This result indicates that the presence of the extra stem loop in leucine tRNAs has an effect on the tertiary structure of the tRNA. When this structure is altered conserved nucleotides, unimportant in its presence, take on an important role. Possible reasons for this effect are discussed.
\r\n" }, { "name": "Whittaker, Kellie Lynn", "degree": "PhD", "year": "1997", "title": "RNA Localization in Drosophila Oogenesis and Early Embryogenesis", "advisor": "Lipshitz, Howard D.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05122026-180430833", "creators": [ { "name": { "family": "Whittaker", "given": "Kellie Lynn" }, "id": "Whittaker-Kellie-Lynn", "display_name": "Whittaker, Kellie Lynn" } ], "advisors": [ { "name": { "family": "Lipshitz", "given": "Howard D." }, "id": "Lipshitz-H-D", "orcid": "0000-0002-7372-4419", "role": "advisor", "display_name": "Lipshitz, Howard D." } ], "committee": [ { "name": { "family": "Lipshitz", "given": "Howard D." }, "id": "Lipshitz-H-D", "orcid": "0000-0002-7372-4419", "role": "chair", "display_name": "Lipshitz, Howard D." }, { "name": { "family": "Abelson", "given": "John N." }, "id": "Abelson-J-N", "orcid": "0000-0002-9040-1910", "role": "member", "display_name": "Abelson, John N." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." } ], "option_major": [ "biodev" ], "doi": "10.7907/wpq8-s423", "abstract": "Localization of maternal mRNAs is an important developmental strategy for\r\nproviding spatially restricted synthesis of the encoded proteins. Much of the work done on\r\nlocalized RN As in Drosophila development has focused on identification of pattern\r\ndeterminants, but not all localized maternal RNAs encode such patterning specifiers.\r\nAdducin-like, also known as hu-li tai shao, represents the only example to date of a\r\ntranscript which is both localized throughout Drosophila oogenesis and encodes a\r\ncytoskeletal protein. Adducin-like encodes the Drosophila homologue of mammalian\r\nadducin, which in humans is a membrane-cytoskeletal protein that contributes to local\r\ncytoskeletal assembly, particularly at sites of cell-cell contact and communication. We show\r\nhere that Adducin-like generates a family of transcripts through alternative mRNA splicing.\r\nThe splice variants of Adducin-like share sequence in the open reading frame (ORF) but are\r\ntruncated at different points and have unique 3' ORF sequences plus different 3'UTRs.\r\nOne of these transcript classes, R1, encodes an Adducin-like protein isoform with a\r\nMARCKS-related element and represents the first Drosophila orthologue of mammalian\r\nadducin. R1 Adducin-like activity is likely to be regulated by signal transduction pathways\r\nsimilar to those affecting human adducins. Another Adducin-like transcript class, N4, is\r\nlocalized specifically to and within the developing Drosophila oocyte and early embryo.\r\nCis-acting signals for N4 RNA localization are contained within the 3'UTR. We have\r\nmapped these signals and identified a unique cis-acting element capable of directing early\r\ntransport of mRNA into the oocyte. swallow is required for the localization of Adducin-like\r\nmRNA, and we present evidence that swallow acts through the Adducin-like N4\r\n3'UTR to promote Adducin-like N4 mRNA localization in mid- to late oogenesis. We\r\nconsider the possible functions of alternative splicing in development.
\r\n\r\nAnother non-pattern-specifying maternal RNA localized in early Drosophila\r\ndevelopment is the mitochondrial 16S large ribosomal RNA (16S RNA). The 16S RNA is \r\nlocalized to the posterior of the early embryo and appears to be a constituent of polar\r\ngranules, since maternal effect mutations which disrupt the polar granules also abolish 16S\r\nRNA localization. We show that 16S RNA localization does not appear to be necessary for\r\npole cell formation or function.
" }, { "name": "Yun, Kyuson", "degree": "PhD", "year": "1997", "title": "Murine Twist is a bHLH Regulator that Inhibits Myogenesis by Multiple Molecular Mechanisms", "advisor": "Wold, Barbara J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:07182025-151320548", "creators": [ { "name": { "family": "Yun", "given": "Kyuson" }, "id": "Yun-Kyuson", "display_name": "Yun, Kyuson" } ], "advisors": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "advisor", "display_name": "Wold, Barbara J." } ], "committee": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "chair", "display_name": "Wold, Barbara J." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Lipshitz", "given": "Howard D." }, "id": "Lipshitz-H-D", "orcid": "0000-0002-7372-4419", "role": "member", "display_name": "Lipshitz, Howard D." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Mann", "given": "Jeffrey R." }, "id": "Mann-J-R", "orcid": "0000-0003-0028-8692", "role": "member", "display_name": "Mann, Jeffrey R." } ], "option_major": [ "biology" ], "doi": "10.7907/kt6j-sg39", "abstract": "Twist is a member of the basic helix-loop-helix (bHLH) class of transcc1iption factors.\r\nFrom its expression patterns during fly and mouse embryogenesis, it was chosen to be\r\nstudied as a potential upstream- or cross-regulator of myogenic regulatory factors\r\n(MRFs). Additional motivation for studying twist in the context of myogenesis comes\r\nfrom the shared structural motif among MRFs, E-proteins (\"universal\" partners for\r\ntissue-specific factors), and twist that would allow direct physical association. Helix-loop-\r\nhelix domain allows combinatorial dimerization that results in distinct DNA-binding\r\ncomplexes. Depending on the partner choice and availability, twist and other\r\nbHLHs can form complexes with different activities.
\r\n\r\nIn E8.5d mouse embryo, twist and myf5 overlap in their expression domains in the\r\ndeveloping somites, indicating the physiological relevance of the twist and MRF\r\ninteraction. This observation suggests that twist and myf5 (MRF) may interact with\r\neach other, directly or indirectly through E-proteins. Supporting data for this idea are\r\npresented in chapter 2. In addition, twist is demonstrated to form active DNA binding\r\ncomplexes with different E-proteins. Since twist titrates available E-proteins from\r\nMyoD in vitro and in vivo, twist acts as a dominant negative regulator of myogenesis.\r\nHowever, the fact that twist:E complex is an active DNA binding factor suggests that\r\ntwist may regulate the expression of downstream target genes.
\r\n\r\nStudies using a tethered dimer between MyoD and E47 (MyoD-E47) support the\r\nlikelihood of transcriptional regulaton by twist, presented in chapter 3. Results from\r\nchapters 2 and 3 suggest that although bHLH competition and MEF2 titration are viable\r\nmechanisms of myogenic inhibition by twist, the most potent inhibitory activity of twist\r\nis likely to involve another mechanism.
\r\n\r\nSince twist inhibits myogenesis initiated by all combinations of MRFs and MEF2 tested\r\nin transfected cells, the cell cycle status of these cells were tested since forced\r\nproliferation would account for failure to differentiate. MyoD expressing cells are\r\nfound to arrest normally in the presence of twist, suggesting twist inhibition is specific\r\nto differentiation and not an overall inactivation of MyoD function. Furthermore, twist\r\ninhibits the onset of myogenin suggesting this early myogenic event is blocked.\r\nHowever, this is not the only restriction point targeted by twist since late twist\r\nexpression (driven by the myogenin promoter) can still inhibit muscle differentiation.\r\nThese observations together show that twist is a potent inhibitor of myogenesis and\r\nsuggest that twist may be involved in regulating proper muscle differentiation in\r\ndeveloping embryos.
" }, { "name": "Anderson, Roger Francis", "degree": "PhD", "year": "1996", "title": "Characterization of the sea urchin homologue of the replication factor A 70 kD subunit and the novel interspersed repeat family to which it binds", "advisor": "Davidson, Eric H.", "url": "https://resolver.caltech.edu/CaltechETD:etd-09212006-131348", "creators": [ { "name": { "family": "Anderson", "given": "Roger Francis" }, "id": "Anderson-R-F", "display_name": "Anderson, Roger Francis" } ], "advisors": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "advisor", "display_name": "Davidson, Eric H." } ], "committee": [ { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "member", "display_name": "Strauss, James H." }, { "name": { "family": "Lipshitz", "given": "Howard D." }, "id": "Lipshitz-H-D", "role": "member", "display_name": "Lipshitz, Howard D." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "chair", "display_name": "Davidson, Eric H." } ], "option_major": [ "biology" ], "doi": "10.7907/dh99-m567", "abstract": "The 5' regulatory region of the Strogylocentrotus purpuratus cytoskeletal actin gene, CyIIIa, contains a 150 bp inverted interspersed repeat. The center of the inverted repeats contains binding sites for a 12 zinc finger DNA binding protein; SpZ12-1. This repeat was also found in the flanking region of the genes for two factors; SpZ2-1 and SpP3A2 which bind to the CyIIIa promoter. The high degree of conservation of the repeats is indicative of potential protein binding sites. Gel-shift assays using sea urchin egg cytoplasmic extracts revealed a second binding activity, on a separate region of the repeat, for a protein which we later identified as the sea urchin homologue of the 70 kD subunit of replication factor A, SpRFA-70.\n\nReplication factor A is a well known single-stranded DNA (ssDNA) binding protein that is required for DNA replication (Kim et al., Mol. Cell. Biol. 12, 3050-3059, 1992). We demonstrate that in addition to its non-specific affinity for ssDNA, SpRFA-70 has a high sequence specific binding affintiy for double-stranded DNA (dsDNA). The equilibrium constant (Keq) for SpRFA-70 binding to ssDNA is 1.7 x 10(10)M-1 and the Keq, for binding to the specific dsDNA site is 1.8 x 10(9)M-1 as determined by protein excess titration. Quantitative gel retardation assays reveal that the SpRFA-70 dsDNA binding activity is two orders of magnitude more prevalent per egg than per whole embryo at 24 hours. These data suggest that SpRFA-70 functions as a regulatory factor predominantly in the egg and early embryo.\n\nA genomic library was screened with short oligo probes to determine the number of sites for the DNA binding factors SpZ12-1 and SpRFA-70. The results of this screen indicate there are ~460 SpRFA-70 sites and ~318 SpZ12-1 sites. These sites match to a consensus sequence by >80%. There are 40-80 instances where these sites occur together. This is 30 fold higher than would be expected by random association. The distance between the two binding sites is also conserved. This arrangement is the same as that which occurs in the CyIIIa regulatory region. That this configuration is so conserved implies a potential regulatory interaction for these two factors." }, { "name": "Annau, Thomas Mark", "degree": "PhD", "year": "1996", "title": "Models of visual feature detection and spike coding in the nervous system", "advisor": "Hopfield, John J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-09212006-152641", "creators": [ { "name": { "family": "Annau", "given": "Thomas Mark" }, "id": "Annau-T-M", "display_name": "Annau, Thomas Mark" } ], "advisors": [ { "name": { "family": "Hopfield", "given": "John J." }, "id": "Hopfield-J-J", "role": "advisor", "display_name": "Hopfield, John J." } ], "committee": [ { "name": { "family": "Hopfield", "given": "John J." }, "id": "Hopfield-J-J", "role": "chair", "display_name": "Hopfield, John J." }, { "name": { "family": "Mead", "given": "Carver" }, "id": "Mead-C-A", "orcid": "0000-0003-4051-0462", "role": "member", "display_name": "Mead, Carver" }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "member", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Abu-Mostafa", "given": "Yaser S." }, "id": "Abu-Mostafa-Y-S", "role": "member", "display_name": "Abu-Mostafa, Yaser S." } ], "option_major": [ "cns" ], "doi": "10.7907/CN6R-WE94", "abstract": "We propose mathematical models to analyze two nervous system phenomena. The first is a model of the development and function of simple cell receptive fields in mammalian primary visual cortex. The model assumes that images are composed of combinations of a limited set of specific visual features and that the goal of simple cells is to detect the presence or absence of these features. Based on a presumed statistical character of images and their visual features, the model uses a constrained Hebbian learning rule to discover the structure of the features, and thus the appropriate response properties of simple cells, by training on a database of photographs. The response properties of the model simple cells agree qualitatively with neurophysiological observation.\r\n\r\nThe second is a model of the coding of information in the nervous system by the rate of axonal voltage spikes. Assuming an integrate-and-fire mechanism for spike generation, we develop a quantization-based model of rate coding and use it to derive the mathematical relationship between the amplitude and temporal resolution of a rate encoded signal. We elaborate the model to include integrator leak in the spike generation mechanism and show that it compactly combines coding and the computation of a threshold function.\r\n" }, { "name": "Apperson, Michelle Louise", "degree": "PhD", "year": "1996", "title": "Molecular Analysis of the Postsynaptic Density: Cloning and Characterization of Densin-lSO, a Novel Postsynaptic Density-Associated Adhesion Molecule", "advisor": "Kennedy, Mary B.", "url": "https://resolver.caltech.edu/CaltechETD:etd-01052007-083947", "creators": [ { "name": { "family": "Apperson", "given": "Michelle Louise" }, "id": "Apperson-Michelle-Louise", "display_name": "Apperson, Michelle Louise" } ], "advisors": [ { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "advisor", "display_name": "Kennedy, Mary B." } ], "committee": [ { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "chair", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Schuman", "given": "Erin Margaret" }, "id": "Schuman-E-M", "orcid": "0000-0002-7053-1005", "role": "member", "display_name": "Schuman, Erin Margaret" }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "role": "member", "display_name": "Dunphy, William G." } ], "option_major": [ "molbio" ], "doi": "10.7907/ze5z-f533", "abstract": "The postsynaptic density (PSD) is an electron dense structure just beneath the postsynaptic membrane. Several functions have been proposed for the PSD including regulating receptor number and clustering, anchoring signal transduction molecules at the synapse and mediating adhesion between the presynaptic and postsynaptic membranes. However, little was known about the proteins that make up the PSD until the biochemical purification of a PSD fraction from brain was established in 1974. Since then, several interesting proteins have been localized to the PSD fraction. The most abundant PSD protein is the [alpha] subunit of the type II calcium/calmodulin dependent protein kinase ([alpha]CaMKII). This protein is likely to play a role in the calcium-mediated signal transduction at the synapse that mediates certain forms of synaptic plasticity. Another major PSD protein is PSD-95, a member of the guanylate kinase family (GUK) of proteins.\r\n\r\nHere, I describe the purification and identification of three additional PSD proteins that comigrate at a molecular weight of 180 kDa on SDS-polyacrylamide gels. First, PSDgp180 is identified as the 2B subunit of the N-methyl-D-aspartate receptor (NR2B). NR2B is a major component of the PSD fraction and binds to PSD-95 in vitro. This interaction may anchor NMDA receptors at the synapse.\r\n\r\nNext, I report the cloning and characterization of densin-180, a 180 kDa PSD protein with a novel adhesion molecule-like sequence. Densin-180 is a brain-specific sialomucin that is enriched in the PSD fraction and localized to the synapse by immunocytochemistry.\r\n\r\nIn order to study the assembly of PSD proteins at the synapse, I use antibodies against [alpha]CaMKII, PSD-95 and densin-180 for double labeling cultured hippocampal neurons. In these cultures, densin-180 protein is the first marker to be expressed and this early densin-180 expression is in a diffuse membrane pattern along dendrites. When synapse formation begins at about 5 days after plating, the densin-180 protein is clustered at synapses and PSD-95 expression is induced. PSD-95 colocalizes with densin-180 clusters. The [alpha]CaMKII protein is expressed later in synapse formation (7 to 9 days in vitro) and may be a marker of mature excitatory neurons.\r\n\r\nIn the brain, densin-180 is localized to the neuropil regions in a punctate pattern likely to represent synaptic staining. In addition, anti-densin-180 is localized to a specific set of cells and that may represent undifferentiated neurons and small processes that may represent dendritic filopodia.\r\n\r\nThe third 180 kDa PSD protein is citron, a recently identified Rho/Rac binding protein. The citron sequence contains numerous motifs found in signal transduction proteins and a myosin-like coiled coil domain. Citron may be a target for Rho/Racdependent signal transduction at the synapse and may mediate physical stabilization of the postsynaptic density." }, { "name": "Arn, Eric", "degree": "PhD", "year": "1996", "title": "The 2'-5' RNA ligase of Escherichia coli : purification, cloning, and investigations of in vivo function", "advisor": "Abelson, John N.", "url": "https://resolver.caltech.edu/CaltechETD:etd-09122006-130201", "creators": [ { "name": { "family": "Arn", "given": "Eric" }, "id": "Arn-E", "display_name": "Arn, Eric" } ], "advisors": [ { "name": { "family": "Abelson", "given": "John N." }, "id": "Abelson-J-N", "role": "advisor", "display_name": "Abelson, John N." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biochem" ], "doi": "10.7907/d36x-7p54", "abstract": "An RNA ligase activity has previously been detected in extracts of E. coli which is capable of joining S. cerevisiae tRNA splicing intermediates in the absence of ATP to form a 2'-5'phosphodiester linkage (36). In order to study the mechanism and function of this unusual enzyme in bacterial RNA metabolism, a purification of the ligase enzyme was undertaken. The ligase was purified to homogeneity from a soluble high-speed extract of E. coli utilizing standard chromatographic techniques and reconstitution of activity following separation by SDS-PAGE. A single active polypeptide of approximately 20 kiloDaltons (kD) was shown to provide RNA ligase activity. This protein was N-terminally sequenced, and the open reading frame (ORF) encoding it was identified by a database search. This ORF, which codes for a novel protein with a predicted molecular weight of 19.9 kD, was cloned by PCR and used for overexpression of active recombinant lipase in E. coli and S. cerevisiae. The single chromosomal gene encoding the ligase was disrupted by insertion, abolishing ligase activity. Cells lacking active ligase are viable and show growth kinetics identical to the parent strain. Either parent strain or ligase knockout expressing high levels of recombinant ligase grow slowly compared to wild type and are temperature sensitive. Computer analysis of the ligase protein sequence allowed prediction of antigenic peptides derived from it. These peptides were synthesized and injected into rabbits to elicit polyclonal anti-ligase antibodies. Such antibodies were purified from rabbit sera by affinity to immobilized ligase, and shown to specifically recognize the ligase protein on Western blots. Ligase minus strains, purified recombinant ligase, and anti-ligase antibodies have been utilized in a variety of experiments to attempt to identify the in vivo substrate of this enzyme.\r\n" }, { "name": "Bair, Wyeth", "degree": "PhD", "year": "1996", "title": "Analysis of temporal structure in spike trains of visual cortical area MT", "advisor": "Koch, Christof", "url": "https://resolver.caltech.edu/CaltechTHESIS:04092013-154919755", "creators": [ { "name": { "family": "Bair", "given": "Wyeth" }, "id": "Bair-W", "display_name": "Bair, Wyeth" } ], "advisors": [ { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "role": "advisor", "display_name": "Koch, Christof" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "cns" ], "doi": "10.7907/8nqb-td88", "abstract": "The temporal structure of neuronal spike trains in the visual cortex can provide\r\ndetailed information about the stimulus and about the neuronal implementation of\r\nvisual processing. Spike trains recorded from the macaque motion area MT in previous\r\nstudies (Newsome et al., 1989a; Britten et al., 1992; Zohary et al., 1994) are\r\nanalyzed here in the context of the dynamic random dot stimulus which was used\r\nto evoke them. If the stimulus is incoherent, the spike trains can be highly modulated\r\nand precisely locked in time to the stimulus. In contrast, the coherent motion\r\nstimulus creates little or no temporal modulation and allows us to study patterns\r\nin the spike train that may be intrinsic to the cortical circuitry in area MT. Long\r\ngaps in the spike train evoked by the preferred direction motion stimulus are found,\r\nand they appear to be symmetrical to bursts in the response to the anti-preferred\r\ndirection of motion. A novel cross-correlation technique is used to establish that the\r\ngaps are correlated between pairs of neurons. Temporal modulation is also found in\r\npsychophysical experiments using a modified stimulus. A model is made that can\r\naccount for the temporal modulation in terms of the computational theory of biological\r\nimage motion processing. A frequency domain analysis of the stimulus reveals\r\nthat it contains a repeated power spectrum that may account for psychophysical and electrophysiological observations.
\r\n\r\nSome neurons tend to fire bursts of action potentials while others avoid burst\r\nfiring. Using numerical and analytical models of spike trains as Poisson processes\r\nwith the addition of refractory periods and bursting, we are able to account for peaks\r\nin the power spectrum near 40 Hz without assuming the existence of an underlying\r\noscillatory signal. A preliminary examination of the local field potential reveals that\r\nstimulus-locked oscillation appears briefly at the beginning of the trial.
\r\n" }, { "name": "Boysen, Anne-Cecilie", "degree": "PhD", "year": "1996", "title": "Analysis of the Human T Cell Receptor \u03b1/\u03b4 Locus: New Approaches to Mapping and Sequencing", "advisor": "Hood, Leroy E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:12162020-194820334", "creators": [ { "name": { "family": "Boysen", "given": "Anne-Cecilie" }, "id": "Boysen-Anne-Cecilie", "display_name": "Boysen, Anne-Cecilie" } ], "advisors": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "advisor", "display_name": "Hood, Leroy E." } ], "committee": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "chair", "display_name": "Hood, Leroy E." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "member", "display_name": "Simon, Melvin I." } ], "option_major": [ "immun" ], "doi": "10.7907/7x0h-qy95", "abstract": "The human T cell receptor (TCR) \u03b1/\u03b4 locus has been mapped and sequenced. This region occupies roughly one megabase (Mb) of DNA or equivalent to one three thousandth of the entire human genome, the longest continuous piece of human DNA yet sequenced. The sequence has provided new insights into the complex organization, structure and evolution of two intermingled multigene families (\u03b1 and \u03b4), and will hopefully in the future help answer interesting questions concerning the complex expression patterns of TCR \u03b1 and \u03b4 chains and about possible associations between specific polymorphisms in the TCR \u03b1/\u03b4 locus and susceptibility to autoimmune diseases. Comparison to cDNA data has provided information about expression of each of the TCR elements and about the striking diversification in the third hypervariable or junctional region. The sequence has contributed a glimpse of closely associated genomic DNA, in that the sequences surrounding the TCR locus, include the defender against death gene as well as five olfactory receptor genes. The sequence also harbors many other stretches of DNA, highly similar to previously identified genes, although in most cases, these have been found to be nonfunctional due to one or a few mutations. Comparison of 130 kilobases (kb) in the 3' region of the human sequence with its murine counterpart, suggests this region is highly conserved. The same 3' region has also been found to be limited in the concentration of genome wide repeats compared to the remainder of the locus. Furthermore, it contains a substantially reduced frequency of DNA variations compared to the rest of the locus. Apart from DNA variations in noncoding sequence, polymorphisms have also been identified in the coding regions of the TCR variable (V) gene segments, where, if they lead to amino acid changes, may alter the function of the TCR.
\r\n\r\nDuring the physical clone mapping and sequencing, new strategies were tested using primarily bacterial artificial chromosome (BAC) clones. These clones proved to be much more reliable and stable than clones currently employed in the human genome project (e.g., cosmids and yeast artificial chromosomes, YACs). BAC inserts can be sequenced completely by the high redundancy shotgun approach. Their insert size, stability, and capacity to be easily sequenced suggests that BAC clones are excellent mapping and sequencing reagents. The ends of BAC clone inserts can be sequenced directly. This has led to the proposal of a new strategy for obtaining the entire DNA sequence of the human genome without physical mapping.
" }, { "name": "Bradley, Jonathan Christopher Robert", "degree": "PhD", "year": "1996", "title": "Molecular Analysis of Olfactory Signal Transduction", "advisor": "Zinn, Kai George", "url": "https://resolver.caltech.edu/CaltechTHESIS:12212020-221414771", "creators": [ { "name": { "family": "Bradley", "given": "Jonathan Christopher Robert" }, "id": "Bradley-Jonathan-Christopher-Robert", "display_name": "Bradley, Jonathan Christopher Robert" } ], "advisors": [ { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "advisor", "display_name": "Zinn, Kai George" } ], "committee": [ { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "chair", "display_name": "Zinn, Kai George" }, { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "role": "member", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "role": "member", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "member", "display_name": "Simon, Melvin I." } ], "option_major": [ "biology" ], "doi": "10.7907/9h9a-zx14", "abstract": "Olfactory receptor neurons respond to odorant stimulation with a rapid and transient increase in intracellular cAMP that opens cyclic nucleotide-gated (cng) cation channels. Cng channels in rat olfactory neurons are activated by cAMP in the low micromolar range and are outwardly rectifying. The cloned rat olfactory cng channel, (rOCNC1), however, is much less sensitive to cAMP and exhibits very weak rectification. We have investigated this discrepancy between native and cloned channels, and have cloned a new rat cng channel subunit, denoted rOCNC2. rOCNC2 does not form functional channels when expressed alone in HEK 293 cells. When rOCNC1 and rOCNC2 are coexpressed, however, an outwardly rectifying cation conductance with cAMP sensitivity near that of the native channel is observed. In situ hybridization with probes specific for the two subunits shows they are coexpressed in olfactory receptor neurons. Further, subunit specific antibodies coimmunoprecipitate the other subunit from olfactory cilia membrane extracts. These data indicate that the native olfactory cng channel is likely to be a hetero-oligomer of the rOCNC1 and rOCNC2 subunits (Bradley et al. Proc. Natl. Acad. Sci. USA 91, 8890-8894 1994).
\r\n\r\nThe olfactory cng channels are also expressed in non sensory neurons in the brain. We have determined by in situ hybridization, immunocytochemistry, and Western blot that the olfactory cng channels are expressed in the hippocampus, cerebellum, and cortex of adult rats. Cultured hippocampal neurons from embryonic day 17 rats also express the olfactory cng channels as detected by immunofluorescence. Whole cell and excised inside-out patch recordings indicate that these cells have cng channels sensitive to 10\u03bcMcAMP, are outwardly rectifying, and insensitive to block by nickel ions. Consistent with the identification of these channels as the two subunits of the olfactory cng channel.
\r\n\r\nIn order to identify and characterize olfactory receptors for specific odorant chemicals, we have developed a method for generating an electrophysiological signal in response to cAMP elevation in Xenopus oocytes. To do this, we expressed the cystic fibrosis transmembrane regulator (CFTR), a chloride channel that is controlled via phosphorylation by cAMP-dependent protein kinase A (Uezono et al. Receptors and Channels 1:223-241 1993). Pools of synthetic mRNAs from dories of putative olfactory receptor genes were coinjected into oocytes together with CFTR mRNA and tested with odorant mixtures. We have preliminary data indicating that single clones can mediate odorant responses. These responses are quite variable and we have determined using immunofluorescence that this is likely due to a trafficking problem of the expressed receptor protein inside the cell. We have observed this trafficking problem in both the Xenopus oocytes and HEK293 cells. To circumvent this problem we isolated the small fraction (1%) of transfected HEK293 cells that express receptor protein on their surface by fluorescence activated cell sorting (FACS). These cells can then be assayed functionally for odorant interaction using a fura based Ca\u00b2\u207a imaging set-up. Here, the reporter is the cng channels which conduct Ca\u00b2\u207a into the cell in response the receptor mediated rise in intracellular cAMP.
" }, { "name": "Lewicki, Michael Samuel", "degree": "PhD", "year": "1996", "title": "Neural Representation of Auditory Temporal Structure", "advisor": "Konishi, Masakazu", "url": "https://resolver.caltech.edu/CaltechTHESIS:04092013-090259511", "creators": [ { "name": { "family": "Lewicki", "given": "Michael Samuel" }, "id": "Lewicki-Michael-Samuel", "display_name": "Lewicki, Michael Samuel" } ], "advisors": [ { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "advisor", "display_name": "Konishi, Masakazu" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "cns" ], "doi": "10.7907/99jm-m070", "abstract": "Neurons in the songbird forebrain nucleus HVc are highly sensitive to auditory temporal\r\ncontext and have some of the most complex auditory tuning properties yet discovered. HVc\r\nis crucial for learning, perceiving, and producing song, thus it is important to understand\r\nthe neural circuitry and mechanisms that give rise to these remarkable auditory response\r\nproperties. This thesis investigates these issues experimentally and computationally.
\r\n\r\nExtracellular studies reported here compare the auditory context sensitivity of neurons\r\nin HV c with neurons in the afferent areas of field L. These demonstrate that there is a\r\nsubstantial increase in the auditory temporal context sensitivity from the areas of field L\r\nto HVc. Whole-cell recordings of HVc neurons from acute brain slices are described which\r\nshow that excitatory synaptic transmission between HVc neurons involve the release of glutamate\r\nand the activation of both AMPA/kainate and NMDA-type glutamate receptors.\r\nAdditionally, widespread inhibitory interactions exist between HVc neurons that are mediated\r\nby postsynaptic GABA_A receptors. Intracellular recordings of HVc auditory neurons\r\nin vivo provides evidence that HV c neurons encode information about temporal structure\r\nusing a variety of cellular and synaptic mechanisms including syllable-specific inhibition,\r\nexcitatory post-synaptic potentials with a range of different time courses, and burst-firing,\r\nand song-specific hyperpolarization.
\r\n\r\nThe final part of this thesis presents two computational approaches for representing\r\nand learning temporal structure. The first method utilizes comput ational elements that are\r\nanalogous to temporal combination sensitive neurons in HVc. A network of these elements\r\ncan learn using local information and lateral inhibition. The second method presents a\r\nmore general framework which allows a network to discover mixtures of temporal features\r\nin a continuous stream of input.
" }, { "name": "Li, Chiang-Shan Ray", "degree": "PhD", "year": "1996", "title": "Macaque Lateral Intraparietal Area and Oculomotor Behaviors", "advisor": "Andersen, Richard A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04102013-115616526", "creators": [ { "name": { "family": "Li", "given": "Chiang-Shan Ray" }, "id": "Li-Chiang-Shan-Ray", "display_name": "Li, Chiang-Shan Ray" } ], "advisors": [ { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "role": "advisor", "display_name": "Andersen, Richard A." } ], "committee": [ { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "role": "member", "display_name": "Perona, Pietro" }, { "name": { "family": "Schlag", "given": "John" }, "id": "Schlag-J", "role": "member", "display_name": "Schlag, John" } ], "option_major": [ "cns" ], "doi": "10.7907/54y6-wq81", "abstract": "Neurons in the primate lateral intraparietal area (area LIP) carry visual,\r\nsaccade-related and eye position activities. The visual and saccade activities\r\nare anchored in a retinotopic framework and the overall response magnitude\r\nis modulated by eye position. It was proposed that the modulation by eye\r\nposition might be the basis of a distributed coding of target locations in a\r\nhead-centered space. Other recording studies demonstrated that area LIP is\r\ninvolved in oculomotor planning. These results overall suggest that area LIP\r\ntransforms sensory information for motor functions. In this thesis I further\r\nexplore the role of area LIP in processing saccadic eye movements by\r\nobserving the effects of reversible inactivation of this area. Macaque monkeys\r\nwere trained to do visually guided and memory saccades and a double\r\nsaccade task to examine the use of eye position signal. Finally, by intermixing\r\nvisual saccades with trials in which two targets were presented at opposite\r\nsides of the fixation point, I examined the behavior of visual extinction.
\r\n\r\nIn chapter 2, I will show that lesion of area LIP results in increased latency\r\nof contralesional visual and memory saccades. Contralesional memory\r\nsaccades are also hypometric and slower in velocity. Moreover, the\r\nimpairment of memory saccades does not vary with the duration of the delay\r\nperiod. This suggests that the oculomotor deficits observed after inactivation\r\nof area LIP is not due to the disruption of spatial memory.
\r\n\r\nIn chapter 3, I will show that lesion of area LIP does not severely affect the\r\nprocessing of spontaneous eye movement. However, the monkeys made\r\nfewer contralesional saccades and tended to confine their gaze to the\r\nipsilesional field after inactivation of area LIP. On the other hand, lesion of\r\narea LIP results in extinction of the contralesional stimulus. When the initial\r\nfixation position was varied so that the retinal and spatial locations of the\r\ntargets could be dissociated, it was found that the extinction behavior could\r\nbest be described in a head-centered coordinate.
\r\n\r\nIn chapter 4, I will show that inactivation of area LIP disrupts the use of eye\r\nposition signal to compute the second movement correctly in the double\r\nsaccade task. If the first saccade steps into the contralesional field, the error\r\nrate and latency of the second saccade are both increased. Furthermore, the\r\ndirection of the first eye movement largely does not have any effect on the\r\nimpairment of the second saccade. I will argue that this study provides\r\nimportant evidence that the extraretinal signal used for saccadic localization\r\nis eye position rather than a displacement vector.
\r\n\r\nIn chapter 5, I will demonstrate that in parietal monkeys the eye drifts\r\ntoward the lesion side at the end of the memory saccade in darkness. This\r\nresult suggests that the eye position activity in the posterior parietal cortex is\r\nactive in nature and subserves gaze holding.
\r\n\r\nOverall, these results further support the view that area LIP neurons encode\r\nspatial locations in a craniotopic framework and is involved in processing\r\nvoluntary eye movements.
\r\n" }, { "name": "Montgomery, John Michael", "degree": "PhD", "year": "1996", "title": "Cell migration domains in the chick telencephalon", "advisor": "Anderson, David J.; Fraser, Scott E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06022011-144716111", "creators": [ { "name": { "family": "Montgomery", "given": "John Michael" }, "id": "Montgomery-J-M", "display_name": "Montgomery, John Michael" } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "advisor", "display_name": "Anderson, David J." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "advisor", "display_name": "Fraser, Scott E." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/G3NH-0V88", "abstract": "Little is known about the process by which the vertebrate forebrain (the diencephalon and telencephalon) becomes regionalized during development. In studies reported here, DiI injections were used to label the embryonic day 3 (stage-16) chick telencephalon in ovo , and the migration patterns of labelled cells were analyzed in relation to various molecular markers, including the regulatory genes Cash-l and Sonic hedgehog (Shh). Cells generated in the ventral telencephalon (basal ventricular ridge, or BVR) were found to migrate widely, but were restricted from crossing into the more dorsal telencephalon (dorsal ventricular ridge, or DVR), and the more caudal\r\ndiencephalon. The cell migration boundary between the BVR and DVR correlates with a Cash-l expression boundary, and the cell migration boundary between BVR and diencephalon correlates with a Shh expression boundary. In addition, cell\r\nmigration patterns were dramatically different in the BVR and DVR territories. These results suggest that the BVR represents a \"cell migration domain,\" or a true unit of\r\ntelencephalic compartmental organization, which is distinct from cell migration domains in both the DVR and diencephalon. In addition, two lines of evidence in the\r\nearly embryo are shown to support the proposal that avian BVR is homologous to mammalian basal ganglia, and that avian DVR is homologous to mammalian cerebral cortex: regulatory gene expression patterns in the chick and mouse telencephalon are very similar; and the cell migration patterns in the chick telencephalon demonstrated here are found to correspond closely to those previously reported in the mouse telencephalon.\r\n\r\nUsing the same DiI labelling technique, a regional fate map of the stage-16 chick telencephalon was derived. This fate map can now be used to guide transplantation or misexpression experiments, and to interpret gene expression\r\npatterns in the stage-16 telencephalon. For example, though Cash-l is expressed in the entire BVR at ES-E7 (stage 24-30), superimposition of its expression pattern onto the stage-16 fate map shows that it is only expressed in a subregion of the presumptive BVR at stage-16.\r\n" }, { "name": "Patapoutian, Ardem", "degree": "PhD", "year": "1996", "title": "The Role of the MyoD Family Genes during Mouse Development", "advisor": "Wold, Barbara J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10042021-225839792", "creators": [ { "name": { "family": "Patapoutian", "given": "Ardem" }, "id": "Patapoutian-Ardem", "orcid": "0000-0003-0726-7034", "display_name": "Patapoutian, Ardem" } ], "advisors": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "advisor", "display_name": "Wold, Barbara J." } ], "committee": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "chair", "display_name": "Wold, Barbara J." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biology" ], "doi": "10.7907/7p08-6588", "abstract": "Myogenesis is studied as an example of vertebrate cell type determination and differentiation mainly due to the cloning and characterization of genes, both regulators and downstream structural genes, specifically expressed in this lineage. The studies presented in this thesis describe the regulation and function of the MyoD family of myogenic regulatory factors (MRFs) in the developing mouse embryo.
\r\n\r\nThere are four known MRFs (MyoD, Myf-5, myogenin, MRF4/herculin/myf6) in vertebrates; all are exclusively expressed in skeletal muscle and their progenitors, but each with a unique and dynamic pattern. The individual function of one of these, MRF4, was tested by gene disruption via homologous recombination. MRF4 is required for proper muscle formation in a specific domain of the axial lineage during embryogenesis. Later in development, the muscle phenotype is rescued apparently by cellular compensation, suggesting partial redundancy between MRF members. However, an unexpected rib pattern formation defect was observed that caused the death of MRF4 null mice at birth. An inductive signal from muscle precursors to rib progenitors is postulated to be the cause for this malformation.
\r\n\r\nA differentiated cell is usually considered to be a terminal phenotype. However, the MRFs, when force expressed, have the unique capacity to transform various differentiated cells into a myogenic phenotype. Such a switch in phenotype seems to occur during normal perinatal development of esophagus muscle, as these cells transdifferentiate from a functional smooth type to skeletal muscle by sequentially expressing the MRFs, and then skeletal muscle-specific structural genes. This is one of the few examples of transdifferentiation that occurs during normal development of vertebrates.
\r\n\r\nThe potent capacity of the MRFs to convert cells to a myogenic phenotype requires tight regulation of MRF expression as well as modulation of their function. Transgenic mice containing certain regulatory sequences from the Myf-5 locus, the first MRF to be expressed in all muscle lineages studied, drives the expression of a marker gene specifically\r\nin the early head but not trunk muscle precursors. This implies distinct regulatory pathways of initiating muscle determination in the two lineages. Furthermore, the head lineage is unique since Myf-5 is expressed at least three days before any of the other MRFs or muscle-specific differentiation genes are detectable, and suggests that Myf-5 function is under negative control.
" }, { "name": "Schoenherr, Christopher John", "degree": "PhD", "year": "1996", "title": "The Neuron Restrictive Silencer Factor: a Coordinate Repressor of Neuronal Genes", "advisor": "Anderson, David J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08112025-210630696", "creators": [ { "name": { "family": "Schoenherr", "given": "Christopher John" }, "id": "Schoenherr-Christopher-John", "display_name": "Schoenherr, Christopher John" } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "advisor", "display_name": "Anderson, David J." } ], "committee": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "chair", "display_name": "Anderson, David J." }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." } ], "option_major": [ "biology" ], "doi": "10.7907/d78s-xd11", "abstract": "The transcriptional regulation of neuronal genes requires the combination of\r\npositive and negative control mechanisms. As a model neuronal gene, we have studied the\r\nneuron-specific gene, SCG10. The expression of SCG10 appears to be restricted to\r\nneurons by selective repression in non-neuronal cells. The upstream regulatory region of\r\nSCG 10 contains a short sequence element that can repress, or silence, the activity of\r\npromoter fusion constructs in all non-neuronal cells assayed. In neuronal cells, this\r\nelement has very little silencing activity. This neuron-restrictive silencer element (NRSE)\r\nwas localized to about 21bp by deletional analysis. We have identified an NRSE binding\r\nprotein that is present only in non-neuronal cell lines, but is absent from neuronal cell lines.\r\nThis protein, the neuron-restrictive silencer factor (NRSF), is likely to mediate the\r\nrepression activity of the NRSE as a double point mutation in the element that eliminates\r\nNRSF binding also eliminates silencing. Intriguingly, a similar element was identified in\r\nthe type II sodium channel gene and shown to bind NRSF. Taken with its wide spread\r\nactivity, this suggests that NRSF may be a coordinate regulator of neuronal gene\r\nexpression in non-neuronal cells.
\r\n\r\nTo determine the role of NRSF in neuronal gene regulation, we have isolated cDNA\r\nclones encoding a portion of human NRSF and the complete mouse homologue. NRSF is\r\na novel protein with nine zinc fingers and several distinctive domains. Using in situ\r\nhybridization, expression of NRSF mRNA was detected in most non-neuronal tissues at\r\nseveral developmental stages, supporting the hypothesis that it functions as a near-global,\r\nsequence-specific repressor of neuronal gene expression. In the nervous system, NRSF\r\nmRNA was detected in neuronal progenitors, but not in postmitotic neurons. Its presence\r\nin precursor cells suggests that relief from NRSF-imposed repression may be an important\r\nevent in the selection or execution of a neuronal differentiation program.
\r\n\r\nFurther support for NRSF's role in neuronal gene regulation and development was\r\nprovided by identification of potential NRSF target genes. Endogenous and recombinant\r\nNRSF represses the activity of NRSE-containing reporter constructs and binds to\r\nconsensus NRSEs in 14 other neuron-specific genes in addition to SCG10 and the type II\r\nsodium channel. At least seven additional neuronal genes were found to have sequences\r\nwith significant similarity to the NRSE which are likely to represent functional binding sites\r\nfor NRSF. These results suggest that one protein can coordinately repress many neuronal\r\ngenes. Included amongst these genes are transcription factors that are implicated in the\r\nactivation of neuronal differentiation, providing further evidence that NRSF may repress\r\nthis process. Potential NRSEs also are found in non-neuronal genes which indicates that\r\nNRSF may have a function beyond the regulation of neuronal genes.
" }, { "name": "Schwarz, Erich Marquard", "degree": "PhD", "year": "1996", "title": "Calx, A Sodium-Calcium Exchanger of Drosophila melanogaster", "advisor": "Benzer, Seymour", "url": "https://resolver.caltech.edu/CaltechTHESIS:10292019-132931068", "creators": [ { "name": { "family": "Schwarz", "given": "Erich Marquard" }, "id": "Schwarz-Erich-Marquard", "orcid": "0000-0003-3151-4381", "display_name": "Schwarz, Erich Marquard" } ], "advisors": [ { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "advisor", "display_name": "Benzer, Seymour" } ], "committee": [ { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "chair", "display_name": "Benzer, Seymour" }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "member", "display_name": "Simon, Melvin I." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "molbio" ], "doi": "10.7907/M2PR-WT51", "abstract": "Calcium extrusion is necessary for cellular survival and suspected to modulate cellular activity. Drosophila phototransduction is a promising system in which to study calcium export, since it is dominated by calcium activity yet, unlike most calcium-dependent signalling pathways, genetically pliable. The multiple roles of calcium flux in Drosophila phototransduction are reviewed in Chapter One.
\r\n\r\nCalx, a Drosophila ortholog of mammalian 3Na+/1Ca2+ exchangers, was isolated and characterized (Chapter Two). Calx's gene product has ~50% identity to its direct mammalian homologs, with more distant similarities to an exchanger-related superfamily. There exist at least seven alternately spliced adult Calx transcripts, with an alternatively spliced miniexon in Calx's protein-coding region. A full-length Calx cDNA of 5408 bp has lengthy, elaborate 5' and 3' UTRs. Calx transcripts are ubiquitously expressed in embryos and adult heads, with one 5.7 kb transcript expressed in photoreceptors; Calx protein is also ubiquitous in adult heads, with a notable presence in photoreceptors and neuropil. Heterologous expression of Calx in Xenopus oocytes shows that it encodes a bona fide sodium-calcium exchanger; unlike mammalian retinal exchangers, it does not depend on potassium for activity. Calx encodes two novel protein motifs, Calx-\u03b1 and Calx-\u03b2. Both are intragenically duplicated, but they probably have different functions: Calx-\u03b1 is likely to encode residues central to calcium export, while Calx-\u03b2 may mediate intracellular signalling or cytoskeletal anchoring.
\r\n\r\n" }, { "name": "Sun, Jennifer Yun-Man", "degree": "PhD", "year": "1996", "title": "Three-Dimensional Shape from Shading: Perception and Mechanisms", "advisor": "Perona, Pietro", "url": "https://resolver.caltech.edu/CaltechETD:etd-09132006-155558", "creators": [ { "name": { "family": "Sun", "given": "Jennifer Yun-Man" }, "id": "Sun-Jennifer-Yun-Man", "display_name": "Sun, Jennifer Yun-Man" } ], "advisors": [ { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "advisor", "display_name": "Perona, Pietro" } ], "committee": [ { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "chair", "display_name": "Perona, Pietro" }, { "name": { "family": "Julesz", "given": "Bela" }, "id": "Julesz-B", "role": "member", "display_name": "Julesz, Bela" }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "member", "display_name": "Koch, Christof" }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" } ], "option_major": [ "biology" ], "doi": "10.7907/t5ng-6v86", "abstract": "In this thesis, we address the issue of 3-D shape from shading by investigating shape perception in humans and the early vision mechanisms that subserve this perception. We first investigated the influence of scale, contour, and reflectance function on shape perception from shading. Our results suggest that subjects can form robust 3-D shape percepts that remain consistent across sittings for shapes of various contours and reflectance functions. We have found that salient 3-D percepts can be formed at the level of early vision mechanisms. Experiments in which a single target pattern is discriminated from multiple background distractors show that certain shaded, 2-D stimuli consistent with a top-lit, convex interpretation can be processed fast (<80 msec) and in parallel. Strong pop-out asymmetries and control experiments involving shaded patterns that do not have familiar 3-D interpretations suggest that such fast, parallel processing is indeed dependent upon perception of 3-D shape. We find that these mechanisms proceed most readily when the stimuli can be interpreted as convex and lit from top-left. These preferences for shape and lighting directions appear to be intrinsic to early vision and cannot be overturned using stereo disparity cues. These early vision 3-D mechanisms can also be influenced by 3-D contextual information. We report that, together with 3-D shape, apparent reflectance is computed fast as well. Moreover, it is apparent reflectance, rather than brightness or perceptual 3-D shape, that is the primary basis for discrimination during the early stages of visual processing." }, { "name": "Hacia, Joseph Gerard", "degree": "PhD", "year": "1995", "title": "The use of modified oligonucleotides to investigate biological applications for triple helix formation", "advisor": "Wold, Barbara J.; Dervan, Peter B.", "url": "https://resolver.caltech.edu/CaltechETD:etd-10122007-074500", "creators": [ { "name": { "family": "Hacia", "given": "Joseph Gerard" }, "id": "Hacia-Joseph-Gerard", "display_name": "Hacia, Joseph Gerard" } ], "advisors": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "advisor", "display_name": "Wold, Barbara J." }, { "name": { "family": "Dervan", "given": "Peter B." }, "id": "Dervan-P-B", "role": "advisor", "display_name": "Dervan, Peter B." } ], "committee": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "chair", "display_name": "Wold, Barbara J." }, { "name": { "family": "Varshavsky", "given": "Alexander J." }, "id": "Varshavsky-A-J", "role": "member", "display_name": "Varshavsky, Alexander J." }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Dervan", "given": "Peter B." }, "id": "Dervan-P-B", "role": "member", "display_name": "Dervan, Peter B." }, { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "role": "member", "display_name": "Dunphy, William G." } ], "option_major": [ "biology" ], "doi": "10.7907/15j7-1b14", "abstract": "Oligonucleotide-directed triple helix formation was assayed for potential biological applications. Unmodified pyrimidine-rich oligodeoxyribo-nucleotides were found to block the progress of primer extension by DNA polymerase in vitro through triple helix formation. Klenow fragment polymerization was obstructed at sites that map near the proximal boundary between duplex and triplex. Among a family of related three-stranded structures, longer triplexes were more effective polymerase inhibitors than shorter complexes. One such complex provided an effective polymerase blockade for at least twenty minutes.\r\n\r\nChemical modifications enhanced both triple helix formation and the nuclease resistance of pyrimidine-rich and purine-rich oligonucleotides. Unmodified purine-rich and pyrimidine-rich oligonucleotides containing a diastereomeric mixture of phosphorothioate or stereoregular (all R[rho]) phosphorothioate linkages were tested for triple helix formation by quantitative DNase I footprinting analysis. Both purine-rich and pyrimidine-rich phosphorothioate oligonucleotides containing modified nucleosides formed triple helical complexes in vitro under physiological ionic conditions. Pyrimidine-rich oligoribonucleotides as well as 2'-OMe oligoribonucleotides bound DNA with high affinity. Only purine-rich oligodeoxyribonucleotides had significant affinity for double-helical DNA.\r\n\r\nSmall uridine-rich oligoribonucleotides were overexpressed in E. coli for in vivo triple helix formation studies using a low-copy number plasmid target site. Micromolar intracellular concentrations of intact oligoribonucleotide were generated; however, in vivo footprinting analysis indicated that no triple helical complexes formed. [Beta]-galactosidase activity assays further indicated that the presence of the uridine-rich RNA did not inhibit transcription of a chimeric lacZ gene in which the triple helix target site was inserted.\r\n\r\nModified nuclease-resistant pyrimidine-rich and purine-rich oligonucleotides were tested for triple helix formation on Xenopus oocyte minichromosomes. In vivo footprinting analysis showed that none of the oligonucleotides formed triple helical complexes with the target site. A modified oligonucleotide, which directed an alkylation reaction to a specific guanosine residue on duplex DNA in vitro, did not react in vivo with the target minichromosomes. Further chemical modifications will have to be made to facilitate in vivo triple helix formation." }, { "name": "Halsell, Susan Richardson", "degree": "PhD", "year": "1995", "title": "Expression and localization of Hsp83 RNA in the early Drosophila embryo", "advisor": "Lipshitz, Howard D.", "url": "https://resolver.caltech.edu/CaltechETD:etd-10092007-075248", "creators": [ { "name": { "family": "Halsell", "given": "Susan Richardson" }, "id": "Halsell-S-R", "display_name": "Halsell, Susan Richardson" } ], "advisors": [ { "name": { "family": "Lipshitz", "given": "Howard D." }, "id": "Lipshitz-H-D", "role": "advisor", "display_name": "Lipshitz, Howard D." } ], "committee": [ { "name": { "family": "Lipshitz", "given": "Howard D." }, "id": "Lipshitz-H-D", "role": "chair", "display_name": "Lipshitz, Howard D." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biochem" ], "doi": "10.7907/cx4q-tz48", "abstract": "The Hsp83 gene encodes the sole Drosophila homolog of the mammalian Hsp90 family of regulatory molecular chaperones. Hsp90 has been implicated in regulating the activity of several signal transduction molecules, including receptor tyrosine kinases, steroid hormone receptors, src family tyrosine kinases, raf kinase, Weel cell cycle kinase, eIF-2[alpha], protein kinase C and casein kinases. We show that Hsp83 is dynamically expressed during Drosophila oogenesis and embryogenesis. Maternally transcribed mRNA is uniformly distributed in the early embryo, but becomes localized to the posterior pole by a unique mechanism of generalized degradation and localized protection. Hsp83 mRNA is a component of the posterior polar plasm, and the polar plasm is necessary and sufficient for the protection of the maternal RNA from degradation. In vivo analysis reveals that the protection is mediated by sequence elements within the 3'UTR of the Hsp83 mRNA. Maternal Hsp83 mRNA is taken up into the germline progenitor cells (the pole cells) as they form, and RNA is detected in the pole cells throughout embryogenesis. Hsp83 is first expressed zygotically throughout stage 3 embryos but this RNA disappears after only 20 minutes and is undetectable in stage 4 embryos. Hsp83 is then expressed in the anterior third of stage 5 embryos under the control of the anterior morphogen, BICOID. Cis-regulatory sequences necessary for transcriptional activation by BICOID map 1124 bp within the single intron of the Hsp83 gene. In vitro gel shift analysis shows that BICOID binds to a site within this sequence, suggesting that Hsp83 is a direct target of BICOID. In addition, we show that there is a later, independent phase of transcription within the anterior of the early embryo. These analyses form the basis for detailed molecular analysis of Hsp83 functions during early embryogenesis.\n" }, { "name": "Huang, Linda S.", "degree": "PhD", "year": "1995", "title": "The Caenorhabditis elegans lin-15 locus", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechETD:etd-10092007-135940", "creators": [ { "name": { "family": "Huang", "given": "Linda S." }, "id": "Huang-L-S", "display_name": "Huang, Linda S." } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" } ], "option_major": [ "biology" ], "doi": "10.7907/pat7-8d95", "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\n\nIn the nematode [...], vulval development involves \nan inductive signalling event mediated by the LIN-3 growth factor and the \nLET-23 receptor tyrosine kinase. The LIN-3 growth factor is sent from the \nanchor cell, a cell in the somatic gonad, to the six multipotent vulval \nprecursor cells (VPCs) in the ventral epidermis. Lack of the signal leads to \nnone of the six VPCs adopting vulval fates. The [...] locus is a negative \nregulator of [...] mediated signalling; animals carrying loss-of\nfunction mutations in [...] have excessive vulval differentiation. Mosaic \nstudies demonstrate this negative regulation to occur non-autonomously. \nFurthermore, the [...] locus has two genetically defined functions, A and B. \nThese A and B functions are shared by other loci with these functions, \ndefining two distinct pathways for negative regulation. The A and B \nfunctions are redundant because animals defective in only one function \ndisplay the wild-type phenotype; only animals defective in both A and B \nfunction display the excessive vulval differentiation phenotype.\n\n\tThis thesis describes the molecular and genetic analysis of the [...] \nlocus. The lin-15 locus was cloned based on its genetic map position. The two \nfunctions of lin-15 are due to two transcripts comprising the [...] locus, one \ntranscript corresponding to the A function, the other, the B function. These \ntwo transcripts are transcribed in the same direction, separated by 105 base \npairs, and possibly transcribed polycistronically. The predicted proteins \ncoded for by both transcripts are novel and hydrophilic. Antibodies raised \nagainst the LIN-15B protein show that LIN-15B is a 170 kD protein that is \nlocalized to the nucleus and broadly expressed. Genetic studies demonstrate\nthat [...] acts upstream of the receptor and in parallel to the inductive \nsignal. When a [...] null allele is used to modulate signalling through \nweakly functioning LET-23 receptors, we see that the activity of the receptor \ncan be correlated to the type of vulval fate specified.\n" }, { "name": "Liu, Katharine S.", "degree": "PhD", "year": "1995", "title": "Male Mating Behavior in Caenorhabditis elegans", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechETD:etd-10232007-110013", "creators": [ { "name": { "family": "Liu", "given": "Katharine S." }, "id": "Liu-Katharine-S", "display_name": "Liu, Katharine S." } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "role": "member", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "member", "display_name": "Benzer, Seymour" } ], "option_major": [ "biology" ], "doi": "10.7907/x5k7-r486", "abstract": "Several key features about the nematode, C. elegans, make it a tractable model system for the analysis of behavior. C. elegans allows genetic analysis of behavior in an animal with few neurons and few connections between these neurons. Male mating behavior in C. elegans requires the coordination of several steps: response to contact with the hermaphrodite; turning around her head or tail; location of the vulva; insertion of the spicules into the vulva; and sperm transfer. I have chosen to study this relatively complex behavior in this simple system to begin to understand how complex behaviors are coordinated.
\r\n\r\nI have taken two approaches to the problem that take advantage of the unique characteristics of C. elegans. First, since all cells are reproducibly identifiable in this system, I lesioned individual structures and neurons to determine which neurons mediate mating behavior. By cell ablation, we have identified participating sensory neurons for each step of mating behavior. The sensory rays mediate response and turning. The neurons of the hook and post-cloacal sensillae mediate vulva location. Two pairs of the spicule neurons mediate spicule insertion, while another pair regulates sperm transfer. In addition, some inter and motor neurons have been identified which participate in these steps.
\r\n\r\nSecond, I screened for mutants impaired in mating behavior. From the screen, we have isolated mutants defective in each step of the behavior. These results independently suggest that the different steps of mating behavior are independently mutable and therefore likely mediated by separable neuronal system. In addition, mutants were isolated that appear to have hermaphrodite specific defects, indicating along with ablation results that a hermaphrodite signal is required for the initiation of sperm transfer.
\r\n\r\nIn conclusion, I have found that the steps in mating behavior are to a large extent separable. Male mating behavior in C. elegans is not entirely innately controlled, but rather the initiation of each step and the integration between steps is highly regulated by sensory feedback.
" }, { "name": "Mazer, James Allan", "degree": "PhD", "year": "1995", "title": "Integration of Parallel Processing Streams in the Inferior Colliculus of the Barn Owl", "advisor": "Konishi, Masakazu", "url": "https://resolver.caltech.edu/CaltechETD:etd-10162007-141005", "creators": [ { "name": { "family": "Mazer", "given": "James Allan" }, "id": "Mazer-James-Allan", "display_name": "Mazer, James Allan" } ], "advisors": [ { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "advisor", "display_name": "Konishi, Masakazu" } ], "committee": [ { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "chair", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "role": "member", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Andersen", "given": "Richard A." }, "id": "Andersen-R-A", "role": "member", "display_name": "Andersen, Richard A." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." } ], "option_major": [ "biology" ], "doi": "10.7907/JVR9-1R69", "abstract": "The study of the neural mechanisms underlying sensation has demonstrated than an intimate relationship exists between behavioral function and neural structure. One common structural theme in sensory processing is the use of parallel processing streams. Parallel processing occurs in virtually every sensory modality and in almost every species studied to date. This work describes the integration of parallel auditory processing streams for time, intensity and frequency in the inferior colliculus of the barn owl, Tyto alba, which leads to the formation of a centrally synthesized map of auditory space.
\r\n\r\nInteraural time differences (ITDs) provide the primary cue for localization in the horizontal plane. ITDs are extracted from the firing patterns of auditory nerve fibers by a coincidence detection circuit located in the owl's brainstem. Interaural level differences, which are used by the owl for vertical plane localization, are computed by an anatomically distinct and parallel circuit. The brainstem and midbrain structures that process time and level differences are independent up to the level of the inferior colliculus. In the lateral shell division of the central nucleus of the inferior colliculus, these two processing streams are combined by single neurons that exhibit spatially restricted auditory receptive fields.
\r\n\r\nThe first part of this thesis characterizes the detailed physiological properties of lateral shell neurons and describes a model of hierarchical integration within the shell that underlies the synthesis of space-specific neurons located in the topographic auditory space map in the external nucleus of the inferior colliculus (ICx). The second part examines the integration of the parallel, narrow band frequency channels arising at the level of the brain stem coincidence detector, nucleus laminaris. The output of single coincidence detector neurons, which encode interaural phase difference, does not unambiguously signal horizontal location. Multiple phase ambiguous narrow band frequency channels are integrated in the lateral shell to eliminate phase ambiguity. Experiments presented here describe the relationship between signal bandwidth and phase ambiguity in an attempt to elaborate the neural circuitry underlying the integration of parallel, narrow band, interaural phase difference channels.
\r\n" }, { "name": "Proctor, Larry P.", "degree": "PhD", "year": "1995", "title": "Characterization of the Auditory Thalamic Nucleus of the Barn Owl", "advisor": "Konishi, Masakazu", "url": "https://resolver.caltech.edu/CaltechETD:etd-10182007-094614", "creators": [ { "name": { "family": "Proctor", "given": "Larry P." }, "id": "Proctor-Larry-P", "display_name": "Proctor, Larry P." } ], "advisors": [ { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "advisor", "display_name": "Konishi, Masakazu" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "neurobio" ], "doi": "10.7907/ze17-3216", "abstract": "The barn owl has the remarkable ability to accurately localize a target on the basis of auditory cues alone. The investigation of the central nervous system mechanisms underlying the sensory aspect of this behavior led to the discovery of neurons which have auditory receptive fields restricted to small regions of the acoustic environment. Neurons with these characteristics were found both in the forebrain and in the inferior colliculus. In the inferior colliculus, neurons which were near one another had receptive fields which were near one another in the sound field; there was a physiological map of auditory space. No such organization was observed in the forebrain. The auditory map of space in the inferior colliculus projects to the optic tectum where it is preserved and placed in register with a visual map of space. The auditory space map in the inferior colliculus and optic tectum have been functionally related to sound localization in a behavioral assay. Recent experiments have demonstrated that the thalamo-telencephalic auditory pathway is sufficient for localization of sound sources, despite the fact that a topographic map of auditory space has not been demonstrated in this neural pathway. The purpose of the work reported upon here was to examine the manner in which the auditory thalamus, nucleus ovoidalis (N.Ov), transforms the neural code which represents the auditory environment. Neurons in nucleus ovoidalis were characterized with respect to their responses to stimulation with sounds presented through earphones. This dichotic stimulation allowed for the independent control of the amplitude, frequency and temporal structure of the sounds delivered to the ear. The anatomy of the afferent and efferent pathways to the auditory thalamus were also investigated.
\r\n\r\nThe activity of extracellularly isolated single-neurons was recorded in the nucleus ovoidalis of the anesthetized barn owl. This nucleus contains two subdivisions based on tonotopic organization. The central and medial portion of the nucleus is organized such that neurons responding well to high frequencies tend to be located dorsally while neurons which respond well to lower frequencies are located progressively more ventral. In the lateral portion of the nucleus, neurons in a dorsoventral electrode penetration have the same best frequency, between 4.5 and 5.0 kHz. Of 114 neurons whose best frequency tuning curves were characterized completely, 14 had two or three clearly distinguishable peaks. The response to sound localization cues at one of the frequency peaks was different from those at the other frequency peak(s). Of 207 neurons which were recorded within N.Ov, all responded to auditory stimulation with broad band noise and all were sensitive to at least one sound localization parameter. In contrast to previously studied auditory nuclei in the medulla and mesencephalon of the barn owl, there was no apparent systematic mapping of either sound localization cue in the dorsoventral, mediolateral or rostrocaudal axes.
\r\n\r\nNeurons within N.Ov had response tuning curves to interaural time difference (ITD), interaural intensity difference (IID) and frequency which were most similar to those found in the lateral shell and core subdivisions of the central nucleus of the inferior colliculus (ICc). However, neurons were found in ovoidalis which had combinations of response tuning curve types not previously observed in the auditory system of the barn owl. Thirteen neurons were classified as space-specific, although their response properties were not always similar to neurons in the space maps of ICx or optic tectum. Six neurons were found which had broad frequency tuning curves but which also had ambiguous ITD or IID tuning curves in response to white noise.
\r\n\r\nInjections of retrograde tracers into N.Ov resulted in stained cell bodies in ICc that were sparsely distributed across the three subdivisions of the nucleus. Labeled neurons were located in various positions along the dorsoventral axis of ICc, along which frequency is mapped. Retrogradely labeled somata were found bilaterally in ICc, though the number of labeled cells was higher in inferior colliculus ipsilateral to the injection site. The widely distributed pattern of retrograde staining in ICc was obtained irrespective of the location of the tracer injection within N.Ov. Anterograde tracers injected into the core/medial shell region of ICc resulted in a pattern of stained axonal terminals in the centromedial N.Ov that was relatively focal and which corresponded well with the tonotopic organization of this region of the nucleus. Injection of anterograde tracers into the lateral shell subdivision of ICc yielded labeled axon terminals in the lateral portion of N.Ov in caudal sections, while the heaviest staining was located along the dorsal aspect in the most rostral sections. Anterograde tracer studies revealed that N.Ov efferents originating from the medial portion of the nucleus have a restricted terminal field in the most medial region of the forebrain area Field L2a. The ventrolateral region of N.Ov, however, sends a wide projection pattern of efferent terminations across the mediolateral extent of caudal Field L2. Labeled axon terminals are quite dense in the lateral portion of Field L2 and rather diffuse in the medial aspect.
\r\n\r\nThe unique combinations of physiological responses found in N.Ov as well as its patterns of afferent and efferent connectivity suggest that ovoidalis is reorganizing the neural information concerning acoustic stimuli. While tuning to sound localization cues is maintained, it is possible that such coding may be of secondary consideration in the thalamo-telencephalic pathway. The foundation for the neural representation of auditory recognition may well begin at the level of the auditory thalamus.
\r\n" }, { "name": "Stack, Jeffrey Herman", "degree": "PhD", "year": "1995", "title": "Protein and Phosphatidylinositol Kinases in Yeast Protein Sorting", "advisor": "Emr, Scott D.", "url": "https://resolver.caltech.edu/CaltechETD:etd-10262007-081900", "creators": [ { "name": { "family": "Stack", "given": "Jeffrey Herman" }, "id": "Stack-Jeffrey-Herman", "display_name": "Stack, Jeffrey Herman" } ], "advisors": [ { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "orcid": "0000-0002-5408-6781", "role": "advisor", "display_name": "Emr, Scott D." } ], "committee": [ { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "orcid": "0000-0002-5408-6781", "role": "chair", "display_name": "Emr, Scott D." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "member", "display_name": "Revel, Jean-Paul" }, { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "orcid": "0000-0001-7598-8939", "role": "member", "display_name": "Dunphy, William G." } ], "option_major": [ "molbiochem" ], "doi": "10.7907/ayzy-yn85", "abstract": "The yeast vps mutants are defective in the delivery of proteins to the vacuole. The products of the VPS15 and VPS34 genes encode \r\nhomologs of a serine/threonine protein kinase and a phosphatidylinositol 3-kinase (Ptdlns 3-kinase), respectively, that are required for the sorting of soluble vacuolar proteins. Mutations altering highly conserved residues in the catalytic domain of either protein result in the missorting and secretion of vacuolar hydrolases such as carboxypeptidase Y, suggesting that protein and lipid phosphorylation reactions are required for the vesicular transport of vacuolar proteins. Biochemical characterization of Vps34p has shown that, in addition to possessing Ptdlns 3-kinase activity, Vps34p undergoes an autophosphorylation reaction, indicating that it is a novel multiple specificity kinase able to phosphorylate both lipid and protein substrates.\r\n\r\nThe Vps15 protein kinase both functionally and physically interacts with the Vps34 PtdIns 3-kinase and the two proteins form a complex \r\nassociated with the cytoplasmic face of an intracellular membrane fraction most likely corresponding to a late Golgi compartment. In addition to recruiting Vps34p to the membrane site of its phospholipid substrate, we have found that Vpsl5p is also required for the activation of Vps34p as Vps34p Ptdlns 3-kinase activity is extremely defective in vps15 mutant strains. Vpsl5p protein kinase activity appears to be responsible for the association with and subsequent activation of Vps34p because vpsl5 kinase domain mutations result in defects in Ptdlns 3-kinase activity and the mutant Vps15 proteins are unable to associate with Vps34p. Together, these results have demonstrated that a functional and stable complex between Vpsl5p and Vps34p is absolutely required for vacuolar protein sorting.\r\n\r\nUse of a temperature-conditional allele of VPS34 that is for both protein sorting and Ptdlns 3-kinase activity has allowed us to demonstrate the direct involvement of Ptdlns 3-kinase in vacuolar protein sorting. Our findings with Vps34p suggest that the functions of mammalian phosphoinositide 3-kinase may include the regulation of membrane trafficking and have led us to propose that Ptdlns(3)P is involved in regulating intracellular protein sorting reactions in all eukaryotic cells." }, { "name": "Wang, David Guo-Wei", "degree": "PhD", "year": "1995", "title": "Identification and characterization of a negative regulator required for spatial control of the territory-specific CyIIIa gene in the sea urchin embryo", "advisor": "Davidson, Eric H.", "url": "https://resolver.caltech.edu/CaltechETD:etd-10242007-082412", "creators": [ { "name": { "family": "Wang", "given": "David Guo-Wei" }, "id": "Wang-D-G", "display_name": "Wang, David Guo-Wei" } ], "advisors": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "advisor", "display_name": "Davidson, Eric H." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/tejh-wh40", "abstract": "The Cyllla cytoskeletal actin gene of the sea urchin Strongylocentrotus purpuratus is activated in late cleavage and expressed exclusively in the aboral ectoderm territory of the embryo. Previous gene transfer studies defined a 2.3 kb cis-regulatory domain that is necessary and sufficient for correct temporal and spatial expression of a Cyllla-CAT fusion gene. In this study, a negative regulatory element within this region was identified that is required for repression of the Cyllla gene in skeletogenic mesenchyme cells. The repression mediated by this element takes place after initial territorial specification.\n\nTo study negative spatial control of the Cylila gene at the molecular level, a cDNA clone encoding a DNA-binding protein with twelve Zn fingers (SpZ12-1) was isolated by probing an expression library with this cis-regulatory element. Deletion analysis of the SpZ12-1 protein confirmed that a DNA-binding domain is located within the zinc finger region. SpZ12-1 is the only DNA-binding protein in embryo nuclear extract that interacts with the specific cis target sites required for repression of Cyllla-CAT in skeletogenic mesenchyme and is likely to be the trans factor that mediates this repression.\n\nSpZ12-1 is present in significant quantities even in unfertilized egg cytoplasm, and in similar quantities in mesenchyme blastula-stage embryo cytoplasm. Taken together with earlier measurements of Calzone et al. (Genes & Dev. 2,1074-1088, 1988), our results indicate that SpZ12-1 enters the embryonic nuclei between late cleavage and mesenchyme blastula stages. A low prevalence of SpZ12-1 mRNA is also present throughout development. Translation of this mRNA could easily account for the complete complement of SpZ12-1 protein in the embryo, as estimated from its DNA binding activity. SpZ12-1 probably functions at several developmental stages, and is evidently of both maternal and embryonic provenance." }, { "name": "Yip, Man Lun Richard", "degree": "PhD", "year": "1995", "title": "Molecular genetic analysis of morphogenesis in Drosophila : functions of the hindsight locus", "advisor": "Lipshitz, Howard D.", "url": "https://resolver.caltech.edu/CaltechETD:etd-10262007-093418", "creators": [ { "name": { "family": "Yip", "given": "Man Lun Richard" }, "id": "Yip-M-R", "display_name": "Yip, Man Lun Richard" } ], "advisors": [ { "name": { "family": "Lipshitz", "given": "Howard D." }, "id": "Lipshitz-H-D", "role": "advisor", "display_name": "Lipshitz, Howard D." } ], "committee": [ { "name": { "family": "Lipshitz", "given": "Howard D." }, "id": "Lipshitz-H-D", "role": "chair", "display_name": "Lipshitz, Howard D." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "member", "display_name": "Simon, Melvin I." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/5kbx-dn94", "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\n\nIn order to form complex three-dimensional body structures, multicellular organisms have to be able to coordinate the activities of all cells involved; in metazoa the control of various morphogenetic movements during gastrulation are particularly important. Drosophila embryogenesis provides an excellent model. The Drosophila hindsight gene function is required for germband retraction. Embryos lacking hindsight activity have a normal body plan and undergo normal morphogenetic movement prior to the onset of germband retraction. However, they fail to retract their geinubands. hindsight encodes a large nuclear protein of 1920 amino acids. Sequence analysis reveals that it contains fourteen [...] type zinc-fingers, arranged in widely spaced clusters. Additional features of the HINDSIGHT protein, such as glutamine-rich and proline-rich domains, suggest it functions as a transcription factor. Embryonic expression of hindsight is complex: it is found in the endoderm (anterior and posterior midgut), amnioserosa, subsets of the central nervous system, the peripheral nervous system and the tracheal system. However, it is the expression of hindsight in the midgut that is important for germband retraction since mutations which abolish hindsight endodermal expression also affect germband retraction. Although hnt is not expressed in ectoderm, it is these cells that undergo the cell shape changes that accomplish germband retraction. We propose that hindsight activity regulates a signal produced by the endodelin that is responsible for the coordination of morphogenetic cell shape changes and movements in ectoderm. In addition, hindsight is also required for normal eye development. HINDSIGHT protein is initially detected in the morphogenetic furrow of the developing Drosophila eye. HINDSIGHT is expressed in all photoreceptor cells as they are recruited into the ommatidial cluster. Mosaic analysis of hindsight in the larval eye disc reveals that homozygous hindsight mutant patches contain regularly spaced ommatidial clusters with variable numbers of photoreceptor cells. Analysis of these photoreceptor cells using cell-specific and general developmental markers indicates that their differentiation is abnoiinal. The presumptive R8 cells fail to express BOSS protein and the presumptive R2-5 cells do not express ROUGH protein. Genetically, hindsight shows synergistic interaction with Star, a gene also involved in photoreceptor specification. Taken together, these results demonstrate an early role for hindsight in photoreceptor development. Furthermore, hindsight is expressed throughout eye development and its activity is required late in pupal development when photoreceptor cells undergo morphological changes, such as apical-basal extension and rhabdomere separation." }, { "name": "Zeller, Robert Werner", "degree": "PhD", "year": "1995", "title": "Transcriptional control of spatially regulated genes in the early sea urchin embryo", "advisor": "Davidson, Eric H.", "url": "https://resolver.caltech.edu/CaltechETD:etd-10232007-134522", "creators": [ { "name": { "family": "Zeller", "given": "Robert Werner" }, "id": "Zeller-Robert-Werner", "display_name": "Zeller, Robert Werner" } ], "advisors": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "advisor", "display_name": "Davidson, Eric H." } ], "committee": [ { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "chair", "display_name": "Fraser, Scott E." }, { "name": { "family": "Lipshitz", "given": "Howard D." }, "id": "Lipshitz-H-D", "orcid": "0000-0002-7372-4419", "role": "member", "display_name": "Lipshitz, Howard D." }, { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "member", "display_name": "Hood, Leroy E." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." } ], "option_major": [ "biology" ], "doi": "10.7907/08kg-x260", "abstract": "The regulatory domains of several Strongylocentrotus purpuratus embryo genes appear to be organized into \"modules\" of closely associated target sites for DNA-binding proteins. The Sp(G/C)F1 protein is a novel DNA-binding protein which binds specifically to G/C rich cis-elements. The protein was purified from embryo nuclear extracts by DNA affinity chromatography and the corresponding cDNA isolated. Five different Sp(G/C)F1 polypeptides, produced from a nested set of AUG initiation codons, are synthesized from a single mRNA template. Each protein shares a common C-terminus and a centrally located DNA-binding domain but incorporates variable amounts of an N-terminal proline-rich region. Since proline-rich regions often serve as transcriptional activation domains, the five Sp(G/C)F1 proteins are likely to possess different transcriptional \"activation potentials.\" Sp(G/C)F1 proteins bind DNA cooperatively, most likely as homodimers, and multiple protein-DNA complexes are formed at high protein to DNA ratios in vitro. When bound to two or more target sites in the CyIIIa regulatory domain, the Sp(G/C)F1 polypeptides fouu protein-protein contacts, and \"loop out\" intervening regions of DNA which could allow the interaction of distant regulatory modules. The Sp(G/C)F1 protein is thus likely to serve as an intermodule communicator in the regulatory domains of many sea urchin embryo genes.\r\n\r\nAntibodies were used to quantitate the nuclear concentrations of two different DNA-binding proteins, P3A1 and P3A2, which bind specifically to P3A cis-elements found in several different regulatory domains. Both proteins are present maternally, but by late gastrula, P3A1 protein has disappeared from the nucleus leaving P3A2 protein levels relatively unchanged at about 10[superscript 4] molecules per nucleus. Since the relative binding affinities of P3A1 protein for different DNA target sites are up to 50X lower than the affinities of P3A2 protein for the same sites, P3A1 protein is therefore likely to be of functional significance only in early to mid-cleavage before genes known to contain P3A target sites, such as CyIIIa, SM50 and Specla, are expressed. The P3A1 and P3A2 proteins can thus serve as an antagonistic regulatory switch at P3A sites in cleavage stage sea urchin embryos." }, { "name": "Chamberlin, Helen Marie", "degree": "PhD", "year": "1994", "title": "Cell fate specification during Caenorhabditis elegans male tail development", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04292013-110424232", "creators": [ { "name": { "family": "Chamberlin", "given": "Helen Marie" }, "id": "Chamberlin-H-M", "display_name": "Chamberlin, Helen Marie" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "member", "display_name": "Lewis, Edward B." }, { "name": { "family": "Lipshitz", "given": "Howard D." }, "id": "Lipshitz-H-D", "role": "member", "display_name": "Lipshitz, Howard D." } ], "option_major": [ "biology" ], "doi": "10.7907/nmjn-dx76", "abstract": "The cells of the specialized mating structures of the nematode\r\nCaenorhabditis elegans adult male tail develop from sex-specific divisions of\r\npostembryonic blast cells. One male-specific blast cell, B, is the precursor to\r\nall the cells of the copulatory spicules. Both cell interactions and autonomous\r\nfate specification mechanisms are utilized in the B lineage to specify fate.
\r\n\r\nDuring development the anterior daughter of B, B.a, generates four\r\ndistinct pairs of cells. Cell ablation experiments indicate that the cells of\r\neach pair respond to positional cues provided by other male-specific blast\r\ncells. F and U promote anterior fates, Y.p promotes some posterior fates, and\r\nthe B.a progeny promote posterior fates. The cells within each pair may also\r\ninteract.
\r\n\r\nThe lin-3/let-23 signalling pathway, identified for its function in C.\r\nelegans hermaphrodite vulval induction, mediates the signal from F and U.\r\nReduction-of-function mutations in lin-3 (EGF-like signal), let-23 (receptor),\r\nsem-5 (adaptor), let-60 (ras), or lin-45 (raf) disrupt the fates of the anterior\r\ncells, and mimic F and U ablation. In addition, ectopically expressed lin-3\r\ndisrupts the fates of the posterior cells, and can promote anterior fates even\r\nin the absence of F and U.
\r\n\r\nA genetic screen of over 9000 mutagenized gametes recovered 22\r\nmutations in 20 loci that disrupt fate specification in male tail lineages.\r\nSeven of these mutations may represent new genes that play a role in male\r\ntail development.
\r\n\r\nThe first division of the B cell is asymmetric. The gene vab-3 is\r\nrequired for specification of B.a fates, and it may represent a factor whose\r\nactivity is localized to the B.a cell via the gene lin-17. lin-17 acts both at the\r\nfirst division of the B cell and at specific other cell divisions in the lineage.
\r\n\r\n" }, { "name": "Chen, Dan", "degree": "PhD", "year": "1994", "title": "Molecular mechanisms of interleukin-2 gene inducibility: developmental control and combinatorial action of transcription factors", "advisor": "Rothenberg, Ellen V.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04262013-142519914", "creators": [ { "name": { "family": "Chen", "given": "Dan" }, "id": "Chen-D", "display_name": "Chen, Dan" } ], "advisors": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "advisor", "display_name": "Rothenberg, Ellen V." } ], "committee": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "member", "display_name": "Strauss, James H." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." } ], "option_major": [ "biology" ], "doi": "10.7907/snb3-5k23", "abstract": "Interleukin-2 is one of the lymphokines secreted by T helper type 1 cells upon\r\nactivation mediated by T-cell receptor (TCR) and accessory molecules. The ability to\r\nexpress IL-2 is correlated with T-lineage commitment and is regulated during T cell\r\ndevelopment and differentiation. Understanding the molecular mechanism of how IL-2\r\ngene inducibility is controlled at each transition and each differentiation process of T-cell\r\ndevelopment is to understand one aspect of T-cell development. In the present study, we\r\nfirst attempted to elucidate the molecular basis for the developmental changes of IL-2 gene\r\ninducibility. We showed that IL-2 gene inducibility is acquired early in immature CD4-\r\nCD8-TCR- thymocytes prior to TCR gene rearrangement. Similar to mature T cells, a\r\ncomplete set of transcription factors can be induced at this early stage to activate IL-2 gene\r\nexpression. The progression of these cells to cortical CD4^+CD8^+TCR^(1o) cells is\r\naccompanied by the loss of IL-2 gene inducibility. We demonstrated that DNA binding\r\nactivities of two transcription factors AP-1 and NF-AT are reduced in cells at this stage.\r\nFurther, the loss of factor binding, especially AP-1, is attributable to the reduced ability to\r\nactivate expression of three potential components of AP-1 and NF-AT, including c-Fos,\r\nFosB, and Fra-2. We next examined the interaction of transcription factors and the IL-2\r\npromoter in vivo by using the EL4 T cell line and two non-T cell lines. We showed an all-or-none phenomenon regarding the factor-DNA interaction, i.e., in activated T cells, the\r\nIL-2 promoter is occupied by sequence-specific transcription factors when all the\r\ntranscription factors are available; in resting T cells or non-T cells, no specific protein-DNA\r\ninteraction is observed when only a subset of factors are present in the nuclei.\r\nPurposefully reducing a particular set of factor binding activities in stimulated T cells using\r\npharmacological agents cyclosporin A or forskolin also abolished all interactions. The\r\nresults suggest that a combinatorial and coordinated protein-DNA interaction is required for\r\nIL-2 gene activation.\r\n\r\nThe thymocyte experiments clearly illustrated that multiple transcription factors are\r\nregulated during intrathymic T-cell development, and this regulation in tum controls the\r\ninducibility of the lineage-specific IL-2 gene. The in vivo study of protein-DNA interaction\r\nstressed the combinatorial action of transcription factors to stably occupy the IL-2 promoter\r\nand to initiate its transcription, and provided a molecular mechanism for changes in IL-2\r\ngene inducibility in T cells undergoing integration of multiple environmental signals." }, { "name": "Fann, Ming-Ji", "degree": "PhD", "year": "1994", "title": "Cytokine control of neuronal phenotype", "advisor": "Patterson, Paul H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04292013-154555817", "creators": [ { "name": { "family": "Fann", "given": "Ming-Ji" }, "id": "Fann-M-J", "display_name": "Fann, Ming-Ji" } ], "advisors": [ { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "advisor", "display_name": "Patterson, Paul H." } ], "committee": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" } ], "option_major": [ "biology" ], "doi": "10.7907/2rm0-fg46", "abstract": "Diffusible proteins regulate neural development at a variety of\r\nstages. Using a novel neuronal culture assay, I have identified several\r\ncytokines that regulate the expression of neurotransmitters and\r\nneuropeptides in sympathetic neurons. These cytokines fall into two\r\nfamilies. The first group is termed the neuropoietic cytokines, while\r\nincluding CDF/LIF, CNTF, OSM and GPA, induces expression of the same\r\nset of neuropeptide mRNAs in cultured sympathetic neurons. These four\r\nfactors not only exhibit similar biological activities; they also share a\r\npredicted secondary structure and bind to a signal-transducing receptor\r\nsubunit in common with IL-6 and IL-11. The latter two cytokines display a\r\nweaker activity in this assay. In addition, I find that several members of\r\nthe TGF-\u03b2 superfamily, activin A, BMP-2, and BMP-6, have a selective\r\noverlap with the neuropoietic family in the spectrum of neuropeptides\r\nthat these cytokines induce in sympathetic neurons. Different patterns of\r\nneuropeptides induced by the TGF-\u03b2 family members, however,\r\ndemonstrate that the activities of these cytokines are distinct from those of\r\nthe neuropoietic family. Another 30 cytokines are without detectable effect\r\nin this neuronal assay.
\r\n\r\nActivin A induces a set of neurotransmitters and neuropeptides that\r\nis somewhat similar to the phenotype of sympathetic neurons innervating\r\nsweat glands in rat footpads. In situ hybridization and RNase protection\r\nwere carried out to test whether activins were involved in the phenotypic\r\ntransition when sympathetic neurons contact sweat glands. I find that\r\nactivin mRNA is present in both cholinergic and noradrenergic targets.\r\nMoreover, homogenates of footpads do not contain activin-like activity in\r\nthe neuronal assay in vitro. Taken together, these data do not support\r\nactivins as the best candidates for the sweat gland factor.
\r\n\r\nSeveral novel factors that regulate neuropeptide expression exist in\r\nheart cell conditioned medium. I attempted to purify these factors in\r\ncollaboration with Dr. Jane Talvenheimo. Our results suggest that these\r\nfactors are sensitive to the storage conditions used. Several modifications\r\nof purification strategy are discussed.
\r\n" }, { "name": "Hill, Russell James", "degree": "PhD", "year": "1994", "title": "The LIN-3 gene of the nematode C. elegans is the vulval-inducing signal", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05082013-083718323", "creators": [ { "name": { "family": "Hill", "given": "Russell James" }, "id": "Hill-R-J", "display_name": "Hill, Russell James" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "member", "display_name": "Benzer, Seymour" }, { "name": { "family": "Lipshitz", "given": "Howard D." }, "id": "Lipshitz-H-D", "role": "member", "display_name": "Lipshitz, Howard D." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." } ], "option_major": [ "biology" ], "doi": "10.7907/8z98-jj03", "abstract": "The development of the vulva of the nematode Caenorhabditis elegans is\r\ninduced by a signal from the anchor cell of the somatic gonad. Activity of the\r\ngene lin-3 is required for the Vulval Precursor Cells (VPCs) to assume vulval\r\nfates. It is shown here that lin-3 encodes the vulval-inducing signal.
\r\n\r\nlin-3 was molecularly cloned by transposon-tagging and shown to encode\r\na nematode member ofthe Epidermal Growth Factor (EGF) family. Genetic\r\nepistasis experiments indicate that lin-3 acts upstream of let-23, which\r\nencodes a homologue of the EGF-Receptor.
\r\n\r\nlin-3 transgenes that contain multiple copies of wild-type lin-3 genomic\r\nDNA clones confer a dominant multivulva phenotype in which up to all six of\r\nthe VPCs assume vulval fates. The properties of these trans genes suggest\r\nthat lin-3 can act in the anchor cell to induce vulval fates. Ablation of the\r\ngonadal precursors, which prevents the development of the AC, strongly\r\nreduces the ability of lin-3 transgenes to stimulate vulval development. A\r\nlin-3 recorder transgene that retains the ability to stimulate vulval\r\ndevelopment is expressed specifically in the anchor cell at the time of vulval\r\ninduction.
\r\n\r\nExpression of an obligate secreted form of the EGF domain of Lin-S from\r\na heterologous promoter is sufficient to induce vulval fates in the absence of\r\nthe normal source of the inductive signal. This result suggests that Lin-S\r\nmay act as a secreted factor, and that Lin-S may be the sole vulval-inducing\r\nsignal made by the anchor cell.
\r\n\r\nlin-3 transgenes can cause adjacent VPCs to assume the 1\u00b0 vulval fate\r\nand thus can override the action of the lateral signal mediated by lin-12 that\r\nnormally prevents adjacent 1\u00b0 fates. This indicates that the production of\r\nLin-3 by the anchor cell must be limited to allow the VPCs to assume the\r\nproper pattern of fates of so 3\u00b0 3\u00b0 2\u00b0 1\u00b0 2\u00b0 3\u00b0.
" }, { "name": "Huber, Andrew Henry", "degree": "PhD", "year": "1994", "title": "A biochemical and structural characterization of Drosophila neuroglian", "advisor": "Bjorkman, Pamela J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05032013-161139911", "creators": [ { "name": { "family": "Huber", "given": "Andrew Henry" }, "id": "Huber-Andrew-Henry", "display_name": "Huber, Andrew Henry" } ], "advisors": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "advisor", "display_name": "Bjorkman, Pamela J." } ], "committee": [ { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "chair", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Abelson", "given": "John N." }, "id": "Abelson-J-N", "role": "member", "display_name": "Abelson, John N." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "member", "display_name": "Zinn, Kai George" } ], "option_major": [ "biology" ], "doi": "10.7907/43ng-md46", "abstract": "A number of cell-cell interactions in the nervous system are mediated by\r\nimmunoglobulin gene superfamily members. For example, neuroglian, a homophilic\r\nneural cell adhesion molecule in Drosophila, has an extracellular portion comprising six C-\r\n2 type immunoglobulin-like domains followed by five fibronectin type III (FnIII) repeats.\r\nNeuroglian shares this domain organization and significant sequence identity with Ll, a\r\nmurine neural adhesion molecule that could be a functional homologue. Here I report the\r\ncrystal structure of a proteolytic fragment containing the first two FnIII repeats of\r\nneuroglian (NgFn 1,2) at 2.0\u00c5. The interpretation of photomicrographs of rotary\r\nshadowed Ng, the entire extracellular portion of neuroglian, and NgFnl-5, the five\r\nneuroglian Fn III domains, is also discussed.
\r\n\r\nThe structure of NgFn 1,2 consists of two roughly cylindrical \u03b2-barrel structural motifs\r\narranged in a head-to-tail fashion with the domains meeting at an angle of ~120, as defined\r\nby the cylinder axes. The folding topology of each domain is identical to that previously\r\nobserved for single FnIII domains from tenascin and fibronectin. The domains of\r\nNgFn1,2 are related by an approximate two fold screw axis that is nearly parallel to the\r\nlongest dimension of the fragment. Assuming this relative orientation is a general property\r\nof tandem FnIII repeats, the multiple tandem FnIII domains in neuroglian and other\r\nproteins are modeled as thin straight rods with two domain zig-zag repeats. When\r\ncombined with the dimensions of pairs of tandem immunoglobulin-like domains from CD4\r\nand CD2, this model suggests that neuroglian is a long narrow molecule (20 - 30 \u00c5 in\r\ndiameter) that extends up to 370\u00c5 from the cell surface.
\r\n\r\nIn photomicrographs, rotary shadowed Ng and NgFn1-5 appear to be highly flexible\r\nrod-like molecules. NgFn 1-5 is observed to bend in at least two positions and has a mean\r\ntotal length consistent with models generated from the NgFn1,2 structure. Ng molecules have up to four bends and a mean total length of 392 \u00c5, consistent with a head-to-tail\r\npacking of neuroglian's C2-type domains.
\r\n" }, { "name": "Lee, Junho", "degree": "PhD", "year": "1994", "title": "Negative regulators of a growth factor-mediated signaling pathway in the nematode Caenorhabditis elegans", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05092013-145747882", "creators": [ { "name": { "family": "Lee", "given": "Junho" }, "id": "Lee-Jun", "display_name": "Lee, Junho" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "role": "member", "display_name": "Dunphy, William G." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Varshavsky", "given": "Alexander J." }, "id": "Varshavsky-A-J", "role": "member", "display_name": "Varshavsky, Alexander J." } ], "option_major": [ "biology" ], "doi": "10.7907/g59t-0b31", "abstract": "Vulval differentiation in C. elegans is mediated by an Epidermal\r\ngrowth factor (EGF)- EGF receptor (EGFR) signaling pathway. I have\r\ncloned unc-101, a negative regulator of vulval differentiation of the nematode\r\nC. elegans. unc-101 encodes a homolog of AP47, the medium chain of the\r\ntrans-Golgi clathrin-associated protein complex. This identity was\r\nconfirmed by cloning and comparing sequence of a C. elegans homolog of\r\nAP50, the medium chain of the plasma membrane clathrin-associated\r\nprotein complex. I provided the first genetic evidence that the trans-Golgi\r\nclathrin-coated vesicles are involved in regulation of an EGF signaling\r\npathway. Most of the unc-101 alleles are deletions or nonsense mutations,\r\nsuggesting that these alleles severely reduce the unc-101 activity. A hybrid\r\ngene that contains parts of unc-101 and mouse AP4 7 rescued at least two\r\nphenotypes of unc-101 mutations, the Unc and the suppression of vulvaless\r\nphenotype of let-23(sy1) mutation. Therefore, the functions of AP47 are\r\nconserved between nematodes and mammals.
\r\n\r\nunc-101 mutations can cause a greater than wild-type vulval\r\ndifferentiation in combination with certain mutations in sli-1, another\r\nnegative regulator of the vulval induction pathway. A mutation in a new\r\ngene, rok-1, causes no defect by itself, but causes a greater than wild-type\r\nvulval differentiation in the presence of a sli-1 mutation. The unc-101; rok-1;\r\nsli-1 triple mutants display a greater extent of vulval differentiation than any\r\ndouble mutant combinations of unc-101, rok-1 and sli-1. Therefore, rok-1\r\nlocus defines another negative regulator of the vulval induction pathway.
\r\n\r\nI analyzed a second gene encoding an AP47 homolog in C. elegans.\r\nThis gene, CEAP47, encodes a protein 72% identical to both unc-101 and\r\nmammalian AP47. A hybrid gene containing parts of unc-101 and CEAP47\r\nsequences can rescue phenotypes of unc-101 mutants, indicating that UNC-\r\n101 and CEAP47 proteins can be redundant if expressed in the same set of\r\ncells.
" }, { "name": "Leiserson, William M.", "degree": "PhD", "year": "1994", "title": "Molecular genetics of the Drosophila eyes absent gene", "advisor": "Benzer, Seymour", "url": "https://resolver.caltech.edu/CaltechTHESIS:05082013-155756716", "creators": [ { "name": { "family": "Leiserson", "given": "William M." }, "id": "Leiserson-W-M", "display_name": "Leiserson, William M." } ], "advisors": [ { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "advisor", "display_name": "Benzer, Seymour" } ], "committee": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Lipshitz", "given": "Howard D." }, "id": "Lipshitz-H-D", "role": "member", "display_name": "Lipshitz, Howard D." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/3jn4-xk56", "abstract": "The Drosophila compound eye has provided a genetic approach to\r\nunderstanding the specification of cell fates during differentiation. The eye is\r\nmade up of some 750 repeated units or ommatidia, arranged in a lattice. The\r\ncellular composition of each ommatidium is identical. The arrangement of the\r\nlattice and the specification of cell fates in each ommatidium are thought to occur\r\nin development through cellular interactions with the local environment. Many\r\nmutations have been studied that disrupt the proper patterning and cell fating in the\r\neye. The eyes absent (eya) mutation, the subject of this thesis, was chosen because\r\nof its eyeless phenotype. In eya mutants, eye progenitor cells undergo\r\nprogrammed cell death before the onset of patterning has occurred. The molecular\r\ngenetic analysis of the gene is presented.
\r\n\r\nThe eye arises from the larval eye-antennal imaginal disc. During the third\r\nlarval instar, a wave of differentiation progresses across the disc, marked by a\r\nfurrow. Anterior to the furrow, proliferating cells are found in apparent disarray.\r\nPosterior to the furrow, clusters of differentiating cells can be discerned, that\r\ncorrespond to the ommatidia of the adult eye. Analysis of an allelic series of eya\r\nmutants in comparison to wild type revealed the presence of a selection point: a\r\nwave of programmed cell death that normally precedes the furrow. In eya\r\nmutants, an excessive number of eye progenitor cells die at this selection point,\r\nsuggesting the eya gene influences the distribution of cells between fates of death\r\nand differentiation.
\r\n\r\nIn addition to its role in the eye, the eya gene has an embryonic function.\r\nThe eye function is autonomous to the eye progenitor cells. Molecular maps of the\r\neye and embryonic phenotypes are different. Therefore, the function of eya in the\r\neye can be treated independently of the embryonic function. Cloning of the gene reveals two cDNA's that are identical except for the use of an alternatively-spliced\r\n5' exon. The predicted protein products differ only at the N-termini. Sequence\r\nanalysis shows these two proteins to be the first of their kind to be isolated.\r\nTrangenic studies using the two cDNA's show that either gene product is able to\r\nrescue the eye phenotype of eya mutants.
\r\n\r\nThe eya gene exhibits interallelic complementation. This interaction is an\r\nexample of an \"allelic position effect\": an interaction that depends on the relative\r\nposition in the genome of the two alleles, which is thought to be mediated by\r\nchromosomal pairing. The interaction at eya is essentially identical to a\r\nphenomenon known as transvection, which is an allelic position effect that is\r\nsensitive to certain kinds of chromosomal rearrangements. A current model for\r\nthe mechanism of transvection is the trans action of gene regulatory regions. The\r\neya locus is particularly well suited for the study of transvection because the\r\nmutant phenotypes can be quantified by scoring the size of the eye.
\r\n\r\nThe molecular genetic analysis of eya provides a system for uncovering\r\nmechanisms underlying differentiation, developmentally regulated programmed\r\ncell death, and gene regulation.
\r\n" }, { "name": "McDonough, Stefan I.", "degree": "PhD", "year": "1994", "title": "Pharmacology and pore-forming domains of the cystic fibrosis transmembrane conductance regulator", "advisor": "Lester, Henry A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05142013-132218781", "creators": [ { "name": { "family": "McDonough", "given": "Stefan I." }, "id": "McDonough-S-I", "display_name": "McDonough, Stefan I." } ], "advisors": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "advisor", "display_name": "Lester, Henry A." } ], "committee": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "chair", "display_name": "Lester, Henry A." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "member", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "member", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "member", "display_name": "Revel, Jean-Paul" } ], "option_major": [ "biology" ], "doi": "10.7907/eyre-8424", "abstract": "The cystic fibrosis transmembrane conductance regulator (CFTR) is a\r\nchloride channel member of the ATP-binding cassette (ABC) superfamily of\r\nmembrane proteins. CFTR has two homologous halves, each consisting of six\r\ntransmembrane spanning domains (TM) followed by a nucleotide binding fold,\r\nconnected by a regulatory (R) domain. This thesis addresses the question of\r\nwhich domains are responsible for Cl^- selectivity, i.e., which domains line the\r\nchannel pore.
\r\n\r\nTo address this question, novel blockers of CFTR were characterized.\r\nCFTR was heterologously expressed in Xenopus oocytes to study the\r\nmechanism of block by two closely related arylaminobenzoates,\r\ndiphenylamine-2-carboxylic acid (DPC) and flufenamic acid (FFA). Block by\r\nboth is voltage-dependent, with a binding site \u2248 40% through the electric field\r\nof the membrane. DPC and FFA can both reach their binding site from either\r\nside of the membrane to produce a flickering block of CFTR single channels.\r\nIn addition, DPC block is influenced by Cl^- concentration, and DPC blocks with\r\na bimolecular forward binding rate and a unimolecular dissociation rate.\r\nTherefore, DPC and FFA are open-channel blockers of CFTR, and a residue of\r\nCFTR whose mutation affects their binding must line the pore.
\r\n\r\nScreening of site-directed mutants for altered DPC binding affinity\r\nreveals that TM-6 and TM-12 line the pore. Mutation of residue 5341 in TM-6\r\nabolishes most DPC block, greatly reduces single-channel conductance, and\r\nalters the direction of current rectification. Additional residues are found in\r\nTM-6 (K335) and TM-12 (T1134) whose mutations weaken or strengthen DPC\r\nblock; other mutations move the DPC binding site from TM-6 to TM-12. The\r\nstrengthened block and lower conductance due to mutation T1134F is\r\nquantitated at the single-channel level. The geometry of DPC and of the\r\nresidues mutated suggest \u03b1-helical structures for TM-6 and TM-12. Evidence is\r\npresented that the effects of the mutations are due to direct side-chain\r\ninteraction, and not to allosteric effects propagated through the protein.\r\nMutations are also made in TM-11, including mutation S1118F, which gives\r\nvoltage-dependent current relaxations. The results may guide future studies on\r\npermeation through ABC transporters and through other Cl^- channels.
\r\n" }, { "name": "Olshausen, Bruno Adolphus", "degree": "PhD", "year": "1994", "title": "Neural Routing Circuits for Forming Invariant Representations of Visual Objects", "advisor": "Anderson, Charles Hammond; Van Essen, David C.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04162013-161719346", "creators": [ { "name": { "family": "Olshausen", "given": "Bruno Adolphus" }, "id": "Olshausen-Bruno-Adolphus", "display_name": "Olshausen, Bruno Adolphus" } ], "advisors": [ { "name": { "family": "Anderson", "given": "Charles Hammond" }, "id": "Anderson-C-H", "role": "advisor", "display_name": "Anderson, Charles Hammond" }, { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "advisor", "display_name": "Van Essen, David C." } ], "committee": [ { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "chair", "display_name": "Van Essen, David C." }, { "name": { "family": "Anderson", "given": "Charles Hammond" }, "id": "Anderson-C-H", "role": "member", "display_name": "Anderson, Charles Hammond" }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "member", "display_name": "Perona, Pietro" }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "member", "display_name": "Koch, Christof" } ], "option_major": [ "cns" ], "doi": "10.7907/PSR1-YH20", "abstract": "This thesis presents a biologically plausible model of an attentional mechanism for forming position- and scale-invariant representations of objects in the visual world. The model relies on a set of control neurons to dynamically modify the synaptic strengths of intra-cortical connections so that information from a windowed region of primary visual cortex (Vl) is selectively routed to higher cortical areas. Local spatial relationships (i.e., topography) within the attentional window are preserved as information is routed through the cortex, thus enabling attended objects to be represented in higher cortical areas within an object-centered reference frame that is position and scale invariant. The representation in V1 is modeled as a multiscale stack of sample nodes with progressively lower resolution at higher eccentricities. Large changes in the size of the attentional window are accomplished by switching between different levels of the multiscale stack, while positional shifts and small changes in scale are accomplished by translating and rescaling the window within a single level of the stack. The control signals for setting the position and size of the attentional window are hypothesized to originate from neurons in the pulvinar and in the deep layers of visual cortex. The dynamics of these control neurons are governed by simple differential equations that can be realized by neurobiologically plausible circuits. In pre-attentive mode, the control neurons receive their input from a low-level \"saliency map\" representing potentially interesting regions of a scene. During the pattern recognition phase, control neurons are driven by the interaction between top-down (memory) and bottom-up (retinal input) sources. The model respects key neurophysiological, neuroanatomical, and psychophysical data relating to attention, and it makes a variety of experimentally testable predictions.\r\n" }, { "name": "Plaxco, Kevin W.", "degree": "PhD", "year": "1994", "title": "Protein-DNA interactions : molecular modeling and energetics", "advisor": "Goddard, William A., III", "url": "https://resolver.caltech.edu/CaltechTHESIS:11112009-144801757", "creators": [ { "name": { "family": "Plaxco", "given": "Kevin W." }, "id": "Plaxco-K-W", "display_name": "Plaxco, Kevin W." } ], "advisors": [ { "name": { "family": "Goddard", "given": "William A., III" }, "id": "Goddard-W-A-III", "role": "advisor", "display_name": "Goddard, William A., III" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/fdk3-5402", "abstract": "The thesis deals with the structural elements involved in and the energetics of sequence specific recognition of DNA.\r\n\r\nChapter 1 of the thesis provides a brief overview on the mechanics and applicability of molecular dynamics based methods for studying the structure and function of molecules of proteins, DNA and other macromolecules of biological relevance.\r\n\r\nChapter 2 presents a constrained molecular dynamics derived model we have developed for the DNA binding domain of the protein Hin Recombinase. Based on a combination of homology modeling and experimentally derived placement constraints we used molecular dynamics to conduct a search of conformation space constrained to remain consistent with the then known experimental characterizations of the protein. The model generated by this approach allowed us to correctly predict the sequence selectivity of the Hin, and lead to a number of insights into the nature of its sequence selectivity.\r\n\r\nChapter 3 discusses a variety of experimental results that have been obtained on the structure of the Hin-hix complex. While these results primarily conform with model based predictions, others have pointed towards further refinements that are possible.\r\n\r\nChapter 4 of the thesis provides a brief overview of the methods and applicability of perturbation thermodynamic analysis as applied to the molecular dynamics based simulation of proteins and DNA in general and some specific issues of concern with regard to the simulations reported in this thesis.\r\n\r\nIn chapter 5 we report on our perturbation thermodynamic molecular dynamics analysis of the relative free energy of solvation of thymine and uricil. This work provides important insights into the role solvation plays in the formation of sequence specific protein-DNA complexes.\r\n\r\nFinally, chapter 6 is a report on our investigations into the mechanisms of sequence specific binding for the minor groove binding peptide Netropsin. Steric, electrostatic and solvation effects are all investigated using a perturbation thermodynamic approach to elucidate the mechanisms involved in complex formation for this important class of DNA binding ligands.\r\n" }, { "name": "Ryckebusch, Sylvie Adrienne", "degree": "PhD", "year": "1994", "title": "The Central Nervous Control of Walking in the Locust Schistocerca americana", "advisor": "Laurent, Gilles J.; Mead, Carver", "url": "https://resolver.caltech.edu/CaltechTHESIS:04112013-092807415", "creators": [ { "name": { "family": "Ryckebusch", "given": "Sylvie Adrienne" }, "id": "Ryckebusch-Sylvie-Adrienne", "display_name": "Ryckebusch, Sylvie Adrienne" } ], "advisors": [ { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "advisor", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Mead", "given": "Carver" }, "id": "Mead-C-A", "orcid": "0000-0003-4051-0462", "role": "co-advisor", "display_name": "Mead, Carver" } ], "committee": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "chair", "display_name": "Lester, Henry A." }, { "name": { "family": "Barr", "given": "Alan H." }, "id": "Barr-A-H", "role": "member", "display_name": "Barr, Alan H." }, { "name": { "family": "Bower", "given": "James M." }, "id": "Bower-J-M", "role": "member", "display_name": "Bower, James M." }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "member", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Mead", "given": "Carver" }, "id": "Mead-C-A", "orcid": "0000-0003-4051-0462", "role": "member", "display_name": "Mead, Carver" } ], "option_major": [ "cns" ], "doi": "10.7907/1aey-8096", "abstract": "Rhythmic motor behaviors in all animals appear to be under the control of \"central pattern generator\" circuits, neural circuits which can produce output patterns appropriate for behavior even when isolated from their normal peripheral inputs. Insects have been a useful model system in which to study the control of legged terrestrial locomotion. Much is known about walking in insects at the behavioral level, but to date there has been no clear demonstration that a central pattern generator for walking exists. The focus of this thesis is to explore the central neural basis for locomotion in the locust, Schistocerca americana.
\r\n\r\nRhythmic motor patterns could be evoked in leg motor neurons of isolated thoracic ganglia of locusts by the muscarinic agonist pilocarpine. These motor patterns would be appropriate for the movement of single legs during walking. Rhythmic patterns could be evoked in all three thoracic ganglia, but the segmental rhythms differed in their sensitivities to pilocarpine, their frequencies, and the phase relationships of motor neuron antagonists. These different patterns could be generated by a simple adaptable model circuit, which was both simulated and implemented in VLSI hardware. The intersegmental coordination of leg motor rhythms was then examined in preparations of isolated chains of thoracic ganglia. Correlations between motor patterns in different thoracic ganglia indicated that central coupling between segmental pattern generators is likely to contribute to the coordination of the legs during walking.
\r\n\r\nThe work described here clearly demonstrates that segmental pattern generators for walking exist in insects. The pattern generators produce motor outputs which are likely to contribute to the coordination of the joints of a limb, as well as the coordination of different limbs. These studies lay the groundwork for further studies to determine the relative contributions of central and sensory neural mechanisms to terrestrial walking.
\r\n" }, { "name": "Tole, Shubha", "degree": "PhD", "year": "1994", "title": "Surface markers of regionalization in the vertebrate nervous system", "advisor": "Patterson, Paul H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05172013-074622662", "creators": [ { "name": { "family": "Tole", "given": "Shubha" }, "id": "Tole-S", "display_name": "Tole, Shubha" } ], "advisors": [ { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "advisor", "display_name": "Patterson, Paul H." } ], "committee": [ { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "chair", "display_name": "Patterson, Paul H." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" } ], "option_major": [ "biology" ], "abstract": "In order to identify new molecules that might play a role in regional\r\nspecification of the nervous system, we generated and characterized\r\nmonoclonal antibodies (mAbs) that have positionally-restricted labeling\r\npatterns.
\r\n\r\nThe FORSE-1 mAb was generated using a strategy designed to produce\r\nmAbs against neuronal cell surface antigens that might be regulated by\r\nregionally-restricted transcription factors in the developing central nervous\r\nsystem (CNS). FORSE-1 staining is enriched in the forebrain as compared to\r\nthe rest of the CNS until E18. Between E11.5-E13.5, only certain areas of the\r\nforebrain are labeled. There is also a dorsoventrally-restricted region of\r\nlabeling in the hindbrain and spinal cord. The mAb labels a large\r\nproteoglycan-like cell-surface antigen (>200 kD). The labeling pattern of\r\nFORSE-1 is conserved in various mammals and in chick.
\r\n\r\nTo determine whether the FORSE-1 labeling pattern is similar to that\r\nof known transcription factors, the expression of BF-1 and Dlx-2 was\r\ncompared with FORSE-1. There is a striking overlap between BF-1 and\r\nFORSE-1 in the telencephalon. In contrast, FORSE-1 and Dlx-2 have very\r\ndifferent patterns of expression in the forebrain, suggesting that regulation by\r\nDlx-2 alone cannot explain the distribution of FORSE-1. They do, however,\r\nshare some sharp boundaries in the diencephalon. In addition, FORSE-1\r\nidentifies some previously unknown boundaries in the developing forebrain.\r\nThus, FORSE-1 is a new cell surface marker that can be used to subdivide the\r\nembryonic forebrain into regions smaller than previously described,\r\nproviding further complexity necessary for developmental patterning.
\r\n\r\nI also studied the expression of the cell surface protein CD9 in the\r\ndeveloping and adult rat nervous system. CD9 is implicated in intercellular\r\nsignaling and cell adhesion in the hematopoetic system. In the nervous\r\nsystem, CD9 may perform similar functions in early sympathetic ganglia,\r\nchromaffin cells, and motor neurons, all of which express the protein. The\r\npresence of CD9 on the surfaces of Schwann cells and axons at the appropriate\r\ntime may allow the protein to participate in the cellular interactions involved\r\nin myelination.
" }, { "name": "Yang-Snyder, Julia Ann", "degree": "PhD", "year": "1994", "title": "Anatomical and developmental patterns of interleukin-2 gene expression in the mouse: analysis of IL-2 expressing cells and partial characterization of interactions involved in mediating IL-2 gene expression in vivo", "advisor": "Rothenberg, Ellen V.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05212013-133238888", "creators": [ { "name": { "family": "Yang-Snyder", "given": "Julia Ann" }, "id": "Yang-Snyder-J-A", "display_name": "Yang-Snyder, Julia Ann" } ], "advisors": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "advisor", "display_name": "Rothenberg, Ellen V." } ], "committee": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "member", "display_name": "Simon, Melvin I." } ], "option_major": [ "biology" ], "doi": "10.7907/s0hy-ng62", "abstract": "Interleukin-2 (IL-2) is an important mediator in the vertebrate immune system. IL-2 is a\r\npotent growth factor that mature T lymphocytes use as a proliferation signal and the production of\r\nIL-2 is crucial for the clonal expansion of antigen-specific T cells in the primary immune response.\r\nIL-2 driven proliferation is dependent on the interaction of the lymphokine with its cognate\r\nmultichain receptor. IL-2 expression is induced only upon stimulation and transcriptional\r\nactivation of the IL-2 gene relies extensively on the coordinate interaction of numerous inducible\r\nand constitutive trans-acting factors. Over the past several years, thousands of papers have been\r\npublished regarding molecular and cellular aspects of IL-2 gene expression and IL-2 function. The\r\nvast majority of these reports describe work that has been carried out in vitro. However,\r\nconsiderably less is known about control of IL-2 gene expression and IL-2 function in vivo.
\r\n\r\nTo gain new insight into the regulation of IL-2 gene expression in vivo, anatomical and\r\ndevelopmental patterns of IL-2 gene expression in the mouse were established by employing in situ\r\nhybridization and immunohistochemical staining methodologies to tissue sections generated from\r\nnormal mice and mutant animals in which T -cell development was perturbed. Results from these\r\nstudies revealed several interesting aspects of IL-2 gene expression, such as (1) induction of IL-2\r\ngene expression and protein synthesis in the thymus, the primary site of T-cell development in the\r\nbody, (2) cell-type specificity of IL-2 gene expression in vivo, (3) participation of IL-2 in the\r\nextrathymic expansion of mature T cells in particular tissues, independent of an acute immune\r\nresponse to foreign antigen, (4) involvement of IL-2 in maintaining immunologic balance in the\r\nmucosal immune system, and (5) potential function of IL-2 in early events associated with\r\nhematopoiesis.
\r\n\r\nExtensive analysis of IL-2 mRNA accumulation and protein production in the murine\r\nthymus at various stages of development established the existence of two classes of intrathymic\r\nIL-2 producing cells. One class of intrathymic IL-2 producers was found exclusively in the fetal\r\nthymus. Cells belonging to this subset were restricted to the outermost region of the thymus. IL-2\r\nexpression in the fetal thymus was highly transient; a dramatic peak ofiL-2 mRNA accumulation was identified at day 14.5 of gestation and maximal IL-2 protein production was observed 12\r\nhours later, after which both IL-2 mRNA and protein levels rapidly decreased. Significantly, the\r\npresence of IL-2 expressing cells in the day 14-15 fetal thymus was not contingent on the\r\ngeneration of T-cell receptor (TcR) positive cells. The second class of IL-2 producing cells was\r\nalso detectable in the fetal thymus (cells found in this class represented a minority subset of IL-2\r\nproducers in the fetal thymus) but persist in the thymus during later stages of development and\r\nafter birth. Intrathymic IL-2 producers in postnatal animals were located in the subcapsular region\r\nand cortex, indicating that these cells reside in the same areas where immature T cells are\r\nconsigned. The frequency of IL-2 expressing cells in the postnatal thymus was extremely low,\r\nindicating that induction of IL-2 expression and protein synthesis are indicative of a rare activation\r\nevent. Unlike the fetal class of intrathymic IL-2 producers, the presence of IL-2 producing cells in\r\nthe postnatal thymus was dependent on to the generation of TcR+ cells. Subsequent examination\r\nof intrathymic IL-2 production in mutant postnatal mice unable to produce either \u03b1\u03b2 or \u03b3\u03b4 T cells\r\nshowed that postnatal IL-2 producers in the thymus belong to both \u03b1\u03b2 and \u03b3\u03b4 lineages.\r\nAdditionally, further studies indicated that IL-2 synthesis by immature \u03b1\u03b2 -T cells depends on the\r\nexpression of bonafide TcR \u03b1\u03b2-heterodimers. Taken altogether, IL-2 production in the postnatal\r\nthymus relies on the generation of \u03b1\u03b2 or \u03b3\u03b4-TcR^+ cells and induction of IL-2 protein synthesis can\r\nbe linked to an activation event mediated via the TcR.
\r\n\r\nWith regard to tissue specificity of IL-2 gene expression in vivo, analysis of whole body\r\nsections obtained from normal neonatal mouse pups by in situ hybridization demonstrated that IL-2\r\nmRNA^+ cells were found in both lymphoid and nonlymphoid tissues with which T cells are\r\nassociated, such as the thymus (as described above), dermis and gut. Tissues devoid of IL-2\r\nmRNA^+ cells included brain, heart, lung, liver, stomach, spine, spinal cord, kidney, and bladder.\r\nAdditional analysis of isolated tissues taken from older animals revealed that IL-2 expression was\r\nundetectable in bone marrow and in nonactivated spleen and lymph nodes. Thus, it appears that\r\nextrathymic IL-2 expressing cells in nonimmunologically challenged animals are relegated to\r\nparticular epidermal and epithelial tissues in which characterized subsets of T cells reside and thatinduction of IL-2 gene expression associated with these tissues may be a result of T-cell activation therein.
\r\n\r\nBased on the neonatal in situ hybridization results, a detailed investigation into possible\r\ninduction of IL-2 expression resulting in IL-2 protein synthesis in the skin and gut revealed that\r\nIL-2 expression is induced in the epidermis and intestine and IL-2 protein is available to drive cell\r\nproliferation of resident cells and/or participate in immune function in these tissues. Pertaining to\r\nIL-2 expression in the skin, maximal IL-2 mRNA accumulation and protein production were\r\nobserved when resident V\u03b3_3^+ T-cell populations were expanding. At this age, both IL-2 mRNA^+\r\ncells and IL-2 protein production were intimately associated with hair follicles. Likewise, at this\r\nage a significant number of CD3\u03b5^+ cells were also found in association with follicles. The\r\ncolocalization of IL-2 expression and CD3\u03b5^+ cells suggests that IL-2 expression is induced when T\r\ncells are in contact with hair follicles. In contrast, neither IL-2 mRNA nor IL-2 protein were\r\nreadily detected once T-cell density in the skin reached steady-state proportions. At this point, T\r\ncells were no longer found associated with hair follicles but were evenly distributed throughout the\r\nepidermis. In addition, IL-2 expression in the skin was contingent upon the presence of mature T\r\ncells therein and induction of IL-2 protein synthesis in the skin did not depend on the expression of\r\na specific TcR on resident T cells. These newly disclosed properties of IL-2 expression in the skin\r\nindicate that IL-2 may play an additional role in controlling mature T-cell proliferation by\r\nparticipating in the extrathymic expansion of T cells, particularly those associated with the\r\nepidermis.
\r\n\r\nFinally, regarding IL-2 expression and protein synthesis in the gut, IL-2 producing cells\r\nwere found associated with the lamina propria of neonatal animals and gut-associated IL-2\r\nproduction persisted throughout life. In older animals, the frequency of IL-2 producing cells in the\r\nsmall intestine was not identical to that in the large intestine and this difference may reflect regional\r\nspecialization of the mucosal immune system in response to enteric antigen. Similar to other\r\ninstances of IL-2 gene expression in vivo, a failure to generate mature T cells also led to an\r\nabrogation of IL-2 protein production in the gut. The presence of IL-2 producing cells in the\r\nneonatal gut suggested that these cells may be generated during fetal development. Examination of\r\nthe fetal gut to determine the distribution of IL-2 producing cells therein indicated that there was a\r\ntenfold increase in the number of gut-associated IL-2 producers at day 20 of gestation compared to\r\nthat observed four days earlier and there was little difference between the frequency of IL-2\r\nproducing cells in prenatal versus neonatal gut. The origin of these fetally-derived IL-2 producing\r\ncells is unclear. Prior to the immigration of IL-2 inducible cells to the fetal gut and/or induction of\r\nIL-2 expression therein, IL-2 protein was observed in the fetal liver and fetal omentum, as well as\r\nthe fetal thymus. Considering that induction of IL-2 protein synthesis may be an indication of\r\nfuture functional capability, detection of IL-2 producing cells in the fetal liver and fetal omentum\r\nraises the possibility that IL-2 producing cells in the fetal gut may be extrathymic in origin and IL-2\r\nproducing cells in these fetal tissues may not belong solely to the T lineage. Overall, these results\r\nprovide increased understanding of the nature of IL-2 producing cells in the gut and how the\r\nabsence of IL-2 production therein and in fetal hematopoietic tissues can result in the acute\r\npathology observed in IL-2 deficient animals.
\r\n" }, { "name": "Zhu, Jian", "degree": "PhD", "year": "1994", "title": "Indentification and characterization of a novel CA^(2+)-binding protein in avian erythrocytes", "advisor": "Lazarides, Elias", "url": "https://resolver.caltech.edu/CaltechTHESIS:05172013-161307081", "creators": [ { "name": { "family": "Zhu", "given": "Jian" }, "id": "Zhu-Jian", "display_name": "Zhu, Jian" } ], "advisors": [ { "name": { "family": "Lazarides", "given": "Elias" }, "id": "Lazarides-E", "role": "advisor", "display_name": "Lazarides, Elias" } ], "committee": [ { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "role": "member", "display_name": "Dunphy, William G." }, { "name": { "family": "Lipshitz", "given": "Howard D." }, "id": "Lipshitz-H-D", "role": "member", "display_name": "Lipshitz, Howard D." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." } ], "option_major": [ "biology" ], "doi": "10.7907/72bt-qx11", "abstract": "A novel Ca^(2+)-binding protein with Mr of 23 K (designated p23) has been\r\nidentified in avian erythrocytes and thrombocytes. p23 localizes to the marginal bands\r\n(MBs), centrosomes and discrete sites around the nuclear membrane in mature avian\r\nerythrocytes. p23 appears to bind Ca^(2+) directly and its interaction with subcellular\r\norganelles seems to be modulated by intracellular [Ca^(2+)]. However, its unique protein\r\nsequence lacks any known Ca^(2+)-binding motif. Developmental analysis reveals that p23\r\nassociation to its target structures occurs only at very late stages of bone marrow\r\ndefinitive erythropoeisis. In primitive erythroid cells, p23 distributes diffusely in the\r\ncytoplasm and lacks any distinct localization. It is postulated that p23 association to\r\nsubcellular structures may be induced in part by decreased intracellular [Ca^(2+)]. In vitro\r\nand in vivo experiments indicate that p23 does not appear to act as a classical\r\nmicrotubule-associated protein (MAP) but p23 homologues appear to be expressed in\r\nMB-containing cells of a variety of species from different vertebrate classes. It has been\r\nhypothesized that p23 may play a regulatory role in MB stabilization in a Ca^(2+)-dependent\r\nmanner.
\r\n\r\nBinucleated (bnbn) turkey erythrocytes were found to express a truncated p23\r\nvariant (designated p21) with identical subcellular localization as p23 except\r\nimmunostaining reveals the presence of multi-centrosomes in bnbn cells. The p21\r\nsequence has a 62 amino acid deletion at the C-terminus and must therefore have an\r\nadditional ~40 amino acids at the N-terminus. In addition, p21 seems to have lost the\r\nability to bind Ca^(2+) and its supramolecular interactions are not modulated by intracellular\r\n[Ca^(2+)]. These apparent differences between p23 and p21 raised the possibility that the p23/p21 allelism could be the Bn/bn genotype. However, genetic analysis suggested that\r\np23/p21 allelism had no absolute correlation with the Bn/bn genotype.
\r\n" }, { "name": "Adolphs, Ralph", "degree": "PhD", "year": "1993", "title": "Processing of Interaural Level Differences in the Auditory Brainstem of the Barn Owl", "advisor": "Konishi, Masakazu", "url": "https://resolver.caltech.edu/CaltechETD:etd-06172004-110344", "creators": [ { "name": { "family": "Adolphs", "given": "Ralph" }, "id": "Adolphs-Ralph", "orcid": "0000-0002-8053-9692", "display_name": "Adolphs, Ralph" } ], "advisors": [ { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "advisor", "display_name": "Konishi, Masakazu" } ], "committee": [ { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "chair", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "member", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "member", "display_name": "Van Essen, David C." } ], "option_major": [ "neurobio" ], "doi": "10.7907/QN2E-YR81", "abstract": "Nervous systems process information about the environment in order to generate adaptive behavior. Sensory information that is obtained through different modalities, gleaned from different interactions of the animal with its surroundings, generated by different neural algorithms, or used to infer different distal stimulus properties, is often processed by distinct pathways in the brain. The auditory system compares sounds at the two ears in order to derive the location of the source. In the barn owl, Tyto alba, this is accomplished by interaural comparisons of the time that a sound reaches each ear, and of the level (intensity) at each ear. Interaural time differences code for horizontal positions of sound sources while interaural level differences, due to a vertical asymmetry in the owl's ears, can encode the vertical position of a sound. These two cues together can assign unique locations to sound sources in space. The barn owl processes time- and level differences in separate neural channels that converge in the inferior colliculus. This structure is the first site of neurons with spatially restricted auditory receptive fields, and with a neural map of auditory space. Downstream projections from here provide the sensory input for accurate sound localization by saccadic head movements.
\r\n\r\nI report that the owl's two auditory processing streams are also segregated histochemically. The pathway that computes level differences stains especially strongly for the enzyme acetylcholinesterase, which may underlay processing of scalar (intensity) information over large dynamic ranges. This staining is complementary to immunohistochemical staining for calbindin, which has been shown previously to stain the pathway that processes interaural time differences.
\r\n\r\nIn further hodological and physiological experiments, I describe the algorithms that generate tuned responses in the inferior colliculus that encode vertical sound source position. This study shows that a lemniscal nucleus, nucleus ventralis lemnisci lateralis pars posterior (VLVp), projects bilaterally to a subdivision of the inferior colliculus (the shell of ICc). This projection appears to preserve tonotopy, and to provide inhibition by sounds of large interaural level difference. This probably GABAergic mechanism leads to the synthesis of neuronal responses in the inferior colliculus that are narrowly tuned to interaural level difference. My methodological strategy was to increase or decrease activity in VLVp by injection of blockers or agonists of GABA-A receptors, and then to record downstream in the inferior colliculus any changes in response tuning that resulted. The study suggests that the bilateral inhibition by VLVp is sufficient to explain the peaked responses to level differences of collicular neurons. Excitatory input to the inferior colliculus is conveyed by fibers of the lateral lemniscus, and may arise from a number of stations, including lemniscal and cochlear nuclei. The circuits I describe determine the tuning of cells to interaural level differences, but are independent of and have no effect on the tuning to interaural time differences, and further support that time and level are processed separately in the owl's brainstem.
\r\n" }, { "name": "Anderson, Brooke P.", "degree": "PhD", "year": "1993", "title": "Various algorithms for optimization and learning in adaptive systems", "advisor": "Hopfield, John J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04102013-153335605", "creators": [ { "name": { "family": "Anderson", "given": "Brooke P." }, "id": "Anderson-B-P", "display_name": "Anderson, Brooke P." } ], "advisors": [ { "name": { "family": "Hopfield", "given": "John J." }, "id": "Hopfield-J-J", "role": "advisor", "display_name": "Hopfield, John J." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "cns" ], "doi": "10.7907/rnjw-jy11", "abstract": "This thesis discusses various methods for learning and optimization in adaptive\r\nsystems. Overall, it emphasizes the relationship between optimization, learning,\r\nand adaptive systems; and it illustrates the influence of underlying hardware upon\r\nthe construction of efficient algorithms for learning and optimization. Chapter 1\r\nprovides a summary and an overview.
\r\n\r\nChapter 2 discusses a method for using feed-forward neural networks to filter\r\nthe noise out of noise-corrupted signals. The networks use back-propagation learning,\r\nbut they use it in a way that qualifies as unsupervised learning. The networks\r\nadapt based only on the raw input data-there are no external teachers providing\r\ninformation on correct operation during training. The chapter contains an analysis\r\nof the learning and develops a simple expression that, based only on the geometry\r\nof the network, predicts performance.
\r\n\r\nChapter 3 explains a simple model of the piriform cortex, an area in the brain\r\ninvolved in the processing of olfactory information. The model was used to explore\r\nthe possible effect of acetylcholine on learning and on odor classification. According\r\nto the model, the piriform cortex can classify odors better when acetylcholine\r\nis present during learning but not present during recall. This is interesting since it\r\nsuggests that learning and recall might be separate neurochemical modes (corresponding\r\nto whether or not acetylcholine is present). When acetylcholine is turned off at all times, even during learning, the model exhibits behavior somewhat similar\r\nto Alzheimer's disease, a disease associated with the degeneration of cells that\r\ndistribute acetylcholine.
\r\n\r\nChapters 4, 5, and 6 discuss algorithms appropriate for adaptive systems implemented\r\nentirely in analog hardware. The algorithms inject noise into the systems\r\nand correlate the noise with the outputs of the systems. This allows them to\r\nestimate gradients and to implement noisy versions of gradient descent, without\r\nhaving to calculate gradients explicitly. The methods require only noise generators,\r\nadders, multipliers, integrators, and differentiators; and the number of devices\r\nneeded scales linearly with the number of adjustable parameters in the adaptive\r\nsystems. With the exception of one global signal, the algorithms require only local\r\ninformation exchange.
\r\n" }, { "name": "Bernander, Jan \u04e6jvind", "degree": "PhD", "year": "1993", "title": "Synaptic Integration and its Control in Neocortical Pyramidal Cells", "advisor": "Koch, Christof", "url": "https://resolver.caltech.edu/CaltechTHESIS:11282012-105047936", "creators": [ { "name": { "family": "Bernander", "given": "Jan \u04e6jvind" }, "id": "Bernander-Jan-\u04e6jvind", "display_name": "Bernander, Jan \u04e6jvind" } ], "advisors": [ { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "advisor", "display_name": "Koch, Christof" } ], "committee": [ { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "chair", "display_name": "Koch, Christof" }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Barr", "given": "Alan H." }, "id": "Barr-A-H", "role": "member", "display_name": "Barr, Alan H." }, { "name": { "family": "Bower", "given": "James M." }, "id": "Bower-J-M", "role": "member", "display_name": "Bower, James M." }, { "name": { "family": "Laurent", "given": "Gilles J." }, "id": "Laurent-G-J", "orcid": "0000-0002-2296-114X", "role": "member", "display_name": "Laurent, Gilles J." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "member", "display_name": "Van Essen, David C." } ], "option_major": [ "cns" ], "doi": "10.7907/rz58-rh86", "abstract": "The main goal of this thesis is to investigate the input/output relationship of single regular-firing neocortical pyramidal neurons and how this relationship can be controlled by external inputs. The thesis can be divided into three main parts. First, a detailed single cell model was developed, based on the morphology of reconstructed cells and experimental values for membrane conductances and synaptic input distributions. This model was used for the following investigations.
\r\n\r\nSecond, the spatio-temporal integration of single and multiple inputs was studied. Several measures for the efficacy and time delay of single synapses were defined and shown to vary dramatically. For example, a somatic synapse was only 2.2 times stronger than a very distal synapse using the charge attenuation measure, but more than 450 times stronger in the voltage attenuation measure. The effect that temporal synchronicity of multiple inputs had on firing rate was shown to vary with the number of inputs: for just-threshold input rates, synchronicity increased firing rate; for large inputs, high synchronicity strongly reduced firing rate, due to inputs being \"wasted\" during the refractory period.
\r\n\r\nThird, a subset of the inputs were considered to constitute a control signal, and their effect on other inputs was studied for three cases. The first case considers the level of synaptic background activity to be a control signal; since each synapse is a small conductance change, and not a voltage-independent current source, the sum total of all \"background\" synapses will constitute the lion's share of the membrane conductance. The background firing rate, f_b, will therefore determine the electrotonic structure of the cell. For f_b in the range of 0-10 H z, a more than 10-fold decrease was seen in both input resistance (50.4-5.1 M\u03a9) and membrane time constant (33.7-1.6 msec). Electrotonic length and resting potential were similarly affected. The second case treats input to the apical trunk as the control signal; when this input was weak and excitatory, the more distal input to the apical tuft could be facilitated, but when this input was strong or combined with inhibition, more distal input were reduced. The third case involves distributing two types of active conductances throughout the apical dendrites. The activation curves of these conductances were \"designed\" to ensure that the current delivered to the soma was linear in the input rate and amplified, since a passive tree strongly attenuates large apical inputs. The linearization was implemented with a persistent potassium conductance in the superficial layer I- III and the amplification with a persistent calcium conductance in the apical trunk (layer IV). The amplification gain could be set arbitrarily by modulating the channel density of either the potassium or calcium conductance.
" }, { "name": "Bhalla, Upinder Singh", "degree": "PhD", "year": "1993", "title": "Information processing in the mammalian olfactory bulb", "advisor": "Unknown, Unknown", "url": "https://resolver.caltech.edu/CaltechTHESIS:12092009-100507031", "creators": [ { "name": { "family": "Bhalla", "given": "Upinder Singh" }, "id": "Bhalla-U-S", "orcid": "0000-0003-1722-5188", "display_name": "Bhalla, Upinder Singh" } ], "advisors": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/3dq4-ad59", "abstract": "A combination of computer modeling and experimental approaches were taken to studying the mammalian olfactory bulb. First, detailed single cell models were developed for the main cell classes in the olfactory bulb. This involved development of simulation techniques and a parameter search method for assigning unknown parameters for neuronal models. This study demonstrated the feasibility of using indirect information, such as spike waveforms, to determine the detailed electrical properties of neurons. The models demonstrated that spikes propagate into the secondary dendrites, which may play a role in long-range spatial interactions in the bulb. Blockage of this spike propagation might be involved in bulbar information processing. Second, a series of recordings were made from neurons in the olfactory bulb of awake unrestrained rats exposed to a cyclical sequence of odorants. These recordings demonstrated a significant amount of variability in the response of individual neurons over time. The neuronal responses were well described as a combination of a consistent component with a component that varied over time. Comparisons made between response properties of the same neuron at different times, between adjacent neurons, between distant neurons and between unrelated neurons showed a clear sequence of increasing difference in the order: same < adjacent < unrelated < distant. However, during sniff periods, the sequence was: adjacent < same < unrelated < distant. This suggests the bulb normally responds evenly to a wide range of odorants, but during sniffing responses to familiar odorants are suppressed so as to preferentially detect novel odorants. The final stage of the study involved the development of detailed models of the bulb as a whole using both the single cell models, and the experimental results previously obtained. It was found that topographical organization of receptor input according to receptor type was not required to produce the range of responses seen in the experiments. The response variability of neurons in the model was much smaller than in experiment. We propose that the bulb can operate in multiple processing modes so as to optimize its responses for different situations and that this leads to variability in single neuron responses.\r\n" }, { "name": "Blanchard, Alan-Philippe", "degree": "PhD", "year": "1993", "title": "Sequence specific effects on the incorporation of dideoxynucleotides by a modified T7 polymerase", "advisor": "Chandy, K. Mani", "url": "https://resolver.caltech.edu/CaltechTHESIS:11122012-105407501", "creators": [ { "name": { "family": "Blanchard", "given": "Alan-Philippe" }, "id": "Blanchard-Alan-Philippe", "display_name": "Blanchard, Alan-Philippe" } ], "advisors": [ { "name": { "family": "Chandy", "given": "K. Mani" }, "id": "Chandy-K-M", "orcid": "0000-0001-9190-1290", "role": "advisor", "display_name": "Chandy, K. Mani" } ], "committee": [ { "name": { "family": "Abu-Mostafa", "given": "Yaser S." }, "id": "Abu-Mostafa-Y-S", "role": "chair", "display_name": "Abu-Mostafa, Yaser S." }, { "name": { "family": "Mead", "given": "Carver" }, "id": "Mead-C-A", "orcid": "0000-0003-4051-0462", "role": "member", "display_name": "Mead, Carver" }, { "name": { "family": "Hopfield", "given": "John J." }, "id": "Hopfield-J-J", "role": "member", "display_name": "Hopfield, John J." }, { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "member", "display_name": "Hood, Leroy E." }, { "name": { "family": "Chandy", "given": "K. Mani" }, "id": "Chandy-K-M", "orcid": "0000-0001-9190-1290", "role": "member", "display_name": "Chandy, K. Mani" } ], "option_major": [ "cns" ], "doi": "10.7907/9vz4-0e68", "abstract": "While incorporating nucleotides onto the end of a DNA molecule, DNA polymerases selectively discriminate against dideoxynucleotides in favor of incorporating deoxynucleotides. The magnitude of this discrimination is modulated by the template DNA sequence near the incorporation site. This effect has been characterized by analyzing the raw data from a large number of DNA sequencing experiments. It is shown that, for bacteriophage T7 polymerase, the 5 contiguous bases extending from 3 bases 3' (on the template strand) from the incorporation site to 1 base 5' of the incorporation site are the most important in modulating dideoxynucelotide discrimination. A table of discrimination ratios for 1007 different 5-mer contexts is presented.
" }, { "name": "Clark, Steven Manning", "degree": "PhD", "year": "1993", "title": "Advances in scanning force microscopy of biological structures", "advisor": "Revel, Jean-Paul; Baldeschwieler, John D.", "url": "https://resolver.caltech.edu/CaltechTHESIS:11192012-103302776", "creators": [ { "name": { "family": "Clark", "given": "Steven Manning" }, "id": "Clark-Steven-Manning", "display_name": "Clark, Steven Manning" } ], "advisors": [ { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "advisor", "display_name": "Revel, Jean-Paul" }, { "name": { "family": "Baldeschwieler", "given": "John D." }, "id": "Baldeschwieler-J-D", "role": "co-advisor", "display_name": "Baldeschwieler, John D." } ], "committee": [ { "name": { "family": "Baldeschwieler", "given": "John D." }, "id": "Baldeschwieler-J-D", "role": "chair", "display_name": "Baldeschwieler, John D." }, { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "member", "display_name": "Revel, Jean-Paul" }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Rees", "given": "Douglas C." }, "id": "Rees-D-C", "orcid": "0000-0003-4073-1185", "role": "member", "display_name": "Rees, Douglas C." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "member", "display_name": "Hood, Leroy E." } ], "option_major": [ "biology" ], "doi": "10.7907/0vhp-pk35", "abstract": "A multifacted approach to the imaging of biological structures by scanning force microscopy is described. The major problems addressed are the distortion of biological samples by excessive forces applied by the cantilever stylus and sample motion relative to the imaging substrate.
\r\n\r\nThe first two chapters discuss the design of digital signal processor based scanning force microscope control electronics and a novel microscope head that eliminates the application of excessive forces to the sample caused\r\nby electronic or vibrational noise.
\r\n\r\nThe third chapter presents a novel use of chemical vapor deposition for application of heterofunctional alkoxysilanes to scanning force microscopy imaging subsrates. This technique provides imaging substrates which have chemical groups that can be used for sample immobilization without compromising substrate smoothness. The use of the chemically derivatized substrates for scanning force microscopy is also explored.
\r\n\r\nThe final chapter presents high resolution images of bovine liver catalase micro-crystals. The images of the protein micro-crystals show resolution on the order of 2 to 3 nanometers allowing the visualization of individual catalase tetramers. To our knowledge this is the first report of images of protein micro-crystals taken by scanning force microscopy which have resolution comparable to that of electron microscopy.
\r\n" }, { "name": "Cramer, Edith Karina Schimmerling", "degree": "PhD", "year": "1993", "title": "Motor Neuron Projection Patterns and Maturation of Motor Unit Types in the Rabbit Soleus Muscle", "advisor": "Van Essen, David C.", "url": "https://resolver.caltech.edu/CaltechTHESIS:11202012-095703986", "creators": [ { "name": { "family": "Cramer", "given": "Edith Karina Schimmerling" }, "id": "Cramer-Edith-Karina-Schimmerling", "display_name": "Cramer, Edith Karina Schimmerling" } ], "advisors": [ { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "advisor", "display_name": "Van Essen, David C." } ], "committee": [ { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "chair", "display_name": "Van Essen, David C." }, { "name": { "family": "Fraser", "given": "Scott E." }, "id": "Fraser-S-E", "orcid": "0000-0002-5377-0223", "role": "member", "display_name": "Fraser, Scott E." }, { "name": { "family": "Bower", "given": "James M." }, "id": "Bower-J-M", "role": "member", "display_name": "Bower, James M." }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." } ], "option_major": [ "biology" ], "doi": "10.7907/4fwg-dk86", "abstract": "The nervous system contains specific connections reflecting different types of projection patterns. Some of these projections have a topographic arrangement, while others have a diffuse pattern. Projections from spinal cord motor pools to different muscles are topographic. At a finer scale, patterns of projections within several muscles are topographic. These muscles tend to be flat and/or segmented. This thesis presents an investigation of the projection pattern in the innervation of the rabbit soleus muscle, which is compact and has only one tendon of origin and one tendon of insertion. Using intracellular recording of endplate potentials, tension overlap between pairs of ventral root filaments, and retrograde labeling of motor neurons following small injections of horseradish peroxidase into different regions within the muscle, it was shown that the soleus receives diffuse innervation from the spinal cord. It is thus likely that topography is related to muscle function, and that it correlates with spatial\r\nheterogeneity within muscles.
\r\n\r\nAnother type of specificity in connections to muscles is that between fast and slow motor neurons and their corresponding muscle fiber types. These connections form distinct types of motor units. We have investigated the\r\nmaturation of motor unit types during postnatal synapse elimination.
\r\n\r\nThe ratio of motor unit tension at polyinnervated ages to that at singly innervated ages has been used to estimate the degree of polyinnervation for fast versus slow muscle fibers. Twitch and tetanic tension yield conflicting results. This contradiction was resolved using latencies to endplate potentials as an indicator of muscle fiber type. We found that fast and slow muscle fibers are polyinnervated to the same extent during both early and intermediate stages of synapse elimination, implying that specific tension, and not polyinnervation, changes differently in fast versus slow muscle fibers. These changes are consistent with those found in twitch/tetanus ratios. During synapse elimination, the twitch/tetanus ratios for fast motor units increase while those for slow motor units decrease. Furthermore, these intracellular recordings suggest a high degree of specificity at birth, which is further refined during synapse elimination.
\r\n" }, { "name": "Ding, Dali", "degree": "PhD", "year": "1993", "title": "Localization of Maternal RNAs in the Early Embryo of Drosophila", "advisor": "Lipshitz, Howard D.", "url": "https://resolver.caltech.edu/CaltechTHESIS:11282012-105220091", "creators": [ { "name": { "family": "Ding", "given": "Dali" }, "id": "Ding-Dali", "display_name": "Ding, Dali" } ], "advisors": [ { "name": { "family": "Lipshitz", "given": "Howard D." }, "id": "Lipshitz-H-D", "role": "advisor", "display_name": "Lipshitz, Howard D." } ], "committee": [ { "name": { "family": "Lipshitz", "given": "Howard D." }, "id": "Lipshitz-H-D", "role": "chair", "display_name": "Lipshitz, Howard D." }, { "name": { "family": "Abelson", "given": "John N." }, "id": "Abelson-J-N", "role": "member", "display_name": "Abelson, John N." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" } ], "option_major": [ "biology" ], "doi": "10.7907/VH4J-8J21", "abstract": "Maternally synthesized localized determinants play an important role in cell fate specification in early Drosophila embryos. Since only a third of the genes in Drosophila have been identified genetically, we carried out a systematic screen for polar localized maternal RNAs in the early embryo as a means of identifying novel molecules that might serve important developmental functions. Anterior-and posterior-specific directional cDNA libraries were constructed using RNA purified from anterior-or posterior-poles cut off early embryos. These libraries were used in a differential screen for cDNAs representing polar-localized maternal RNAs. Five such clones were identified, encoding cyclin B RNA, Hsp83 RNA, 28S rRNA, mitochondrial COl RNA and mitochondria/16S lrRNA (16S RNA). Maternal Hsp83 transcripts are localized to the posterior pole of the early embryo by a novel mechanism involving generalized RNA degradation and local protection at the posterior. This protection is dependent on the integrity of the polar plasm, suggesting that Hsp83 RNA is a component of the polar plasm. Results from antisense oligodeoxynucleotide injection experiments suggest that Hsp83 is required for the formation/maintenance of germ cells. The 16S RNA is highly concentrated at the posterior pole of newly fertilized Drosophila eggs, a process dependent on the integrity of the polar plasm. The localization pattern of 16S RNA does not correlate well with either the distribution or the activity of mitochondria in early embryos. Taken together with previously published data, these results suggest that the 16S RNA is exported from the mitochondria into the posterior polar plasm and that it functions in pole cell formation. In addition to the localized RNAs identified in the differential screen, a novel anteriorly localized RNA was identified. This mRNA encodes a Drosophila homolog of mammalian adducin, a membrane-cytoskeletal protein that functions in the assembly of the spectrin-actin network. A comparison of the spatial distribution of bicoid and Adducin-like transcripts in maternal effect RNA-localization mutants indicates that different genetic pathways exist for localization of mRNAs to the anterior pole of the oocyte and early embryo.
\r\n" }, { "name": "Garrity, Paul Allen", "degree": "PhD", "year": "1993", "title": "The in vivo examination of transcriptional control mechanisms in mammalian cells", "advisor": "Wold, Barbara J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:12112012-085623512", "creators": [ { "name": { "family": "Garrity", "given": "Paul Allen" }, "id": "Garrity-P-A", "display_name": "Garrity, Paul Allen" } ], "advisors": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "advisor", "display_name": "Wold, Barbara J." } ], "committee": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" } ], "option_major": [ "biology" ], "doi": "10.7907/kt02-n508", "abstract": "In the investigations described in this thesis I have examined various\r\nmechanisms involved in transcriptional control. The first chapter is a general\r\nexamination of the mechanisms used in transcriptional control. The second\r\nchapter addresses issues associated with the selection of transcriptional start\r\nsite. This work shows that core promoter usage can be altered in a tissue specific\r\nand environmentally-responsive manner. The third chapter of this\r\nwork describes a significant improvement in the technique of ligation mediated\r\nPCR-aided in vivo footprinting and genomic sequencing. This\r\nimprovement in the quality of in vivo footprint data allows the pattern of\r\nprotein : DNA interactions to be obtained with greater signal to noise and for a\r\nlarger group of DNA sequences. The fourth chapter of this thesis uses these in\r\nvivo footprinting techniques to investigate the mechanisms controlling the\r\ntranscription of the mouse interleukin-2 gene upon T cell stimulation. T cell\r\nstimulation was shown to result in the coordinated occupancy of a number of\r\nmajor groove binding proteins to the previously unoccupied IL-2 regulatory\r\nregion in vivo. Finally, the appendix describes in vivo footprints at the\r\ndifferentiated-muscle-specific promoter of the delta-subunit of the nicotinic\r\nacetylcholine receptor. Multiple protein : DNA interactions were seen at the\r\npromoter both before and after muscle cell differentiation, suggesting that\r\ntranscriptional regulation of this gene occurs at the level of protein\r\nreplacement or an alteration of the ability of the assembled proteins to activate\r\ntranscription." }, { "name": "Hamilton, Bruce A.", "degree": "PhD", "year": "1993", "title": "Assessing molecular function in the Drosophila nervous system: a reverse genetic approach", "advisor": "Meyerowitz, Elliot M.; Zinn, Kai George", "url": "https://resolver.caltech.edu/CaltechTHESIS:12072012-092856340", "creators": [ { "name": { "family": "Hamilton", "given": "Bruce A." }, "id": "Hamilton-B-A", "display_name": "Hamilton, Bruce A." } ], "advisors": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "advisor", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "advisor", "display_name": "Zinn, Kai George" } ], "committee": [ { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "chair", "display_name": "Zinn, Kai George" }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "orcid": "0000-0002-5408-6781", "role": "member", "display_name": "Emr, Scott D." }, { "name": { "family": "Lipshitz", "given": "Howard D." }, "id": "Lipshitz-H-D", "orcid": "0000-0002-7372-4419", "role": "member", "display_name": "Lipshitz, Howard D." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "display_name": "Meyerowitz, Elliot M." } ], "option_major": [ "biology" ], "doi": "10.7907/aqes-7j89", "abstract": "I describe the application of new methods for reverse genetic analyses in Drosophila melanogaster to genes expressed in the nervous system. The methods can be devided into two classes: tools for molecular analysis and tools for genetic analysis.
\r\n\r\nThe first set of methods is designed to facilitate rapid, large-scale molecular analysis of cDNA clones. This set of tools begins with a family of bacteriophage \u03bb cDNA cloning vectors and E. coli host cell strains that allow automatic plasmid subcloning by in vivo site-specific recombination. A high density filter hybridization and diagnostic PCR assay technique applied to size-selected libraries then substantially simplifies the isolation of full-length cDNA clones in these vectors. Next, a transposon \u03b3\u03b4-facilitated DNA sequencing procedure minimizes the labor required to isolate a nested set of initiation sites for chain termination sequencing of each strand of a cloned DNA segment. I also describe the characterization of 250 cDNA clones isolated from adult heads on the basis of gross expression patterns in the embryonic ventral nerve cord and larval fat body.
\r\n\r\nThe second set of procedures facilitates the isolation of mutations in the chromosomal genes that correspond to isolated cDNA clones. I describe three such experiments. A plasmid rescue and hybridization strategy allowed the isolation of PlacW elements inserted adjacent to 4 cloned genes in an array of nearly 700. Modifications of this procedure to take advantage of P-element local transposition allowed the isolation and characterization of an apparent null mutation in the receptor-linked protein tyrosine phosphatase gene DPTP99A. In a pilot study, I demonstrate the feasibility of isolating chemically induced mutations that remove targeted restriction enzyme cleavage sites with a PCR-based assay. In addition, I describe the serendipitous isolation of a PlacW-induced mutation, encumbered, that affects the morphogenesis of imaginal wing discs as well as adult longevity, activity, and fertility.
\r\n" }, { "name": "Hsu, Hsiaolan S.", "degree": "PhD", "year": "1993", "title": "Properties of the first genetically engineered neuron.", "advisor": "Lester, Henry A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:12062012-091333466", "creators": [ { "name": { "family": "Hsu", "given": "Hsiaolan S." }, "id": "Hsu-Hsiaolan-S", "display_name": "Hsu, Hsiaolan S." } ], "advisors": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "advisor", "display_name": "Lester, Henry A." } ], "committee": [ { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Yang", "given": "Changhuei" }, "id": "Yang-Changhuei", "role": "member", "display_name": "Yang, Changhuei" } ], "option_major": [ "biology" ], "doi": "10.7907/svss-ye57", "abstract": "Electrically excitable channels were expressed in Chinese hamster ovary cells using a vaccinia virus vector system. In cells expressing rat brain IIA Na^+ channels, brief pulses (< 1ms) of depolarizing current resulted in action potentials with a prolonged (0.5-3s) depolarizing plateau; this plateau was caused by slow and incomplete Na^+ channel inactivation. In cells expressing both Na^+ and Drosophila Shaker H4 transient K^+ channels, there were neuron-like action potentials. In cells with appropriate Na^+/K^+ current ratios, maintained stimulation produced repetitive firing over a 10-fold range of frequencies but eventually led to \"lockup\" of the potential at a positive value after several seconds of stimulation; the latter effect was due primarily to slow inactivation of the K^+ currents. Numerical simulations of modified Hodgkin-Huxley equations describing these currents, using parameters from voltage-clamp kinetics studied in the same cells, accounted for most features of the voltage trajectories. The present study shows that insights into the mechanisms for generating action potentials and trains of action potentials in real excitable cells can be obtained from the analysis of synthetic excitable cells that express a controlled repertoire of ion channels. This model system provides a direct control of complexity of neuronal behavior, and a tool for studying various forms of neural modulation at molecular and cellular levels.
" }, { "name": "Hunkapiller, Tim", "degree": "PhD", "year": "1993", "title": "Diversity and Evolution of the Immunoglobulin Gene Superfamily", "advisor": "Hood, Leroy E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:12182012-093217041", "creators": [ { "name": { "family": "Hunkapiller", "given": "Tim" }, "id": "Hunkapiller-Tim", "display_name": "Hunkapiller, Tim" } ], "advisors": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "advisor", "display_name": "Hood, Leroy E." } ], "committee": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "chair", "display_name": "Hood, Leroy E." }, { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "member", "display_name": "Strauss, James H." }, { "name": { "family": "Brokaw", "given": "Charles J." }, "id": "Brokaw-C-J", "role": "member", "display_name": "Brokaw, Charles J." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." } ], "option_major": [ "biology" ], "doi": "10.7907/mcbv-n026", "abstract": "The Immunoglobulin Gene Superfamily is characterized by a common protein\r\nhomology unit that is present in arguably the largest and most diverse set of genes and\r\ngene families of any protein motif. This distribution indicates that the homology unit is\r\na remarkably versatile functional unit. Its central role in defining the complex\r\nphenotypes of the immune and nervous systems, likewise, is testament to the ability of\r\nthe motif to support an amazing and unique degree of diversification. Understanding\r\nmore about the function, structure and evolution of the Immunoglobulin Gene\r\nSuperfamily can provide insights into both the general issues of complex system\r\nevolution as well as the specific nature of the various systems the superfamily plays a\r\ncentral role in. This thesis is a collection of work aimed at a more thorough\r\nunderstanding of these elements. Particularly, these works summarize much of our\r\ncurrent understanding of the members of the Immunoglobulin Gene Superfamily along\r\nwith speculations on their evolutionary history as well as both the evolutionary and\r\nsomatic mechanisms responsible for their diversity. This work includes initial\r\ndescriptions of several features relevant to somatic diversification of rearranging\r\nimmune receptors, including: l) the role of joining imprecision in the generation of\r\njunctional diversity in immunoglobulin kappa chain; 2) the initial description of the T-cell\r\nbeta chain J/C locus; 3) the translation of T-cell beta chain D gene segments in all\r\nthree reading frames; 4) the occurrence of a cryptic rearrangement signal in most\r\nrearranging V families; 5) the first description of the mechanisms of class switching\r\nbetween heavy chain mu and delta genes; 6) the limited diversity of germline T-cell\r\nbeta chains; 7) the shared complementary determining region structure of T-cell beta\r\nchains and immunoglobulin heavy chains. Also, from these efforts, new members of\r\nthe superfamily have been identified including MHC class I molecules, L3T4 and\r\nMyelin Associated Glycoprotein. Various observations concerning the evolutionary\r\nrelationships of these molecules and motifs have been made. Particularly, a variation\r\non the basic homology unit motif has been proposed that probably more nearly\r\nrepresents the primordial sequence and function.
\r\n\r\nAs a result of these discoveries, a new, comprehensive picture of the\r\nimmunoglobulin superfamily is emerging that has implications for interpreting current\r\nfunctional relationships in the context of the evolutionary history of the members.\r\nParticularly, it is suggested from this work that the ability of the homology unit to\r\naccommodate diversity has made possible the evolution of the superfamily. Given the\r\ntremendous diversity within the superfamily, it might be assumed that selective\r\npressures favoring diversity have driven its evolution. However, much of the analysis\r\nwithin this collection suggests that, on the contrary, diversity is an inherent feature of\r\nthe conserved protein and gene structure of the homology unit and that it was the a\r\npriori diversity itself that drove and shaped the evolution of the complex systems that\r\nemploy the homology unit today. This basic diversity is the consequence of three\r\ncharacteristics of the homology unit. First, the tertiary structure of the protein motif is\r\nsuch that homology units tend to interact preferentially to form homo- or heterodimers,\r\nforming the basis of many of the receptors and the receptor/ligand interactions common\r\nwithin the superfamily. These combinatorial associations increase both the somatic and\r\nevolutionary potential for diversification. This can lead to the rather sudden\r\nappearance of new functional associations between existing members of the superfamily\r\npreadapted for otherwise unrelated functions. Second, except for a minimal number of\r\namino acid residues involved in critical intra- and interchain interactions, the primary\r\nstructure of these units can vary dramatically and still provide for essentially the same\r\ntertiary structure. This has been borne out by various crystallographic studies. The\r\nvariability is particularly true of the loop structures normally identified with antigen\r\nspecificity, but seen in other extended families as well. Reduced constraints on\r\nstructural sequences inherently promote the establishment of variation within\r\npopulations. Third, with very few exceptions the genes of the superfamily, the\r\nhomology units are not only encoded by discrete exons, but these exons have a shared\r\n1/2 splicing rule. That is, each is begun with the second 2 bases of a codon and ended\r\nwith the first base. This allows the in-frame splicing of any number of tandem\r\nhomology units, while maintaining functional protein domains. This rule generally\r\napplies to the non-homology unit exons of member genes as well. This allows, through\r\nrelatively simple genetic events, the development of new contexts for homology unit\r\nexpression, both by simple expansion and contraction of homology unit number and\r\nexon shuffling. This is probably at work, as well, in the frequent occurrence and\r\nutilization of alternative transcripts seen throughout the superfamily. Many of the\r\nrecognized occurrences of alternative splicing, such as that between membrane-bound\r\nand secreted forms, indicate that this gene structure provides for a further level of\r\nfunctional diversity and the expansion of the virtual genetic information.
\r\n\r\nBeyond the explicit discussion of the superfamily members, this work also\r\nspeaks to various issues of evolution in general. In particular, the history of the\r\nsuperfamily suggests the importance of canalization and non-gradual episodes of\r\nevolutionary change. It can contribute, as well, to the discussion of adaptive versus\r\nneutral change.
" }, { "name": "Jongeward, Gregg D.", "degree": "PhD", "year": "1993", "title": "Negative regulators of the let-23 EGF receptor in Caenorhabditis elegans vulval differentiation", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:12132012-153519784", "creators": [ { "name": { "family": "Jongeward", "given": "Gregg D." }, "id": "Jongeward-G-D", "display_name": "Jongeward, Gregg D." } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "chair", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Lipshitz", "given": "Howard D." }, "id": "Lipshitz-H-D", "orcid": "0000-0002-7372-4419", "role": "member", "display_name": "Lipshitz, Howard D." }, { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "member", "display_name": "Lewis, Edward B." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "orcid": "0000-0002-5408-6781", "role": "member", "display_name": "Emr, Scott D." } ], "option_major": [ "biology" ], "doi": "10.7907/87ea-tr80", "abstract": "Vulval induction in C. elegans is an example of the use of an EGF\r\n(Epidermal Growth Factor) mediated signal transduction system. At least\r\nfive genes are involved in the negative regulation of vulval induction.
\r\n\r\nMutations at the silent locus sli-1 (suppressor of lineage defect) are\r\nsufficient to suppress all of the phenotypes associated with hypomorphic\r\nalleles of let-23. sli-1 functions to modify the activity of let-23 but muations\r\nat sli-1 do not bypass the requirement for let-23. Based on the phenotypes of\r\nanimals bearing mutations at sli-1 and other genes, sli-1 may function at or\r\nnear the let-23 or sem-5 step of vulval differentiation.
\r\n\r\nNull alleles of the pleiotropic locus unc-101 cause a number of mutant\r\nphenotypes including neural defects and suppression of the vulval defects\r\nassociated with some weak let-23 mutations. These unc-101 mutations\r\ninteract with mutations in other genes required for proper vulval\r\ndifferentiation but do not act as generalized suppressors. This locus has been\r\ncloned and encodes the C. elegans homolog of the Golgi-associated clathrin\r\nadaptor protein AP47.
\r\n\r\nAnimals mutant for both unc-101 and sli-1 display excessive vulval\r\ndifferentiation. Animals mutant at only one of these loci display no vulval\r\nabnormalities. This excessive vulval differentiation requires the inductive\r\nsignal and functionallet-23, suggesting that sli-1 and unc-101 function to\r\nnegatively regulate the response to the inductive signal, rather than the\r\nbasal activity of let-23.
\r\n\r\nRare mutant alleles at lin-2, lin-7, and let-23 result in excessive vulval\r\ndifferentiation. These alleles are genetically similar to more common alleles\r\nof these genes which result in the failure to differentiate vulval tissue. These\r\nthree genes apparently are required for the activation of both positive and negative regulators of vulval differentiation.
\r\n\r\nA number of negative regulators function to control the activity of let-23.\r\nAt least three pathaways of negative regulation have been genetically\r\nidentified. These negative regulators act to limit the response to a growth or\r\ndifferentiation factor.
\r\n" }, { "name": "Komatsoulis, George A.", "degree": "PhD", "year": "1993", "title": "Recognition of tRNA^(Cys) by the E. coli cysteinyl-tRNA synthetase: in vivo and in vitro studies", "advisor": "Abelson, John N.", "url": "https://resolver.caltech.edu/CaltechTHESIS:12062012-143754549", "creators": [ { "name": { "family": "Komatsoulis", "given": "George A." }, "id": "Komatsoulis-G-A", "display_name": "Komatsoulis, George A." } ], "advisors": [ { "name": { "family": "Abelson", "given": "John N." }, "id": "Abelson-J-N", "role": "advisor", "display_name": "Abelson, John N." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/c5zd-j733", "abstract": "A study of the recognition of tRNA^(Cys) by E. coli cysteinyl-tRNA synthetase using in vivo and in vitro methods was performed. All three anticodon nucleotides, the discriminator U73, and some element(s) within the\r\ntertiary domain (the D stem/loop, the T\u03a8C stem/loop and extra loop) are important for recognition; the anticodon stem and acceptor stem appear to contain no essential elements. A T7 RNA polymerase transcribed tRNA^(Cys) is a\r\n5.5-fold worse substrate than native tRNA^(Cys)(in terms of the selectivity constant, k_cat/K_m) mainly due to an increase in K_m. This may reflect recognition of modified nucleotides or subtle effects on the folding of the tRNA. The greatest loss of specificity caused by mutation of a single nucleotide occurs when the discriminator U73 is changed; k_cat/K_m declines 3 to 4 orders of magnitude\r\ndepending on the substitution. Mutations in the wobble nucleotide of the anticodon also cause reductions in the selectivity constant of 3 orders of magnitude, while mutations in the other anticodon nucleotides caused lesser\r\neffects. Interestingly, a C35A mutation had no effect on aminoacylation by the cysteinyl-tRNA synthetase. Several amber suppressor tRNAs were constructed whose in vivo identity did not correlate with their in vitro specificity, indicating the need for both types of experiments to understand the factor(s) which maintain tRNA specificity. Future in vitro experiments will attempt to explain the\r\nin vivo discrimination between the glycine, phenylalanine, and cysteine tRNAs by the cysteinyl-tRNA synthetase. Finally, these results suggest that the notion that a small set of isoacceptor specific elements define tRNA identity (the socalled \"second genetic code\") is incorrect. A better model is based on competition between synthetases for tRNA substrates which contain differing amounts of partially overlapping identity determinants.\r\n" }, { "name": "Meier, Joseph Thomas", "degree": "PhD", "year": "1993", "title": "A Biological Arms Race: Site Specific DNA Recombination in Competing Immunofunctional Proteins", "advisor": "Hood, Leroy E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:12262012-130125197", "creators": [ { "name": { "family": "Meier", "given": "Joseph Thomas" }, "id": "Meier-Joseph-Thomas", "orcid": "0009-0005-2996-9768", "display_name": "Meier, Joseph Thomas" } ], "advisors": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "advisor", "display_name": "Hood, Leroy E." } ], "committee": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "chair", "display_name": "Hood, Leroy E." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Barbour", "given": "Alan G." }, "id": "Barbour-A-G", "orcid": "0000-0002-0719-5248", "role": "member", "display_name": "Barbour, Alan G." } ], "option_major": [ "biology" ], "doi": "10.7907/fd8q-cb34", "abstract": "This thesis is a compilation of inquiries into the molecular biology of two disparate\r\norganisms, each using site-specific recombination to generate diversity in and regulate the\r\nproduction of a protein. Coincidentally, each of these proteins functions in the context of the\r\nvertebrate immune system: one is the major defensive weapon of a eubacterial pathogen, and\r\nthe other the sine qua non of the system designed to recognize and destroy infectious agents, the\r\nantibody. The first section describes a series of experiments designed to explore the molecular\r\nbasis for antigenic variation in Borrelia hermsii, the eubacterial agent responsible for relapsing\r\nfever. A serotype 7 vmp gene fragment was cloned using mixed sequence oligonucleotide\r\nprobes derived from the sequencing of CNBr peptides from VMP 7. Use of this fragment in\r\nnorthern and southern blot experiments demonstrated that B. hermsii DNA sequences duplicate\r\nand rearrange, and that these duplications correlate with differential expression of VMPs (in a\r\npattern remarkably reminiscent of the trypanosomes). These striking results formed the basis\r\nfor several subsequent studies, which are also discussed. The final section details two separate\r\nprojects involving V(D)J recombination. Inital effort was directed at producing a non-lymphoid\r\ncell line capable of performing V(D)J recombination. Our strategy was based upon the ability of\r\nretroviruses to transcriptionally activate genes distant from the site of integration. Due to\r\nreports of the cloning of RAG-1 and -2, the project was discontinued, but not before producing\r\none line with an interesting phenotype. Following largely anecdotal reports of a previously\r\nunnoticed pattern of base addition during V(D)J recombination, we decided to perform a\r\nrigorous examination of the hypothesis, using both experiment and a detailed examination of\r\npublished data. While we were able to confirm the existence of palindromic, non-templated\r\nbases, our results contradicted other reports with regard to the origins and characteristics of\r\nthese inserts. Some surprises arose, most notably in the influence primary DNA sequence has\r\non the spectrum of product molecules; this adds a new dimension to a process previously\r\nthought to be well understood. 1his work represents the most thorough study of P nucleotide\r\naddition to date." }, { "name": "Stemple, Derek Lyle", "degree": "PhD", "year": "1993", "title": "Isolation of a mammalian neural crest stem cell and environmental control of cell fate choices", "advisor": "Anderson, David J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01082013-104637928", "creators": [ { "name": { "family": "Stemple", "given": "Derek Lyle" }, "id": "Stemple-D-L", "display_name": "Stemple, Derek Lyle" } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "advisor", "display_name": "Anderson, David J." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/g1cd-aj32", "abstract": "One question central to the study of developmental biology is: How does\r\nphenotypic diversity arise? The neural crest provides an excellent model system for\r\ninvestigations into the nature of cell-fate decisions and generation of lineage diversity.\r\nIn the studies described here, I have examined two aspects of this question in cell\r\nculture. The first aspect concerns the cellular dynamics underlying the cell-fate\r\ndecisions. A necessary prerequisite to the study lineage diversification is the reliable\r\nproduction of differentiated cells from undifferentiated precursors in a controllable\r\nenvironment. I have developed a system for the growth of rat neural crest cells. The\r\nsystem allows the serial propagation of a defined sub-population of neural crest cells at\r\nclonal density. In the system neural crest cells can differentiate into at least two\r\nidentifiable cell types; peripheral neurons and Schwann cells. By sub-cloning I have\r\nbeen able to address specific predictions made by a stem cell model of neural crest\r\ndevelopment, and found neural crest cells to possess multipotency, self-renewal and the\r\ncapacity to divide asymmetrically.
\r\n\r\nThe second aspect of the question addressed in this study concerns the ability of\r\nthe neural crest cell environment to control the choice of cell fate. I have examined\r\nvarious culture conditions for their ability to affect the choice of cell fate both in clonal\r\ncultures and in mass cultures. In the clonal cultures I found that the fate of neural crest\r\ncell clones can be altered in an instructive fashion by the composition of the substrate,\r\nor by the presence of fetal bovine serum. In mass cultures, we have examined the\r\neffect of medium composition on the expression of adrenergic traits and on the\r\nexpression of a transcription factor, MASH-1, thought to participate in neural\r\ndetermination. Finally, in mass cultures of neo-natal adrenal chromaffin cells, we have\r\nexamined the effects of basic fibroblast growth factor on neuronal differentiation,\r\nmitosis and acquisition of trophic dependence.
\r\n" }, { "name": "Wang, Yukang", "degree": "PhD", "year": "1993", "title": "Transcriptional Regulation of T Cell Receptor Genes by a Novel CACCC Box Binding Protein", "advisor": "Hood, Leroy E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01102013-132313493", "creators": [ { "name": { "family": "Wang", "given": "Yukang" }, "id": "Wang-Yukang", "display_name": "Wang, Yukang" } ], "advisors": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "advisor", "display_name": "Hood, Leroy E." } ], "committee": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "chair", "display_name": "Hood, Leroy E." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "orcid": "0000-0002-6706-5605", "role": "member", "display_name": "Zinn, Kai George" }, { "name": { "family": "Parker", "given": "Carl Stevens" }, "id": "Parker-C-S", "orcid": "0000-0001-9795-4211", "role": "member", "display_name": "Parker, Carl Stevens" }, { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "member", "display_name": "Strauss, James H." } ], "option_major": [ "immun" ], "doi": "10.7907/x9cm-6a16", "abstract": "The vertebrate immune response consists of humoral and cellular immune\r\nreactions, which are mediated mainly by immunoglobulins (Ig) and Tcell receptors (TCR)\r\nrespectively. The organization of Ig and TCR genes has been well established. Each of\r\nthe lg and TCR genes consists of multiple germ line gene segments that rearrange during\r\nlymphocyte development to generate diverse receptor structures expressed on mature B\r\nand T cells. The transcriptional regulation of Ig genes has been well studied. The\r\noctamer motif in the lg gene promoter or enhancer, the E-box and the KB site have been\r\nfunctionally characterized. The regulation of transcription factors that bind to these\r\nsites is well understood. The transcriptional regulation of TCR genes is not as well\r\nstudied as that of lg genes. TCR-\u03b1, -\u03b2, -\u03b3, -\u03b4 gene enhancers and a TCR \u03b1 gene\r\nsilencer have been reported. Some of the transcription factors that bind to these ciselements\r\nhave been cloned. A T cell-specific transcription factor, GATA-3, may play\r\nan important regulatory role on the expression of TCR genes in T cells. The promoters\r\nof TCR genes also have been investigated, however, the transcription factors that\r\ninteract with them have not been characterized. The aim of this thesis was to isolate\r\nand characterize transcription factors that function in TCR gene transcription.
\r\n\r\nA eDNA clone ht\u03b2, encoding a zinc finger protein that binds to the promoter\r\nregion of the human TCR gene V\u03b28.1, was cloned from a human peripheral blood T cell\r\nlibrary. The region of this protein containing four zinc fingers of the class Cys_2-X_(12)-\r\nHis_2 may be responsible for DNA binding to the TCR V\u03b28.1 promoter sequence\r\nGAAGTTGGGGGTGGTG. A putative transcriptional activation domain that is highly\r\nnegatively charged has also been found in ht\u03b2. Analysis of expression of ht\u03b2 mRNA\r\nreveales similar expression levels in Hela cells, Jurkat T cells, Ramos B cells and U -937\r\nmonocyte line. In addition to binding to the human TCR V\u03b28.1 promoter, ht\u03b2 also can\r\nbind to the mouse TCR gene \u03b1 silencer. The comparison of ht\u03b2 binding sites between\r\nthe human TCR V\u03b28.1 promoter and the mouse TCR gene \u03b1 silencer reveals a core\r\nsequence of the CACCC box. Gel-shift assay analysis of five repeats of the CACCC\r\nbox with bacterially expressed ht\u03b2 protein indicates that ht\u03b2 can bind to the CACCC\r\nbox. Gel-shift assays of the CACCC box with nuclear extracts from various cell lines\r\nreveal four common bands in T cell, B cell, monocyte and Hela cell lines, and one extra\r\nband in Hela cell extracts. CAT assay analysis indicates the CACCC box is essential for\r\nefficient transcription of the V\u03b28.l promoter. Cotransfection with a ht\u03b2 expression\r\nplasmid and a reporter plasmid show that ht\u03b2 can activate human TCR V\u03b28.1 gene\r\ntranscription. Ht\u03b2 also is able to counteract the silencing effect of the TCR \u03b1 silencer.\r\nHtl3 may have an interaction with the cAMP response element binding protein (CREB)\r\nto negatively regulate human V\u03b28.1 gene transcription in Hela cells, and that negative\r\neffect is not significant in Jurkat T cells. The CACCC box has been found in almost all\r\nV\u03b28 subfamily members (4 of 5 V\u03b28 members in human, and 2 of 3 V\u03b28 members in\r\nmouse), and both TCR \u03b1 and \u03b2 enhancers in human and mouse. These results suggest\r\nthat the CACCC box binding protein may have an important function in the immune\r\nsystem.
\r\n\r\nA murine zinc finger protein (M-zif) has been isolated and characterized. It has\r\nfour fingers in the zinc finger domain, the putative DNA binding domain. Also, a\r\nglutamine-rich region was found, which may be involved in transcriptional activation.\r\nA previously reported eDNA molecule was shown to contain in opposite orientatioins the\r\ncoding regions of both the interleukin-2 receptor \u03b1 (IL-2R\u03b1.) and M-zif genes. The\r\nresults presented here indicate that the cDNA is a chimeric molecule resulting from\r\ncloning artifact. The zinc finger domain of M-zif is highly homologous to that of ht\u03b2, a\r\nhuman T cell receptor V\u03b28.1 promoter binding protein. They may have a similar DNA\r\nbinding site. M-zif is not the mouse equivalent of ht\u03b2.
" }, { "name": "Westaway, Shawn Kathleen", "degree": "PhD", "year": "1993", "title": "Structure and function of yeast tRNA ligase", "advisor": "Abelson, John N.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01102013-143442193", "creators": [ { "name": { "family": "Westaway", "given": "Shawn Kathleen" }, "id": "Westaway-S-K", "display_name": "Westaway, Shawn Kathleen" } ], "advisors": [ { "name": { "family": "Abelson", "given": "John N." }, "id": "Abelson-J-N", "role": "advisor", "display_name": "Abelson, John N." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/fasc-j790", "abstract": "The gene for yeast tRNA ligase has been sequenced and its transcription start sites have been mapped. Three other open reading frames in the vicinity of the tRNA ligase\r\ngene were characterized. One open reading frame, ORF4, is the yeast ARG3 gene. ORF1 is probably not transcribed or translated in yeast. ORF2 is an unidentified but essential\r\ngene in yeast.
\r\n\r\nA deletion of the central 200 amino acids has been engineered in the ligase protein. This deletion protein, designated DAC, was characterized in the in vitro tRNA splicing reaction with regard to the structure of the joined tRNA product. Cofactor requirements for tRNA joining activity and polynucleotide kinase activity were also determined. DAC possesses a GTP-dependent joining activity that is not manifested by wild-type ligase. In addition, both the wild-type and DAC proteins exhibit polynucleotide kinase activities that are more efficient with GTP than with ATP. Joining reactions with wild-type ligase indicate that joining of tRNA halves is more efficient in the presence of both GTP and ATP than with either cofactor alone. Wild-type tRNA ligase can incorporate they-phosphate of GTP into the splice junction of joined tRNA, but only when ATP is also provided. The ligase protein contains two distinct nucleotide triphosphate binding sites- one specific for GTP and one specific for ATP. A revised mechanism for tRNA splicing in yeast is presented.
\r\n" }, { "name": "Aroian, Raffi V", "degree": "PhD", "year": "1992", "title": "The let-23 gene of the nematode C. elegans : genetics and molecular biology of a member of the EGF receptor tyrosine kinase family", "advisor": "Sternberg, Paul W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:09142011-114200593", "creators": [ { "name": { "family": "Aroian", "given": "Raffi V" }, "id": "Aroian-Raffi-V", "display_name": "Aroian, Raffi V" } ], "advisors": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "advisor", "display_name": "Sternberg, Paul W." } ], "committee": [ { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "member", "display_name": "Benzer, Seymour" }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "orcid": "0000-0002-5408-6781", "role": "member", "display_name": "Emr, Scott D." }, { "name": { "family": "Lipshitz", "given": "Howard D." }, "id": "Lipshitz-H-D", "orcid": "0000-0002-7372-4419", "role": "member", "display_name": "Lipshitz, Howard D." } ], "option_major": [ "biology" ], "doi": "10.7907/q10c-b807", "abstract": "Genetic studies indicate that the let-23 gene affects several developmental decisions in the nematode\r\nCaenorhabditis elegans. let-23 is required for the proper development of the hermaphrodite vulva, the male tail, and the posterior ectoderm. In addition, let-23 mutations can cause lethality and hermaphrodite sterility. These five let-23 functions can be independently mutated, suggesting that the let-23 protein encodes tissuespecific functions . Furthermore, let-23 controls two opposing pathways: one that stimulates and another that inhibits vulval\r\ndevelopment. These two pathways ensure that the proper level of vulval development occurs. Twenty let-23 alleles exist: 14 eliminate function (null), three reduce function in all tissues (hypomorphic), and three reduce function in certain tissues (tissue-specific). In addition, two of these alleles are defective in the inhibitory vulval pathway.
\r\n\r\nThe let-23 primary structure resembles that of the mammalian epidermal growth factor receptor (EGFR). The let-23 protein possesses putative ligand binding,\r\ntransmembrane, and tyrosine kinase domains, as well as cysteine-rich regions, all with the characteristics of the EGFR family. Like let-23, mammalian EGFR is multifunctional, encodes tissuespecific functions, and functions in stimulatory and inhibitory pathways. let-23 may be the receptor in the vulva for the anchor-cell\r\ninductive signal. Furthermore, genetic data indicate let-23 acts upstream of the let-60 ras gene, supporting mammalian studies that suggest a link between EGFR and ras.
\r\n\r\nTo investigate how EGFR primary structure relates to function, mutations in eight let-23 alleles have been\r\nsequenced. Five null alleles alter sequences in both the kinase and the extracellular domains. These alterations suggest that let-23 has kinase activity and that the extra cysteine domain found only in invertebrate EGFRs is important. A strong hypomorphic allele mutates one of the conserved extracellular cysteines close to the ligand binding domain. A tissue-specific allele mutates an\r\nintronlexon boundary in the C-terminus. This mutation suggests that the C-terminus can provide tissue-specific information. Finally, a hypomorphic allele that is defective in the let\u202223 inhibitory vulval pathway alters a different intron/exon boundary in the C-terminus. This mutation results in numerous, unexpected transcripts. Models are suggested to account for the behavior of this allele.
\r\n" }, { "name": "Funkhouser, William Keith Jr.", "degree": "PhD", "year": "1992", "title": "Demyelinating autoimmunity: murine T cell epitopes of MBP and primate T cell receptor V\u03b2 variation", "advisor": "Wise, Mark B.; Hood, Leroy E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08302011-103945566", "creators": [ { "name": { "family": "Funkhouser", "given": "William Keith Jr." }, "id": "Funkhouser-William-Keith-Jr", "display_name": "Funkhouser, William Keith Jr." } ], "advisors": [ { "name": { "family": "Wise", "given": "Mark B." }, "id": "Wise-M-B", "orcid": "0000-0002-9125-801X", "role": "advisor", "display_name": "Wise, Mark B." }, { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "co-advisor", "display_name": "Hood, Leroy E." } ], "committee": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "chair", "display_name": "Hood, Leroy E." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "orcid": "0000-0002-5408-6781", "role": "member", "display_name": "Emr, Scott D." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Wise", "given": "Mark B." }, "id": "Wise-M-B", "orcid": "0000-0002-9125-801X", "role": "member", "display_name": "Wise, Mark B." } ], "option_major": [ "immun" ], "doi": "10.7907/kmkw-9619", "abstract": "Autoimmune diseases result from inappropriate self-reactivity by lymphocytes. The long-term goal is to generate specific therapies for autoimmune diseases of humans, the success of which hinges on the definition of specific therapeutic targets. Experimental allergic encephalomyelitis (EAE) is a good animal model for the human demyelinating autoimmune disease, multiple sclerosis (MS). Risk for these diseases stratifies by major\r\nhistocompatibility complex (MHC) allele, as well as by T\r\ncell receptor (TCR) locus RFLP, in the case of MS. These\r\ndata suggest that (TCR-self peptide-MHC) complexes are\r\nassociated, possibly causally, with pathogenesis. This work\r\nfocused on the self peptide and TCR components of this\r\ncomplex. One specific aim was to document and characterize\r\nthe T cell epitopes of the autoantigen, myelin basic protein\r\n(MBP), in the EAE-susceptible mouse strain, B10.PL. Inbred\r\nB10.PL mice which were immunized with self MBP in complete\r\nFreund's adjuvant activated lymphocytes specific for\r\nepitopes estimated by peptides MBP(NAc1-20), MBP(31-50), and\r\nMBP(121-140). These mice generated the bulk of their immune\r\nresponse to the MBP(NAc1-20) epitope. The responses to self\r\nMBP immunization of B10.PL wildtype and MBP null \"shiverer\"\r\nmice were compared, and it was found that MBP(12I-140) is\r\ntolerogenic in animals which express MBP. A similar result\r\nwas observed in BALB/c wildtype and shiverer mice. These\r\ndata demonstrate that MBP is not a sequestered antigen, that\r\nmultiple epitopes tolerize T cells independently, and that\r\nincomplete, rather than absent, tolerance is present in mice\r\nsusceptible to EAE. A second specific aim was to document\r\nthe degree of variation in the primate TCR V\u03b2 8 subfamily,\r\nthree members of which are adjacent to a BamHI RFLP\r\nrestriction site linked to multiple sclerosis (MS) disease\r\nrisk. V\u03b2 8.1 and 8.2 were compared in a number of primates.\r\nIt was found that the overall coding sequences, but not the\r\nCDR coding sequences, were conserved compared with adjacent\r\nnon-coding flanking sequences. CDR coding sequences were\r\nnot demonstrably positively selected compared with noncoding\r\nflanking sequences or with synonymous coding sequences. A comparison of unrelated normal humans failed to demonstrate any non-synonymous Substitutions within V\u03b2 8.1 and 8.2, and demonstrated a single non-synonymous Substitution in V\u03b2 8.3. These data demonstrate that germline V\u03b2 8 gene segments are conserved and minimally polymorphic, implying that final TCR protein diversity derives from other mechanisms. Occasional allelism has been demonstrated in other V\u03b2 subfamilies, and our data does not rule out that certain TCR V\u03b2 alleles may ultimately be found to contribute to autoimmunity disease risk.\r\n" }, { "name": "Garyantes, Tina Kramer", "degree": "PhD", "year": "1992", "title": "The Effect of Electrical Stimulation on Neuronal Outgrowth and the Development of a New Method for Chronic Long-Term Stimulation and Recording from Groups of Neurons in Culture", "advisor": "Pine, Jerome; Baldeschwieler, John D.; Berg, Howard C.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08192011-093115822", "creators": [ { "name": { "family": "Garyantes", "given": "Tina Kramer" }, "id": "Garyantes-Tina-Kramer", "display_name": "Garyantes, Tina Kramer" } ], "advisors": [ { "name": { "family": "Pine", "given": "Jerome" }, "id": "Pine-J", "role": "advisor", "display_name": "Pine, Jerome" }, { "name": { "family": "Baldeschwieler", "given": "John D." }, "id": "Baldeschwieler-J-D", "role": "advisor", "display_name": "Baldeschwieler, John D." }, { "name": { "family": "Berg", "given": "Howard C." }, "id": "Berg-Howard-C", "role": "advisor", "display_name": "Berg, Howard C." } ], "option_major": [ "biology" ], "doi": "10.7907/4CQ9-7F40", "abstract": "In this dissertation, I shall examine the response of neurite outgrowth from cultured rat superior cervical ganglion (SCG) neurons to electrical stimulation and to changes in cytoplasmic calcium. Previous studies have shown that suprathreshold electrical stimulation arrests axonal growth from mouse dorsal root ganglion (DRG) and Helisoma neurons (Fields et al., 1990; Cohan and Kater, 1986). Cohan and collaborators (1987) have attributed the arrest of neurite outgrowth from Helisoma neurons to a rise in the growth-cone calcium concentration, [Ca]gc. In the experiments presented in this dissertation, neurite outgrowth from neonatal rat SCG neurons continued unabated during continuous suprathreshold electrical stimulation at 10 Hz for up to one hour. As in previous studies, the internal calcium concentration rose during stimulation. Fura-2 measurements showed that growth cone calcium levels rose from about 100 nM to greater than 500 nM, before settling at about 350 nM during stimulation. Despite this increase, neurite outgrowth continued. My results suggest that electrical activity is not a universal signal for neurons to stop growing and that a rise in internal calcium does not always arrest the migration of growth cones.
\r\n\r\nI was also able to record from and stimulate rat SCG neurons using a new device that allows maintained two-way communication between neurons and electronic circuitry. The new device or \"neuron well array\" holds individual neurons in surface micromachined holes. A self-supporting overhanging grillwork restrains the neurons in the holes. Each hole has an electrical contact which allows recording from and stimulation of the cell trapped therein. Neurons that grow in the holes appeared to suffer no observable ill effects of entrapment. Future neuronal development studies are planned with the wells.
\r\n" }, { "name": "Kayyem, Jon Faiz", "degree": "PhD", "year": "1992", "title": "Bravo, a novel immunoglobulin superfamily member in the developing avian nervous system, is identified using a new method", "advisor": "Unknown, Unknown", "url": "https://resolver.caltech.edu/CaltechETD:etd-11222006-090419", "creators": [ { "name": { "family": "Kayyem", "given": "Jon Faiz" }, "id": "Kayyem-J-F", "display_name": "Kayyem, Jon Faiz" } ], "advisors": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "molbio" ], "doi": "10.7907/3gk0-m777", "abstract": "Cell-surface molecules play an essential role in guiding axons to their targets. We have developed a method to generate monoclonal antibodies (MAbs) which recognize cell-surface molecules of defined molecular weight that are expressed during the development of the chicken retinotectal system. The antigen distribution on optic fibers recognized by one of these MAbs, Bravo, is restricted to retinal ganglion cell axons in the retina, and absent from these same axons in the tectum.\n\nThe complete derived sequence of Bravo including four putative alternatively spliced regions has been obtained. It reveals a close relationship to the neural members of the immunoglobulin superfamily, with closest relationship to chicken Ng-CAM and mouse L1. Like Ng-CAM and L1, Bravo contains six immunoglobulin domains, five fibronectin type III repeats, a transmembrane domain and a highly conserved but functionally uncharacterized cytoplasmic region. Like the other neural members of the immunoglobulin superfamily, Bravo carries the HNK-1 carbohydrate epitope, and specifically like L1 and Ng-CAM, Bravo is found predominantly in the form of a heterodimer, an intact chain cleaved in identical locations in all three molecules into two non-covalently associating parts.\n\nThese data present an interesting view of retinotectal optic fiber outgrowth: As optic fibers grow in dense fascicles towards the optic nerve exit, they express both the known cell adhesion molecule Ng-CAM, and the closely related Bravo. As these fibers pass through the optic chiasm, Bravo is reduced on their surfaces, coincidentally in the same place and time that these fibers noticeably defasciculate, presumably to allow independent target seeking by individual axons.\n\nFurthermore, Bravo staining of retinal glial processes has been detected. The close relationhip of Bravo to Ng-CAM coupled to Ng-CAM's known homophilic binding capacity suggest Bravo may be the heterophilic glial ligand for Ng-CAM long postulated to exist. Bravo is identical in sequence with the recently characterized Ng-CAM related glycoprotein, Nr-CAM." }, { "name": "Mahowald, Michelle A. (Misha)", "degree": "PhD", "year": "1992", "title": "VLSI Analogs of Neuronal Visual Processing: A Synthesis of Form and Function", "advisor": "Mead, Carver", "url": "https://resolver.caltech.edu/CaltechTHESIS:09122011-094355148", "creators": [ { "name": { "family": "Mahowald", "given": "Michelle A. (Misha)" }, "id": "Mahowald-Michelle-A-Misha", "display_name": "Mahowald, Michelle A. (Misha)" } ], "advisors": [ { "name": { "family": "Mead", "given": "Carver" }, "id": "Mead-C-A", "orcid": "0000-0003-4051-0462", "role": "advisor", "display_name": "Mead, Carver" } ], "committee": [ { "name": { "family": "Mead", "given": "Carver" }, "id": "Mead-C-A", "orcid": "0000-0003-4051-0462", "role": "chair", "display_name": "Mead, Carver" }, { "name": { "family": "Perona", "given": "Pietro" }, "id": "Perona-P", "orcid": "0000-0002-7583-5809", "role": "member", "display_name": "Perona, Pietro" }, { "name": { "family": "Martin", "given": "Alain J." }, "id": "Martin-A-J", "role": "member", "display_name": "Martin, Alain J." }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Pine", "given": "Jerome" }, "id": "Pine-J", "role": "member", "display_name": "Pine, Jerome" } ], "option_major": [ "cns" ], "doi": "10.7907/4bdw-fg34", "abstract": "This thesis describes the development and testing of a simple visual system fabricated using complementary metal-oxide-semiconductor (CMOS) very large scale integration\r\n(VLSI) technology. This visual system is composed of three subsystems. A silicon retina, fabricated on a single chip, transduces light and performs signal processing in a manner similar to a simple vertebrate retina. A stereocorrespondence chip uses bilateral retinal input to\r\nestimate the location of objects in depth. A silicon optic nerve allows communication between chips by a method that preserves the idiom of action potential transmission in the\r\nnervous system. Each of these subsystems illuminates various aspects of the relationship between VLSI analogs and their neurobiological counterparts. The overall\r\nsynthetic visual system demonstrates that analog VLSI can capture a significant portion of the function of neural structures at a systems level, and concomitantly, that incorporating neural architectures leads to new engineering approaches to computation in VLSI. The relationship\r\nbetween neural systems and VLSI is rooted in the shared limitations imposed by computing in similar physical media. The systems discussed in this text support the belief that the physical limitations imposed by the computational medium significantly affect the evolving algorithm. Since circuits are essentially physical structures, I advocate the use of analog VLSI as powerful medium of abstraction, suitable for understanding and expressing the function of real neural systems. The working chip elevates the circuit description to a kind of synthetic formalism. The behaving physical circuit provides a formal test of theories of\r\nfunction that can be expressed in the language of circuits.\r\n" }, { "name": "Michelsohn, Arie M.", "degree": "PhD", "year": "1992", "title": "Sequential Steps in the Determination of Chromaffin Cell Fate by Glucocorticoids", "advisor": "Anderson, David J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08242011-162533018", "creators": [ { "name": { "family": "Michelsohn", "given": "Arie M." }, "id": "Michelsohn-Arie-M", "display_name": "Michelsohn, Arie M." } ], "advisors": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "advisor", "display_name": "Anderson, David J." } ], "committee": [ { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." } ], "option_major": [ "neurobio" ], "doi": "10.7907/pbh4-ts59", "abstract": "The development of the sympathoadrenal (SA) lineage has been studied as a model system in which to investigate the mechanisms that control the timing of environmental influences on cell fate. Glucocorticoids (GC) play a key role in the fate of the SA progenitor, causing it to differentiate to an adrenal chromaffin cell, rather than to a sympathetic neuron. Previously, it has been shown that GC exert both positive and negative effects on developing chromaffin cells: they promote cell survival and the expression of an adrenergic phenotype, and inhibit the expression of neuronal properties. However, the time at which GC first influence cell fate, and the mechanism(s) which underlie its effect(s), have remained matters of controversy. In this thesis, it is shown that the positive and negative effects of GC on SA progenitors during development are temporally separated and pharmacologically distinct. Most SA progenitors are competent to respond to GC by inhibition of process outgrowth two days before they are competent to respond by induction of PNMT, a chromaffm-specific marker. Competence to express PNMT appears to be acquired according to a cell-autonomous \"clock\". The early inhibition of neuronal differentiation may be a prerequisite to subsequent PNMT expression, since sympathetic neuroblasts rapidly lose the capacity to express PNMT. The two effects of GC are both mediated via the type-II glucocorticoid receptor (GCR). However, lower concentrations of GC are required to inhibit neuronal differentiation than to promote the expression of PNMT, and the two effects show differential responsiveness towards the receptor-specific antagonist RU38486. That the two effects of GC in this system are pharmacologically separable suggests that they may be mediated via different interactions of the GCR with endogenous cellular transcription machinery. Such differential interactions may explain how the two effects of GC in this system are temporally separated. Taken together, the results presented here provide precedent for an inductive developmental event in which the timing of the effects of an instructive signal on a bipotential progenitor are controlled neither by the schedule of appearance of the signal, nor of its receptor, but rather by cell-intrinsic, developmental changes in the response properties of the cell." }, { "name": "Minor, Joseph E.", "degree": "PhD", "year": "1992", "title": "Evolution of the sea urchin sperm protein bindin", "advisor": "Davidson, Eric H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08312011-095256471", "creators": [ { "name": { "family": "Minor", "given": "Joseph E." }, "id": "Minor-J-E", "display_name": "Minor, Joseph E." } ], "advisors": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "advisor", "display_name": "Davidson, Eric H." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/zbbb-kf36", "abstract": "The sperm protein bindin is responsible for the species-specific adhesion of the sperm to the egg. The role of\r\nbindin in the establishment of reproductive isolation in the\r\nspecies Strongylocentrotus franciscanus and S.purpuratus is\r\nconsidered. Evolutionary changes in the bindin molecule are\r\ndescribed from an analysis of new cDNA sequences obtained\r\nfrom the species Strongylocentrotus franciscanus and\r\nLytechinus variegatus. These sequences are compared to the\r\npreviously obtained bind in sequence from S.purpuratus. The\r\nmiddle third of the mature bindin sequence is highly\r\nconserved in all three species, and the flanking sequences\r\nshare short repeated sequences that vary in number between\r\nthe species. This sequence comparison identified the regions\r\nof bindin that differ between the species, and that are\r\ntherefore likely to be responsible for the species-specific\r\nproperties of bindin.\r\n\r\nThe regions of the bindin molecule responsible for\r\nforming the contact between the sperm and the egg were\r\ninvestigated by assaying the ability of bindin-derived\r\npeptides to inhibit fertilization. Twenty-four peptides were\r\nstudied: seven based on the Strongylocentrotus purpuratus\r\nbindin sequence, eleven based on the S.franciscanus bindin\r\nsequence, and six control peptides. Values for IC_(50), the\r\nconcentration of peptide required to inhibit 50% of the\r\nproductive sperm contacts, were extracted from experimental\r\nmeasurements of the extent of fertilization in the presence\r\nof various concentrations of these peptides. The IC_(50) value averaged 220 \u00b5M for the control peptides. Five subregions of bindin are represented by peptides that had IC_(50) values less than 20 \u00b5M; the most potent peptide (SfO) had an IC_(50) value of 2.2 \u00b5M. Peptide SfR, derived from a region of the S.franciscanus bindin that differs from the S.purpuratus bindin, inhibited fertilization species-specifically. The peptides inhibit fertilization with a steep dose-response relationship, which probably reflects a requirement for the engagement of multiple bindin monomers in the initiation of the sperm-egg bond. These results demonstrate that a few specific regions of the bindin molecule are involved in the sperm-egg contact, and that these regions mediate the species-specificity of the interaction.\r\n" }, { "name": "Moore, Andrew John", "degree": "PhD", "year": "1992", "title": "Spatial Filtering in Tone Reproduction and Vision", "advisor": "Goodman, Rodney M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04112013-082009623", "creators": [ { "name": { "family": "Moore", "given": "Andrew John" }, "id": "Moore-Andrew-John", "display_name": "Moore, Andrew John" } ], "advisors": [ { "name": { "family": "Goodman", "given": "Rodney M." }, "id": "Goodman-R-M", "role": "advisor", "display_name": "Goodman, Rodney M." } ], "committee": [ { "name": { "family": "Goodman", "given": "Rodney M." }, "id": "Goodman-R-M", "role": "chair", "display_name": "Goodman, Rodney M." }, { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "member", "display_name": "Van Essen, David C." }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Mead", "given": "Carver" }, "id": "Mead-C-A", "orcid": "0000-0003-4051-0462", "role": "member", "display_name": "Mead, Carver" }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "member", "display_name": "Koch, Christof" } ], "option_major": [ "cns" ], "doi": "10.7907/YVBY-QN45", "abstract": "This thesis is concerned with spatial filtering. What is its utility in tone reproduction? Does it exist in vision, and if so, what constraints does it impose on the nervous system?
\r\n\r\nTone reproduction is just the art and science of taking a picture and then displaying it. The sensors available to capture an image have a greater dynamic range than the media that may be used to display it. Conventionally, spatial filtering is used to boost contrast; it ameliorates the loss of contrast that results when the sensor signal range is scaled down to fit the display range. In this thesis, a type of nonlinear spatial filtering is discussed that results in direct range reduction without range scaling. This filtering process is instantiated in a real-time image processor built using analog CMOS VLSI.
\r\n\r\nSpatial filtering must be applied with care in both artificial and natural vision systems. It is argued that the nervous system does not simply filter linearly across an image. Rather, the way that we see things implies that the nervous system filters nonlinearly. Further, many models for color vision include a high-pass filtering step in which the DC information is lost. A real-time study of filtering in color space leads to the conclusion that the nervous system is not that simple, and that it maintains DC information by referencing to white.
" }, { "name": "Robinson, Murray O.", "degree": "PhD", "year": "1992", "title": "Gene regulation during spermatogenesis: transcriptional and translational control of phosphoglycerate kinase 2 in transgenic mice", "advisor": "Simon, Melvin I.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08302011-152155117", "creators": [ { "name": { "family": "Robinson", "given": "Murray O." }, "id": "Robinson-M-O", "display_name": "Robinson, Murray O." } ], "advisors": [ { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "advisor", "display_name": "Simon, Melvin I." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/a6rw-je84", "abstract": "The work presented in this thesis is an analysis of the\r\ntranscriptional and translational control of the testis-specific gene for phosphoglycerate kinase (Pgk-2) in an effort to understand the mechanisms of gene regulation during spermatogenesis.\r\n\r\nTo address transcriptional control (Chapter two), a genomic\r\nfragment containing the human gene for PGK-2 was expressed in transgenic mice, and then a 323 bp region of the promoter was shown to be necessary for tissue-specific, developmentally regulated expression.\r\n\r\nIn Chapter three, a novel quantitation technique based on\r\nthe polymerase chain reaction was developed and used to\r\nmeasure the accumulation of transgenic and endogenous Pgk-2\r\ntranscripts. The human PGK-2 transgene, the PGK-2/CAT\r\ntransgene and the endogenous Pgk-2 gene all had similar levels and patterns of expression. Transcript levels of round spermatid-expressed protamine 2 were tenfold higher and showed delayed accumulation kinetics, implying that the peak expression of the Pgk-2-promoted genes occurred in pachytene spermatocytes.\r\n\r\nIn Chapter four, the translational regulation of the Pgk-2\r\nmessage was investigated, and the cis-acting sequences for\r\nproper translation of the PGK-2 message were shown to reside in the 5' untranslated region of the gene. Transgenes containing the PGK-2 promoter, the CAT coding sequence, and either the PGK-2 3' or SV40 3' sequences were both shown to behave like the translationally regulated endogenous Pgk-2 locus. Polysomal profiles of the PGK-2/CAT/SV40 3' construct demonstrated that\r\nthe message shifts from a translationally inactive state to a translationally active state between post natal day 20 and day 33. This developmental shift occurs with the same timing as that of the endogenous Pgk-2 message.\r\n\r\nThe Appendix investigates the relative levels of a number\r\nof transcripts in separated spermatogenic cells. Notably, the levels of Pgk-2 and the X-linked Pgk-1 were analyzed during spermatogenesis. Pgk-1 transcript levels diminish in the spermatogonial stages, whereas the Pgk-2 transcript first appears in early meiotic cells. This pattern of gene activity is consistent with the hypothesis that Pgk-2 expression substitutes for the inactivated Pgk-1 locus during the later stages of spermatogenesis.\r\n" }, { "name": "Strobel, Scott A.", "degree": "PhD", "year": "1992", "title": "Site specific cleavage of genomic DNA mediated by triple helix formation", "advisor": "Dervan, Peter B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:09092011-112420199", "creators": [ { "name": { "family": "Strobel", "given": "Scott A." }, "id": "Strobel-S-A", "display_name": "Strobel, Scott A." } ], "advisors": [ { "name": { "family": "Dervan", "given": "Peter B." }, "id": "Dervan-P-B", "role": "advisor", "display_name": "Dervan, Peter B." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/7TYF-9326", "abstract": "Physical isolation of large segments of chromosomal DNA is a major goal of human genetics. This would be greatly assisted by a generalizable technique for the cleavage of chromosomal DNA at a single site. Pyrimidine oligonucleotide directed triple helix formation is a generalizable motif for the site specific recognition of duplex DNA. The pyrimidine oligodeoxyribonucleotide is bound in the major groove parallel to the purine strand through formation of specific Hoogsteen hydrogen bonds. Specificity is derived from thymine (T) recognition of adenine-thymine (AT) base pairs (T\u2022AT triplet) and N3 protonated cytosine (C+) recognition of guaninecytosine (GC triplet). Theoretically, the binding site size is sufficient to specify a unique site in gigabase DNA, yet the binding motif is sufficiently generalizable to recognize an endogenous site every 1000 base pairs.
\r\n\r\nThis thesis describes the application of oligonucleotide directed triple helix formation to bind unique target sites in bacteriophage \u03bb, yeast, and human genomic DNA. Cleavage at the binding sites are achieved by affinity\r\ncleaving with EDTA\u2022Fe(II) derivatized oligonucleotides, alkylation with bromoacetyl derivatized oligonucleotides, and by site specific triple helix mediated methylase inhibition followed by digestion with the cognate endonuclease. Cleavage of genomic substrates with progressively greater complexity is described. Bacteriophage \u03bb genomic DNA (48.5 kilobase pairs) was targeted at a single endogenous homopurine site within the origin of replication. This substrate was also used to demonstrate cooperative binding of heterologous oligonucleotides to duplex DNA at contiguous binding sites.\r\nAn engineered target site on yeast chromosome III (340 kilobase pairs) was cut quantitatively at a single site within total yeast genomic DNA (14 megabase pairs) by both chemical and enzymatic techniques.
\r\n\r\nTechniques for the identification of endogenous triple helix target sites within unsequenced genetic markers were developed and successfully used to characterize a target site on human chromosome 4, proximal to the Huntington disease gene. As a test for the site specific cleavage of gigabase DNA, this site near the end of human chromosome 4 was cleaved by triple helix mediated enzymatic cleavage. This generated a specific 3.6 Mb fragment in greater than 80% yield that contained the entire candidate region for the\r\nHuntington mutation.
" }, { "name": "Tavtigian, Sean Vahram", "degree": "PhD", "year": "1992", "title": "The regulatory capacity of the protooncogene c-myc", "advisor": "Wold, Barbara J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:09142011-101754107", "creators": [ { "name": { "family": "Tavtigian", "given": "Sean Vahram" }, "id": "Tavtigian-S-V", "display_name": "Tavtigian, Sean Vahram" } ], "advisors": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "advisor", "display_name": "Wold, Barbara J." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biochem", "molbio" ], "doi": "10.7907/gqf9-4979", "abstract": "Myc is a crucial regulatory gene capable of altering normal cellular proliferation and differentiation. Its mechanism of action is largely unknown. The cloning and identification of downstream myc regulatory targets constitutes a key step towards deeper understanding.
\r\n\r\nBeginning with a conditional c-myc expression system and a physiologic setting where conditional myc expression produced clear phenotypic effects at both cell cycle progression and mRNA expression levels, I cloned a set of myc regulated genes. The frequency with which myc targets were identified \"among a panel of cDNAs subject to either up\r\nor downregulation during G_l of the first cell cycle\r\nfollowing serum stimulated emergence from growth arrest suggested that only about one-third of such genes may be myc targets. Consequently, this work has extended the myc target gene class to include several extracellular matrix proteins, one anabolic and one catabolic enzyme, a\r\ndifferentiation marker, several important cell proliferation regulators, and an assortment of unidentified genes.
\r\n\r\nWhile that cloning effort was in progress, two groups identified max, a gene encoding multiple bHLHzip proteins that can form DNA binding oligomers either alone or in a heterotypic complex with myc. After incorporating\r\nconditional max expression into the experimental paradigm\r\nof conditional myc expression, reexamination of the myc target gene set identified individual members that are cooperatively upregulated by myc and max and members that are regulated in opposite directions by myc and by max. In addition, we made the entirely unexpected observation that\r\nmax is a regulator of a specific subset of the immediate early serum response gene class.
\r\n\r\nBased on the results of these studies, I propose an integrative model accounting for the diverse effects of myc and max on cellular\r\nfunction.
\r\n" }, { "name": "Vernooij, Bernardus Theodorus Maria", "degree": "PhD", "year": "1992", "title": "The Mouse T Cell Receptor Gamma Genes", "advisor": "Hood, Leroy E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:09212011-131628448", "creators": [ { "name": { "family": "Vernooij", "given": "Bernardus Theodorus Maria" }, "id": "Vernooij-Bernardus-Theodorus-Maria", "display_name": "Vernooij, Bernardus Theodorus Maria" } ], "advisors": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "advisor", "display_name": "Hood, Leroy E." } ], "committee": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "chair", "display_name": "Hood, Leroy E." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "member", "display_name": "Simon, Melvin I." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Bjorkman", "given": "Pamela J." }, "id": "Bjorkman-P-J", "orcid": "0000-0002-2277-3990", "role": "member", "display_name": "Bjorkman, Pamela J." } ], "option_major": [ "molbio" ], "doi": "10.7907/wnmy-rm49", "abstract": "Murine T cells express either of 2 antigen receptors on\r\ntheir surface: \u03b1\u03b2 or \u03b3\u03b4 T cell receptors. The \u03b3\u03b4 T cell\r\npopulation contains subsets which show tissue specific\r\nlocalization, invariant T cell receptors and/or specificity\r\nfor stress antigens. This makes these T cells unlike \u03b1\u03b2 T\r\ncells.
\r\n\r\nThis thesis describes the genomic organization of the entire mouse T cell receptor gamma locus. It contains 4 clusters of gene segments, each with a C, a J and 1 to 4 V gene segments. Compared to other T cell receptor and\r\nimmunoglobulin loci, this is an unusual organization. The\r\nC\u03b32 cluster is in an orientation that is opposite to that of\r\nall other clusters.
\r\n\r\nTwo new \u03b3 enhancer-like elements were identified in the\r\nlocus. Also shown is that the hinge region of C\u03b34 is encoded\r\nby at least 2 exons. This is similar to the gene\r\norganization of the human C\u03b32 gene segment, and different\r\nfrom the other mouse and human C\u03b3 gene segments.\r\nSequence comparison of the T cell receptor \u03b3 gene segments\r\nof various mammals reveals structural conservation during\r\nevolution. The C region is most conserved, except in the\r\nhinge region. This subdomain is variable in length and in\r\nsequence. The extracellular domain is well conserved and\r\ncontains amino acid residues which are also conserved in the\r\nother T cell receptor and immunoglobulin proteins.
\r\n\r\nThe V\u03b3 gene segments are less well conserved, but several\r\namino acid residues are found which are (nearly) invariant.\r\nDuring evolution, the 2 studied mammals each appear to have\r\nlost certain V gene segments relative to a hypothetical\r\nancestor.
\r\n" }, { "name": "Altman, Elliot Charles", "degree": "PhD", "year": "1991", "title": "Characterization of the SecB Protein, a Chaperone that Facilitates Protein Secretion in Escherichia coli", "advisor": "Emr, Scott D.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06152007-080238", "creators": [ { "name": { "family": "Altman", "given": "Elliot Charles" }, "id": "Altman-Elliot-Charles", "orcid": "0000-0002-0721-0022", "display_name": "Altman, Elliot Charles" } ], "advisors": [ { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "orcid": "0000-0002-5408-6781", "role": "advisor", "display_name": "Emr, Scott D." } ], "committee": [ { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "orcid": "0000-0002-5408-6781", "role": "chair", "display_name": "Emr, Scott D." }, { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "member", "display_name": "Simon, Melvin I." }, { "name": { "family": "Abelson", "given": "John N." }, "id": "Abelson-J-N", "role": "member", "display_name": "Abelson, John N." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "orcid": "0000-0001-8291-5551", "role": "member", "display_name": "Campbell, Judith L." } ], "option_major": [ "biology" ], "doi": "10.7907/tq2t-3k47", "abstract": "It has become increasingly clear that in Escherichia coli, most exported proteins are translocated either posttranslationally or late in their synthesis, and that a component of the export apparatus, SecB, facilitates the export of a subset of the secreted proteins by maintaining them in an export-competent, unfolded form. In an effort to understand how SecB functions as an antifolding factor, we mapped and characterized the sites of SecB interaction in the outer membrane protein LamB. We found that the interaction of SecB with LamB was dependent on the LamB signal sequence as well as on a region in the mature LamB protein. The simplest interpretation of these findings is that SecB binds to both the LamB signal sequence and a mature region in LamB, and that this interaction promotes the antifolding activity of SecB.
\r\n\r\nGiven the fact that several heat-shock proteins have also been shown to function as antifolding factors, we wanted to investigate whether heat-shock proteins might act in a manner analogous to SecB in facilitating the export process. We found that induction of the heat-shock response could substitute for SecB function (SecB is not a heat-shock protein), and that a basal level of heat-shock proteins was necessary for the cell to survive in the absence of SecB protein. These results suggested that heat-shock proteins might indeed be involved in the secretory process and function in a manner similar to that of SecB.
\r\n\r\nIn an attempt to identify these proteins, suppressors of a secB null mutation were isolated and characterized. Not unexpectedly, most of these suppressors mapped to the rpoH locus. Since rpoH encodes \u03c3\u00b3\u00b2, the heat-shock transcription factor, it is likely that these suppressors affect the synthesis levels of heat-shock proteins, which can substitute for SecB function. The remaining suppressors did not map to any known heat-shock or export genes, and potentially represent unidentified heat-shock proteins or export factors that act in a manner similar to SecB in facilitating the export process in E. coli.
" }, { "name": "Bowman, John L.", "degree": "PhD", "year": "1991", "title": "Molecular genetics of flower development in Arabidopsis thaliana", "advisor": "Meyerowitz, Elliot M.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06212007-131544", "creators": [ { "name": { "family": "Bowman", "given": "John L." }, "id": "Bowman-J-L", "display_name": "Bowman, John L." } ], "advisors": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "advisor", "display_name": "Meyerowitz, Elliot M." } ], "committee": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "chair", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "member", "display_name": "Revel, Jean-Paul" }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "orcid": "0000-0002-5408-6781", "role": "member", "display_name": "Emr, Scott D." } ], "option_major": [ "biology" ], "doi": "10.7907/VJCE-Z966", "abstract": "Flowers of Arabidopsis thaliana consist of a precise pattern of organs arranged in four concentric whorls, with each whorl containing a different type of floral organ in characteristic positions and numbers. Arabidopsis flowers begin their development as small outgrowths of cells on the flank of the inflorescence meristem. Cells within each flower primordium must somehow assess their position relative to others and subsequently differentiate accordingly. Homeotic mutations at four loci (AGAMOUS, APETALA2, APETALA3, PISTILLATA) identified in Arabidopsis appear to cause cells in two adjacent whorls of the developing flower primordium to misinterpret their position and differentiate inappropriately. The development of wild-type flowers and that of several alleles of each locus is analyzed as well as the development of double and triple mutant combinations between the homeotic mutations. Based on this genetic data, a model is proposed for how this limited number of floral homeotic genes, by being expressed in overlapping fields of cells in the meristem of the developing flower primordium, and acting alone and in combination, could specify the identity of each of the whorls. AGAMOUS and APETALA2 are proposed to negatively regulate each other's activity with the result that they are expressed in mutually exclusive domains, APETALA2 in the outer two whorls of the flower and AGAMOUS in the inner two whorls. The activities of APETALA3 and PISTILLATA are proposed to be localized to the second and third whorls, with another gene, SUPERMAN, negatively regulating their activities in the fourth whorl. By protein sequence homology to known transcriptions factors, SRF of humans and MCM1 of yeast, AGAMOUS encodes a putative transcription factor. In support of the proposed model, RNA tissue in situ hybridizations to developing flowers show that AGAMOUS RNA is spatially localized to the inner two whorls in developing floral buds. Furthermore, in apetala2 mutant flowers, AGAMOUS RNA is detected in all floral whorls suggesting that APETALA2 negatively regulates AGAMOUS expression in the outer two whorls at the trancriptional level. Expression patterns of AGAMOUS late during flower development suggest that AGAMOUS may also play a role in cell fate specification during cellular differentiation of stamens and carpels." }, { "name": "Emerling, Michael Roy", "degree": "PhD", "year": "1991", "title": "Enzymatic hydrolysis of the amide bond : mutagenic studies of the mechanisms of [alpha]-lytic protease and [beta]-lactamase", "advisor": "Richards, John H.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06202007-140449", "creators": [ { "name": { "family": "Emerling", "given": "Michael Roy" }, "id": "Emerling-M-R", "display_name": "Emerling, Michael Roy" } ], "advisors": [ { "name": { "family": "Richards", "given": "John H." }, "id": "Richards-J-H", "role": "advisor", "display_name": "Richards, John H." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/mbnb-c645", "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\n\nThe enzymatic hydrolysis of amide bonds was studied in two systems by site-specific mutagenic techniques. In the first study, I developed an expression system for the serine protease, [alpha]-lytic protease, from Lysobacter enzymogenes 496, using a previously constructed synthetic gene, which contained numerous unique restriction sites. Since the wild type enzyme is expressed as a zymogen, which is self-processed into the mature proteolytic enzyme, a unique chimeric expression system utilizing the concomitant expression of the pro-domain from the wild type enzyme was created. The system expresses the properly-folded, mature protease-domain of the enzyme, thus allowing for production of active-site mutants of [alpha]-lytic protease, that otherwise could not be obtained in the mature form.\n\nThe ability of the enzymatic machinery to enhance the nucleophilicity of a chemical group other than the active-site serine hydroxyl was investigated through the mutation of serine 195 to an alanine. The removal of the serine hydroxyl was hypothesized to provide sufficient volume in the active site to allow a water molecule to bind and possibly function as the attacking nucleophile in the hydrolysis. The structure of the enzyme would be minimally disturbed by the removal of the single atom. The mutant enzyme was assayed for activity on the serine protease inhibitor diethyl [...]-nitrophenyl phosphate. No enzymatic hydrolysis of the substrate was detected. Analysis of structural constraints of the enzyme suggests that a serine 195 glycine mutation might provide a more hydrophilic environment in the active site for the binding of a water molecule.\n\nIn the second study, I investigated the mechanism of RTEM-1 [beta]-lactamase, an enzyme which hydrolyzes the amide bond of [beta]-lactam antibiotics, conferring antibiotic resistance to bacterial cells. Serine 130, a conserved active-site residue in the class A [beta]-lactamases, has been proposed to be involved in positioning the conserved lysine 234 through a hydrogen bond interaction (Moews et al. (1990) Proteins 7 156). The function of lysine 234 is known to be one solely of substrate binding (D. M. Long (1991) Ph.D. Thesis, California Institute of Technology). I performed site-saturation mutagenesis of serine 130, and the resulting 20 mutant enzymes were assayed for the ability to confer resistance to E. coli towards several [beta]-lactam antibiotics. Four mutants (Ser 130 Gly, Ser 130 Thr, Ser 130 Asn, and Ser 130 Gln) conferred notable resistance to the penam antibiotics. These four mutants were purified to homogeneity, and the steady-state kinetic parameters for hydrolysis of benzylpenicillin were measured for each. The values of KM for all four mutants were no more than ten-fold more than the wild type value. However, values of kcat for the four mutants were decreased at least 1000-fold from that of the wild type, demonstrating clearly the involvement of serine 130 in catalysis. These results, along with an analogy to structural mutants of Thr 157 in T4 lysozyme, suggest how the four serine 130 mutants might maintain the native hydrogen bonding interactions of the serine with lysine 234, as well as participate in the catalytic mechanism." }, { "name": "Harris, John Gregory", "degree": "PhD", "year": "1991", "title": "Analog Models for Early Vision", "advisor": "Koch, Christof", "url": "https://resolver.caltech.edu/CaltechETD:etd-06202007-093347", "creators": [ { "name": { "family": "Harris", "given": "John Gregory" }, "id": "Harris-John-Gregory", "display_name": "Harris, John Gregory" } ], "advisors": [ { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "advisor", "display_name": "Koch, Christof" } ], "committee": [ { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "chair", "display_name": "Koch, Christof" }, { "name": { "family": "Barr", "given": "Alan H." }, "id": "Barr-A-H", "display_name": "Barr, Alan H." }, { "name": { "family": "Mead", "given": "Carver" }, "id": "Mead-C-A", "orcid": "0000-0003-4051-0462", "role": "member", "display_name": "Mead, Carver" }, { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "member", "display_name": "Van Essen, David C." }, { "name": { "family": "Goodman", "given": "Rodney M." }, "id": "Goodman-R-M", "role": "member", "display_name": "Goodman, Rodney M." } ], "option_major": [ "cns" ], "doi": "10.7907/CTH4-5074", "abstract": "Analog models provide a novel framework for understanding and developing algorithms for computer vision. This thesis introduces several extensions to well-known resistive network techniques for solving early vision problems. First, constraint boxes are developed as a general methodology for mapping regularization-based algorithms onto stable analog hardware. These multiterminal resistor systems solve low-level vision problems by minimizing a global Lyapunov energy. Second, a circuit element called the resistive fuse is introduced to extend these networks for discontinuity detection. This is the first hardware circuit that explicitly implements line-process discontinuities. Since resistive fuse networks must minimize a non-convex energy function that may contain local minima, complex annealing or continuation methods are necessary for adequate solutions of the problem. Third, the tiny-tanh network is proposed as a new mechanism for discontinuity detection that is not plagued by problems with local minima. A piece-wise constant segmentation is performed through minimization of a convex Lyapunov energy." }, { "name": "Herman, Paul Kenneth", "degree": "PhD", "year": "1991", "title": "Protein Sorting in the Eukaryotic Secretory Pathway: An Essential Role for a Novel Yeast Protein Kinase", "advisor": "Emr, Scott D.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06202007-083735", "creators": [ { "name": { "family": "Herman", "given": "Paul Kenneth" }, "id": "Herman-Paul-Kenneth", "display_name": "Herman, Paul Kenneth" } ], "advisors": [ { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "orcid": "0000-0002-5408-6781", "role": "advisor", "display_name": "Emr, Scott D." } ], "committee": [ { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "orcid": "0000-0002-5408-6781", "role": "chair", "display_name": "Emr, Scott D." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." }, { "name": { "family": "Dunphy", "given": "William G." }, "id": "Dunphy-W-G", "orcid": "0000-0001-7598-8939", "role": "member", "display_name": "Dunphy, William G." }, { "name": { "family": "Brokaw", "given": "Charles J." }, "id": "Brokaw-C-J", "role": "member", "display_name": "Brokaw, Charles J." }, { "name": { "family": "Tanouye", "given": "Mark" }, "id": "Tanouye-M", "role": "member", "display_name": "Tanouye, Mark" } ], "option_major": [ "molbiochem" ], "doi": "10.7907/38ba-0w26", "abstract": "The yeast vps mutants are defective for the intracellular sorting of proteins to the vacuolar compartment. Mutants from two particular vps complementation groups, vpsl5 and vps34, share a common set of phenotypes that suggested that the VPS15 and VPS34 gene products might be functioning at a similar step of the vacuolar protein sorting pathway. vpsl5 and vps34 mutants exhibit specific defects in the sorting of soluble hydrolases to the vacuolar compartment. Whereas soluble hydrolases such as carboxypeptidase Y are almost quantitatively mislocalized to the cell surface, vacuolar membrane proteins appear to be properly localized to the vacuole.\r\n\r\nThe wild-type VPS15 and VPS34 genes were both cloned from yeast genomic DNA libraries by complementation of temperature-sensitive growth defects associated with mutations in these genes. Haploid yeast strains carrying a disruption of either locus were viable but exhibited a severe ts growth defect indicating that both genes are essential for vegetative growth at elevated temperatures. The vps34 null mutant was also found to exhibit a defect in the segregation of the vacuolar compartment upon cell division.\r\n\r\nThe predicted sequence of the VPS15 gene product exhibits significant similarity to the catalytic domains of the serine/threonine family of protein kinases. Point mutations altering specific amino acid residues of Vpsl5p that are highly conserved in all protein kinases result in the biological inactivation of Vpsl5p. The kinase domain mutants exhibit severe vacuolar protein sorting and ts growth defects. In addition, Vpsl5p is phosphorylated in vivo in a reaction that requires a wild-type Vpsl5p kinase domain. Subcellular fractionation experiments indicate that Vpsl5p is peripherally associated with the cytoplasmic face of a late Golgi or vesicle compartment. A vpsl5 mutant that encodes a protein lacking 30 carboxy-terminal amino acids exhibits a severe ts defect in vacuolar protein delivery. At the restrictive temperature, carboxpeptidase Y accumulates in a specific intracellular compartment that may represent a normal transport intermediate between the Golgi and vacuolar compartments. The vacuolar delivery defect in this mutant has an extremely rapid rate of onset suggesting that Vps15p is directly involved in the sorting of soluble proteins to the vacuole. Altogether, these data suggest that Vpsl5p regulates specific protein phosphorylation reactions in vivo that are required for the delivery of soluble hydrolases to the vacuole." }, { "name": "Hoh, Jan Hakan", "degree": "PhD", "year": "1991", "title": "Studies on the structure and molecular diversity of the gap junction", "advisor": "Revel, Jean-Paul", "url": "https://resolver.caltech.edu/CaltechETD:etd-06212007-075739", "creators": [ { "name": { "family": "Hoh", "given": "Jan Hakan" }, "id": "Hoh-Jan-Hakan", "display_name": "Hoh, Jan Hakan" } ], "advisors": [ { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "advisor", "display_name": "Revel, Jean-Paul" } ], "committee": [ { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "chair", "display_name": "Revel, Jean-Paul" }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "orcid": "0000-0002-5408-6781", "role": "member", "display_name": "Emr, Scott D." }, { "name": { "family": "Lipshitz", "given": "Howard D." }, "id": "Lipshitz-H-D", "orcid": "0000-0002-7372-4419", "role": "member", "display_name": "Lipshitz, Howard D." }, { "name": { "family": "Tanouye", "given": "Mark" }, "id": "Tanouye-M", "display_name": "Tanouye, Mark" } ], "option_major": [ "biology" ], "doi": "10.7907/8n7d-2t98", "abstract": "An improved method for the isolation of hepatic gap junctions that substantially shortens preparation time and improves the yield of previous methods is described. The topology of the 28 kD protein component (connexin-32, Cx32) of gap junctions isolated with this method is examined using proteases and antibodies against specific peptides. These experiments are consistent with the current model for the organization of the protein in the membrane, but reveal that an unexpectedly large part of the carboxy-terminus is protected from proteolytic attack. Together with data from comparisons of the Cx32 protein sequence with other channel proteins, a modified topological model is proposed.\r\n\r\nThe structure of the gap junction is further studied by atomic force microscopy. Using this new technology, high resolution images of a gap junction in phosphate buffered saline are obtained, and after \"force dissection,\" which removes half the plaque, the extracellular domains of individual connexons in a hexagonal array with lattice constant of 9.1 nm are revealed. These are the first images of an ion channel by atomic force microscopy, and the observations open the door for a variety of new experiments not previously possible.\r\n\r\nLow stringency screening of a rat genomic library produced genomic clones for Cx32 and a new member of the gene family, connexin-31 (Cx31) or [beta]3. Cx31 has a unique distribution and is found in the eye, Harderian gland, skin, and placenta. Comparison of the Cx31 with the other known connexins, reveals unique and conserved domains in the protein sequences. This comparison is extended to a phylogenetic analysis of the entire gene family that shows two major branches of connexins that diverged 1.3-1.9 billion years ago. Comparison with other ion channels reveals a short sequence similarity between the connexins and channels such as the voltage activated K+ channel. In K+ channels the sequence has been shown to line the aqueous pore, and the model for connexin organization is modified to account for this possibility. The similarity also suggests that gap junctions are part of a superfamily of ion channels." }, { "name": "Knierim, James Julius", "degree": "PhD", "year": "1991", "title": "Neural Responses to Texture Patterns in Area V1 of the Alert Monkey", "advisor": "Van Essen, David C.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06252007-155847", "creators": [ { "name": { "family": "Knierim", "given": "James Julius" }, "id": "Knierim-James-Julius", "display_name": "Knierim, James Julius" } ], "advisors": [ { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "advisor", "display_name": "Van Essen, David C." } ], "committee": [ { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "chair", "display_name": "Van Essen, David C." }, { "name": { "family": "Bower", "given": "James M." }, "id": "Bower-J-M", "role": "member", "display_name": "Bower, James M." }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Pine", "given": "Jerome" }, "id": "Pine-J", "role": "member", "display_name": "Pine, Jerome" } ], "option_major": [ "neurobio" ], "doi": "10.7907/2gs8-m769", "abstract": "Much of what our visual systems infers about a scene is based on cues derived from the visual texture of objects and surfaces in the scene. Areas of texture contrast are particularly salient and can automatically draw our attention. We recorded responses from cells in area V1 of the alert macaque monkey to texture stimuli in order to study the neurophysiology of texture segregation. A single oriented bar was placed in the center of a cell's classical receptive field (CRF) and we recorded the response to that center bar when it was alone against a blank background and when it was embedded in a texture of orthogonally oriented bars (orientation contrast texture) or identically oriented bars (uniform orientation texture). We found that the addition of the texture background suppressed the response to the center bar by an average of around 35%. In addition, for many cells there was a differential amount of suppression induced by the two texture backgrounds, such that the cell responded more strongly to the orientation contrast texture than to the uniform orientation texture. Such response properties correlate with the perceptual salience of the central bar.
\r\n\r\nThe same pattern of results was obtained when cells were tested with another noncontrast stimulus, a field of randomly oriented bars. The suppression from outside the CRF was shown to originate from areas on all sides of the CRF. A temporal analysis of the population responses to the center bar and the texture stimuli showed that both the general suppression and the orientation contrast effects are evident very early after stimulus onset. However, they both seem to take some small amount of time to develop, with the general suppression effect appearing about 7-10 msec after the onset of the population response and the orientation contrast effect appearing about 10-15 msec later. This short latency is consistent with the short presentation time sufficient in psychophysical studies of the popout effect for subjects to detect the presence of a target element differing in orientation from a field of distractors. The physiological response properties discussed here may underlie this perceptual ability.
" }, { "name": "Lochrie, Michael Alan", "degree": "PhD", "year": "1991", "title": "Molecular biology of G protein alpha subunits from bovine photoreceptors and the nematode Caenorhabditis elegans", "advisor": "Simon, Melvin I.", "url": "https://resolver.caltech.edu/CaltechETD:etd-07092007-132214", "creators": [ { "name": { "family": "Lochrie", "given": "Michael Alan" }, "id": "Lochrie-M-A", "display_name": "Lochrie, Michael Alan" } ], "advisors": [ { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "advisor", "display_name": "Simon, Melvin I." } ], "committee": [ { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "chair", "display_name": "Simon, Melvin I." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Tanouye", "given": "Mark" }, "id": "Tanouye-M", "role": "member", "display_name": "Tanouye, Mark" }, { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "role": "member", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/zqph-5443", "abstract": "This thesis examines the molecular biology of G protein alpha subunits from bovine photoreceptors and the nematode Caenorhabditis elegans.\n\nChapter one describes the nucleotide sequence of the bovine cone photoreceptor transducin alpha subunit (Tc[alpha]). Analysis of the sequence defined regions homologous to other GTP binding proteins which may be involved in guanine nucleotide binding, allowed the positions of amino acids which are ADP-ribosylated by pertussis toxin and cholera toxin to be determined, and led to the prediction that G proteins are posttranslationally modified with lipids, which serve to anchor G protein alpha subunits to membranes. Comparison of the Tc[alpha] amino acid sequence with other alpha subunit sequences as they became available provided the first indications that G proteins would be more numerous and diverse than previously thought. The diversity observed among G protein subunits and its structural and functional implications are reviewed in the introduction.\n\nIn Chapter 2 the characterization of G protein alpha subunits in the nematode C. elegans is described. Two genes were isolated and their DNA sequences were determined. The protein products of these genes appear to be unique to C. elegans. A cDNA encoding a homolog of Go[alpha] was also isolated and sequenced. Thus, C. elegans has identifiable homologs of mammalian G proteins as well as G proteins that may be unique to it. The chromosomal positions of the genes were determined. Each maps to a unique location near mutations that could be in G protein alpha subunits.\n\nIn Appendix 1 the characterization of photoreceptor specific gene expression in human retinoblastoma cultures is described. These cells express cone photoreceptor-specific genes, but not rod photoreceptor specific genes. Therefore they may provide a system for studying the DNA elements required for the expression of genes specifically in cone cells.\n\nAppendix 2 surveys systems for the heterologous expression of transducin alpha subunits in E. coli, yeast, and insect cells. The E. coli expression system offers the most promise for obtaining adequate amounts of active, pure protein." }, { "name": "Mahanthappa, Nagesh Kalyana", "degree": "PhD", "year": "1991", "title": "Functional and biochemical studies on neuronal Thy-1", "advisor": "Patterson, Paul H.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06212007-133431", "creators": [ { "name": { "family": "Mahanthappa", "given": "Nagesh Kalyana" }, "id": "Mahanthappa-N-K", "display_name": "Mahanthappa, Nagesh Kalyana" } ], "advisors": [ { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "advisor", "display_name": "Patterson, Paul H." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/qa3k-hw80", "abstract": "Thy-1, a cell surface glycoprotein, is one of the smallest members of the immunoglobulin superfamily. It is expressed abundantly in nervous systems of many species and is expressed at highest levels by long projection axons after the major period of neurite outgrowth and synaptogenesis. In this study, I demonstrate that three distinct perturbations that either remove or prevent expression of cell surface Thy-1 result in enhanced neurite outgrowth and initiation of sprouting: (i) Binding by soluble anti-Thy-1 monoclonal antibodies results in increased numbers of neurite-bearing neurons, chromaffin cells, and PC12 cells in culture. Sprouting requires multivalent binding (anti-Thy-1 Fab fragments do not give this effect), and results in shedding of Thy-1 from the cell surface. (ii) Chromaffin and PC12 cells exhibit enhanced sprouting when exposed to phosphotidylinositol-specfic phospholipase C, a treatment that removes cell surface Thy-1. This effect is blocked by anti-Thy-1 Fabs. (iii) When mutagenized and selected for Thy-l-deficiency, 90% of resultant PC12 cell mutants sprout spontaneously. These pertubations suggest that the normal function of Thy-1 is to stabilize neurites and inhibit sprouting, and that removal or prevention of Thy-1 expression disinhibits such mechanisms.\n\nBiochemical analysis reveals that Thy-1 exists in homomultimeric forms in situ and in cultured neurons. Neuronal Thy-1 is distributed equally in monomeric, dimeric, and hexameric forms; the latter being composed of monomers and dimers. The dimer does not give rise to monomers when boiled in the presence of disulfide reducing agents and SDS. In PC12 cells, the multimeric forms of Thy-1 predominate, but treatment with agents that cause sprouting make the Thy-1 distribution more neuronal. The homology between Thy-1 and immunoglobulin variable domain allowed peptides to be synthesized that correspond to candidate sites of intermolecular association. These were tested for their effects on neurite outgrowth and Thy-1 multimerization. One peptide induces a decrease in Thy-1 multimerization, as well as enhancing outgrowth. Thus, the stabilization function of Thy-1 may be mediated by homomultimerization on the cell surface, and its removal from the surface, or dissolution into monomers and dimers, permits outgrowth and sprouting.\n" }, { "name": "McCormack, Ken", "degree": "PhD", "year": "1991", "title": "Structure-function studies of Drosophila shaker potassium channels", "advisor": "Tanouye, Mark", "url": "https://resolver.caltech.edu/CaltechETD:etd-07182007-074159", "creators": [ { "name": { "family": "McCormack", "given": "Ken" }, "id": "McCormack-K", "display_name": "McCormack, Ken" } ], "advisors": [ { "name": { "family": "Tanouye", "given": "Mark" }, "id": "Tanouye-M", "role": "advisor", "display_name": "Tanouye, Mark" } ], "committee": [ { "name": { "family": "Tanouye", "given": "Mark" }, "id": "Tanouye-M", "role": "chair", "display_name": "Tanouye, Mark" }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." } ], "option_major": [ "biology" ], "doi": "10.7907/63q7-hw24", "abstract": "Voltage-dependent ion channels mediate electrical signals in the nervous system; many sodium (Na+), calcium (Ca++) and potassium (K+) selective channels are structurally related, and thus represent a family. These proteins undergo interesting conformational changes in response to alterations in transmembrane potential. However, the functional determinants involved in these transitions are not well understood. Chapters 2A and 2B describe the identification and characterization of an amino acid sequence motif (a leucine-heptad repeat) that is evolutionarily conserved among this family of voltage-dependent ion channels. Conservative, single amino-acid substitutions within this region of Drosophila Shaker (Sh) proteins have substantial effects on the voltage-dependence of activation. The observed alterations suggest that the heptad-repeat region is an important determinant in the conformational transitions leading to channel opening.\n\nNa+ and Ca++ channels are composed of four homologous domains, each of which is equivalent to a single K+ channel subunit. Thus, K+ channels are thought to be functional multimers. Furthermore, there are a large number of different voltage-dependent K+ genes and alternatively spliced products that potentially can be expressed in the same cell. Therefore, the potential number of different K+ channel multimers could be quite extensive. Chapter 3 describes the physiological characteristics of combinations of K+ channels belonging to the Sh family that have been coexpressed in Xenopus oocytes. Members of the same molecular class of Sh channel form heteromultimers with novel functional properties, adding to the diversity of K+ channel function. Members of different molecular classes do not form heteromultimeric channels, suggesting that there are distinct K+ channel systems. The Appendix describes an alternative exon in the \"constant\" region of the Drosophila Sh gene, the existence of which suggests, that the molecular diversity of this gene is greater than previously determined." }, { "name": "Miner, Jeffrey H.", "degree": "PhD", "year": "1991", "title": "Factors regulating skeletal muscle development : cell culture and transgenic mouse studies", "advisor": "Wold, Barbara J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-07172007-153729", "creators": [ { "name": { "family": "Miner", "given": "Jeffrey H." }, "id": "Miner-J-H", "display_name": "Miner, Jeffrey H." } ], "advisors": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "advisor", "display_name": "Wold, Barbara J." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/dqmw-tw31", "abstract": "The final steps of vertebrate skeletal muscle development involve withdrawal of determined muscle precursors from the cell cycle, expression of muscle-specific genes encoding myofibril components, and cell fusion to form terminally differentiated multinucleated myotubes. A great deal of this process has been adapted to and studied in cell culture for decades. While much has been learned about how a cell's environment influences differentiation decisions and what those decisions involve, only in the last few years has progress been made in understanding how the observed drastic changes in gene expression which accompany differentiation are regulated in the nucleus. This progress was spurred by the cloning of MyoD, a nuclear protein which can convert a variety of nonmyogenic cell types to skeletal muscle. Much evidence suggests that MyoD and its three relatives, myogenin, Myf-5, and MRF4-herculin-Myf-6, play critical roles in the transcriptional activation of skeletal muscle differentiation genes.\n\nThe studies presented in this thesis have addressed several important aspects of murine skeletal muscle development. Herculin, a novel member of the MyoD family of myogenic regulators, was cloned and characterized. Of the four known MyoD family members, herculin is the most abundant in adult skeletal muscle and therefore may be crucial for maintaining and/or enacting the mature muscle phenotype. Also, the c-myc proto-oncogene was shown to inhibit the ability of both MyoD and myogenin to initiate myogenic differentiation, even under conditions which normally promote it. This result is relevant for rationalizing, in part, why proliferating myoblasts which express MyoD do not spontaneously differentiate: c-myc is normally expressed in proliferating myoblasts but is down-regulated upon differentiation, perhaps allowing MyoD to become fully transcriptionally active. Finally, transgenic mice which express MyoD ectopically in the heart were produced. Transgenic hearts have morphological abnormalities and express myogenin and sarcomeric genes usually specific to skeletal muscle. This was the first demonstration that MyoD can function during murine embryogenesis and, more generally, that targeted misexpression of a tissue-specific regulator during mammalian development can activate genes normally transcribed in an unrelated tissue." }, { "name": "Molloy, Sean S.", "degree": "PhD", "year": "1991", "title": "A study of the type II CA2+/calmodulin-dependent protein kinase on hippocampal neurons", "advisor": "Kennedy, Mary B.", "url": "https://resolver.caltech.edu/CaltechETD:etd-08232007-130039", "creators": [ { "name": { "family": "Molloy", "given": "Sean S." }, "id": "Molloy-S-S", "display_name": "Molloy, Sean S." } ], "advisors": [ { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "role": "advisor", "display_name": "Kennedy, Mary B." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/nanb-2p11", "abstract": "Characterization of the type II Ca2+/calmodulin-dependent protein (CaM) kinase in vitro has revealed several intriguing physical and biochemical properties, including the induction of Ca2+-independent activity by autophosphorylation. This thesis describes our attempts to determine the importance of autophosphorylation to the regulation of the kinase in hippocampal neurons. In order to study the type II CaM kinase in these neurons, we established long-term cultures of rat hippocampal slices. We used these cultures to address several questions regarding the phosphorylation of the CaM kinase in the intact neurons, namely: 1) is the CaM kinase phosphorylated in the cultures under basal conditions, 2) if so, is phosphate incorporated into the sites previously characterized in vitro, 3) can phosphorylation of the CaM kinase be modulated in the neurons. Incubation of slice cultures with radiolabeled phosphate in situ showed that both the [alpha] and [beta] subunits of the kinase incorporate phosphate under basal conditions in intact neurons. Furthermore, HPLC analysis of tryptic fragments derived from [alpha] subunit radiolabeled in the cultures in situ revealed that the majority of phosphate was incorporated into Thr286 (the site which controls Ca2+-independent activity in vitro). Measurements of Ca2+-independent activity in homogenates showed that approximately one third of the kinase is autophosphorylated and constitutively active in the cultures. The proportion of Ca2+-independent enzyme in the cultures decreased by 80-90% following removal of external Ca2+. Application of the membrane permeant kinase inhibitors H7 and W7 also caused a substantial decrease in Ca2+-independent kinase activity, while the phosphatase inhibitor, okadaic acid, increased the proportion of Ca2+-independent kinase. Therefore, the resting level of Ca2+-independent CaM kinase apparently reflects a balance between continual Ca2+ dependent autophosphorylation and dephosphorylation by phosphatases. Homogenates of rat forebrains and hippocampi also had substantial levels of Ca2+-independent CaM kinase. These results suggest that the autophosphorylation mechanism acts to maintain a relatively high proportion of constitutively active kinase under conditions of low resting Ca2+ in neurons. This finding is in direct contrast to some models of kinase regulation in hippos pal neurons which predicted that the enzyme would only autophosphorylate following prolonged, synaptically driven increases in intracellular Ca2+. Furthermore, these studies indicate that pharmacological agents could either up or down regulate the level of constitutively active CaM kinase locally, at or near the synapse, by affecting the rate of autophosphorylation or dephosphorylation.\n" }, { "name": "Mooney, Richard Daniel", "degree": "PhD", "year": "1991", "title": "The Development of Connectivity and the Nature of Synaptic Transmission between Avian Song Control Nuclei", "advisor": "Konishi, Masakazu", "url": "https://resolver.caltech.edu/CaltechETD:etd-07192007-074811", "creators": [ { "name": { "family": "Mooney", "given": "Richard Daniel" }, "id": "Mooney-Richard-Daniel", "display_name": "Mooney, Richard Daniel" } ], "advisors": [ { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "advisor", "display_name": "Konishi, Masakazu" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/SBMB-1A95", "abstract": "Song is a learned vocalization unique to passerine birds. Young songbirds hear and memorize their father's song and then, as they mature, match their own song to the memorized model using auditory feedback. Song is controlled by a specialized neural circuit in the songbird's brain. The primary song control circuit is involved in vocal motor control, and is essential to song production in the adult. An \"accessory\" portion of the circuit appears unnecessary for adult song production, but is required for normal song development. The discrete nature of the song circuit, plus the stereotyped nature of song development, promise that the acquisition and refinement of a learned behavior can be understood on a cellular level.
\r\n\r\nThe robust nucleus of the archistriatum (RA), located in the songbird's caudal forebrain, is part of the primary pathway, and projects to motoneurons innervating the vocal musculature. RA is innervated by nucleus HVc, another primary structure, and by nucleus MAN, part of the accessory pathway.
\r\n\r\nThis study examines the changes in connectivity and the nature of synaptic transmission between these nuclei during development. MAN terminals were present within RA fifteen days after hatching, although HVc axons did not enter the nucleus until ten days later. The physiology of MAN and HVc synapses within RA was examined in an in vitro brain slice preparation. Before day 25, stimulation of MAN, but not HVc, fibers evoked excitatory synaptic potentials from virtually all RA neurons. MAN EPSPs were glutamatergic and activated NMDA-receptors on RA neurons. In slices prepared from male birds 40 to 70 days old, stimulation of MAN and HVc fibers evoked synaptic responses from most RA neurons. The properties of MAN EPSPs resembled those observed before day 25. Unlike the MAN EPSPs, HVc EPSPs were largely mediated by non-NMDA glutamate receptors. Both EPSPs could be enhanced for long periods of time by certain patterns of electrical stimulation.
\r\n\r\nTherefore, connections important to song development form before those essential to adult song production. These two pathways exhibit distinct pharmacological properties that may be related to their specific roles in song acquisition and production.
" }, { "name": "Patton, Bruce Lowell", "degree": "PhD", "year": "1991", "title": "Autophosphorylation Sites of the Type II Ca\u00b2\u207a/Calmodulin-Dependent Protein Kinase: Identification, Regulation of Kinase Activity, and Site-Specific Antibodies", "advisor": "Kennedy, Mary B.", "url": "https://resolver.caltech.edu/CaltechETD:etd-07232007-144526", "creators": [ { "name": { "family": "Patton", "given": "Bruce Lowell" }, "id": "Patton-Bruce-Lowell", "display_name": "Patton, Bruce Lowell" } ], "advisors": [ { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "advisor", "display_name": "Kennedy, Mary B." } ], "committee": [ { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "chair", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "member", "display_name": "Van Essen, David C." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Tanouye", "given": "Mark" }, "id": "Tanouye-M", "role": "member", "display_name": "Tanouye, Mark" }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." } ], "option_major": [ "neurobio" ], "doi": "10.7907/ete7-xp75", "abstract": "Biochemical and immunological approaches have been developed to study the regulation of the rat neuronal type II Ca\u00b2\u207a/calmodulin-dependent protein kinase (type II CaM kinase) by autophosphorylation. This thesis describes the identification of in vitro autophosphorylation sites on the CaM kinase and their role in regulating the catalytic activity of the CaM kinase. In addition, this thesis describes the development of antibodies against the type II CaM kinase that specifically recognize either the autophosphorylated kinase or the nonphosphorylated kinase.
\r\n\r\nThe autophosphorylation sites on in vitro autophosphorylated type II CaM kinase were identified by tryptic phosphopeptide mapping using reverse phase HPLC to isolate individual autophosphorylation sites. The sequence of the purified phosphopeptides was determined by gas phase microsequencing and compared to the known sequences of the kinase subunits, deduced from the cDNAs encoding them. The rates of site-specific autophosphorylation, or dephosphorylation by protein phosphatases, was compared with the rate of change in the Ca\u00b2\u207a/calmodulin-dependence of kinase catalytic activity. In the presence of Ca\u00b2\u207a and calmodulin, type II CaM kinase autophosphorylated an homologous residue in the \u03b1 and \u03b2 subunits of the type II CaM kinase, Thr\u00b2\u2078\u2076 and Thr\u00b2\u2078\u2077, respectively. Phosphorylation of this site correlated with the generation of Ca\u00b2\u207a-independent catalytic activity. Removal of free Ca\u00b2\u207a ion from the autophosphorylation reaction resulted in the autophosphorylation of two pairs of homologous residues, Thr\u00b3\u2070\u2075 and Ser\u00b3\u00b9\u2074 in the a subunit a and Thr\u00b3\u2070\u2076 and Ser\u00b3\u00b9\u2075 in the kinase catalytic activity. In the presence of Ca\u00b2\u207a and calmodulin, type II CaM kinase autophosphorylated an homologous residue in the \u03b2 subunit. Ser\u00b3\u00b9\u2074/\u00b3\u00b9\u2075 is resistant to dephosphorylation by purified protein phosphatases 1 and 2A. Selective dephosphorylation of the Thr\u00b3\u2070\u2075/\u00b3\u2070\u2076 autophosphorylation site demonstrated that the presence of phosphate on Thr\u00b3\u2070\u2075/\u00b3\u2070\u2076 inhibits Ca\u00b2\u207a/calmodulin-stimulated catalytic activity. The presence of phosphate on Ser\u00b3\u00b9\u2074/\u00b3\u00b9\u2075 slightly decreases the sensitivity of the kinase to Ca\u00b2\u207a/calmodulin.
\r\n\r\nAntibodies that bind to the type II CaM kinase at the Thr\u00b2\u2078\u2076/\u00b2\u2078\u2077 autophosphorylation site were produced in note and rabbits by immunization with thiophosphorylated and nonphosphorylated peptide haptens. A monoclonal antibody was obtained that specifically recognized the autophosphorylated type IICaM kinase. The monoclonal antibody recognized the Thr\u00b2\u2078\u2076/\u00b2\u2078\u2077 autophosphorylation site. A polyclonal antisera was obtained that, when affinity purified, specifically recognized the nonphosphorylated type II CaM kinase. Autophosphorylation of type II CaM kinase on Thr\u00b2\u2078\u2077 potently inhibited binding of the polyclonal antibodies. The monoclonal antibody and polyclonal antisera recognized type II CaM kinase in immunocytochemical sections and were used to assess the extent and distribution type II CaM kinase autophosphorylation in organotypic cultures of rat brain hippocampal slices. Double immunofluorescence immunocytochemistry with the antibodies specific for phosphorylated and nonphosphorylated type II CaM kinase indicated that most neurons and dendrites contain a mixture of phosphorylated and nonphosphorylated kinase, in varying proportions. Removal of extracellular Ca\u00b2\u207a greatly reduced the immunoreactivity specific for the phosphorylated kinase, implying that the type II CaM kinase phosphorylation state is in dynamic equilibrium in neurons.
" }, { "name": "Rao, Mahendra S.", "degree": "PhD", "year": "1991", "title": "Comparison of the properties of cholinergic differentiation factors and examination of their possible role in vivo", "advisor": "Patterson, Paul H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04112011-104242807", "creators": [ { "name": { "family": "Rao", "given": "Mahendra S." }, "id": "Rao-M-S", "display_name": "Rao, Mahendra S." } ], "advisors": [ { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "advisor", "display_name": "Patterson, Paul H." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/9ffk-0t58", "abstract": "We have compared the immunological, biochemical, and biological properties of three previously described cholinergic factors, cholinergic differentiation factor/Leukemia inhibitory factor (CDF/UF), ciliary neurotrophic factor (CNTF), and membrane-associatedneurotransmitter-inducing substance (MANS). CDF/U F differs from CNTF and MANS in that it does not have any ciliary neurotrophic activity. Further, antibodies generated against the N-terminal sequence of CDF/LIF do not precipitate cholinergic activity from sciatic nerve (CNTF) or spinal cord (MANS) preparations indicating that CDF/UF is a distinct molecule. MANS preparations contain a 24 kD molecule immunologically related to CNTF and CNTF antisera also, immunoprecipitate the cholinergic differentiation activity present in MANS fractions. CNTF, like CDF/LlF, affects neuropeptide expression. Neuropeptide Y levels are reduced and vasoactive intestinal peptide, somatostatin and Substance P levels are elevated in a dose-dependent fashion. Unlike CDF/LlF, the effects of CNTF on cholineacetyltransferase and peptide induction are not antagonized by depolarization. In addition, CNTF, in contrast to CDF/LlF, does not modulate peptide levels in DRG neuronal cultures. Thus, at least two distinct factors, CNTF and CDF/LlF, exist and have distinct but overlapping functions. We have investigated the possible role of CDF (cholinergic differentiation factor from skeletal muscle), CDF/LIF CNTF, and MANS in mediating the target directed noradrenergic to cholinergic switch that characterizes sweat gland innervation. Sweat gland extracts contain a cholinergic and peptidergic differentiation activity for cultured sympathetic neurons. Extracts from tabby mice (which lack sweat glands) and noradrenergic sympathetic targets have significantly reduced cholinergic differentiation activity. Expression of the differentiation activity in footpads occurs at a time period appropriate for a role for this factor(s) in vivo. Comparison with other differentiation molecules suggests that it is distinct from MANS, CDF and the heparin binding cholinergic factor. Immunological and biochemical analysis indicate that the major cholinergic-inducing activity is not LlF but is a CNTF-like molecule. Western blots, northern blot and in situ hybridization analysis fail to detect CNTF or CNTF message in footpads. The possible relationship between CNTF, LlF and sweat gland cholinergic differentiation factor(s) is discussed.\r\n" }, { "name": "Robinson, Jane Suzanna", "degree": "PhD", "year": "1991", "title": "Genetic, Molecular and Biochemical Studies of Vacuole Biogenesis and Maintenance in the Yeast Saccharomyces cerevisiae", "advisor": "Emr, Scott D.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04112011-112805564", "creators": [ { "name": { "family": "Robinson", "given": "Jane Suzanna" }, "id": "Robinson-Jane-Suzanna", "display_name": "Robinson, Jane Suzanna" } ], "advisors": [ { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "orcid": "0000-0002-5408-6781", "role": "advisor", "display_name": "Emr, Scott D." } ], "committee": [ { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "orcid": "0000-0002-5408-6781", "role": "chair", "display_name": "Emr, Scott D." }, { "name": { "family": "Abelson", "given": "John N." }, "id": "Abelson-J-N", "role": "member", "display_name": "Abelson, John N." }, { "name": { "family": "Brokaw", "given": "Charles J." }, "id": "Brokaw-C-J", "role": "member", "display_name": "Brokaw, Charles J." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/zfzc-0035", "abstract": "Using a selection for spontaneous mutants that mislocalize a vacuolar CPY-Inv\r\nfusion protein to the cell surface, 505 Saccharomyces cerevisiae mutants with defects in\r\nprotein sorting were identified. Seventeen of these mutants were dominant; the others\r\ndefined 25 new vps (for vacuolar protein sorting) complementation groups. Alleles of\r\neach vps complementation group exhibit defects in the targeting and final processing of\r\nsoluble vacuolar enzymes (CPY, PrA and PrB). Two of the genes, VPS17 and VPS15,\r\nmap to ChXV and ChII, respectively. The vpsll, vps16, vps18 and vps33 mutants\r\nexhibit morphological defects, their cells containing debris of a membranous nature and\r\nhighly abnormal vacuole remnants. Intercrosses with other vacuole function-defective\r\nmutants (vpl, pep, sip and end), revealed genetic overlaps. It is evident that more than\r\n50 gene products are involved in biogenesis and maintenance of the yeast vacuole.\r\nAlleles of 7 of the vps complementation groups are temperature-sensitive for vegetative\r\ncell growth at 37\u00b0C, and this recessive, Ts phenotype cosegregated with the vps defect\r\nin each case. This easily complemented phenotype has facilitated cloning of six of the\r\ngenes.\r\nThe VPS18 gene was chosen for further studies. A plasmid complementing the\r\nTs growth defect of vps18-1 was isolated and shown by integrative mapping to carry\r\nDNA from the VPS18 locus. Yeast strains with a deletion of the entire VPS18 coding\r\nregion (\u0394vps18), are viable and exhibit the same phenotypes as vps18-1. \u0394vps18, \u03b1 strains have smaller \u03b1-factor halos on sst2,a lawns than do VPS18, \u03b1 strains.\r\nImmunoprecipitation of \u03b1-factor indicated that it is secreted from the \u0394vps18 mutant in\r\nprecursor form. Several of the other severely defective vps mutants also show this \u03b1-factor\r\nprocessing defect. DNA sequencing of VPs18 showed an open reading frame\r\nencoding a 918aa protein, hydrophilic in nature. The protein sequence revealed a zinc finger like, cysteine-rich motif in its C-terminal region. A synthetic mutant with a\r\ncysteine to serine alteration has a temperature-conditional CPY sorting defect with very\r\nrapid onset. Therefore, Vps18p may be a zinc-binding protein, directly necessary for\r\ncorrect vacuolar protein sorting and proper functioning of the Golgi compartment that\r\ncontains Kex2p.\r\n" }, { "name": "Strathmann, Michael Paul", "degree": "PhD", "year": "1991", "title": "G protein diversity", "advisor": "Unknown, Unknown", "url": "https://resolver.caltech.edu/CaltechTHESIS:04112011-133018554", "creators": [ { "name": { "family": "Strathmann", "given": "Michael Paul" }, "id": "Strathmann-M-P", "display_name": "Strathmann, Michael Paul" } ], "advisors": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/wmg0-x120", "abstract": "The flow of information from hormone receptors through heterotrimeric G proteins to intracellular effectors constitutes a basic form of signal transduction that has been found in every eukaryotic cell examined. This mode of signaling is the basis for intercellular communication in one-celled organisms, such as S. cerevisiae, and simple eukaryotes that undergo limited development, e.g., Dictyostelium. We examined G protein diversity in the mouse to explore how G protein-mediated signal transduction has adapted to the complex signaling processes that define a multicellular organism. We found that the diversity of G protein alpha subunits is generated by a large number of distinct genes and by products of alternative splicing. Five new genes were found to encode alpha subunits that fall into two new classes. These classes, which are defined by amino acid sequence identity, have been conserved in distantly related animals; the four classes known to exist in mammals are also present in Drosophila, and three of these classes have been found in nematodes. The gene encoding G\u03b1_o in mammals, an alpha subunit earlier characterized by biochemical means, was found to undergo alternative splicing to produce transcripts encoding two forms of the protein. In addition to the diversity among alpha subunits, we found a novel beta subunit. This was particularly surprising because biochemical evidence suggested that beta-gamma dimers are interchangeable. The possibility of combinatorial associations of alpha, beta, and gamma subunits to produce functionally distinct heterotrimers must be considered. The considerable task of analyzing gene families by cloning and sequencing necessitated the development of new techniques for these purposes. We used the polymerase chain reaction to amplify cDNA with degenerate oligonucleotide primers. The primers were designed to hybridize to regions of DNA that are conserved in all members of the gene family. In addition, we developed a technique for randomly inserting sequencing primer sites throughout a cloned region of DNA. For this purpose, we used the transposon \u03b3\u03b4 coupled with an efficient scheme for isolating and characterizing the insertions." }, { "name": "Wang, Kang-Sheng", "degree": "PhD", "year": "1991", "title": "Molecular characterization of a receptor for the Togavirus Sindbis virus", "advisor": "Strauss, James H.; Strauss, Ellen G.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04112011-142633680", "creators": [ { "name": { "family": "Wang", "given": "Kang-Sheng" }, "id": "Wang-Kang-Sheng", "display_name": "Wang, Kang-Sheng" } ], "advisors": [ { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "advisor", "display_name": "Strauss, James H." }, { "name": { "family": "Strauss", "given": "Ellen G." }, "id": "Strauss-E-G", "role": "advisor", "display_name": "Strauss, Ellen G." } ], "committee": [ { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "chair", "display_name": "Davidson, Norman R." }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "chair", "display_name": "Patterson, Paul H." }, { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "orcid": "0000-0002-5408-6781", "role": "chair", "display_name": "Emr, Scott D." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "chair", "display_name": "Rothenberg, Ellen V." } ], "option_major": [ "biology" ], "doi": "10.7907/qdr9-p250", "abstract": "The first step in any virus entry process is binding to the plasma membrane of the host cell. The nature of this obligatory step depends upon both the viral and cellular components and may be quite diverse among viruses. The entry of Sindbis virus into a host cell is reported to occur via receptor-mediated endocytosis. We have used several approaches to isolate and characterize the receptor(s) for Sindbis virus. In one such approach, we searched for monoclonal antibodies (mAb) that could interfere with Sindbis virus attachment to and infection of baby hamster kidney (BHK) cells. Mice were immunized with multiple injections of whole BHK cells or with BHK cell membranes. Hybridomas were prepared and supernatants from approximately 3600 hybridoma clones were screened by a plaque reduction assay for their ability to interfere with virus infection. One IgM mAb from a mouse immunized with whole BHK cells inhibited Sindbis virus attachment to BHK cells by 80% at 20 \u00b5g/ml, and immunoprecipitated a 68 kDal membrane protein from BHK cells. This mAb also inhibits virus attachment to two other mammalian cell lines tested, Vero cells (monkey) and SW13 cells (human), and also immunoprecipitates a 68 Kd protein from these cells. The mAb does not interfere with virus infection of chicken cells but did immunoprecipitate a 71 kDal protein from chicken cells. This mAb was used to screen 10^6 plaques from a \u03bbgt11 cDNA library from BHK cells, and 15 reactive phages were found. Six of the fifteen were shown by sequence analysis to react with overlapping regions of a protein that was identical in sequence to the mouse high affinity laminin receptor. By rescreening with a probe from one of these reactive phages, other lambda phages containing the remaining regions of the gene were found. The complete sequence of this protein was deduced by sequence analysis of the cDNA clones and was identical to that of the mouse laminin receptor and 99% identical to the human laminin receptor. A full length cDNA clone of the gene was constructed and inserted into a high efficiency expression vector. BHK cell lines stably transfected with vector expressing the plus sense BHK laminin receptor cDNA are 3-5 fold more susceptible to infection by Sindbis virus as measured by plaque assay, and overexpress the receptor protein on their surface as assayed by flow cytometry analysis. Conversely, cell lines transfected with vector expressing antisense laminin receptor cDNA are only about one half as susceptible to infection by Sindbis virus as the nontransformed BHK cells, and expression of laminin receptor on the cell surface is reduced as measured by flow cytometry analysis. In a second study we looked for Sindbis virus receptors on the surface of chicken cells, using specific molecular mimicry to identify receptor molecules. It has been postulated that viral receptors may share structural features (idiotypes) with antibodies directed against the cell attachment protein of virus. Using antiidiotypic antibodies directed against Sindbis-specific neutralization antibodies, we have demonstrated that an antiidiotypic antibody to a neutralizing mAb reactive with the E2 glycoprotein of Sindbis virus specifically interferes with the binding of wild type Sindbis virus to chicken cells. This antiidiotypic antibody also immunoprecipitates a 63 kDal protein from chicken cells and binds to the surface of these cells. This 63 kDal protein is presumably a receptor for Sindbis virus in chicken cells. The relationship between this protein and the laminin receptor used as a Sindbis receptor in mammalian cells remains to be determined. We also wished to determine the domains of the virus envelope proteins that are responsible for attachment to the cell membrane. The Sindbis virus envelope contains two species of integral membrane glycoproteins, El and E2, which assemble into heterodimers. Each spike on the surface of the virion is a trimer of these dimeric units. We attempted to map the neutralization epitopes on the surface of the virus, including epitopes implicated in virus binding to cells by the antiidiotypic antibody results described above. A \u03bbgt11 expression library was constructed containing cDNA inserts 100-300 nucleotides in length obtained by randomly primed synthesis on Sindbis genomic RNA. This library was probed with several neutralizing monoclonal antibodies specific for E2 and one neutralizing antibody specific for El. Four positive clones, all of which contained inserts from the region of the Sindbis genome that encodes amino acids 173 to 220 of glycoprotein E2, were found from the screening with mAb 23. No reactive clones could be identified using any of the other antibodies. We hypothesize that this domain of E2 centered at residue 200 forms part of the virus binding site for attachment to the cell to initiate infection." }, { "name": "Wilson, Matthew A.", "degree": "PhD", "year": "1991", "title": "An analysis of olfactory cortical behavior and function using computer simulation techniques", "advisor": "Bower, James M.", "url": "https://resolver.caltech.edu/CaltechETD:etd-08182006-133038", "creators": [ { "name": { "family": "Wilson", "given": "Matthew A." }, "id": "Wilson-M-A", "display_name": "Wilson, Matthew A." } ], "advisors": [ { "name": { "family": "Bower", "given": "James M." }, "id": "Bower-J-M", "role": "advisor", "display_name": "Bower, James M." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/B85N-BK43", "abstract": "This thesis presents the results of computer simulations of olfactory cortex designed to explore the role of biological mechanisms in the behavior and function of cerebral cortical networks.\n\nChapter 1 provides the basic description of the model of piriform cortex including simulation methodology and parameters. The results of network simulations which reproduce three characteristic macroscopic evoked cortical responses are described with the suggestion that the simulated temporal dynamics which underlie these responses may reflect the operation of a fundamental computational strategy used in the storage and retrieval of olfactory information.\n\nUsing both single cell and network simulations, chapter 2 looks in detail at the patterns of synaptic currents produced along the dendritic tree of single cells and compares simulated results with actual experimental measurements. This technique provides the means to identify the relative synaptic contributions and provides an important constraint on the selection of synaptic weight distribution parameters in the network model. The results identify the sources of synaptic inputs underlying characteristic macroscopic evoked events and thus provide additional insights into the results of chapter 1.\n\nUsing the basic model outlined in chapter 1, chapter 3 explores the effects of incorporating a mechanism for activity dependent modification of synaptic weights along selected interconnection pathways. This is done in the context of storage and retrieval of patterned inputs to the model intended to simulate the patterns of activity which represent actual olfactory input. The results indicate that the basic model which reproduces known physiological responses can also be made to store and retrieve patterned input indicating that the dynamics of the simulated cortex are compatible with a continuous mechanism of Hebbian synaptic plasticity.\n\nIn chapter 4 the structure of the basic piriform cortex model is modified slightly to reflect more neocortical-like features. The dynamics of this modified network are compared with experimental observations of coherent oscillatory behavior in primary visual cortex. These simulations indicate that the observed behavior are characteristic of the network architecture and do not necessarily represent the encoding of stimulus specific information.\n\nChapter 5 provides a general overview of the simulation system used to implement all of the simulations used in this research.\n\nAppendix 1 examines the effects of critical parameter variations on simulated EEGs.\n\nAppendices 2 and 3 contain the complete GENESIS scripts describing the network and single cell models used in this work.\n" }, { "name": "Banta, Lois Margaret", "degree": "PhD", "year": "1990", "title": "Vacuolar Protein Sorting in Yeast: Characterization of Mutants and Identification of a Protein Required for Vacuole Biogenesis", "advisor": "Emr, Scott D.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06112007-104332", "creators": [ { "name": { "family": "Banta", "given": "Lois Margaret" }, "id": "Banta-Lois-Margaret", "display_name": "Banta, Lois Margaret" } ], "advisors": [ { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "orcid": "0000-0002-5408-6781", "role": "advisor", "display_name": "Emr, Scott D." } ], "committee": [ { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "orcid": "0000-0002-5408-6781", "role": "chair", "display_name": "Emr, Scott D." }, { "name": { "family": "Abelson", "given": "John N." }, "id": "Abelson-J-N", "role": "member", "display_name": "Abelson, John N." }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." }, { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "member", "display_name": "Revel, Jean-Paul" } ], "option_major": [ "biology" ], "doi": "10.7907/TZHF-3J73", "abstract": "The lysosome-like vacuole of the yeast Saccharomyces cerevisiae is an acidic compartment containing a number of hydrolytic glycoproteins including carboxypeptidase Y (CPY), proteinase A (PrA) and proteinase B (PrB). A gene fusion-based selection scheme was utilized to isolate ~600 mutants defective in the localization and processing of vacuolar proteins. These vacuolar protein sorting (vps) mutants define >33 complementation groups and exhibit hybrid protein-independent defects in the sorting of CPY, PrA, and PrB. Light and electron microscopic analyses of the vacuole morphology revealed three distinct classes of vps mutants. The class A mutants (26 complementation groups) contain 1-3 large vacuoles that resemble those of the parental strain. One class A mutant is sensitive to low pH and exhibits a defect in vacuole acidification. Consistent with a role for vacuolar pH in protein sorting, perturbation of vacuole acidification resulted in the missorting and secretion of CPY and PrA in wild-type cells. Mutants in the three class B complementation groups exhibit a fragmented vacuole morphology. The class C vps mutants (four complementation groups) lack any compartment resembling a wild-type vacuole, but accumulate vesicles and other membranous structures. Many class C strains exhibit genetically linked defects including temperature-sensitivity and sensitivity to osmotic stress. Unlike other vps mutants, these mutants secrete up to 50% of a vacuolar membrane marker enzyme. The gene defined by one class C mutant, vps33, has been cloned. The predicted VPS33 gene product is hydrophilic and shares sequence similarity with a family of ATP-binding proteins. Disruption of VPS33 is not lethal but results in temperature-sensitive growth. Vps33p-specific antisera recognize a cytosolic protein of ~75 kD. One temperature-sensitive vps33 mutant carrying a missense mutation contains apparently normal vacuoles at the permissive temperature, but lacks vacuoles specifically in the bud at the nonpermissive temperature. We propose that the abnormalities in vacuole morphology and inheritance in vps33 mutants are a consequence of a primary defect in Golgi-to-vacuole protein delivery. A second VPS gene, VPS28, has also ben cloned. Our data suggest that the VPS28 gene product only indirectly affects vacuole protein sorting, but may function in a late protein modification process.
" }, { "name": "Battiti, Roberto", "degree": "PhD", "year": "1990", "title": "Multiscale Methods, Parallel Computation, and Neural Networks for Real-Time Computer Vision", "advisor": "Fox, Geoffrey C.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06072007-074441", "creators": [ { "name": { "family": "Battiti", "given": "Roberto" }, "id": "Battiti-Roberto", "display_name": "Battiti, Roberto" } ], "advisors": [ { "name": { "family": "Fox", "given": "Geoffrey C." }, "id": "Fox-G-C", "role": "advisor", "display_name": "Fox, Geoffrey C." } ], "committee": [ { "name": { "family": "Hopfield", "given": "John J." }, "id": "Hopfield-J-J", "role": "chair", "display_name": "Hopfield, John J." }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Fox", "given": "Geoffrey C." }, "id": "Fox-G-C", "role": "member", "display_name": "Fox, Geoffrey C." }, { "name": { "family": "Furmanski", "given": "Wojtek" }, "id": "Furmanski-Wojtek", "role": "member", "display_name": "Furmanski, Wojtek" }, { "name": { "family": "Koch", "given": "Christof" }, "id": "Koch-C", "orcid": "0000-0001-6482-8067", "role": "member", "display_name": "Koch, Christof" } ], "option_major": [ "cns" ], "doi": "10.7907/4q6t-2b57", "abstract": "This thesis presents new algorithms for low and intermediate level computer vision.
\r\n\r\nThe guiding ideas in the presented approach are those of hierarchical and adaptive processing, concurrent computation, and supervised learning.
\r\n\r\nProcessing of the visual data at different resolutions is used not only to reduce the amount of computation necessary to reach the fixed point, but also to produce a more accurate estimation of the desired parameters. The presented adaptive multiple scale technique is applied to the problem of motion field estimation. Different parts of the image are analyzed at a resolution that is chosen in order to minimize the error in the coefficients of the differential equations to be solved. Tests with video-acquired images show that velocity estimation is more accurate over a wide range of motion with respect to the homogeneous scheme. In some cases introduction of explicit discontinuities coupled to the continuous variables can be used to avoid propagation of visual information from areas corresponding to objects with different physical and/or kinematic properties.
\r\n\r\nThe human visual system uses concurrent computation in order to process the vast amount of visual data in \"real-time.\" Although with different technological constraints, parallel computation can be used efficiently for computer vision. All the presented algorithms have been implemented on medium grain distributed memory multicomputers with a speed-up approximately proportional to the number of processors used. A simple two-dimensional domain decomposition assigns regions of the multiresolution pyramid to the different processors. The inter-processor communication needed during the solution process is proportional to the linear dimension of the assigned domain, so that efficiency is close to 100% if a large region is assigned to each processor.
\r\n\r\nFinally, learning algorithms are shown to be a viable technique to engineer computer vision systems for different applications starting from multiple-purpose modules. In the last part of the thesis a well known optimization method (the Broyden-Fletcher-Goldfarb-Shanno memoryless quasi-Newton method) is applied to simple classification problems and shown to be superior to the \"error back-propagation\" algorithm for numerical stability, automatic selection of parameters, and convergence properties.
" }, { "name": "Brorson, Kurt Andrew", "degree": "PhD", "year": "1990", "title": "Analysis of Expression, Structure and Evolution of Non-Classical Class I Major Histocompatibility Complex Genes", "advisor": "Hood, Leroy E.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06132007-074646", "creators": [ { "name": { "family": "Brorson", "given": "Kurt Andrew" }, "id": "Brorson-Kurt-Andrew", "display_name": "Brorson, Kurt Andrew" } ], "advisors": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "advisor", "display_name": "Hood, Leroy E." } ], "committee": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "chair", "display_name": "Hood, Leroy E." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "member", "display_name": "Owen, Ray David" } ], "option_major": [ "molbio" ], "doi": "10.7907/5NHE-7929", "abstract": "Class I major histocompatibility molecules (MHC) are 45 kilodalton (kD) glycoproteins that associate with a smaller 12 kD polypeptide, \u03b2\u2082-microglobulin. In the BALB/c mouse, there are three classical class I molecules, H-2K\u1d48, D\u1d48, and L\u1d48, which are expressed throughout the body and present viral antigens to cytotoxic T lymphocytes (CTLs). In addition to the genes that encode the three classical class I antigens, the BALB/c genome contains 32 genes that structurally resemble the classical class I genes, and therefore possibly encode class I molecules. A few of the non-classical class I genes have been shown to encode molecules, TL, Qa-1, Qa-2, Q10, Qb-1, and Hmt, which are expressed in a generally tissue-specific manner, and probably do not act as restriction elements. However, it is unclear what function these molecules play, or why such a large gene family is maintained if only three viral antigen-presenting restriction elements are required by the murine immune system.
\r\n\r\nDNA sequences were obtained from each of the 35 class I genes of the BALB/c mouse of the transmembrane domain-encoding fifth exon. Based on nucleotide sequence similarity, the fifth exons could be divided into seven groups that share little similarity with each other. In addition, the majority of the fifth exons are able to encode a transmembrane domain that can be separated into a proline-rich connecting peptide, a hydrophobic transmembrane segment, and a ctyoplasmic portion that includes basic anchoring residues. Since this conservation occurs in spite of extensive variation of nucleotide sequence in these exons, it is likely that selective pressure exists to maintain a functional structure in the majority of class I genes.
\r\n\r\nA cDNA library was constructed from a thymus from a five-week-old BALB/c mouse. From this library, 69 class I cDNA transcripts from 15 different class I genes were isolated and analyzed. Included were three novel transcripts from Tla subregion genes, the T9\u1d9c, T17\u1d9c, and T18\u1d9c genes. Sequence analysis of these clones reveals that the T9\u1d9c gene is probably a pseudogene, while the T18\u1d9c gene has an open reading frame in at least exons 2, 3, 4, and 5. A fourth cDNA clone was a transcript from the Thy19.4 gene, a gene that had not been previously isolated on a recombinant DNA clone. The isolation of transcripts from such a relatively large number of genes suggests that the number of expressed and perhaps functionally important class I genes may be larger than previously believed, and that expression of class I recognition structures may be important for cell-cell interactions within the thymus.
\r\n\r\nTo further pursue the characterization of the Thy19.4 gene, a genomic clone containing this gene was isolated from a size-selected insert library, and the DNA sequence of the Thy19.4 gene was obtained. The Thy19.4 gene contains an open reading frame, and in several aspects resembles the genes that encode the transplantation antigens. These similarities include a shared exon/intron structure and shared amino acid sequence motifs. In addition, PCR amplification experiments using tissue cDNA demonstrates that the Thy19.4 gene is expressed in a variety of tissues. However, unlike the classical transplantation antigens, the Thy19.4 gene maps distal to the H-2 region, in the Hmt region.
\r\n\r\nThese studies have demonstrated that class I gene transcription is more extensive than previously believed. Some of the expressed genes, like the T18\u1d9c and Thy19.4 genes, appear to be able to encode class I molecules which may share structural characteristics with the classical transplantation antigens and may possibly serve as recognition structures in cell-cell interaction events. In addition, examination of the transmembrane domain exon of each of the 35 class I genes suggests that some selective constraint is acting on the majority of members of this family of genes, thus raising the possibility that many of the nonclassical class I genes encode functionally important products.
" }, { "name": "Carman, George John", "degree": "PhD", "year": "1990", "title": "Mappings of the Cerebral Cortex", "advisor": "Van Essen, David C.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06112007-103643", "creators": [ { "name": { "family": "Carman", "given": "George John" }, "id": "Carman-George-John", "display_name": "Carman, George John" } ], "advisors": [ { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "advisor", "display_name": "Van Essen, David C." } ], "committee": [ { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "chair", "display_name": "Van Essen, David C." }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Bower", "given": "James M." }, "id": "Bower-J-M", "role": "member", "display_name": "Bower, James M." }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" } ], "option_major": [ "biology" ], "doi": "10.7907/8aad-ym19", "abstract": "The mammalian cerebral cortex is organized into a variety of two-dimensional areas whose anatomical and physiological organization is obscured by the folding of the cortical mantle in three dimensions. This organization can be revealed by unfolding the cortex so as to produce a two-dimensional representation or map of its surface. To produce such mappings, we have developed algorithms for computational cartography which maximally preserve intrinsic geometry while unfolding the cortical surface. Our computational algorithms were used to produce the first computational maps of the entire primary visual cortex of the macaque monkey (Carman and Van Essen, 1985), and the first completely noninvasive mapping of in vivo human visual cortex (Carman and Mora, 1989).
\r\n\r\nIn order to measure the geometry of the region of cortex to be mapped, a reconstruction of a surface or layer of cortex must be obtained from a typically sparse sample of contours of section. We obtain a solution to this reconstruction problem by computing a flow which fuses pairs of images containing successive contours of the surface. These flows are governed by a pair of complex harmonic potentials which represent translations, rotations, and scalings which combine to produce a conformal mapping of the two images onto a third fused and interpolated image. Since these potentials are harmonic, their values over a region of the images can be computed from samples taken only along the boundary of that region by solution of the Dirchlet problem. Thus, a coarse to fine series of samples on concentric annuli, similar to the sampling of the primate retina, can be used to compute such flows at a continuum of spatial scales. A number of visual problems arising in the analysis of motion, stereo, and shape information are formally equivalent to this reconstruction problem and can therefore also be solved by computing such flows. Remarkably, the equations which determine these flows can reproduce many aspects of the topography of the first stages of the primate visual pathway, suggesting that such flows may also be computed by the mappings of the cerebral cortex.
" }, { "name": "Fors, Lance", "degree": "PhD", "year": "1990", "title": "Analysis of the Structure, Expression and Evolution of the Shark Myelin Proteins and Genes", "advisor": "Hood, Leroy E.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06072007-082551", "creators": [ { "name": { "family": "Fors", "given": "Lance" }, "id": "Fors-Lance", "display_name": "Fors, Lance" } ], "advisors": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "advisor", "display_name": "Hood, Leroy E." } ], "committee": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "chair", "display_name": "Hood, Leroy E." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Tanouye", "given": "Mark" }, "id": "Tanouye-M", "role": "member", "display_name": "Tanouye, Mark" }, { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "orcid": "0000-0002-5408-6781", "role": "member", "display_name": "Emr, Scott D." } ], "option_major": [ "biology" ], "doi": "10.7907/840h-0p67", "abstract": "Myelin is a compacted multilamellar membrane which encases axons to provide electrical insulation and facilitates the rapid transmission of nerve impulses. The major myelin structural proteins produced by oligodendrocyctes in the central nervous system (CNS) of mammals are proteolipid protein (PLP) and myelin basic protein (MBP). In contrast, the major myelin structural proteins produced by the Schwann cells in the peripheral nervous system (PNS) of mammals are protein zero (Po) and MBP. Sharks (class Chondrichthyes) are the oldest living vertebrates that have a concentric multilamellar \"mammalian-like\" myelin structure around axons. In addition to this structural similarity, the shark and mammalian myelin proteins appeared to be distantly related biochemically and immunologically even though they diverged from each other about 400 million years ago. Logically those regions in the shared proteins, genes and promoters which are most similar between sharks and mammals are likely to be functionally important to both. Therefore by analyzing these elements in shark myelin and comparing them to what is already known about mammalian myelin we could learn about shark myelin, its evolution and what regions are essential for the proper function and expression of mammalian myelin. This thesis contains an analysis of the structure, expression and evolution of the shark myelin proteins and genes.
\r\n\r\nThe first chapter (Saavedra, R., Fors, L., Aebersold, R., Arden, B., Horvath, S., Sanders, J., and Hood, L. J. Mol. Evol. 29:149) describes the isolation and sequencing of the two major shark CNS proteins Po and MBP and their corresponding cDNAs. This study shows that the myelin proteins of the shark brain are similar to the myelin proteins of the mammalian peripheral nervous system in both primary and secondary structures.
\r\n\r\nThe second chapter (Fors, L., Saavedra, R., and Hood, L. Nuc. Acids Res., Submitted) contains a novel genomic walking technique that was developed to clone the shark Po and MBP promoters. Using this technique it was possible to clone approximately 400 nucleotides immediately upstream of the shark Po and MBP transcription initiation sites. This genomic walking technique will be generally useful for cloning promoters or other sequences of interest without the need for constructing or screening genomic libraries.
\r\n\r\nThe third chapter presents and discusses the similarity between these shark Po and MBP promoters, the JC virus enhancer (which directs tissue-specific expression in oligodendrocytes), and the mouse Po and MBP promoters. The implications of these findings on nervous system specific and CNS vs. PNS specific gene expression are discussed.
\r\n\r\nLastly, the appendix describes the current status of Shiverer transgenic mouse experiments in which constructs bearing the shark MBP gene are injected into mouse eggs. These transgenic experiments are testing if the structural similarity between shark and mammalian MBPs translates into any measurable functional similarity in vivo.
" }, { "name": "Mathers, Peter Hiram", "degree": "PhD", "year": "1990", "title": "Developmental regulation and chromosomal decondensation of the 68C glue gene cluster in Drosophila melanogaster", "advisor": "Meyerowitz, Elliot M.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06122007-144634", "creators": [ { "name": { "family": "Mathers", "given": "Peter Hiram" }, "id": "Mathers-P-H", "display_name": "Mathers, Peter Hiram" } ], "advisors": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "advisor", "display_name": "Meyerowitz, Elliot M." } ], "committee": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "chair", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Lipshitz", "given": "Howard D." }, "id": "Lipshitz-H-D", "role": "member", "display_name": "Lipshitz, Howard D." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." } ], "option_major": [ "biology" ], "doi": "10.7907/qm6d-4135", "abstract": "The larval salivary gland secretion genes (Sgs-3, Sgs-7 and Sgs-8) at chromosome position 68C in Drosophila melanogaster, are developmentally regulated and coordinately expressed. Each gene codes for a component of the mucoprotein glue which is synthesized in the third instar salivary glands. Expression of these three genes is associated with the 68C intermolt puff present in the polytene chromosomes of the salivary gland secretory cells. Expression occurs only in the salivary glands of third instar larvae, and requires the steroid hormone ecdysterone. We show that synthesis of the 68C glue gene RNAs is prevented in larvae carrying trans-acting mutations within the Broad-Complex (BR-C), while a puff persists at 68C in these mutant larvae. We use the l(1)su(f)[superscript ts67g] mutation (which has reduced ecdysterone levels and also prevents 68C glue gene expression) and a mutation in the BR-C (nprl[superscript 3]) to study the cis-acting elements responsible for interaction with trans-acting factors by analyzing the protein:DNA contacts which occur upstream of the Sgs-3 glue gene during active synthesis, using in vivo DMS-footprinting. The proximal promoter can direct tissue- and stage-specific expression and is shown to possess three protein-binding domains. Comparison of contacts at these three domains between expressing and non-expressing tissues (including salivary glands from (l)su(f)[superscript ts67g] and nprl[superscript 3] mutant larvae) identifies a single binding domain responsible for controlling developmental expression of Sgs-3.\n\nWe also analyze the cis-acting sequences required for the chromosomal puffing associated with 68C glue gene expression. Examination of various fragments of 68C DNA reintroduced into the Drosophila chromosomes by P-element transformation identifies a region of 152 basepairs between Sgs-7 and Sgs-8 which is necessary, but not sufficient, to promote puffing. Only when this region is accompanied by an adjacent promoter element is there a puff. The insertion sites containing these reintroduced fragments fail to puff in mutant larvae; therefore, formation of the 68C intermolt puff requires the products of the su(f) and BR-C loci, and the puff at 68C in mutant larvae is not the same puff as that associated with expression of Sgs-3, Sgs-7 and Sgs-8." }, { "name": "Mueller, Paul R.", "degree": "PhD", "year": "1990", "title": "In vivo analysis of interactions between trans-acting factors and their target genes", "advisor": "Wold, Barbara J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:07282014-134702796", "creators": [ { "name": { "family": "Mueller", "given": "Paul R." }, "id": "Mueller-P-R", "display_name": "Mueller, Paul R." } ], "advisors": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "advisor", "display_name": "Wold, Barbara J." } ], "committee": [ { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "member", "display_name": "Simon, Melvin I." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Zinn", "given": "Kai George" }, "id": "Zinn-K-G", "role": "member", "display_name": "Zinn, Kai George" } ], "option_major": [ "biology" ], "doi": "10.7907/yv7h-9335", "abstract": "The investigations presented in this thesis use various in vivo techniques to understand how trans-acting factors control gene expression. The first part addresses the transcriptional regulation of muscle creatine kinase (MCK). MCK expression is activated during the course of development and is found only in differentiated muscle. Several in vivo footprints are observed at the enhancer of this gene, but all of these interactions are limited to cell types that express MCK. This is interesting because two of the footprints appear to represent muscle specific use of general transcription factors, while the other two correspond to sites that can bind the myogenic regulator, MyoD1, in vitro. MyoD1 and these general factors are present in myoblasts, but can bind to the enhancer only in myocytes. This suggests that either the factors themselves are post-translationally modified (phosphorylation or protein:protein interactions), or the accessibility of the enhancer to the factors is limited (changes in chromatin structure). The in vivo footprinting study of MCK was performed with a new ligation mediated, single-sided PCR (polymerase chain reaction) technique that I have developed.
\r\n\r\nThe second half of the thesis concerns the regulation of mouse metallothionein (MT). Metallothioneins are a family of highly conserved housekeeping genes whose expression can be induced by heavy metals, steroids, and other stresses. By adapting a primer extension method of genomic sequencing to in vivo footprinting, I've observed both metal inducible and noninducible interactions at the promoter of MT-I. From these results I've been able to limit the possible mechanisms by which metal responsive trans-acting factors induce transcription. These interpretations correlate with a second line of experiments involving the stable titration of positive acting factors necessary for induction of MT. I've amplified the promoter of MT to 10^2-10^3 copies per cell by fusing the 5' and 3' ends of the MT gene to the coding region of DHFR and selecting cells for methotrexate resistance. In these cells, there is a metal-specific titration effect, and although it acts at the level of transcription, it appears to be independent of direct DNA binding factors.
\r\n" }, { "name": "Novak, Thomas John", "degree": "PhD", "year": "1990", "title": "Isolation of the murine interleukin 2 gene and characterization of its regulatory architecture", "advisor": "Rothenberg, Ellen V.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03192015-094028652", "creators": [ { "name": { "family": "Novak", "given": "Thomas John" }, "id": "Novak-T-J", "display_name": "Novak, Thomas John" } ], "advisors": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "advisor", "display_name": "Rothenberg, Ellen V." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/w4je-df30", "abstract": "Interleukin 2 (IL2) is the primary growth hormone used by mature T cells\r\nand this lymphokine plays an important role in the magnification of cell-mediated\r\nimmune responses. Under normal circumstances its expression is limited to\r\nantigen-activated type 1 helper T cells (TH1) and the ability to transcribe this\r\ngene is often regarded as evidence for commitment to this developmental\r\nlineage. There is, however, abundant evidence than many non-TH1 T cells, under\r\nappropriate conditions, possess the ability to express this gene. Of paramount\r\ninterest in the study of T-cell development is the mechanisms by which\r\ndifferentiating thymocytes are endowed with particular combinations of cell\r\nsurface proteins and response repertoires. For example, why do most helper T\r\ncells express the CD4 differentiation antigen?
\r\n\r\nAs a first step in understanding these developmental processes the gene\r\nencoding IL2 was isolated from a mouse genomic library by probing with a\r\nconspecific IL2 cDNA. The sequence of the 5' flanking region from + 1 to -2800\r\nwas determined and compared to the previously reported human sequence.\r\nExtensive identity exists between +1 and -580 (86%) and sites previously shown to\r\nbe crucial for the proper expression of the human gene are well conserved in both\r\nsequence location in the mouse counterpart.
\r\n\r\nTransient expression assays were used to evaluate the contribution of\r\nvarious genomic sequences to high-level gene expression mediated by a cloned IL2\r\npromoter fragment. Differing lengths of 5' flanking DNA, all terminating in the 5'\r\nuntranslated region, were linked to a reporter gene, bacterial chloramphenicol\r\nacetyltransferase (CAT) and enzyme activity was measured after introduction into\r\nIL2-producing cell lines. No CAT was ever detected without stimulation of the\r\nrecipient cells. A cloned promoter fragment containing only 321 bp of upstream\r\nDNA was expressed well in both Jurkat and EL4.El cells. Addition of intragenic\r\nor downstream DNA to these 5' IL2-CAT constructs showed that no obvious\r\nregulatory regions resided there. However, increasing the extent of 5' DNA from\r\n-321 to -2800 revealed several positive and negative regulatory elements. One\r\nnegative region that was well characterized resided between -750 and -1000 and\r\nconsisted almost exclusively of alternating purine and pyrimidines. There is no\r\nsequence resembling this in the human gene now, but there is evidence that there\r\nmay have once been.
\r\n\r\nNo region, when deleted, could relax either the stringent induction-dependence\r\non cell-type specificity displayed by this promoter. Reagents that\r\nmodulated endogenous IL2 expression, such as cAMP, cyclosporin A, and IL1,\r\naffected expression of the 5' IL2-CAT constructs also. For a given reagent,\r\nexpression from all expressible constructs was suppressed or enhanced to the same\r\nextent. This suggests that these modulators affect IL2 expression through\r\nperturbation of a central inductive signal rather than by summation of the effects\r\nof discrete, independently regulated, negative and positive transcription factors.
" }, { "name": "Preugschat, Frank", "degree": "PhD", "year": "1990", "title": "Proteolytic processing of the nonstructural proteins of dengue 2 virus", "advisor": "Strauss, James H.; Strauss, Ellen G.", "url": "https://resolver.caltech.edu/CaltechTHESIS:07282014-141222100", "creators": [ { "name": { "family": "Preugschat", "given": "Frank" }, "id": "Preugschat-F", "display_name": "Preugschat, Frank" } ], "advisors": [ { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "advisor", "display_name": "Strauss, James H." }, { "name": { "family": "Strauss", "given": "Ellen G." }, "id": "Strauss-E-G", "role": "co-advisor", "display_name": "Strauss, Ellen G." } ], "committee": [ { "name": { "family": "Brokaw", "given": "Charles J." }, "id": "Brokaw-C-J", "role": "member", "display_name": "Brokaw, Charles J." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." } ], "option_major": [ "biology" ], "doi": "10.7907/nn0p-gt03", "abstract": "The genomes of many positive stranded RNA viruses and of all\r\nretroviruses are translated as large polyproteins which are proteolytically\r\nprocessed by cellular and viral proteases. Viral proteases are structurally\r\nrelated to two families of cellular proteases, the pepsin-like and trypsin-like\r\nproteases. This thesis describes the proteolytic processing of several\r\nnonstructural proteins of dengue 2 virus, a representative member of the\r\nFlaviviridae, and describes methods for transcribing full-length genomic\r\nRNA of dengue 2 virus. Chapter 1 describes the in vitro processing of the\r\nnonstructural proteins NS2A, NS2B and NS3. Chapter 2 describes a system\r\nthat allows identification of residues within the protease that are directly or\r\nindirectly involved with substrate recognition. Chapter 3 describes\r\nmethods to produce genome length dengue 2 RNA from cDNA templates.
\r\n\r\nThe nonstructural protein NS3 is structurally related to viral trypsinlike\r\nproteases from the alpha-, picorna-, poty-, and pestiviruses. The\r\nhypothesis that the flavivirus nonstructural protein NS3 is a viral\r\nproteinase that generates the termini of several nonstructural proteins was\r\ntested using an efficient in vitro expression system and antisera specific for\r\nthe nonstructural proteins NS2B and NS3. A series of cDNA constructs\r\nwas transcribed using T7 RNA polymerase and the RNA translated in\r\nreticulocyte lysates. Proteolytic processing occurred in vitro to generate\r\nNS2B and NS3. The amino termini of NS2B and NS3 produced in vitro\r\nwere found to be the same as the termini of NS2B and NS3 isolated from\r\ninfected cells. Deletion analysis of cDNA constructs localized the protease\r\ndomain necessary and sufficient for correct cleavage to the first 184 amino\r\nacids of NS3. Kinetic analysis of processing events in vitro and experiments\r\nto examine the sensitivity of processing to dilution suggested that an\r\nintramolecular cleavage between NS2A and NS2B preceded an\r\nintramolecular cleavage between NS2B and NS3. The data from these\r\nexpression experiments confirm that NS3 is the viral proteinase\r\nresponsible for cleavage events generating the amino termini of NS2B and\r\nNS3 and presumably for cleavages generating the termini of NS4A and NS5\r\nas well.
\r\n\r\nBiochemical and genetic experiments using viral proteinases have\r\ndefined the sequence requirements for cleavage site recognition, but have\r\nnot identified residues within proteinases that interact with substrates. A\r\nbiochemical assay was developed that could identify residues which were\r\nimportant for substrate recognition. Chimeric proteases between yellow\r\nfever and dengue 2 were constructed that allowed mapping of regions\r\ninvolved in substrate recognition, and site directed mutagenesis was used\r\nto modulate processing efficiency.
\r\n\r\nExpression in vitro revealed that the dengue protease domain\r\nefficiently processes the yellow fever polyprotein between NS2A and NS2B\r\nand between NS2B and NS3, but that the reciprocal construct is inactive.\r\nThe dengue protease processes yellow fever cleavage sites more efficiently\r\nthan dengue cleavage sites, suggesting that suboptimal cleavage efficiency\r\nmay be used to increase levels of processing intermediates in vivo. By\r\nmutagenizing the putative substrate binding pocket it was possible to\r\nchange the substrate specificity of the yellow fever protease; changing a\r\nminimum of three amino acids in the yellow fever protease enabled it to\r\nrecognize dengue cleavage sites. This system allows identification of\r\nresidues which are directly or indirectly involved with enzyme-substrate\r\ninteraction, does not require a crystal structure, and can define the\r\nsubstrate preferences of individual members of a viral proteinase family.
\r\n\r\nFull-length cDNA clones, from which infectious RNA can be\r\ntranscribed, have been developed for a number of positive strand RNA\r\nviruses, including the flavivirus type virus, yellow fever. The technology\r\nnecessary to transcribe genomic RNA of dengue 2 virus was developed in\r\norder to better understand the molecular biology of the dengue subgroup. A\r\n5' structural region clone was engineered to transcribe authentic dengue\r\nRNA that contains an additional 1 or 2 residues at the 5' end. A 3'\r\nnonstructural region clone was engineered to allow production of run off\r\ntranscripts, and to allow directional ligation with the 5' structural region\r\nclone. In vitro ligation and transcription produces full-length genomic\r\nRNA which is noninfectious when transfected into mammalian tissue\r\nculture cells. Alternative methods for constructing cDNA clones and\r\nrecovering live dengue virus are discussed.
" }, { "name": "Ramaswami, Mani", "degree": "PhD", "year": "1990", "title": "Molecular genetic studies on voltage-gated ion channels", "advisor": "Tanouye, Mark", "url": "https://resolver.caltech.edu/CaltechTHESIS:07282014-102712405", "creators": [ { "name": { "family": "Ramaswami", "given": "Mani" }, "id": "Ramaswami-M", "display_name": "Ramaswami, Mani" } ], "advisors": [ { "name": { "family": "Tanouye", "given": "Mark" }, "id": "Tanouye-M", "role": "advisor", "display_name": "Tanouye, Mark" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/ycfq-1k97", "abstract": "Several different methods have been employed in the study of voltage-gated ion\r\nchannels. Electrophysiological studies on excitable cells in vertebrates and molluscs have\r\nshown that many different voltage-gated potassium (K+) channels and sodium channels\r\nmay coexist in the same organism. Parallel genetic studies in Drosophila have identified\r\nmutations in several genes that alter the properties of specific subsets of physiologically\r\nidentified ion channels. Chapter 2 describes molecular studies that identify two Drosophila\r\nhomologs of vertebrate sodium-channel genes. Mutations in one of these Drosophila\r\nsodium-channel genes are shown to be responsible for the temperature-dependent paralysis\r\nof a behavioural mutant parats. Evolutionary arguments, based on the partial sequences of\r\nthe two Drosophila genes, suggest that subfamilies of voltage-gated sodium channels in\r\nvertebrates remain to be identified.
\r\n\r\nIn Drosophila, diverse voltage-gated K+ channels arise from alternatively spliced\r\nmRNAs generated at the Shaker locus. Chapter 3 and the Appendices describe the isolation\r\nand characterization of several human K+-channel genes, similar in sequence to Shaker.\r\nEach of these human genes has a highly conserved homolog in rodents; thus, this K+-channel\r\ngene family probably diversified prior to the mammalian radiation. Functional K+\r\nchannels encoded by these genes have been expressed in Xenopus oocytes and their\r\nproperties have been analyzed by electrophysiological methods. These studies demonstrate\r\nthat both transient and noninactivating voltage-gated K+ channels may be encoded by\r\nmammalian genes closely related to Shaker. In addition, results presented in Appendix 3\r\nclearly demonstrate that independent gene products from two K+-channel genes may\r\nefficiently co-assemble into heterooligomeric K+ channels with properties distinct from\r\neither homomultimeric channel. This finding suggests yet another molecular mechanism for\r\nthe generation of K+-channel diversity.
" }, { "name": "Topol, Joanne", "degree": "PhD", "year": "1990", "title": "Transcriptional control of the drosophila segmentation gene fushi tarazu", "advisor": "Parker, Carl Stevens", "url": "https://resolver.caltech.edu/CaltechETD:etd-08292007-085427", "creators": [ { "name": { "family": "Topol", "given": "Joanne" }, "id": "Topol-J", "display_name": "Topol, Joanne" } ], "advisors": [ { "name": { "family": "Parker", "given": "Carl Stevens" }, "id": "Parker-C-S", "role": "advisor", "display_name": "Parker, Carl Stevens" } ], "committee": [ { "name": { "family": "Parker", "given": "Carl Stevens" }, "id": "Parker-C-S", "role": "chair", "display_name": "Parker, Carl Stevens" }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "role": "member", "display_name": "Emr, Scott D." } ], "option_major": [ "biology" ], "doi": "10.7907/mvhc-0a34", "abstract": "The Drosophila segmentation gene fushi tarazu (ftz) is expressed in a characteristic pattern of seven stripes during early embryogenesis. The promoter sequences sufficient to direct this stripe pattern are located within the 670 base pairs (bp) proximal to the ftz transcriptional start site. When we extract nuclear proteins from 0-12 hour Drosophila embryos, we find sequence-specific DNA binding proteins that recognize multiple sites within the 670 by zebra stripe promoter. This observation suggests that the control of ftz zebra stripe expression may require a complex array of transcriptional regulators. The results of our P element-mediated transformation experiments using ftz promoter/ [beta]-galactosidase fusion genes confirm this notion. They demonstrate that the zebra stripe promoter contains multiple, distinct activator and repressor recognition elements responsible for the formation of ftz stripes. The transformation studies also reveal that a pattern of general activation, that is, a continuous band of gene expression throughout the germ band, can be generated when repressor recognition sites are deleted from the fusion gene promoter and activator sites are retained. This result strongly supports a mechanism for ftz stripe formation involving general transcriptional activation and localized repression.\n\nStudies with constructs in which individual protein binding sites have been deleted or added to the ftz promoter correlate protein recognition elements with regulatory functions. We characterized two distinct interband repressor sites and two distinct general activator sites with this approach. One activator site recognizes the product of the homeobox gene caudal (cad); the other contains a DNA sequence motif found in the cis-activators of a number of Drosophila genes. As would be expected for general activators, both these sites are able to mediate expression throughout most of the germ band. We also demonstrate that when multiple copies of two distinct repressor recognition sites are independently ligated to a portion of the ftz promoter, they transform the continuous band of gene expression generated by a group of endogenous cis-activators into the characteristic seven stripe pattern of ftz expression. Finally, we find that multiple copies of these repressor elements are more capable of mediating repression than single copies.\n" }, { "name": "Eakle, Kurt Andrew", "degree": "PhD", "year": "1989", "title": "Characterization of the SEC18 Gene of S. cerevisiae: Identification of a Protein Involved in Yeast Secretion", "advisor": "Emr, Scott D.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05302007-153631", "creators": [ { "name": { "family": "Eakle", "given": "Kurt Andrew" }, "id": "Eakle-Kurt-Andrew", "display_name": "Eakle, Kurt Andrew" } ], "advisors": [ { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "orcid": "0000-0002-5408-6781", "role": "advisor", "display_name": "Emr, Scott D." } ], "committee": [ { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "orcid": "0000-0002-5408-6781", "role": "chair", "display_name": "Emr, Scott D." }, { "name": { "family": "Abelson", "given": "John N." }, "id": "Abelson-J-N", "role": "member", "display_name": "Abelson, John N." }, { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "member", "display_name": "Revel, Jean-Paul" }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "member", "display_name": "Strauss, James H." } ], "option_major": [ "biology" ], "doi": "10.7907/6aqb-xd83", "abstract": "SEC18 gene function is required for secretory protein transport between the endoplasmic reticulum and the Golgi complex. We have cloned the SEC18 gene by complementation of the secl8-1 mutation. Deletion/disruption of this gene has shown that SEC18 is essential for yeast cell growth. Sequence analysis of the gene revealed a 2271 by open reading frame which would code for a protein of 83.9 kd. The predicted protein sequence showed no significant homology to other known protein sequences. In vitro transcription and translation of SEC18 led to the synthesis of two proteins of approximately 84 and 82 kd. Antisera raised against a Sec18-?-galactosidase fusion protein, detects two proteins from in vivo 35S labeled yeast cells identical in size to those seen by in vitro translation. Although potential sites for N-linked glycosylation are present in the Sec 18p sequence, the sizes of the in vivo SEC18 gene products are unaffected by the drug tunicamycin. Hydrophobicity analysis indicated that the protein is hydrophilic in nature and lacks any region that would be predicted to serve as a signal sequence or transmembrane anchor. These results suggest that the Secl8p resides in the cell cytoplasm. Pulse-chase experiments indicate that the two forms of Sec 18 protein are not the result of post-translational processing. Mapping of the 5' end of the SEC18 mRNA revealed only one major start site for transcription, which indicates that the multiple forms of Sec 18 protein do not arise from mRNAs with different 5' ends. We suggest that translation initiating at different in-frame AUG start codons is likely to account for the presence of two forms of the Sec 18 protein. While cell fractionation studies show that the Sec 18p are not associated with ER or Golgi compartments, association with a 100,000 x g pellet fraction has been observed suggesting that Sec 18p may bind transiently to small vesicles such as those presumed to participate in ER to Golgi transport.
" }, { "name": "Hahn, Young Shin Lim", "degree": "PhD", "year": "1989", "title": "Functional Analysis of Viral Nonstructural and Structural Proteins", "advisor": "Strauss, James H.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06072007-075259", "creators": [ { "name": { "family": "Hahn", "given": "Young Shin Lim" }, "id": "Hahn-Young-Shin-Lim", "display_name": "Hahn, Young Shin Lim" } ], "advisors": [ { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "advisor", "display_name": "Strauss, James H." } ], "committee": [ { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "chair", "display_name": "Strauss, James H." }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "role": "member", "display_name": "Emr, Scott D." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/3trk-k698", "abstract": "The genera Alphavirus and Flavivirus contain enveloped RNA viruses which are similar in size, morphology, and RNA content, and were once considered to belong to the same family, the Togaviridae. Recently the flaviviruses were reclassified as a separate family, the Flaviviridae, because they differ markedly from alphaviruses in replication strategy and mode of assembly as well as genome organization. In this thesis, representatives of both groups have been studied in order to understand the functions of the various virus-encoded proteins in replication and pathogenesis. Part I describes the mapping of temperature-sensitive (ts) RNA- mutants of the alphavirus Sindbis virus, to elucidate the function of each nonstructural protein during RNA replication. Part II includes the determination of the complete nucleotide sequence of the flavivirus dengue 2, comparative analysis of conserved elements in the 3' untranslated region of various flaviviruses, and expression of dengue 2 structural proteins in a recombinant vaccinia virus.
\r\n\r\nDuring alphavirus replication, the parental 49S plus-strand RNA is transcribed into a complementary minus-strand RNA which serves as the template for the synthesis of both plus-strand 49S genomic RNA and 26S subgenomic RNA. The nonstructural proteins, which are involved in viral RNA replication, are translated from the genomic 49S RNA as two polyprotein precursors that are processed by cotranslational or posttranslational cleavage into four final polypeptide products.
\r\n\r\nts RNA- mutants of Sindbis virus have been isolated previously and grouped by complementation into four groups (A, B, F, G); these mutants fail to make RNA after infection at a nonpermissive temperature and were presumed to contain ts lesions in the nonstructural proteins. Over a number of years, work in our own and other laboratories has established the details of the RNA- phenotypes of these mutants, which include defects in RNA chain elongation, in initiation of genomic and 26S RNA synthesis, in regulation of minus-strand template synthesis, and in processing of polyprotein precursors. However, it is only with the mapping described here that it has been possible to unambiguously assign these functions to particular nonstructural polypeptides. The mutations responsible for the is phenotype of Sindbis complementation group F mutants ts6, tsl10, and tsl18, have been determined. ts6 and ts110 have a single base substitution in nsP4 resulting in a replacement of Gly by Glu at position 153 or position 324, respectively. It is of interest that nsP4 contains the Gly-Asp-Asp motif characteristic of a number of viral replicases, and this together with the fact that all RNA synthesis in ts6-infected cells is shut off upon shift-up from the permissive to the nonpermissive temperature suggests that nsP4 is the viral RNA polymerase or elongation enzyme. tsl18 is a double mutant where one mutation is in nsP2 (the Val at residue 425 is changed to Ala). This mutation alone causes the formation of minute plaques at the nonpermissive condition without a reduction in the plaque number. The second change (Gln-93 of nsP4 changed to Arg) has little apparent phenotype on its own, but in combination with the change in nsP2 it leads to a temperature-sensitive phenotype. This suggests that nsP2 and nsP4 interact with one another in a complex.
\r\n\r\nWe also have mapped representatives of RNA- complementation groups A, B, and G. Mutants belonging to groups A and G have been found to map in nsP2, suggesting that this protein is required for initiation of 26S RNA synthesis, proteolytic processing of the nonstructural precursor, and shut off of minus-strand synthesis. tsl1, the only member of group B, has a mutation in nsP1, indicating that this protein is responsible for initiation of minus-strand synthesis.
\r\n\r\nTo understand the role of viral structural proteins in the pathogenesis of flaviviruses, we studied one of the dengue viruses, which constitute a worldwide health problem of increasing dimensions, and determined the complete nucleotide sequence of the PRl59-S1 strain of dengue 2 virus except for l5 nucleotides at the 5' end. There is one long open reading frame which is translated to give the structural proteins, capsid (C), membrane-like protein (M), and envelope protein (E), followed by nonstructural proteins, NS1, NS2, NS3, NS4, and NS5. The individual proteins appear to be produced by posttranslational cleavage of a precursor polyprotein. There are nucleotide sequences in the 5' terminal region (in the coding region for the capsid protein) and in the 3' terminal region (in the 3' untranslated sequence) that are invariant among flaviviruses examined to date and that may be involved in cyclization of the RNA. These sequences are presumed to be important for viral replication. In addition, the 3' terminal 79 nucleotides are capable of forming a hairpin structure.
\r\n\t\r\nWe have expressed the structural proteins of dengue 2 using a recombinant vaccinia virus to study the role of these proteins in the immunological response. The vaccinia recombinant containing a cDNA copy of the 5' region of the dengue genome virus expressed dengue structural proteins which are correctly cleaved and modified. This suggests that the sequences encoding the structural proteins specify all the necessary catalytic activities or recognition signals required to ensure the proper synthesis and maturation of the polypeptides. And also, this recombinant can generate dengue-specific antibodies in mice which neutralize viral infectivity.
" }, { "name": "Normanly, Jennifer", "degree": "PhD", "year": "1989", "title": "An in vivo Approach to tRNA Identity", "advisor": "Abelson, John N.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06032013-145407392", "creators": [ { "name": { "family": "Normanly", "given": "Jennifer" }, "id": "Normanly-Jennifer", "display_name": "Normanly, Jennifer" } ], "advisors": [ { "name": { "family": "Abelson", "given": "John N." }, "id": "Abelson-J-N", "role": "advisor", "display_name": "Abelson, John N." } ], "committee": [ { "name": { "family": "Abelson", "given": "John N." }, "id": "Abelson-J-N", "role": "chair", "display_name": "Abelson, John N." }, { "name": { "family": "Dervan", "given": "Peter B." }, "id": "Dervan-P-B", "orcid": "0000-0001-8852-7306", "role": "member", "display_name": "Dervan, Peter B." }, { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "role": "member", "display_name": "Emr, Scott D." }, { "name": { "family": "Richards", "given": "John H." }, "id": "Richards-J-H", "role": "member", "display_name": "Richards, John H." }, { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "member", "display_name": "Simon, Melvin I." } ], "option_major": [ "biology" ], "doi": "10.7907/s9xt-6f20", "abstract": "A leucine-inserting tRNA has been transformed into a serine-inserting tRNA by changing 12 nucleotides. Only 8 of the 12 changes are required to effect the conversion of the leucine tRNA to serine tRNA identity. The 8 essential changes reside in basepair 11-24 in the D stem, basepairs 3-70, 2-71 and nucleotides 72 and 73, all of the acceptor stem.
\r\n\r\nFunctional amber suppressor tRNA genes were generated for 14 species of tRNA in E. coli, and their amino acid specificities determined. The suppressors can be classified into three groups, based upon their specificities. Class I suppressors, tRNAAla2CUA, tRNAGlyUCUA, tRNAHisACUA, tRNALysCUA, and tRNAProHCUA, inserted the predicted amino acid. The Class II suppressors, tRNAGluACUA, tRNAGlyTCUA, and tRNAIle1CUA were either partially or predominantly mischarged by the glutamine aminoacyl tRNA synthetase (AAS). The Class III suppressors, tRNAArgCUA, tRNAAspMCUA, tRNAIle2CUA, tRNAThr2CUA, tRNAMet(m)CUA and tRNAValCUA inserted predominantly lysine.
" }, { "name": "Renfranz, Patricia Jean", "degree": "PhD", "year": "1989", "title": "Molecular Neurogenetics of Eye Development in Drosophila", "advisor": "Benzer, Seymour", "url": "https://resolver.caltech.edu/CaltechTHESIS:08262013-140100846", "creators": [ { "name": { "family": "Renfranz", "given": "Patricia Jean" }, "id": "Renfranz-Patricia-Jean", "display_name": "Renfranz, Patricia Jean" } ], "advisors": [ { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "advisor", "display_name": "Benzer, Seymour" } ], "committee": [ { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "chair", "display_name": "Benzer, Seymour" }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "member", "display_name": "Simon, Melvin I." }, { "name": { "family": "Sternberg", "given": "Paul W." }, "id": "Sternberg-P-W", "orcid": "0000-0002-7699-0173", "role": "member", "display_name": "Sternberg, Paul W." } ], "option_major": [ "biology" ], "doi": "10.7907/15zs-fh27", "abstract": "The compound eye of Drosophila melanogaster begins to differentiate during the late third larval instar in the eye-antennal imaginal disc. A wave of morphogenesis crosses the disc from posterior to anterior, leaving behind precisely patterned clusters of photoreceptor cells and accessory cells that will constitute the adult ommatidia of the retina. By the analysis of genetically mosaic eyes, it appears that any cell in the eye disc can adopt the characteristics of any one of the different cell types found in the mature eye, including photoreceptor cells and non-neuronal accessory cells such as cone cells. Therefore, cells within the prospective retinal epithelium assume different fates presumably via information present in the environment. The sevenless\u207a (sev\u207a) gene appears to play a role in the expression of one of the possible fates, since the mutant phenotype is the lack of one of the pattern elements, namely, photoreceptor cell R7. The sev\u207a gene product had been shown to be required during development of the eye, and had also been shown in genetic mosaics to be autonomous to presumptive R7. As a means of better understanding the pathway instructing the differentiation R7, the gene and its protein product were characterized.
\r\n\r\nThe sev\u207a gene was cloned by P-element transposon tagging, and was found to encode an 8.2 kb transcript expressed in developing eye discs and adult heads. By raising monoclonal antibodies (MAbs) against a sev\u207a-\u03b2-galactosidase fusion protein, the expression of the protein in the eye disc was localized by immuno-electronmicroscopy. The protein localizes to the apical cell membranes and microvilli of cells in the eye disc epithelium. It appears during development at a time coincident with the initial formation of clusters, and in all the developing photoreceptors and accessory cone cells at a time prior to the overt differentiation of R7. This result is consistent with the pluripotency of cells in the eye disc. Its localization in the membranes suggests that it may receive information directing the development of R7. Its localization in the apical membranes and microvilli is away from the bulk of the cell contacts, which have been cited as a likely regions for information presentation and processing. Biochemical characterization of the sev\u207a protein will be necessary to describe further its role in development.
\r\n\r\nOther mutations in Drosophila have eye phenotypes. These were analyzed to find which ones affected the initial patterning of cells in the eye disc, in order to identify other genes, like sev, whose gene products may be involved in generating the pattern. The adult eye phenotypes ranged from severe reduction of the eye, to variable numbers of photoreceptor cells per ommatidium, to sub de defects in the organization of the supporting cells. Developing eye discs from the different strains were screened using a panel of MAbs, which highlight various developmental stages. Two identified matrix elements in and anterior to the furrow, while others identified the developing ommatidia themselves, like the anti-sev MAb. Mutation phenotypes were shown to appear at many stages of development. Some mutations seem to affect the precursor cells, others, the setting up of the pattern, and still others, the maintenance of the pattern. Thus, additional genes have now been identified that may function to support the development of a complex pattern.
" }, { "name": "Sivertsen, Dave William", "degree": "PhD", "year": "1989", "title": "The Physiology of High-Order Visual Neurons in the Jumping Spider (Salticidae) and the Vocalizations of Free Ranging Owl Monkeys", "advisor": "Allman, John Morgan", "url": "https://resolver.caltech.edu/CaltechETD:etd-12092008-091408", "creators": [ { "name": { "family": "Sivertsen", "given": "Dave William" }, "id": "Sivertsen-Dave-William", "display_name": "Sivertsen, Dave William" } ], "advisors": [ { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "advisor", "display_name": "Allman, John Morgan" } ], "committee": [ { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "chair", "display_name": "Allman, John Morgan" }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "member", "display_name": "Van Essen, David C." }, { "name": { "family": "Tanouye", "given": "Mark" }, "id": "Tanouye-M", "role": "member", "display_name": "Tanouye, Mark" }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "member", "display_name": "Owen, Ray David" } ], "option_major": [ "neurobio" ], "doi": "10.7907/7V43-WY98", "abstract": "Jumping spiders have a highly specialized visual system for the detection and analysis of prey. The retina of each of the anterior medial eyes has a compact, high-resolution, three-dimensional array of receptors that can scan, saccade, and engage in slow tracking. Qualitative study, computer-controlled data collection, and randomized stimulus presentation were used in an electrophysiological study of the properties of high-order visual neurons.
\r\n\r\nReceptive field mapping revealed neurons with well-defined, positionally stable, receptive fields. Some of these receptive fields had an angular extent greater than that of the static retinal array. The stability with respect to the cephalothorax and the angular extent of these receptive fields suggest integration of retinal information and eye position, to yield position constancy. Most neurons had receptive fields of constant angular subtense as the distance to the tangent screen varied. Some neurons have receptive fields that remain relatively constant in absolute physical size (changing in angular extent) as screen distance varies. Such size constant cells may be useful in assessing prey suitability. These characteristics require integration of depth information. These cells are driven monocularly, ruling out disparity cues. Jumping spiders do not use muscular accommodation. Instead, the retina has a layered, tiered structure. Depth of focus cues are probably the source of this information, as hypothesized previously by Land (1969a).
\r\n\r\nQuantitative assessment of velocity preferences revealed cells with medial eye input responding optimally to velocities of 16\u00b0/second, and other cells responding to higher velocities. Tuning for direction of movement orientation was also observed and quantified. The degree of tuning correlates with a measure of latency.
\r\n\r\nResults are also presented in a separate section from a primate behavior study on the vocalizations of the owl monkey, in which the vocal repertoire of two populations was recorded. Field recordings were analyzed for temporal and spectral properties of both the vocalizations and the acoustic context. We found no systematic variations of calls between populations, except for the number of syllables in the loud call. We examined behavioral context and probable function of inter and intragroup vocalizations. We found that the loud call may carry gender information, with a bimodal distribution of the spectral bandwidth of the fundamental.
\r\n\r\nThis work was supported in part by a National Research Service Award (T32 GM 07737) from the National Institute of General Medical Sciences, and a Lawrence A. Hanson Fellowship.
" }, { "name": "Sucov, Henry Michael", "degree": "PhD", "year": "1989", "title": "Characterization and Developmental Regulation of a Gene Expressed Specifically in the Skeletogenic Lineage of the Sea Urchin Embryo", "advisor": "Davidson, Eric H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08262013-094712996", "creators": [ { "name": { "family": "Sucov", "given": "Henry Michael" }, "id": "Sucov-Henry-Michael", "display_name": "Sucov, Henry Michael" } ], "advisors": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "advisor", "display_name": "Davidson, Eric H." } ], "committee": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "chair", "display_name": "Davidson, Eric H." }, { "name": { "family": "Lazarides", "given": "Elias" }, "id": "Lazarides-E", "role": "member", "display_name": "Lazarides, Elias" }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." } ], "option_major": [ "biology" ], "doi": "10.7907/zm4z-4942", "abstract": "The sea urchin embryonic skeleton, or spicule, is deposited by mesenchymal progeny of four precursor cells, the micromeres, which are determined to the skeletogenic pathway by a process known as cytoplasmic localization. A gene encoding one of the major products of the skeletogenic mesenchyme, a prominent 50 kD protein of the spicule matrix, has been characterized in detail. cDNA clones were first isolated by antibody screening of a phage expression library, followed by isolation of homologous genomic clones. The gene, known as SM50, is single copy in the sea urchin genome, is divided into two exons of 213 and 1682 bp, and is expressed only in skeletogenic cells. Transcripts are first detectable at the 120 cell stage, shortly after the segregation of the skeletogenic precursors from the rest of the embryo. The SM50 open reading frame begins within the first exon, is 450 amino acids in length, and contains a loosely repeated 13 amino acid motif rich in acidic residues which accounts for 45% of the protein and which is possibly involved in interaction with the mineral phase of the spicule.
\r\n\r\nThe important cis-acting regions of the SM50 gene necessary for proper regulation of expression were identified by gene transfer experiments. A 562 bp promoter fragment, containing 438 bp of 5' promoter sequence and 124 bp of the SM50 first exon (including the SM50 initiation codon), was both necessary and sufficient to direct high levels of expression of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene specifically in the skeletogenic cells. Removal of promoter sequences between positions -2200 and -438, and of transcribed regions downstream of +124 (including the SM50 intron), had no effect on the spatial or transcriptional activity of the transgenes.
\r\n\r\nRegulatory proteins that interact with the SM50 promoter were identified by the gel retardation assay, using bulk embryo mesenchyme blastula stage nuclear proteins. Five protein binding sites were identified and mapped to various degrees of resolution. Two sites are homologous, may be enhancer elements, and at least one is required for expression. Two additional sites are also present in the promoter of the aboral ectoderm specific cytoskeletal actin gene CyIIIa; one of these is a CCAA T element, the other a putative repressor element. The fifth site overlaps the binding site of the putative repressor and may function as a positive regulator by interfering with binding of the repressor. All of the proteins are detectable in nuclear extracts prepared from 64 cell stage embryos, a stage just before expression of SM50 is initiated, as well as from blastula and gastrula stage; the putative enhancer binding protein may be maternal as well.
" }, { "name": "Vijayraghavan, Usha", "degree": "PhD", "year": "1989", "title": "A Genetic and Biochemical Analysis of pre-mRNA Splicing in Saccharomyces cerevisiae", "advisor": "Abelson, John N.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10172013-115606044", "creators": [ { "name": { "family": "Vijayraghavan", "given": "Usha" }, "id": "Vijayraghavan-Usha", "display_name": "Vijayraghavan, Usha" } ], "advisors": [ { "name": { "family": "Abelson", "given": "John N." }, "id": "Abelson-J-N", "role": "advisor", "display_name": "Abelson, John N." } ], "committee": [ { "name": { "family": "Abelson", "given": "John N." }, "id": "Abelson-J-N", "role": "chair", "display_name": "Abelson, John N." }, { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "role": "member", "display_name": "Campbell, Judith L." }, { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "role": "member", "display_name": "Emr, Scott D." }, { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "member", "display_name": "Simon, Melvin I." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." } ], "option_major": [ "biology" ], "doi": "10.7907/cxmk-nj42", "abstract": "Pre-mRNA splicing requires interaction of cis- acting intron sequences with trans -acting factors: proteins and small nuclear ribonucleoproteins (snRNPs). The assembly of these factors into a large complex, the spliceosome, is essential for the subsequent two step splicing reaction. First, the 5' splice site is cleaved and free exon 1 and a lariat intermediate (intron- exon2) form. In the second reaction the 3' splice site is cleaved the exons ligated and lariat intron released. A combination of genetic and biochemical techniques have been used here to study pre-mRNA splicing in yeast.
\r\n\r\nYeast introns have three highly conserved elements. We made point mutations within these elements and found that most of them affect splicing efficiency in vivo and in vitro, usually by inhibiting spliceosome assembly.
\r\n\r\nTo study trans -acting splicing factors we generated and screened a bank of temperature-sensitive (ts) mutants. Eleven new complementation groups (prp17 to prp27) were isolated. The four phenotypic classes obtained affect different steps in splicing and accumulate either: 1) pre-mRNA, 2) lariat intermediate, 3) excised intron or 4) both pre-mRNA and intron. The latter three classes represent novel phenotypes. The excised intron observed in one mutant: prp26 is stabilized due to protection in a snRNP containing particle. Extracts from another mutant: prpl8 are heat labile and accumulate lariat intermediate and exon 1. This is especially interesting as it allows analysis of the second splicing reaction. In vitro complementation of inactivated prp18 extracts does not require intact snRNPs. These studies have also shown the mutation to be in a previously unknown splicing protein. A specific requirement for A TP is also observed for the second step of splicing. The PRP18 gene has been cloned and its polyadenylated transcript identified.
" }, { "name": "Callaway, Edward Matthew", "degree": "PhD", "year": "1988", "title": "Regulation of Competitive Interactions During Neuromuscular Synapse Elimination", "advisor": "Van Essen, David C.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01172013-084333489", "creators": [ { "name": { "family": "Callaway", "given": "Edward Matthew" }, "id": "Callaway-Edward-Matthew", "display_name": "Callaway, Edward Matthew" } ], "advisors": [ { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "advisor", "display_name": "Van Essen, David C." } ], "committee": [ { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "chair", "display_name": "Van Essen, David C." }, { "name": { "family": "Bower", "given": "James M." }, "id": "Bower-J-M", "role": "member", "display_name": "Bower, James M." }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." }, { "name": { "family": "Tanouye", "given": "Mark" }, "id": "Tanouye-M", "role": "member", "display_name": "Tanouye, Mark" } ], "option_major": [ "biology" ], "doi": "10.7907/s60x-n119", "abstract": "The subsequent chapters of this thesis address a number of issues related to the phenomena that occur during neuromuscular synapse elimination and to the rules and mechanisms that govern them. The results they describe are therefore based on observations of developmental processes in both normal animals and in those whose normal developmental interactions have been perturbed by alteration of activity for some of the elements involved.
\r\n\r\nChapter 2 addresses the question of whether the rate of neuromuscular synapse elimination might normally depend on the level of postsynaptic activity. Previous studies had strongly implicated activity in regulation of synapse elimination rate; e.g., increased activity evoked by chronic stimulation increased the elimination rate; but these studies did not differentiate between pre- and postsynaptic activity. Duxson (1982) treated the rat soleus muscle with \u03b1-bungarotoxin (\u03b1-BGT), completely blocking postsynaptic activity during the normal period of synapse elimination and reported that the number of terminal profiles per endplate observed in electron micrographs did not decrease as it does normally. I did not consider this study to be conclusive because complete activity blockade might invoke influences that are not normally present in active muscles during synapse elimination, and because the assay was indirect - an increase in the number of terminal profiles per endplate might reflect an increase in terminal complexity rather than maintenance of more terminals.
\r\n\r\nFor the experiments described in Chapter 2, postsynaptic activity was partially blocked by \u03b1-BGT superfusion of the neonatal rabbit soleus muscle. The toxin treatment resulted in slower synapse elimination, as assessed both physiologically and anatomically, even for muscle fibers whose activity was not completely blocked. While the interpretation of this result is dependent on the possibility that \u03b1-BGT has influences other than decreased activity, it appears quite likely that a partial block of postsynaptic activity can slow the rate of neuromuscular synapse elimination.
\r\n\r\nChapter 3 describes a separate series of experiments in which motor unit twitch tensions were assayed for the soleus muscles of neonatal rabbits. Synapse loss could be assayed separately for fast and slow populations by separating the motor units, based on their twitch rise times. Estimates of the rate of synapse elimination for the two populations suggested that slow muscle fibers were initially more heavily polyinnervated than fast fibers and that they lost synapses at a faster rate, so that both populations of fibers became predominantly singly innervated at about the same time. The remainder of the issues in Chapter 3 are related to the question of whether there are particular attributes of motor neurons that might place the inputs from some neurons at a competitive advantage over others.
\r\n\r\nThe first such issue was whether motor neurons with relatively large axonal arbors are at an advantage or disadvantage in the competition for synaptic sites. If this were the case, it would be expected that the diversity in motor unit sizes would decrease during synapse elimination if a large arbor were a disadvantage, and thediversity would increase if it were an advantage. Contrary to both hypotheses, no significant change in the diversity of motor unit sizes was observed.
\r\n\r\nThe next issue was whether motor neurons from particular positions in the spinal cord were at an advantage or disadvantage compared to the others. To test this issue, mean sizes of motor units from both rostral and caudal extremes of the soleus motor pool were compared to the mean sizes for those from the middle of the pool. At the earliest age tested, when the soleus is still heavily polyinnervated, the motor units from the extremes were no smaller than those from the middle. However, just four days later, the units from each extreme were significantly smaller than those from the middle; this difference persisted in older, singly innervated muscles. There was no significant difference between the rostral and caudal motor units at any age tested. It is concluded that motor neurons from rostral and caudal extremes are at a disadvantage when in competition with those from the middle of the motor pool during synapse elimination in the rabbit soleus.
\r\n\r\nFinally, a small portion of the rabbit soleus motor neurons was inactivated by implantation of a tetrodotoxin-laden Silastic plug during synapse elimination. Since there was nearly complete overlap between the inactivated and active motor units at the time of the implant, this allowed a test of whether the level of activity of a motor neuron can influence the ability of its terminals to compete for sole occupancy of endplates. It was found that the inactive motor units ended up significantly larger than their active counterparts in normal and control implanted animals, and remained larger even after the endplates were virtually all singly innervated. It is concluded that inactivity can result in a significant competitive advantage during synapse elimination. The generality of these conclusions and their implications in terms of the ways in which neuromuscular synapse elimination might be regulated are discussed in detail.
" }, { "name": "Chang, Caren", "degree": "PhD", "year": "1988", "title": "Molecular Genetic Studies in Arabidopsis thaliana: I. Cloning and Characterization of the Alcohol Dehydrogenase Gene; II. Complementation of an Alcohol Dehydrogenase Mutant; III. A Restriction Fragment Length Polymorphism Map of the Genome\r ", "advisor": "Meyerowitz, Elliot M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01182013-085552595", "creators": [ { "name": { "family": "Chang", "given": "Caren" }, "id": "Chang-Caren", "display_name": "Chang, Caren" } ], "advisors": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "advisor", "display_name": "Meyerowitz, Elliot M." } ], "committee": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "chair", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "member", "display_name": "Simon, Melvin I." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." } ], "option_major": [ "molbio" ], "doi": "10.7907/H8DS-A792", "abstract": "This thesis examines several genetic aspects of the flowering plant Arabidopsis thaliana which has previously been shown to possess several attributes for molecular genetic experiments such as a short life cycle, small size, fecundity, and a strong background of classical genetics. In particular, the nuclear genome is remarkably small and consists almost entirely of single copy sequences.
\r\n\r\nTwo approaches are established for the isolation and study of plant genes using Arabidopsis as a model system. One approach demonstrates complementation of a mutant phenotype using the Arabidopsis alcohol dehydrogenase (ADH) gene. The ADH gene was first isolated from a genomic DNA library by cross-hybridization with a maize ADH1 gene probe. The gene structure was studied by DNA sequence analysis and mapping of the transcript. The gene conferred wild-type ADH activity to an Arabidopsis ADH null mutant when introduced by Agrobacterium-mediated transformation. Transformed plants were subjected to genetic and molecular analyses. The ADH gene may have utility as a gene tag in transformation experiments.
\r\n\r\nThe second topic of this thesis is the construction of a genetic linkage map of restriction fragment length polymorphisms (RFLPs). The map provides an approach to cloning genes about which nothing more is known than a mutant phenotype and a map location because the RFLP markers can serve as starting points for the isolation of overlapping clones from a genomic DNA library. The map contains 90 RFLP markers distributed randomly throughout the nuclear genome. Since the genome consists of about 70,000 kilobase pairs, the markers are at an average physical spacing of approximately 780 kilobase pairs. The map is based on meiotic segregation of RFLPs in two different crosses detected with the restriction enzymes EcoRI, BglII, and XbaI. The RFLP linkage groups have been aligned with the standard genetic map of approximately 80 mutation markers.
" }, { "name": "Garfinkel, Mark David", "degree": "PhD", "year": "1988", "title": "Structural and Functional Studies of the 68C Glue Protein Gene Cluster of Drosophila melanogaster", "advisor": "Meyerowitz, Elliot M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01222013-140636948", "creators": [ { "name": { "family": "Garfinkel", "given": "Mark David" }, "id": "Garfinkel-Mark-David", "display_name": "Garfinkel, Mark David" } ], "advisors": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "advisor", "display_name": "Meyerowitz, Elliot M." } ], "committee": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "chair", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "member", "display_name": "Lewis, Edward B." }, { "name": { "family": "Parker", "given": "Carl Stevens" }, "id": "Parker-C-S", "role": "member", "display_name": "Parker, Carl Stevens" }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." } ], "option_major": [ "biology" ], "doi": "10.7907/gjtw-k928", "abstract": "The 68C locus of the Drosophila melanogaster polytene chromosomes contains the structural genes for three glue polypeptides (sgs-3, sgs-7, and sgs-8) synthesized in the third instar larval salivary glands. The three 68C glue mRNAs are coded in a gene cluster of less than 5000 base-pairs, and are expressed coordinately under the control of the steroid hormone ecdysterone. Neither amplification nor DNA rearrangement of the locus occurs in the salivary gland. The nucleotide sequence of genomic DNA that includes the entire gene cluster was determined, as were the structures of each of the three glue protein mRNAs. Analysis of the sequences revealed that the three glue proteins form a diverged gene family. Each member of the gene family contains an amino-terminal hydrophobic block of amino acids, which is absent in the mature, secreted glue proteins, and a cysteine-rich carboxyl terminal module. sgs-3 differs from sgs-7 and sgs-8 by containing a third module between the other two, comprised largely of tandem repeats of the five amino acids Pro-Thr-Thr-Thr-Lys.
\r\n\r\nTwo of the genes Sgs-7 and Sgs-8 are divergently transcribed with 475 base-pairs separating the two 5' ends. A transcriptional fusion gene was constructed by joining the 5' untranslated region of Sgs-7 to the 5' untranslated region of the D. melanogaster Adh gene. A translational fusion gene was constructed by joining the Sgs-8 gene to the Escherichia coli lacZ gene. When the fusion genes are placed in their normal divergently transcribed arrangement and reintroduced into D. melanogaster using P element gene transfer, third instar larval salivary gland expression of both alcohol dehydrogenase activity and \u03b2-galactosidase activity was observed. Expression of the two fusion genes requires the l(1)npr-1\u207a gene product, which is known to regulate the 68C glue protein genes, supporting the proposal that this trans-acting factor affects glue protein gene transcription. Normal tissue, stage, and quantity of Sgs-7\u2014Adh fusion gene expression is observed when 211 bp of the 5' flanking sequence is present. An Sgs-7\u2014Adh fusion gene with 92 base-pairs upstream is non-functional. Third instar larval salivary gland expression of the Sgs-8\u2014lacZ fusion gene is observed when 432 base-pairs of the intergenic region are present, while 415 base-pairs of 5' flanking sequence permits normal tissue and stage of expression at levels at least twentyfold reduced. The experiments suggest that a single region functioning bidirectionally, located closer to the Sgs-7 gene, is required for expression of both genes.
" }, { "name": "Hahn, Chang Soo", "degree": "PhD", "year": "1988", "title": "Structure-Function Relationships in the Structural Proteins and in the RNAs of Alphaviruses and Flaviviruses", "advisor": "Strauss, James H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01222013-140535393", "creators": [ { "name": { "family": "Hahn", "given": "Chang Soo" }, "id": "Hahn-Chang-Soo", "display_name": "Hahn, Chang Soo" } ], "advisors": [ { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "advisor", "display_name": "Strauss, James H." } ], "committee": [ { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "chair", "display_name": "Strauss, James H." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "member", "display_name": "Simon, Melvin I." } ], "option_major": [ "biology" ], "doi": "10.7907/n3rf-9306", "abstract": "The RNA virus families Togaviridae and Flaviviridae were considered one family as recently as 1983. These two families contain more than 100 members, many of which are important pathogens for humans and domestic animals. Studies on members of both families which were undertaken to increase our understanding of the functions of the virus structural proteins and of RNA sequence elements that interact with virus proteins, and of the evolution of these two families of RNA viruses, are presented in this thesis. These investigations include two on the nature and function of a virus-encoded self-protease that functions in the processing of the structural proteins, several studies on the role of the virus structural glycoproteins in assembly of progeny virions and viral virulence, and studies on the evolution of these viruses, including the demonstration that recombination has occurred in the Togaviridae to produce an important new pathogen, and that RNA sequence elements have been conserved during the evolution of the Flaviviridae.
\r\n\r\nAlphavirus structural proteins are translated from a subgenomic messenger RNA as a polyprotein, which is cleaved to the final products by proteolytic processing. This processing was studied by comparative sequence analysis of three temperature sensitive mutants of Sindbis virus (the type alphavirus) which have a defect in processing of the polyprotein at the nonpermissive temperature. These mutations were localized in the C-terminal region of the capsid protein. From the position of these mutations and from sequence similarities between the alphavirus capsid proteins and animal serine proteses, we hypothesized that the capsid protein was a serine autoprotease whose active site is formed by His-141, Asp- 147 and Ser-215. To study this capsid protein protease activity in more detail, we have altered the proposed catalytic triad of the protease by site-directed mutagenesis. We have assayed the protease activity in the mutagenized capsid proteins by in vitro transcription and translation, and attempted to rescue virus from mutagenized full-length \"infectious\" clones. The results supported our hypothesis.
\r\n\r\nSindbis virus matures when preformed nucleocapsids acquire their envelopes by budding through virus-modified areas of the cell surface membrane. ts103 is a mutant of Sindbis virus which has a defect in this late maturation step such that it generates multicored particles, and it has provided a good system for studying structure-function relationships during viral assembly and maturation. Hybrid genomes were constructed that were formed from a full-length cDNA clone of wild type Sindbis in which restriction fragments were replaced with cDNA from ts103. Virus rescued from these constructs were used to determine the protein responsible for the multicored phenotype and to map the mutation. tsl03 was found to have a single amino acid substitution in glycoprotein E2. The implications of this mutation for our understanding of virus assembly are\r\ndiscussed.
\r\n\r\nVirus surface glycoproteins are believed to be important determinants of virulence and tissue tropism. Ne urovirulence of Sindbis virus for mice has been used as an animal model system in which to explore the effects of each individual protein or of a particular domain of a given protein, or even of a single amino acid residue, on neurovirulence. By constructing hybrid genomes among various strains of Sindbis virus at the cDNA level and rescuing virus in vitro using in vitro transcription and transfection, it was possible to evaluate the effect of each protein on neurovirulence. From these studies, we concluded that Sindbis virus glycoproteins are important determinants of neurovirulence, but not the sole determinants. The virulence phenotypes of various recombinant viruses in both weanling mice and suckling mice are discussed, with reference to the role of particular residues in producing the neurovirulent phenotype.
\r\n\r\nWe have also undertaken a study of virulence in flaviviruses. The 17D vaccine strain of yellow fever virus, the type flavivirus, is one of the most reliable and stable live virus vaccines ever developed. By comparison of the nucleotide sequences of the 17D vaccine strain and its parental virulent Asibi virus we have located all of the changes which occurred during the attentuation of yellow fever to produce the 17D vaccine. This comparison led us to the conclusion that changes in the viral envelope protein play an important role in attenuation.
\r\n\r\nThe 25 members of the genus Alphavirus have for the most part diverged by linear descent from a common ancestor. We have now found that Western equine encephalitis virus is an exception to this. Western equine encephalitis virus is a close relative of Sindbis virus as determined by immunological cross-reaction, but it is a New World virus that causes encephalitis in humans and horses, whereas Sindbis virus is an Old World virus not normally associated with encephalitis. The nucleotide sequence and deduced amino acid sequence of the structural proteins of Western equine encephalitis virus reveal that it arose by recombination between Eastern equine encephalitis virus (or a recent ancestor of it) and a virus closely related to Sindbis virus. The importance of recombination in the evolution of RNA viruses and in the generation of new potentially pathogenic virus strains, as well as the implications of the amino acid changes which have occurred in Western equine encephalitis virus (subsequent to the initial recombination event) for our understanding of the interaction between the structural proteins of alphaviruses, are discussed.
\r\n\r\nTo study evolution in flaviviruses, sequences at the 5'and 3' ends of several flaviviruses have been compared. Conserved structures or sequence elements have been identified, one pair of which could result in cyclization of flavivirus RNA. The significance of these sequences is discussed.
" }, { "name": "Kamb, Alexander", "degree": "PhD", "year": "1988", "title": "Molecular Biology of Shaker, a Drosophila Gene that Encodes Multiple Potassium Channel Components", "advisor": "Tanouye, Mark", "url": "https://resolver.caltech.edu/CaltechTHESIS:01222013-094834563", "creators": [ { "name": { "family": "Kamb", "given": "Alexander" }, "id": "Kamb-Alexander", "display_name": "Kamb, Alexander" } ], "advisors": [ { "name": { "family": "Tanouye", "given": "Mark" }, "id": "Tanouye-M", "role": "advisor", "display_name": "Tanouye, Mark" } ], "committee": [ { "name": { "family": "Tanouye", "given": "Mark" }, "id": "Tanouye-M", "role": "chair", "display_name": "Tanouye, Mark" }, { "name": { "family": "Abelson", "given": "John N." }, "id": "Abelson-J-N", "role": "member", "display_name": "Abelson, John N." }, { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "member", "display_name": "Lewis, Edward B." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." } ], "option_major": [ "biology" ], "doi": "10.7907/a32z-ye28", "abstract": "Shaker (Sh) mutants of Drosophila suffer from a characteristic leg-shaking behavioral defect. Previous genetic and physiological experiments suggested that Sh encodes at least one component of a fast, transient, or A-type K\u207a channel. To address questions pertaining to the structure, function and heterogeneity of K\u207a channels, we have undertaken a molecular analysis of Sh. We have isolated molecular clones for the genomic region encompassing Sh as part of a 350 kb chromosomal walk. Using a combination of classical and molecular genetics, we have mapped several Sh mutations within this region, and localized the Sh gene. Sh mutations scatter over at least 65 kb of genomic DNA, and the Sh gene itself is large, spanning at least 95 kb. Comparative studies on a collection of Sh cDNA clones show that Sh encodes a diverse array of gene products. The basis for this diversity is a mechanism that generates a limited number of different 5' and 3' end segments, and splices these segments onto a central constant region. This differential splicing mechanism produces at least 10, and possibly 28 or more, predicted Sh proteins that differ at the carboxyl and/or amino terminus. The primary structures of Sh proteins deduced from the cDNAs reveal two general types of polypeptide: a protein that contains seven potential membrane-spanning domains, including a positively charged segment that is similar to a sequence called S4 in Na\u207a channels, and a smaller protein that lacks S4 and contains only three potential membrane-spanning regions. Variants of the flrst protein type range in size from 493 a.a. to 656 a.a., whereas variants of the second type range from 303 a.a. to 337 a.a. These polypeptides may assemble as homomultimers and/or as heteromultimers to produce K\u207a channels with different features.
" }, { "name": "King, Michael Paul", "degree": "PhD", "year": "1988", "title": "Studies of Human Mitochondria. I. Steady-State Levels and Metabolic Properties of the Mitochondrial tRNAs. II. Injection of Mitochondria into Human Cells Leads to a Rapid Replacement of the Endogenous Mitochondrial DNA", "advisor": "Attardi, Giuseppe", "url": "https://resolver.caltech.edu/CaltechTHESIS:02212013-094740326", "creators": [ { "name": { "family": "King", "given": "Michael Paul" }, "id": "King-Michael-Paul", "display_name": "King, Michael Paul" } ], "advisors": [ { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "advisor", "display_name": "Attardi, Giuseppe" } ], "committee": [ { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "chair", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "member", "display_name": "Revel, Jean-Paul" }, { "name": { "family": "Abelson", "given": "John N." }, "id": "Abelson-J-N", "role": "member", "display_name": "Abelson, John N." }, { "name": { "family": "Tanouye", "given": "Mark" }, "id": "Tanouye-M", "role": "member", "display_name": "Tanouye, Mark" } ], "option_major": [ "biology" ], "doi": "10.7907/45zh-5315", "abstract": "The steady-state levels and metabolic properties of mitochondrial tRNAs have been analyzed in Hela cells and correlated with the function of the tRNAs for organelle-specific protein synthesis. DNA excess hybridization experiments utilizing separated strands of mitochondrial DNA (mtDNA) and purified tRNA samples from exponential cells long-term labeled with [\u00b3\u00b2P] orthophosphate have revealed a steady-state level of 6 x 10\u2075 tRNA molecules per cell, with three-fourths being encoded in the heavy (H)-strand and one-fourth in the light (L)-strand. Hybridization of the tRNAs with a panel of M13 clones of human mtDNA containing, in most cases, single tRNA genes and a quantitation of two-dimensional electrophoretic fractionations of the tRNAs have shown that the steady-state levels of tRNAF and tRNAV are two to three times higher than the average level of the other H-strand-encoded tRNAs and three to four times higher than the average level of the L-strand-encoded tRNAs. Similar experiments carried out with tRNAs from cells labeled with very short pulses of [5-\u00b3H] uridine have indicated that the rates of formation of the individual tRNA species are proportional to their steady state amounts. Therefore, the 15-fold to 60-fold higher rate of transcription of the tRNAV and tRNAF genes (transcribed with the rDNA transcription unit) relative to the other H-strand tRNA genes (transcribed with the whole H-strand transcription unit) and the 13-fold to 20-fold higher rate of transcription of the L-strand tRNA genes relative to the H-strand tRNA genes (other than tRNAV and tRNAF genes) are not reflected in the rates of formation of the corresponding tRNAs. The available data indicate that the majority of tRNAV and tRNAF transcribed from the rDNA transcription unit are degraded as they are excised from the primary transcripts. It also seems likely that the majority of the L-strand-encoded tRNAs are degraded before they are excised from the short-lived polycistronic transcripts. Furthermore, a role of the aminoacyl tRNA synthetases in stabilizing the different tRNA species at relatively uniform levels is suggested. A comparison of the steady-state levels of the individual tRNAs with the corresponding codon usage for protein synthesis, as determined from the DNA sequence and the rates of synthesis of the various polypeptides, has not revealed any significant correlation between the two parameters.
\r\n\r\nIn other experiments, isolated human mitochondria containing a mitochondrial DNA (mtDNA)-coded chloramphenicol resistance marker were injected at an average dose of less than one into sensitive human cells partially depleted of their mtDNA by ethidium bromide treatment. Under selective conditions, the mitochondria became established in the recipient cells with a frequency greater than 2 to 3 x 10\u207b\u00b3). A rapid and, in some cases, complete replacement of the resident mtDNA by the exogenous mtDNA took place in the transformants, as shown by multiple mtDNA and nuclear DNA polymorphisms. Intracellular mtDNA selection played a crucial role in this replacement, with significant implications for mitochondrial genetics.
\r\n" }, { "name": "Martin, Christopher Hayes", "degree": "PhD", "year": "1988", "title": "Evolution and Expression at the 68C Glue Gene Cluster of Drosophila", "advisor": "Meyerowitz, Elliot M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02152013-142530976", "creators": [ { "name": { "family": "Martin", "given": "Christopher Hayes" }, "id": "Martin-Christopher-Hayes", "display_name": "Martin, Christopher Hayes" } ], "advisors": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "advisor", "display_name": "Meyerowitz, Elliot M." } ], "committee": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "chair", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "role": "member", "display_name": "Emr, Scott D." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Abelson", "given": "John N." }, "id": "Abelson-J-N", "role": "member", "display_name": "Abelson, John N." }, { "name": { "family": "Tanouye", "given": "Mark" }, "id": "Tanouye-M", "role": "member", "display_name": "Tanouye, Mark" } ], "option_major": [ "biology" ], "doi": "10.7907/p2n6-q185", "abstract": "This thesis describes investigations on the evolution of a region containing a cluster of three glue genes located at chromosomal site 68C in the Drosophila melanogaster genome. These studies have used a set of five closely related Drosophila species, all members of the melanogaster species subgroup. The first chapter serves as an introduction and summarizes this work. The second chapter describes the initial characterization of the glue gene clusters and the surrounding regions in the five Drosophila species. The third chapter describes the characterization at the sequence level of the boundary that was found between adjacent blocks of rapidly and slowly evolving sequences located at the 68C glue gene cluster. The fourth chapter describes the evolution of the largest of the three glue genes in the 68C glue gene cluster: Sgs-3. Together, these studies reveal that this region of the genome is evolving as a mosaic, with adjacent regions evolving at different rates and in very different ways.
" }, { "name": "Miller, Stephen G.", "degree": "PhD", "year": "1988", "title": "Brain Type II Calcium and Calmodulin-Dependent Protein Kinase: Characterization of a Brain-Region Specific Isozyme and Regulation by Autophosphorylation", "advisor": "Kennedy, Mary B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03142013-100553837", "creators": [ { "name": { "family": "Miller", "given": "Stephen G." }, "id": "Miller-Stephen-G", "display_name": "Miller, Stephen G." } ], "advisors": [ { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "role": "advisor", "display_name": "Kennedy, Mary B." } ], "committee": [ { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "role": "chair", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." }, { "name": { "family": "Aswad", "given": "Dana W." }, "id": "Aswad-Dana-W", "role": "member", "display_name": "Aswad, Dana W." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." } ], "option_major": [ "biology" ], "doi": "10.7907/w18z-4r52", "abstract": "A variety of molecular approaches have been used to investigate the structural and enzymatic properties of rat brain type ll Ca\u00b2\u207a and calmodulin-dependent protein kinase (type ll CaM kinase). This thesis describes the isolation and biochemical characterization of a brain-region specific isozyme of the kinase and also the regulation the kinase activity by autophosphorylation.
\r\n\r\nThe cerebellar isozyme of the type ll CaM kinase was purified and its biochemical properties were compared to the forebrain isozyme. The cerebellar isozyme is a large (500-kDa) multimeric enzyme composed of multiple copies of 50-kDa \u03b1 subunits and 60/58-kDa \u03b2/\u03b2' subunits. The holoenzyme contains approximately 2 \u03b1 subunits and 8 \u03b2 subunits. This contrasts to the forebrain isozyme, which is also composed of \u03b1 and \u03b2/\u03b2' subunits, but they are assembled into a holoenzyme of approximately 9 \u03b1 subunits and 3 \u03b2/\u03b2' subunits. The biochemical and enzymatic properties of the two isozymes are similar. The two isozymes differ in their association with subcellular structures. Approximately 85% of the cerebellar isozyme, but only 50% of the forebrain isozyme, remains associated with the particulate fraction after homogenization under standard conditions. Postsynaptic densities purified from forebrain contain the forebrain isozyme, and the kinase subunits make up about 16% of their total protein. Postsynaptic densities purified from cerebellum contain the cerebellar isozyme, but the kinase subunits make up only 1-2% of their total protein.
\r\n\r\nThe enzymatic activity of both isozymes of the type II CaM kinase is regulated by autophosphorylation in a complex manner. The kinase is initially completely dependent on Ca\u00b2\u207a/calmodulin for phosphorylation of exogenous substrates as well as for autophosphorylation. Kinase activity becomes partially Ca\u00b2\u207a-independent after autophosphorylation in the presence of Ca\u00b2\u207a/calmodulin. Phosphorylation of only a few subunits in the dodecameric holoenzyme is sufficient to cause this change, suggesting an allosteric interaction between subunits. At the same time, autophosphorylation itself becomes independent of Ca\u00b2\u207a These observations suggest that the kinase may be able to exist in at least two stable states, which differ in their requirements for Ca\u00b2\u207a/calmodulin.
\r\n\r\nThe autophosphorylation sites that are involved in the regulation of kinase activity have been identified within the primary structure of the \u03b1 and \u03b2 subunits. We used the method of reverse phase-HPLC tryptic phosphopeptide mapping to isolate individual phosphorylation sites. The phosphopeptides were then sequenced by gas phase microsequencing. Phosphorylation of a single homologous threonine residue in the \u03b1 and \u03b2 subunits is correlated with the production of the Ca\u00b2\u207a-independent activity state of the kinase. In addition we have identified several sites that are phosphorylated only during autophosphorylation in the absence of Ca\u00b2\u207a/calmodulin.
" }, { "name": "Soha, James Martin", "degree": "PhD", "year": "1988", "title": "Specificity and Competition During Maturation of Neuromuscular Synapses", "advisor": "Van Essen, David C.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03122013-111504785", "creators": [ { "name": { "family": "Soha", "given": "James Martin" }, "id": "Soha-James-Martin", "display_name": "Soha, James Martin" } ], "advisors": [ { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "advisor", "display_name": "Van Essen, David C." } ], "committee": [ { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "chair", "display_name": "Van Essen, David C." }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." }, { "name": { "family": "Bower", "given": "James M." }, "id": "Bower-J-M", "role": "member", "display_name": "Bower, James M." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." } ], "option_major": [ "biology" ], "doi": "10.7907/3ebg-mv36", "abstract": "Fast and slow contracting fibers in neonatal mammalian skeletal muscle are each innervated in a highly specific manner by motor neurons of the corresponding type, even at an age when polyinnervation is widespread. Chemospecific recognition is a possible mechanism by which this pattern of innervation could be established. I have investigated this possibility by studying the degree of specificity during reinnervation of neonatal rabbit soleus muscle. Fiber type composition was assayed by measuring the twitch rise times of motor units within two days of the onset of functional reinnervation. In contrast to the broad, bimodal distribution of single motor unit twitch rise times seen in normal muscles, motor units in reinnervated muscles yielded a narrower, unimodal distribution of rise times. Rise times of reinnervated units were intermediate to those of normal fast and slow units, suggesting that reinnervated units were composed of a mixture of fast and slow contracting muscle fibers. An alternative possibility, that specific reinnervation was masked by contractile de-differentiation of muscle fibers, was examined by maintaining a transmission blockade induced by botulinum toxin poisoning for an equivalent interval. Twitch rise times of treated motor units exhibited the distinctly bimodal distribution characteristic of normal muscles, suggesting that muscle fibers can retain contractile diversity during a transient period of denervation. Computer simulations were employed to estimate the amount of rise time diversity induced by varying degrees of specificity during reinnervation. Based on this analysis, I conclude that there is little if any selective reinnervation of muscle fiber types at the ages studied.
\r\n\r\nIn a second experiment, I compared the development of fast and slow motor innervation in the neonatal rabbit soleus, a muscle which contains two distinct motor unit types during the early period of polyneuronal innervation. The innervation state of individual muscle fibers was ascertained using an intracellular electrode; a fluorescent dye was then injected into particular fibers to permit subsequent identification of histochemical type. No significant difference in the time course of synapse elimination was observed for fast and slow motor units as judged by the percentage of fibers remaining polyneuronally innervated at two ages: 7-8 days, when most fibers are multiply innervated, and 10-11 days, when the level of polyinnervation is low.
\r\n\r\nIn a third experiment, I examined a phenomenon in which compound endplate potentials were occasionally seen in muscle fibers at an age (17-23 days) well past the major episode of synapse elimination. Several lines of evidence indicate that this apparent polyinnervation in fact derives from an electrode-induced electrical coupling artifact, and that genuinely polyinnervated fibers are very rare at this stage, if present at all.
\r\n\r\nA computer model of neuromuscular synapse elimination was developed to serve as an analytical tool in exploring the potential roles of candidate mechanisms in regulating the normal process and in shaping its response to experimental perturbations. Synapse elimination is a complex process likely to involve the dynamic interaction of several specific mechanisms. This situation limits the reliability of a strictly inductive theoretical investigation into how these mechanisms might act. Three mechanisms which have been previously proposed and discussed in the literature are simulated, including a synaptic stabilization molecule, a muscle derived trophic factor, and a hypothesized intrinsic tendency of motor neurons to limit their arbor. The model is stochastic rather than deterministic in character, and is also dynamic, tracing the growth and retraction of individual presynaptic terminals at each iteration as they compete for limited synaptic space.
\r\n\r\nNine experimental observations were selected to guide development of the model and evaluate its performance. All but one of the experimental observations canbe simulated by at least one of the mechanisms studied. No single mechanism, however, is adequate to duplicate the entire body of experimental evidence. A relative advantage for larger terminals appears critical for convergence in both the scaffolding and trophic factor mechanisms. Several alternative roles for activity are compared.
" }, { "name": "Erondu, Ngozi Emmanuel", "degree": "PhD", "year": "1987", "title": "Regional Distribution and Subcellular Associations of Type II Calcium and Calmodulin-Dependent Protein Kinase in Rat Brain", "advisor": "Kennedy, Mary B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04152019-165931960", "creators": [ { "name": { "family": "Erondu", "given": "Ngozi Emmanuel" }, "id": "Erondu-Ngozi-Emmanuel", "display_name": "Erondu, Ngozi Emmanuel" } ], "advisors": [ { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "advisor", "display_name": "Kennedy, Mary B." } ], "committee": [ { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "chair", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Lazarides", "given": "Elias" }, "id": "Lazarides-E", "role": "member", "display_name": "Lazarides, Elias" }, { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "member", "display_name": "Revel, Jean-Paul" }, { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "member", "display_name": "Benzer, Seymour" }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." } ], "option_major": [ "biology" ], "doi": "10.7907/30tg-qa85", "abstract": "Four monoclonal antibodies generated against the Type II CaM kinase have been characterized. Two of these antibodies were used to confirm that both alpha and beta subunits were part of the holoenzyme complex. I also developed liquid phase and solid phase radioimmunoassays for the kinase.
\r\n\r\nWith the solid phase radioimmunoassay, the distribution of the kinase in rat brain was examined. This study revealed that the concentration of the kinase varies markedly in different brain regions. It is most highly concentrated in the telencephalon where it comprises approximately 2% of total hippocampal protein, 1.3% of cortical protein and 0.7% of striatal protein. It is less concentrated in lower brain regions ranging from 0.3% of hypothalamic protein to 0.1% of protein in the pons/medulla. The unusually high concentration of the kinase in telencephalic regions may confer upon their neurons specialized responses to calcium that are different from those of neurons in lower brain regions.
\r\n\r\nThe association of the kinase with elements of the cytoskeleton was also investigated. The results of this study showed that autophosphorylation causes an increase in the association of the enzyme with taxol-polymerized microtubules and F-actin. This increase in association was reversed by dephosphorylating phosphokinase with protein phosphatase. These results suggest that autophosphorylation could constitute a mechanism for the regulation of the subcellular associations of the Type II CaM kinase by neuronal activity.
" }, { "name": "Matthews, Beverley Bond", "degree": "PhD", "year": "1987", "title": "Structure and Expression of the Actin Gene Family of Drosophila melanogaster", "advisor": "Meyerowitz, Elliot M.; Davidson, Norman R.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02152019-094650551", "creators": [ { "name": { "family": "Matthews", "given": "Beverley Bond" }, "id": "Matthews-Beverley-Bond", "display_name": "Matthews, Beverley Bond" } ], "advisors": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "advisor", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "co-advisor", "display_name": "Davidson, Norman R." } ], "committee": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "chair", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "member", "display_name": "Lewis, Edward B." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." } ], "option_major": [ "molbio" ], "doi": "10.7907/05zz-5w73", "abstract": "We have isolated the six actin genes of Drosophila melanogaster from a Drosophila genomic DNA library and have compared structural features of the genes by restriction mapping, electron microscopy and DNA sequencing. We found that at least two of the actin genes contain intervening sequences which interrupt the genes at different positions. Several of the genes were shown to be lacking intervening sequences in the analogous positions. This nonconservation of intron position is in striking contrast to the strong conservation of intron positions seen in other gene families. The DNA sequences of the protein coding regions of the genes are highly conserved while the intron and untranslated sequences are not. The primary sequences of all the Drosophila actins resemble mammalian cytoplasmic actins more than mammalian muscle actins.
\r\n\r\nWe studied the distribution of actin mRNAs in different developmental stages and in different dissected body parts with the use of gene specific hybridization probes which we isolated from the 3' untranslated portions of the genes. We found that the genes fall into three main categories with respect to their patterns of expression in Drosophila. Trancripts from two of the genes are found throughout Drosophila development. They are expressed at higher levels in ovaries and embryonic cultured cells than in muscle containing tissue and are thought to be cytoplasmic actins. Two others encode thoracic muscle actins. Their transcripts accumulate predominantly in the thoracic regions of the adult where the flight and jump muscles are found. The other two genes are most active in larvae and in adult abdomens. They are thought to encode actins used in the larval, pupal, and adult intersegmental muscles.
\r\n\r\nWe studied the structure of the cytoplasmic actin gene, act5C, in detail and found that it encodes at least six different mRNAs. At the 5' end there are two nonhomologous leader exons which are alternately spliced to the remainder of the gene. At the 3' end of the gene, three sites of polyadenylation are used. The 3' variation is the principal cause of the transcript length heterogeneity observed in the transcripts. In whole animal RNA, the two leader exons are expressed with the same pattern through development and with all three polyadenylation sites. There is some developmental variability in the use of the three polyadenylation sites.
\r\n\r\nIn order to determine if each exon is preceded by a functional promoter and to identify sequences important for transcription initiation from each exon, we made fusions between act5C promoter fragments and the bacterial chloramphenicol acetyltransferase (CAT) gene and tested these for promoter activity in transient assays in Kc cells. We found that each exon is preceded by a separate, functional promoter. At least two regions of DNA sequences are necessary for optimal expression from exon 1. One of these lies greater than 1.9 kb upstream from the exon 1 cap site. All of the sequences required for exon 2 transcription lie within 450 bases of its cap site. There is evidence from some constructions that transcription initiation from exon 1 may inhibit transcription initiation from ex on 2.
" }, { "name": "Mattox, William Wayne", "degree": "PhD", "year": "1987", "title": "The Molecular Structure of the Beadex and Heldup-A Loci of Drosophila melanogaster", "advisor": "Lewis, Edward B.; Davidson, Norman R.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10212019-143142376", "creators": [ { "name": { "family": "Mattox", "given": "William Wayne" }, "id": "Mattox-William-Wayne", "display_name": "Mattox, William Wayne" } ], "advisors": [ { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "advisor", "display_name": "Lewis, Edward B." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "co-advisor", "display_name": "Davidson, Norman R." } ], "committee": [ { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "chair", "display_name": "Lewis, Edward B." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "member", "display_name": "Simon, Melvin I." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." } ], "option_major": [ "biology" ], "doi": "10.7907/c2k4-a231", "abstract": "Genetic studies indicate that excess-of-function Beadex mutations and loss-of-function heldup-a mutations affect different parts of a single bipartite genetic unit. In order to investigate the molecular nature of the Beadex and heldup-a mutations we isolated DNA from a 49 kilobase region surrounding the sites at which these mutations occur. We found gross structural alterations associated with one heldup-a mutation and each of the 13 different Beadex mutations that we examined. As expected from previous genetic studies these loci are separated by only a short molecular distance which is at most 1.5 kilobases. The structural alterations associated with the thirteen Beadex excess-of-function mutations examined are clustered within a three kilobase region. Several of these mutations are found to be associated with the deletion of part of an 800 bp region. This indicates that an element that normally represses gene activity is located within this region: A 200 base pair segment which is required for the function of the wild-type heldup-a locus is defined using a heldup-a mutation which results from a small deletion.
\r\n\r\nTwo RNA transcripts have been found which span this 200 base pair segment. One of these two transcripts, a four kilobase RNA, is expressed during stages in which the Beadex structural gene product is expected to be active. The structure of this transcript is affected by one heldup-a mutation and each of five Beadex mutations that were examined. However, only one of the Beadex mutant alleles expresses significantly elevated levels of this RNA. Other RNA species in the region surrounding the the Beadex and heldup-a loci were not affected either in amount or size by Beadex mutations.
\r\n\r\nWe have also reintroduced a wild-type 10.4 kilobase fragment, which includes the region m which Beadex and heldup-a mutations map, into the Drosophila genome by P element mediated transformation. This introduced fragment fails both to complement loss-of-function heldup-a mutations and to enhance an excess-of-function phenotype. This result indicates that some sequences required for the normal heldup-a function must be located outside this 10.4 kilobase region.
" }, { "name": "Ngai, John J.", "degree": "PhD", "year": "1987", "title": "Expression of the Genes Encoding the Cytoskeletal Proteins Vimentin and Protein 4.1", "advisor": "Lazarides, Elias", "url": "https://resolver.caltech.edu/CaltechTHESIS:10222019-120541581", "creators": [ { "name": { "family": "Ngai", "given": "John J." }, "id": "Ngai-John-J", "orcid": "0000-0002-1191-8971", "display_name": "Ngai, John J." } ], "advisors": [ { "name": { "family": "Lazarides", "given": "Elias" }, "id": "Lazarides-E", "role": "advisor", "display_name": "Lazarides, Elias" } ], "committee": [ { "name": { "family": "Lazarides", "given": "Elias" }, "id": "Lazarides-E", "role": "chair", "display_name": "Lazarides, Elias" }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "member", "display_name": "Simon, Melvin I." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "member", "display_name": "Wold, Barbara J." } ], "option_major": [ "biology" ], "doi": "10.7907/qh54-ka26", "abstract": "The investigations presented in this thesis represent an effort to understand the regulated expression of cytoskeletal proteins in differentiating cell systems. Vimentin is an intermediate filament protein whose expression is regulated during the differentiation of a variety of cell types. I have isolated DNA probes specific for chicken vimentin and utilized them for the study of vimentin gene regulation. The single chicken vimentin gene encodes multiple mRNAs that differ in the lengths of their 3' untranslated regions. These mRNAs are differentially expressed in a tissue-specific manner. Furthermore, vimentin mRNA increases to high levels during chicken embryonic erythropoiesis, underlying similar changes in vimentin protein accumulation.
\r\n\r\nUnlike nucleated avian erythrocytes, mammalian erythrocytes are devoid of intermediate filaments. I show that cultured murine erythroleukemia (MEL) cells repress the levels of vimentin mRNA during inducer-mediated differentiation, resulting in a subsequent loss of vimentin filaments. The expression of vimentin in these cells reflects the disappearance of vimentin filaments during mammalian erythropoiesis in vivo. To examine the molecular basis for divergent vimentin gene regulation in avian and mammalian erythropoiesis, I have studied the behavior of chicken and hamster vimentin genes introduced into MEL cells. During MEL cell differentiation, RNA encoded by transfected chicken vimentin genes significantly increases in abundance, whereas RNAs arising from either transfected hamster vimentin genes or the endogenous mouse vimentin gene are repressed. The results suggest that the difference in vimentin expression in avian and mammalian erythropoiesis is due to a divergence of cis-linked vimentin sequences.
\r\n\r\nProtein 4.1 is an extrinsic membrane protein that facilitates the interaction of spectrin and actin in the erythroid membrane skeleton. Previous studies have shown that chicken protein 4.1 exists as a multiplet of related polypeptides that are differentially expressed during erythropoiesis. I have isolated cloned cDNA probes for chicken protein 4.1, and have found that a single protein 4.1 gene encodes multiple mRNAs by differential processing; the ratios of protein 4.1 mRNAs change during erythroid development. In vitro translation experiments demonstrate that while the expression of protein 4.1 polypeptides is specified initially at the mRNA level by RNA processing, the ultimate expression of protein 4.1 variants is further determined translationally.
" }, { "name": "Yu, Lei", "degree": "PhD", "year": "1987", "title": "The Nicotinic Acetylcholine Receptor: Gene Expression and Ion Channel Function", "advisor": "Davidson, Norman R.; Lester, Henry A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10312019-114338923", "creators": [ { "name": { "family": "Yu", "given": "Lei" }, "id": "Yu-Lei", "display_name": "Yu, Lei" } ], "advisors": [ { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "advisor", "display_name": "Davidson, Norman R." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "co-advisor", "display_name": "Lester, Henry A." } ], "committee": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "chair", "display_name": "Lester, Henry A." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Anderson", "given": "David J." }, "id": "Anderson-D-J", "orcid": "0000-0001-6175-3872", "role": "member", "display_name": "Anderson, David J." }, { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "member", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "member", "display_name": "Van Essen, David C." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." } ], "option_major": [ "biology" ], "doi": "10.7907/fj5c-4h83", "abstract": "The nicotinic acetylcholine receptor (AChR) is a complex protein, which functions as a ligand-gated ion channel on the postsynaptic membrane at the neuromuscular junction and mediates signal transmission from neuron to muscle. Research on the AChR has had a long history and has benefited from the endeavors of scientists from many disciplines. The intensive, multidisciplinary studies have yielded valuable knowledge about this molecule, which serves as a model for the understanding of many fundamental questions in biological sciences. Chapter 1 presents a review of the AChR.
\r\n\r\nAs a tissue-specific and developmental stage-specific molecule, AChR is under temporal and spatial control for its synthesis. Chapter 2 reports a qualitative and quantitative study of AChR gene activity during muscle cell differentiation, using a cDNA clone isolated from a murine muscle cell line, which codes for the \u03b3 subunit of the mouse AChR. The results indicate that the regulation of mRNA accumulation levels is a major mechanism in the differential synthesis of the AChR.
\r\n\r\nThe marriage between AChR and molecular biology resulted in many cDNA clones which, after being introduced to African frogs, produced the next generation \u2014 Xenopus oocytes with exotic AChRs on them. Chapter 3 describes the attempt to localize \"determinants\" that specify species subunit identity in the AChR by constructing chimeric cDNA clones composed of fragments from different origins, taking advantage of the Xenopus oocyte expression system. The results from surface toxin-binding assay and two-electrode voltage-clamp recording suggested that while the species specificity can be dictated by certain subunits, the determination of subunit identity does not reside at a defined locus in the fragments tested.
\r\n\r\nDoes the complex composition of multisubunits in the AChR bear any functional significance? Chapter 4 addresses this question through the study of mouse-Torpedo AChR hybrids. The complete substitution of AChR subunits between mouse and Torpedo receptors generated all 16 combinations, and a systematic analysis of these hybrids revealed an interesting pattern with respect to the voltage sensitivity in the ACh-induced response: The identity of the \u03b2 subunit determines, while the interaction between the \u03b2 and \u03b4 subunits modulates, the AChR voltage sensitivity. The results, therefore, suggest that different subunits of AChR may play a central role in different functional properties.
\r\n\r\nPatch-clamp technique has offered an opportunity for analyzing transmembrane current flow with the high resolution of single-channel recording. Chapter 5 describes such a study on homologous and hybrid AChRs. Voltage influence on the three parameters were evaluated, and the results indicate that the single channel conductance is independent of membrane potential and that the channel closing and opening rates together constitute the basis for the voltage sensitivity in whole-cell recording with the closing rate making the major contribution. Also investigated were the subunit roles in species specificity of channel-open duration and voltage dependence. The results are in agreement with those reported on channel duration and support the conclusions of our previous work on the subunit involvement in determining the voltage sensitivity of the AChR response.
" }, { "name": "Bennett, Mark Knowles", "degree": "PhD", "year": "1986", "title": "Brain Type II Calcium and Calmodulin-Dependent Protein Kinase: Purification, Characterization and Molecular Cloning", "advisor": "Kennedy, Mary B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04122019-162032124", "creators": [ { "name": { "family": "Bennett", "given": "Mark Knowles" }, "id": "Bennett-Mark-Knowles", "display_name": "Bennett, Mark Knowles" } ], "advisors": [ { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "advisor", "display_name": "Kennedy, Mary B." } ], "committee": [ { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "chair", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "member", "display_name": "Simon, Melvin I." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." }, { "name": { "family": "Tanouye", "given": "Mark" }, "id": "Tanouye-M", "role": "member", "display_name": "Tanouye, Mark" } ], "option_major": [ "neurobio" ], "doi": "10.7907/a6p9-vd59", "abstract": "A combination of biochemical, immunochemical, and molecular biological techniques have been employed to purify and characterize a rat brain Ca2+/calmodulin-dependent protein kinase. The enzyme, named type II Ca2+/calmodulin-dependent protein kinase (type II CaM kinase), was identified in rat brain homogenates by its ability to phosphorylate site II on the synaptic vesicle associated protein synapsin I.
\r\n\r\nType II CaM kinase has been purified 290 fold over crude homogenates and is found to be composed of multiple copies of two different subunits. Both subunits copurify with kinase activity and are coprecipitated with kinase activity by an anti-kinase monoclonal antibody. The two subunits have molecular weights of 50,000 (\u03b1) and 58,000/60,000 (\u03b2), and are present in a 3:1 \u03b1:\u03b2 ratio. The type II CaM kinase holoenzyme has a sedimentation coefficient of 16.4 S, a Stokes radius of 95 \u00c5, and a calculated molecular weight of 650,000. A dodecameric holoenzyme consisting of 9 \u03b1 subunits and 3 \u03b2 subunits has been proposed. The purified type II CaM kinase phosphorylates several substrates, in addition to synapsin I, at a significant rate, and may therefore be responsible for a number of neuronal responses to Ca2+.
\r\n\r\nThe \u03b1 subunit of type II CaM kinase has a number of biochemical characteristics which are similar to the major protein component of a subcellular fraction which is derived from brain postsynaptic densities (PSDs). A direct comparison between the a subunit of type II CaM kinase and the major PSD protein using immunochemical and biochemical techniques has revealed that they are in fact very similar or identical proteins.
\r\n\r\nTwo approaches have been taken to further characterize the subunits of type II CaM kinase at a molecular level. The first approach has been to isolate cDNA clones which code for the \u03b2 subunit. A number of clones have been isolated and sequenced. The ammo acid sequence for the \u03b2 subunit (predicted from the cDNA sequence) is homologous to several other protein kinases. Southern blot analysis with a \u03b2 subunit cDNA indicates the existence of a type II CaM kinase multigene family. The second approach to the molecular characterization of the type II CaM kinase subunits has been to determine the amino acid sequence of peptides derived from the \u03b1 subunit. Two regions of \u03b1 subunit sequence have been determined, and both are found to be homologous to regions of \u03b2 subunit amino acid sequence deduced from \u03b2 subunit cDNA clones.
\r\n\r\nThe molecular characterization of neuronal type II CaM kinase in vitro has both provided insight into the possible function of the enzyme in vivo and suggested experimental approaches which may eventually allow its in vivo function to be directly addressed.
" }, { "name": "Crosby, Madeline Anne", "degree": "PhD", "year": "1986", "title": "Regulation of the Drosophila Glue Gene SGS-3: Sequences Required for Puffing and Transcription", "advisor": "Meyerowitz, Elliot M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02152013-085205353", "creators": [ { "name": { "family": "Crosby", "given": "Madeline Anne" }, "id": "Crosby-Madeline-Anne", "display_name": "Crosby, Madeline Anne" } ], "advisors": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "advisor", "display_name": "Meyerowitz, Elliot M." } ], "committee": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "chair", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "member", "display_name": "Lewis, Edward B." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Parker", "given": "Carl Stevens" }, "id": "Parker-C-S", "orcid": "0000-0001-9795-4211", "role": "member", "display_name": "Parker, Carl Stevens" } ], "option_major": [ "molbio" ], "doi": "10.7907/5HDH-D305", "abstract": "The 68C intermolt puff of Drosophila melanogaster contains a cluster of\r\nthree glue protein genes, Sgs-3, Sgs-7, and Sgs-8. Analyses of chromosomal\r\nrearrangments which break near the glue gene cluster show that a region of no\r\nmore than 20 kilobase-pairs (kb) is required for expression of the genes and for\r\nformation of the 68C puff. This result is supported by P-element-mediated-transformation\r\nexperiments in which defined segments of the 68C region are\r\nintroduced back into the fly genome. Based on the criteria of correct tissue- and\r\nstage-specific expression, transcription of an RNA of appropriate size and\r\nabundance, and production of an sgs-3 protein, the correctly regulated expression\r\nof the Sgs-3 gene requires less than 3.4 kb of total flanking sequences,\r\napproximately 2.3 kb 5' and 1.1 kb 3'. When upstream sequences are truncated at\r\n130 base-pairs, low levels of Sgs-3 expression are observed in some cases, with\r\nnormal tissue- and stage-specific expression retained. Formation of a new\r\nintermolt puff at the site of insertion is observed for transformants in which the\r\nintroduced DNA contains all three 68C glue genes, but not for those which contain\r\nonly an introduced Sgs-3 gene, even for cases in which the gene is abundantly\r\nexpressed.
\r\n\r\n\r\nAn attempt was made to recover lethal mutations in genes closely flanking\r\nthe 68C glue protein genes by screening mutagenized chromosomes over large\r\ndeficiencies which delete the 68AC region. Although a large number of lethal and\r\nsemi-lethal mutations were recovered, including many which define new\r\ncomplementation groups, none maps close to the region of the 68C glue gene\r\ncluster.
\r\n" }, { "name": "Fisher, Douglas Arthur", "degree": "PhD", "year": "1986", "title": "Class I Genes of the Major Histocompatibility Complex: Structural Studies on Genes of the Tla Locus", "advisor": "Hood, Leroy E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10102019-163948204", "creators": [ { "name": { "family": "Fisher", "given": "Douglas Arthur" }, "id": "Fisher-Douglas-Arthur", "display_name": "Fisher, Douglas Arthur" } ], "advisors": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "advisor", "display_name": "Hood, Leroy E." } ], "committee": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "chair", "display_name": "Hood, Leroy E." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." } ], "option_major": [ "biology" ], "doi": "10.7907/ymba-8s86", "abstract": "This thesis contains investigations into the structure of molecules encoded within the mouse major histocompatibility complex. The first chapter [Steinmetz, M., J. G. Frelinger, D. Fisher, T. Hunkapiller, D. Pereira, S. M. Weissman, S. G. Nathenson, and L. Hood, Cell 24: 125] describes the isolation and characterization of the first cDNA clones encoding murine transplantation (H-2) antigens. This study showed that H-2 antigens contain DNA and protein sequences related to immunoglobulin (Ig) molecules, but that the similarity does not include the great somatic diversity characteristic of Ig molecules.
\r\n\r\nThe second chapter contains methods for cloning and sequencing in M13 bacteriophage vectors. Included is a novel method of generating overlapping subclones for DNA sequencing by making a family of deletions in a DNA insert cloned in M13.
\r\n\r\nIn the third chapter [Fisher, D. A., S. W. Hunt, and L. Hood J. Exp. Med. 162: 528], the complete structure of a gene encoding a serologically defined thymus leukemia (TL) antigen is elucidated. TL antigen is encoded in a gene, gene Tl3c, closely related to H-2 antigens, and appears to have undergone a gene conversion event with an H-2 gene. Tla-specific probes subcloned from T13C enabled us to examine the organization of the eighteen cross hybridizing class I genes of the Tla region.
\r\n\r\nThe last chapter contains the sequence of another gene, gene T1C, previously identified as encoding TL antigen. However, the T1C gene is a non-functional pseudogene, and was probably mis-identified. There is an apparent site of recombination in the T1C gene that occurs precisely at a B2 Alu repeat sequence.
" }, { "name": "Gaines, George Loweree, III", "degree": "PhD", "year": "1986", "title": "The Control of RNA Synthesis in Isolated HeLa Cell Mitochondria", "advisor": "Attardi, Giuseppe", "url": "https://resolver.caltech.edu/CaltechTHESIS:04172019-094731539", "creators": [ { "name": { "family": "Gaines", "given": "George Loweree, III" }, "id": "Gaines-George-Loweree-III", "display_name": "Gaines, George Loweree, III" } ], "advisors": [ { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "advisor", "display_name": "Attardi, Giuseppe" } ], "committee": [ { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "chair", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Emr", "given": "Scott D." }, "id": "Emr-S-D", "role": "member", "display_name": "Emr, Scott D." }, { "name": { "family": "Abelson", "given": "John N." }, "id": "Abelson-J-N", "role": "member", "display_name": "Abelson, John N." }, { "name": { "family": "Parker", "given": "Carl Stevens" }, "id": "Parker-C-S", "orcid": "0000-0001-9795-4211", "role": "member", "display_name": "Parker, Carl Stevens" }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." } ], "option_major": [ "biology" ], "doi": "10.7907/1hj5-em11", "abstract": "The transcription of DNA and processing of RNA in mitochondria was investigated using isolated HeLa cell mitochondria. The intact organelles transcribe their DNA and process their RNA both qualitatively and quantitatively similar to the in vivo situation. Changing the conditions for the transcription reactions allowed the identification of a processing pathway for the small ribosomal RNA species, and an RNA species from the region surrounding the origin of light-strand replication with novel electrophoretic properties. Removal of the mitochondria from the cellular environment simplified the investigations of the nuclear-cytoplasmic influences upon the mitochondrial transcription. Differential sensitivities of all three transcription events, synthesizing the rRNAs, mRNAs, and light-strand RNAs, was shown to exist to a small molecular weight factor(s) present in the cytoplasm, and the availability of energetic substrates. These sensitivities indicate a link between cytoplasmic and respiratory/oxidative phosphorylative control of mitochondrial transcription.
" }, { "name": "Gao, Boning", "degree": "PhD", "year": "1986", "title": "The Structure and Expression of the Gene Coding for Bindin, a Species Specific Sea Urchin Protein", "advisor": "Davidson, Eric H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04122019-155653613", "creators": [ { "name": { "family": "Gao", "given": "Boning" }, "id": "Gao-Boning", "orcid": "0000-0002-1382-395X", "display_name": "Gao, Boning" } ], "advisors": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "advisor", "display_name": "Davidson, Eric H." } ], "committee": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "chair", "display_name": "Davidson, Eric H." }, { "name": { "family": "Brokaw", "given": "Charles J." }, "id": "Brokaw-C-J", "role": "member", "display_name": "Brokaw, Charles J." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "member", "display_name": "Strauss, James H." } ], "option_major": [ "molbio" ], "doi": "10.7907/h0pw-fh17", "abstract": "Bindin is a major protein of the sea urchin sperm acrosome granule which mediates the species-specific adhesion and binding of sperm to the egg. Bindin protein has been purified from the sperm of the sea urchin Strongylocentrotus purpuratus (S. purpuratus) and the protein has been partially sequenced. The work presented in this thesis is the isolation and the sequence analysis of bindin cDNA and gene, and the study of the expression of the bindin gene.
\r\n\r\nA \u03bbgt10 cDNA library was constructed from S. purpuratus testes poly(A)+ RNA. The library which was screened with a synthetic DNA probe prepared according to the known protein sequence yielded clones representing bindin cDNA. One of these clones containing an 1873 base pair (bp) insert was sequenced and found to code for a bindin precursor (prebindin) which is twice as large as the mature bindin protein. Upon immunoprecipitation with bindin antibody, the testes poly(A)+ RNA in vitro translation product yields a larger precursor. The bind in cDNA was used to study the tissue specificity of expression. The results show that bindin is a sperm specific protein -- its messenger RNA is not detected in the eggs, ovaries, early embryos, coelomocytes, tubefeet, lantern tissue or intestine that we tested.
\r\n\r\nSperm DNA isolated from several individuals was probed with bindin cDNA and reveals that there is one bindin gene per haploid genome. Haploid female coelomocyte DNA also possesses one bindin gene.
\r\n\r\nBindin cDNA was used to screen an EcoRI partially digested sea urchin S. purpuratus DNA charon 4A library. A clone containing a 14 kilobase (Kb) insert hybridized to the 3' end of the bindin cDNA. It also has overlapping restriction enzyme recognition sites with the 3' end of the bindin cDNA. There is an intron in the genomic clone upstream of this overlapping region since it did not hybridize with bindin cDNA. A \u03bbgt10 mini-genomic library was made and a 1.3 Kb genomic DNA clone which hybridized with bindin cDNA has been characterized and partially sequenced. It contains a 219 bp exon in which the 5' end lies 2 bp upstream of the AUG translation initiation codon. This exon is flanked by introns on either side. Thus, there are at least three introns in the bindin gene.
" }, { "name": "Lee, James Joseph", "degree": "PhD", "year": "1986", "title": "The Genomic Organization and Expression of the Strongylocentrotus purpuratus Actin Gene Family", "advisor": "Davidson, Eric H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04172019-114252554", "creators": [ { "name": { "family": "Lee", "given": "James Joseph" }, "id": "Lee-James-Joseph", "display_name": "Lee, James Joseph" } ], "advisors": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "advisor", "display_name": "Davidson, Eric H." } ], "committee": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "chair", "display_name": "Davidson, Eric H." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Lazarides", "given": "Elias" }, "id": "Lazarides-E", "role": "member", "display_name": "Lazarides, Elias" }, { "name": { "family": "Parker", "given": "Carl Stevens" }, "id": "Parker-C-S", "orcid": "0000-0001-9795-4211", "role": "member", "display_name": "Parker, Carl Stevens" } ], "option_major": [ "biology" ], "doi": "10.7907/b4m8-v194", "abstract": "The actin gene family of the sea urchin Strongylocentrotus purpuratus was studied in detail, using subcloned probes specific to the 3'-terminal nontranslated actin gene sequences. By determining the often polymorphic restriction fragment band pattern displayed in genomic blots by each probe, the actin genes in this species could be classified. The evidence presented here shows that the S. purpuratus genome contains eight actin genes, and these can be assigned to four subtypes. Studies of the expression of the genes show that the actin genes of three of these subtypes code for cytoskeletal actins (Cy), while the fourth gives rise to a muscle-specific actin (M). There is a single CyI actin gene, three CyII genes (CyIIa, CyIIb, and CyIIc), three CyIII genes (CyIIIa, CyIIIb, and CyIIIc), and a single M actin gene. Pnmary sequence data shows that two of these genes, CyIIc and CyIIIc, have anomalous structures and are probably pseudogenes.
\r\n\r\nRNA gel blots, using the 3'-trailer probes, show that during embryogenesis, as well as in a number of adult tissues, the CyI, CyII, and M subtype genes are differentially expressed. Genes of the CyIII subtype are not active (i.e., transcripts do not accumulate) in any adult tissues. Moreover, molecular titration experiments, using Sp6 polymerase-generated RNA probes, measured the absolute prevalence of transcripts from five of the six active actin genes at different points in development. This analysis showed that actin transcripts begin to accumulate after seven hours postfertilization and are not prevalent in the maternal RNA pool. In situ hybridizations of sections through the embryo establish that the accumulation of actin transcripts occurs in a cell lineage specific manner. Together with the molecular titrations these analyses provide per-cell prevalence data for transcripts from each actin gene. In vitro nuclear run-off experiments show that actin transcripts accumulate as the result of the activation and continued transcription of each actin gene during embryogenesis. Additional in vivo kinetic labeling experiments were used to measure the rates of nuclear synthesis and decay. This data shows that once activated each actin gene is transcribed at a constant rate and these rates are the prominent kinetic parameters determining actin transcript prevalence levels.
" }, { "name": "Mayne, Katherine Mixter", "degree": "PhD", "year": "1986", "title": "Structural and Functional Studies on the Subunits of the Nicotinic Acetylcholine Receptor", "advisor": "Davidson, Eric H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04152019-153632089", "creators": [ { "name": { "family": "Mayne", "given": "Katherine Mixter" }, "id": "Mayne-Katherine-Mixter", "display_name": "Mayne, Katherine Mixter" } ], "advisors": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "advisor", "display_name": "Davidson, Eric H." } ], "committee": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "chair", "display_name": "Davidson, Eric H." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "member", "display_name": "Benzer, Seymour" } ], "option_major": [ "molbio" ], "doi": "10.7907/nvv4-qt08", "abstract": "An introduction to the work in the study of the nicotinic acetylcholine receptor is presented. The author reviews the field to place the work of the present volume in its proper context The major developments in studying the protein biochemistry of the receptor are reviewed, including the subunit makeup, ligand binding, and protein sequences. These studies led to the cloning and sequencing of many of the subunits as cDNA or genomic DNA constructions. This wealth of sequence information has allowed the formulation of detailed models of receptor structure. Current work centers on testing various aspects of these models and expanding the scope of the field into different species and tissues that utilize this receptor.
\r\n\r\nPartial cDNA clones specific for the \u03b2 and \u03b4 subunits of the acetylcholine receptor of Torpedo californica were isolated by the following method. A cDNA library was constructed from electric organ poly( A)+ RNA and enriched by screening for clones more abundantly represented in electric organ than in brain or-liver mRNA preparations. These clones were tested by hybridization selection of clone specific mRNA which was then translated in vitro. Protein products were immunoprecipitated and analyzed by gel electrophoresis. The isolated clones were used to screen a library of Torpedo genomic DNA which resulted in the isolation of the gene for the Torpedo \u03b4 subunit. The \u03b4 gene was found to be single copy in Torpedo, and it contains at least four introns.
\r\n\r\nA cDNA library was constructed in \u03bbgt10 from membrane bound poly(A)+ RNA from mouse BC3H-1 cells. This library was screened with cDNA encoding the complete protein region of the Torpedo \u03b3 and \u03b4 subunits. Positively hybridizing clones isolated with the Torpedo \u03b3 subunit were sequenced and compared with published data. The deduced amino acid sequence was more highly homologous to the Torpedo \u03b4 than to the Torpedo \u03b3 and on this basis the mouse clone was tentatively identified as a \u03b4 subunit of the acetylcholine receptor. The mouse nucleotide sequence has several stretches of strong homology with the Torpedo \u03b3 subunit cDNA, but no such homology with the Torpedo \u03b4 subunit . A genomic blotting experiment indicated that there is probably one, but at most two chromosomal genes encoding this or closely related sequences.
\r\n\r\nIn order to test the assignment of the mouse \u03b4 cDNA by a more functional criterion than simple amino acid homology, the following experiment was done. The phage SP6 transcription system was used to transcribe mRNA from the four individual Torpedo subunits and from the mouse \u03b4. When the four Torpedo subunit specific mRNAs were injected into Xenopus oocytes, functional receptors appeared in the oocyte membrane. If the \u03b2 or \u03b3 subunit RNA was omitted, no response to acetylcholine was detected, while a small response was detected if the \u03b4 subunit RNA was omitted. When mouse \u03b4 specific RNA was injected in place of the Torpedo \u03b4, a 3-4 fold larger response was measured in response to acetylcholine under voltage clamp conditions. The replacement of Torpedo \u03b3 RNA with mouse \u03b4 RNA gave no detectable response. Surface binding of \u03b1-bungarotoxin was not significantly altered by exchanging the \u03b4 subunits, which indicates that the difference is intrinsic to the channel rather than a matter of stability or synthesis rates. Examination of the amino acid sequences of the two \u03b4 subunits and the Torpedo \u03b3 did not identify an obvious region of subunit specific homology. The amino acid features necessary to determine a specific subunit are not obvious from simple homology comparisons.
\r\n\r\nWe have constructed a series of chimeric subunits to try to localize subunit determining regions of the acetylcholine receptor polypeptides. Each chimera was tested in the oocyte system by replacing its RNA for each of the parent RNAs in turn. None of the chimeras we have constructed retained enough of either parental subunit characteristics to function fully in place of that parent subunit to form an acetylcholine receptor that is responsive to acetylcholine. We conclude that a minimum of two subunit-specific regions are widely dispersed over the subunit length. These data are also consistent with the conclusion that there are no discrete regions that determine subunit identity, but instead that this information is rather evenly distributed along subunit length. In some combinations, the chimeras were incorporated into surface AchRs, although these complexes were only weakly responsive to Ach. We further conclude that there are regions needed for efficient function of these subunits that are not necessary for the formation of surface complexes. We have demonstrated that the \u03b1 subunits of both mouse and chick form functional receptors in the Xenopus oocyte system in combination with the \u03b2 and \u03b3 subunits from Torpedo and a \u03b4 from either Torpedo or mouse. The responses of these hybrid AchRs are smaller than the response from the Torpedo AchR. In contrast, the mouse \u03b3 subunit did not form functional AchRs in any combination of the subunits mentioned above.
\r\n\r\nThe present author spent the early part of her career studying the molecular biology of the actin genes of Drosophila melanogaster. Portions of each of the six actin genes were sequenced. These sequences revealed that the amino acid sequence of actin is highly conserved but that the positions of introns in these genes are strikingly nonconserved. Further, each of the Drosophila actins resembles the cytoplasmic isoforms from vertebrates, while none resemble the muscle isoforms.
" }, { "name": "Meyer, Paul Wells", "degree": "PhD", "year": "1986", "title": "The Avoidance Response of Phycomyces in a Controlled Environment", "advisor": "Berg, Howard C.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04162019-154502622", "creators": [ { "name": { "family": "Meyer", "given": "Paul Wells" }, "id": "Meyer-Paul-Wells", "display_name": "Meyer, Paul Wells" } ], "advisors": [ { "name": { "family": "Berg", "given": "Howard C." }, "id": "Berg-Howard-C", "role": "advisor", "display_name": "Berg, Howard C." } ], "committee": [ { "name": { "family": "Berg", "given": "Howard C." }, "id": "Berg-Howard-C", "role": "chair", "display_name": "Berg, Howard C." }, { "name": { "family": "Brokaw", "given": "Charles J." }, "id": "Brokaw-C-J", "role": "member", "display_name": "Brokaw, Charles J." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Pine", "given": "Jerome" }, "id": "Pine-J", "role": "member", "display_name": "Pine, Jerome" }, { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "member", "display_name": "Revel, Jean-Paul" } ], "option_major": [ "biochem" ], "doi": "10.7907/s6zv-r568", "abstract": "If an object is placed 1 mm away from the growing zone of a Phycomyces sporangiophore growing in air, then after 2 to 6 min the sporangiophore bends away from the object, without ever touching it, at a rate of about 1\u00b0/min. The sporangiophore stops bending after about 30 min. This is called the avoidance response of Phycomyces.
\r\n\r\nHow does the sporangiophore detect the object? It seems likely that a chemical mechanism is involved, since other physical stimuli (light, electric and magnetic fields) have already been ruled out.
\r\n\r\nA simple mechanism was proposed 10 years ago, in which the ambient air currents near the surface of an object modify the distribution of a hypothetical, short-lived effector gas emitted by the sporangiophore. However, the avoidance response occurs at its usual rate in the complete absence of ambient air currents. Thus, the suppression of air currents near the surface of a solid object cannot provide the signal for the response.
\r\n\r\nThe avoidance rate depends significantly on the recent history of the experimental chamber, on the length of time the sporangiophore has spent inside the experimental chamber, and on other factors. By carefully controlling environmental variables, the variation in avoidance rate of different sporangiophores in successive experiments can be held to less than \u00b110%. This allows accurate determination of the distance dependence of the response, and accurate comparison of different types of barriers.
\r\n\r\nThe rate of the response falls off above 90 % relative humidity - but does not fall to zero. Surprisingly, the sporangiophore avoids a thin, 120 \u00b5m diameter parallel wire placed 0.5 mm away at about the same rate as it avoids another sporangiophore placed at the same distance. Also, the distance dependence of the avoidance response appears to be much weaker than previously reported, and the response may depend on the chemical composition of the object, in contrast to previous reports. These findings, combined with the results of calculations presented in Appendix 3, argue strongly against the hypothesis that the barrier acts merely by reflecting a diffusible substance emitted by the sporangiophore.
\r\n\r\nThe only remaining viable chemical mechanism for the avoidance response requires that the signal molecule emitted by the sporangiophore be adsorbed by the surface of the avoided object for a nonzero length of time, and not just be reflected by it. Three new versions of this hypothesis are presented which are consistent with the experimental results.
" }, { "name": "Pruitt, Robert Edwin", "degree": "PhD", "year": "1986", "title": "Characterization of the Genome of Arabidopsis thaliana", "advisor": "Meyerowitz, Elliot M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02142013-150009074", "creators": [ { "name": { "family": "Pruitt", "given": "Robert Edwin" }, "id": "Pruitt-Robert-Edwin", "orcid": "0000-0003-4817-5806", "display_name": "Pruitt, Robert Edwin" } ], "advisors": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "advisor", "display_name": "Meyerowitz, Elliot M." } ], "committee": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "chair", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "member", "display_name": "Simon, Melvin I." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." } ], "option_major": [ "molbio" ], "doi": "10.7907/mefh-yn26", "abstract": "The work described in this thesis consists of two different types of characterizations of the genome of Arabidopsis thaliana. The first part (Chapter 2) is concerned with the organization of the genome as a whole while the second part (Chapter 3) is concerned with a detailed analysis of two pairs of genes which are expressed in the developing Arabidopsis seed. The analysis presented in Chapter 2 is based on a characterization of the DNA sequences found in 50 randomly selected recombinant lambda clones. The clones were characterized by various blotting and restriction digestion techniques to determine their content of unique and repetitive DNA and the interspersion pattern of these two types of sequences. The various repetitive sequences which were identified were characterized further as to the exact nature of the repeated sequence. The primary conclusion which can be drawn from this analysis is that the Arabidopsis genome consists predominantly of long contiguous blocks of single-copy sequences. The analysis presented in Chapter 3 is concerned with two pairs of genes which are expressed specifically and abundantly in the developing seed of Arabidopsis. These genes are characterized with respect to the time of their expression during seed development, the organization of the regions of the genome containing the genes and the directions the genes are transcribed. The nucleotide sequences of the genes and flanking regions are also presented.
" }, { "name": "Sun, Yi Henry", "degree": "PhD", "year": "1986", "title": "Organization and Evolution of the Class I Genes in the Murine Major Histocompatibility Complex", "advisor": "Hood, Leroy E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10252019-115550207", "creators": [ { "name": { "family": "Sun", "given": "Yi Henry" }, "id": "Sun-Yi-Henry", "orcid": "0000-0001-8279-5270", "display_name": "Sun, Yi Henry" } ], "advisors": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "advisor", "display_name": "Hood, Leroy E." } ], "committee": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "chair", "display_name": "Hood, Leroy E." }, { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "member", "display_name": "Simon, Melvin I." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." } ], "option_major": [ "molbio" ], "doi": "10.7907/qfw5-kk48", "abstract": "This thesis contains studies of the organization and evolution of the class I gene family in the murine major histocompatibility complex (the H-2 complex).
\r\n\r\nThe first chapter presents the molecular characterization of the H-2dm1 mutation. The mutant gene is shown to be formed by the fusion of the 5' part of the Dd gene and the 3' part of the Ld gene, with the region in between deleted.
\r\n\r\nChapter Two describes the results of chromosome walking experiments and presents a molecular map of 500 kb of cloned DNA, which links the H-2D and Q\u03b1 regions and contains five D region and eight Q\u03b1 region class I genes.
\r\n\r\nChapter Three presents the DNA sequences of the transmembrane exon from 20 class I genes, and the use of 23 low copy-number flanking-region probes to detect homology between the regions containing each gene. The sequence comparison and the hybridization patterns indicate that multiple recombinational events, notably gene duplication and gene conversion, have occurred during the evolution of this large gene family.
\r\n\r\nChapter Four presents a rapid method of restriction site mapping of cosmids and plasmids. The method was developed due to the need of mapping a large number of clones during the course of this study.
" }, { "name": "Winoto, Astar", "degree": "PhD", "year": "1986", "title": "Structure and Function of the Murine T-Cell Receptor Genes and the Murine Class I Genes of the Major Histocompatability Complex", "advisor": "Hood, Leroy E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03012019-111236038", "creators": [ { "name": { "family": "Winoto", "given": "Astar" }, "id": "Winoto-Astar", "orcid": "0000-0003-4363-4591", "display_name": "Winoto, Astar" } ], "advisors": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "advisor", "display_name": "Hood, Leroy E." } ], "committee": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "chair", "display_name": "Hood, Leroy E." } ], "option_major": [ "molbio" ], "doi": "10.7907/gmnb-h561", "abstract": "This thesis can be divided into three parts: Chapter Two and Chapter Three contain study on the class I genes of the major histocompatibility complex (MHC), Chapter Four and Chapter Five contain study on the T-cell receptor genes, and the appendices deal with my initial attempt to clone the eDNA encoding the T-cell receptor \u03b1 chain and various other research projects that I have been involved in to some extent (mouse MHC class II genes, PDGF genes and rat class I and class II genes).
\r\n\r\nThe first part of my thesis describes the study of the organization of the genes encoding the mouse class I MHC molecule. 54 cosmid clones containing 36 class I genes were isolated and, by restriction enzyme mapping, the 54 clones could be divided into 13 clusters. Using low-copy probes isolated from each cosmid cluster and the restriction enzyme site polymorphism of those probes, I was able to map each of the class I gene clusters into the precise location of the mouse MHC. Surprisingly, most of the class I genes map into the Tla region, only five class I genes (three cosmid clusters) map into the classical H-2 region. The functions of these class I genes in the Tla region are still largely unknown.
\r\n\r\nThe remainder of my thesis contains the study on the T-cell receptor genes. In an effort to isolate the cDNA clone encoding the T-cell receptor \u03b1 chain, I have isolated 64 T\u2013cell specific cDNA clones, using a T-cell minus B-cell subtractive cDNA probe. The T-cell receptor \u03b1 and \u03b2 chain cDNA clones were among these 64 clones. Using the T-cell receptor \u03b1 chain cDNA as a probe, I subsequently isolated clones encoding a germline variable(V) gene segment and cosmid clones spanning 120 kb of DNA encoding the joining(J) and constant(C) gene segments of the T-cell receptor \u03b1 chain. Analysis of these clones, including sequencing of one germline V\u03b1 and six germline J\u03b1 gene segments, showed that the DNA recognition sequence for the \u03b1 chain DNA rearrangment is similar to that of the \u03b2 chain counterpart. In contrast to the general J gene segment organization in the \u03b2 chain, \u03b3 chain and the immunoglobulin gene families, I showed that the 18 J\u03b1 gene segments I analyzed were spread over 60 kb of DNA and lay as far as 63 kb 5' to the C\u03b1 gene.
\r\n\r\nIn a step to dissect the structure function relationship of the T-cell receptor molecules, I have cloned and determined the nucleotide sequences of seven functional \u03b1 chains and six \u03b2 chains of the T-cell receptor genes from nine T-helper hybridomas specific for the C-terminal peptide of pigeon cytochrome c and the E class II molecule. Northern blot analyses using the isolated V\u03b1 and V\u03b2 gene segments were performed on the RNAs isolated from a total of 15 T\u2013helper hybridomas specific for the C-terminal peptide of cytochrome c. A single V\u03b1 gene segment is predominantly used in these 15 T-helper hybridomas, whereas at least five different V\u03b2 gene segments are utilized. I conclude that the V\u03b1 gene segment mis important for the cytochrome c response and might provide most of the contact residues with the C-terminal region of cytochrome c. I also found that the junctional sequences of the \u03b2 chain may alter the antigen fine specificity of the T-cell clones. Finally, somatic hypermutation does not appear to play a crucial role in generating diversity for the T-cell receptor \u03b1 or \u03b2 chains.
" }, { "name": "Kim, Stuart Kilsu", "degree": "PhD", "year": "1985", "title": "Antibody Genes, Oncogenes and Antisense Genes", "advisor": "Wold, Barbara J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01312019-124340304", "creators": [ { "name": { "family": "Kim", "given": "Stuart Kilsu" }, "id": "Kim-Stuart-Kilsu", "display_name": "Kim, Stuart Kilsu" } ], "advisors": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "advisor", "display_name": "Wold, Barbara J." } ], "committee": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "role": "chair", "display_name": "Wold, Barbara J." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "role": "member", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "member", "display_name": "Simon, Melvin I." } ], "option_major": [ "biology" ], "doi": "10.7907/xsdv-c915", "abstract": "There are a number of mechanisms involved in producing a diversity of antibodies including multiple germline genes, somatic gene rearrangement, somatic hypermutation and combinatorial association. By the process of somatic hypermutation, one immunoglobulin gene in the germline can be mutated to produce many different genes in B cells. In chapter 2, this process is characterized. It was found that phosphorylcholine binding antibodies are encoded by one germline VH gene segment. In B cells, this VH gene segment may have extensive point mutations, many of which are silent, indicating the presence of some somatic hypermutational mechanism. Only the VH gene was found to be mutated indicating that the mutational mechanism was specific for VH genes.
\r\n\r\nOne way to study somatic immunoglobulin gene rearrangements, presented in chapter 3, might be to characterize rearrangements which are not easily explained. Immunoglobulin gene rearrangement was thought to exclusively involve immunoglobulin genes. However, some immunoglobulin genes can reproducibly rearrange with other DNA sequences. Insight into the basis of these rearrangements was uncovered by identifying the chromosomal origin of the nonimmunoglobulin rearranging DNA. This DNA originated on chromosome 15 whereas the immunoglobulin gene originated on chromosome 12. The juxtaposition of these sequences is common in plasmacytomas but rare or absent in normal B cells suggesting that it is involved in tumorigenesis. For example, it may be that aberrant immunoglobulin rearrangements can activate a cellular oncogene resulting in a plasmacytoma. This possibility was supported by results from other laboratories when it was found that the non-immunoglobulin rearranging DNA contained the cellular homologue of the myc oncogene.
\r\n\r\nTo understand lymphocyte tumorigenesis, it would be useful to understand the function of the c-myc gene product in normal and transformed cells. One way to begin is to determine which types of cells express the c-myc gene. This approach was employed in chapter 5 and it was found that the c-myc gene is expressed in dividing, but not resting, lymphocytes. One possible function for the c-myc gene product is that it functions in cellular proliferation.
\r\n\r\nAnother way to study the function of the c-myc gene product would be to prevent expression of the rearranged c-myc gene in plasmacytomas. For example, it may be possible to inhibit the synthesis of the c-myc gene product by antisense c-myc RNA. If the antisense RNA can hybridize to the c-myc RNA in vivo, synthesis of myc protein may be prevented. A test case, in which antisense TK RNA is used to inhibit TK expression, is presented in chapter 6. In L cells, high levels of antisense TK RNA expression were capable of inhibiting TK activity. The mechanism of inhibition involves RNA:RNA hybridization since double stranded RNA was formed. If this test case can be applied to other instances, it may be possible to use antisense RNA to inhibit the synthesis of a particular gene product and thus study its cellular function.
" }, { "name": "Levy, David E.", "degree": "PhD", "year": "1985", "title": "Expression of Endogenous Retroviruses in Inbred Mice : Coordinate Regulation and Structure of Multiple Transcription Units", "advisor": "Davidson, Norman R.; Rothenberg, Ellen V.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01302019-123859876", "creators": [ { "name": { "family": "Levy", "given": "David E." }, "id": "Levy-David-E", "orcid": "0000-0002-7320-7788", "display_name": "Levy, David E." } ], "advisors": [ { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "advisor", "display_name": "Davidson, Norman R." }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "co-advisor", "display_name": "Rothenberg, Ellen V." } ], "committee": [ { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "chair", "display_name": "Rothenberg, Ellen V." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "member", "display_name": "Hood, Leroy E." }, { "name": { "family": "Lerner", "given": "R." }, "id": "Lerner-R", "role": "member", "display_name": "Lerner, R." }, { "name": { "family": "Wilson", "given": "M." }, "id": "Wilson-M", "role": "member", "display_name": "Wilson, M." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." } ], "option_major": [ "molbio" ], "doi": "10.7907/165r-9v89", "abstract": "The control of expression of the murine antigen Gix and of other products of endogenous retroviruses, in strain 129 mice and in its congeneic partner strain 129 Gix-, is an example of the coordinate expression of a dispersed family of independent transcription units. In order to provide a molecular description of the Gix phenotype, evidence is presented, from DNA and RNA hybridization analyses using heterologous viral probes, indicating that this phenotype is specified by a distinct regulatory gene, defined genetically, that acts in trans to control the levels of accumulation of specific mRNA species. The steady state levels of several, structurally distinct polyadenylated RNA species are reduced in Gix- mice, and a major reduction in transcription of these sequences accompanies this drop in abundance. Tissue specific patterns of accumulation of different sized RNA species were detected in numerous organs of the mouse, and the majority of these distinct transcripts were collectively regulated.
\r\n\r\nThe isolation and characterization of cDNA copies of these endogenous retroviral transcripts demonstrated that they were derived from multiple, distinct transcription units. Differences among these RNA species were detected by S1 nuclease protection analyses , which confirmed the tissue specific patterns of RNA accumulation. The nucleotide sequences of endogenous virus cDNA clones fully documented the expression of distinct genes, the nature of the sequence heterogeneity, and the relationship of these normal cellular constituents to exogenous, infectious virus. The polymorphism was found to result from both single nucleotide changes and from deletions of different lengths of coding and non-coding information. Comparison of these sequences with exogenous virus demonstrated that the endogenous transcripts are closely related to the recombinant sequences of eukemogenic, mink cell focus forming viruses.
\r\n\r\n" }, { "name": "Lewis, Richard Sheridan", "degree": "PhD", "year": "1985", "title": "The Ionic Basis of Frequency Selectivity in Hair Cells of the Bullfrog's Sacculus", "advisor": "Hudspeth, A. James", "url": "https://resolver.caltech.edu/CaltechTHESIS:04262016-091328237", "creators": [ { "name": { "family": "Lewis", "given": "Richard Sheridan" }, "id": "Lewis-Richard-Sheridan", "orcid": "0000-0002-6010-7403", "display_name": "Lewis, Richard Sheridan" } ], "advisors": [ { "name": { "family": "Hudspeth", "given": "A. James" }, "id": "Hudspeth-A-J", "role": "advisor", "display_name": "Hudspeth, A. James" } ], "committee": [ { "name": { "family": "Berg", "given": "Howard C." }, "id": "Berg-Howard-C", "role": "chair", "display_name": "Berg, Howard C." }, { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "member", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Hudspeth", "given": "A. James" }, "id": "Hudspeth-A-J", "role": "member", "display_name": "Hudspeth, A. James" } ], "option_major": [ "neurobio" ], "doi": "10.7907/MZGR-KW63", "abstract": "Hair cells from the bull frog's sacculus, a vestibular organ responding to substrate-borne vibration, possess electrically resonant membrane properties which maximize the sensitivity of each cell to a particular frequency of mechanical input. The electrical resonance of these cells and its underlying ionic basis were studied by applying gigohm-seal recording techniques to solitary hair cells enzymatically dissociated from the sacculus. The contribution of electrical resonance to frequency selectivity was assessed from microelectrode recordings from hair cells in an excised preparation of the sacculus.
\r\n\r\nElectrical resonance in the hair cell is demonstrated by damped membrane-potential oscillations in response to extrinsic current pulses applied through the recording pipette. This response is analyzed as that of a damped harmonic oscillator. Oscillation frequency rises with membrane depolarization, from 80-160 Hz at resting potential to asymptotic values of 200-250 Hz. The sharpness of electrical tuning, denoted by the electrical quality factor, Qe, is a bell-shaped function of membrane voltage, reaching a maximum value around eight at a membrane potential slightly positive to the resting potential.
\r\n\r\nIn whole cells, three time-variant ionic currents are activated at voltages more positive than -60 to -50 mV; these are identified as a voltage-dependent, non-inactivating Ca current (Ica), a voltage-dependent, transient K current (IA), and a Ca-dependent K current (Ic). The C channel is identified in excised, inside-out membrane patches on the basis of its large conductance (130-200 pS), its selective permeability to Kover Na or Cl, and its activation by internal Ca ions and membrane depolarization. Analysis of open- and closed-lifetime distributions suggests that the C channel can assume at least two open and three closed kinetic states.
\r\n\r\nExposing hair cells to external solutions that inhibit the Ca or C conductances degrades the electrical resonance properties measured under current-clamp conditions, while blocking the A conductance has no significant effect, providing evidence that only the Ca and C conductances participate in the resonance mechanism. To test the sufficiency of these two conductances to account for electrical resonance, a mathematical model is developed that describes Ica, Ic, and intracellular Ca concentration during voltage-clamp steps. Ica activation is approximated by a third-order Hodgkin-Huxley kinetic scheme. Ca entering the cell is assumed to be confined to a small submembrane compartment which contains an excess of Ca buffer; Ca leaves this space with first-order kinetics. The Ca- and voltage-dependent activation of C channels is described by a five-state kinetic scheme suggested by the results of single-channel observations. Parameter values in the model are adjusted to fit the waveforms of Ica and Ic evoked by a series of voltage-clamp steps in a single cell. Having been thus constrained, the model correctly predicts the character of voltage oscillations produced by current-clamp steps, including the dependencies of oscillation frequency and Qe on membrane voltage. The model shows quantitatively how the Ca and C conductances interact, via changes in intracellular Ca concentration, to produce electrical resonance in a vertebrate hair cell.
" }, { "name": "Livant, Donna Lucy", "degree": "PhD", "year": "1985", "title": "The Size of a Murine Heavy Chain Variable Region Gene Family: Implications for the Magnitude and Evolution of the V\u2095 Locus in Mouse", "advisor": "Hood, Leroy E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01242019-174640334", "creators": [ { "name": { "family": "Livant", "given": "Donna Lucy" }, "id": "Livant-Donna-Lucy", "orcid": "0000-0002-6164-6580", "display_name": "Livant, Donna Lucy" } ], "advisors": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "advisor", "display_name": "Hood, Leroy E." } ], "committee": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "chair", "display_name": "Hood, Leroy E." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "member", "display_name": "Lewis, Edward B." }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "member", "display_name": "Owen, Ray David" } ], "option_major": [ "biology" ], "doi": "10.7907/22vp-vt51", "abstract": "The problem of how much antibody diversity is encoded in the germline as variable region genes has long been of interest to immunologists. We have measured the size of the J558 VH family in the BALB/c mouse by a probe excess titration method, and found that the family contains approximately 1000 members. As a control for systematic error, we used the same method to measure the number of class I MHC genes in BALB/c. We found that the third domain of the class I Dd gene detects 36-40 class I genes. Dot blots and genome blots with copy number controls give results consistent with a J558 family size of 500-1000 VH genes. We note that each band evident on genomic blots of DNA from several mouse strains contains multiple VH genes, and that a significant fraction of these bands are polymorphic among the mouse strains tested. We discuss the implications of this result for both the size and evolution of the VH locus in mouse.
" }, { "name": "Masters, Jeffrey Nelson", "degree": "PhD", "year": "1985", "title": "The Organization and Expression of the Human Dihydrofolate Reductase Gene in Methotrexate-Resistant and Methotrexate-Sensitive Cell Lines", "advisor": "Attardi, Giuseppe", "url": "https://resolver.caltech.edu/CaltechTHESIS:01312019-142248860", "creators": [ { "name": { "family": "Masters", "given": "Jeffrey Nelson" }, "id": "Masters-Jeffrey-Nelson", "display_name": "Masters, Jeffrey Nelson" } ], "advisors": [ { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "advisor", "display_name": "Attardi, Giuseppe" } ], "committee": [ { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "orcid": "0000-0001-8291-5551", "role": "chair", "display_name": "Campbell, Judith L." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "member", "display_name": "Lewis, Edward B." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" } ], "option_major": [ "molbio" ], "doi": "10.7907/jaa8-jx74", "abstract": "The analysis of several derivatives of the human cell lines HeLa and VA2-B, selected for resistance to methotrexate (MTX), shows a striking variability in the dihydrofolate reductase (DHFR) enzyme levels, chromosome constitution and growth characteristics in the absence of MTX. In contrast to the mouse system, the number of double minutes of the human cells does not correlate with either the increased DHFR levels or the instability of the amplified phenotype.
\r\n\r\nThe isolation of human DHFR eDNA by differential hybridization or by phenotypic expression in E. coli, facilitates the characterization of the human DHFR coding region and its multiple mRNAs. Comparison of the DNA sequences of several DHFR cDNAs shows a high degree of homology between the coding regions of the human and mouse genes (89%) with no obvious identity in the 3'-untranslated region. The analysis also demonstrates that cDNAs of the 3 identified mRNAs are colinear in the 3'-end sequence, and that polyadenylation occurs at different sites.
\r\n\r\nHybridization of EcoRI digested nuclear DNA from the MTX-resistant 6A3 cells with DHFR cDNAs shows EcoRI fragments that are either unamplified or amplified relative to the same fragments of human sperm, HeLa cell, and VA2-B cell DNA. Two of the unamplified fragments were isolated from a cosmid library made from human sperm DNA. The DNA sequence analysis shows that these two fragments contain a DHFR intronless pseudogene including the EcoRI site found in the DHFR cDNAs. If an RNA intermediate directs the formation of this pseudogene, an RNA larger than the 1.0 kb DHFR mRNA must be involved. In contrast to the unamplified DNA fragments, the amplified fragments contain the exons of the human DHFR gene. The gene is about 29 kb in length, with five introns interrupting the DHFR coding region in the same positions found in the mouse gene. The DHFR mRNAs were mapped as a major 5'-end at position -71 of the human DHFR gene. In addition, six minor 5'-ends of DHFR-specific polysomal RNA were mapped from positions -449 to -480 and represent about 1% of the major transcripts. The upstream transcripts are relatively enriched in the nuclear RNA fraction, indicating a different regulation of expression for these minor transcripts.
" }, { "name": "Parker, Vann Phillips", "degree": "PhD", "year": "1985", "title": "Studies of Gene Structure: I. Expression of Human \u03b1-Globin Genes in COS Cells. II. Isolation and Characterization of the Myosin Light Chain Genes from Drosophila melanogaster", "advisor": "Davidson, Norman R.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01222019-155306456", "creators": [ { "name": { "family": "Parker", "given": "Vann Phillips" }, "id": "Parker-Vann-Phillips", "display_name": "Parker, Vann Phillips" } ], "advisors": [ { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "advisor", "display_name": "Davidson, Norman R." } ], "committee": [ { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "chair", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "member", "display_name": "Hood, Leroy E." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "member", "display_name": "Strauss, James H." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." } ], "option_major": [ "biology" ], "doi": "10.7907/msv6-0s82", "abstract": "The first part of this thesis describes the establishment of COS cells as an effective host for the replication and expression of genes cloned into plasmids containing the SV40 viral replication origin. The human \u03b1-globin gene was cloned into a pBR322 derivative which contained a 311 base pair SV40 fragment. These plasmids were replicated to high copy number. The \u03b1-globin gene was transcribed to produce high levels of RNA which was indistinguishable from human \u03b1-globin mRNA. Deletion mutants were generated which defined the region from 55 to 87 base pairs upstream from the mRNA cap site as necessary for high levels of transcription of this gene. This region includes the conserved sequence CCAAT found in a similar position upstream from many eukaryotic genes.
\r\n\r\nThe second part of this thesis describes the cloning of muscle specific genes from Drosophila melanogaster and a detailed analysis of the two myosin light chain genes. A genomic library was screened with RNA isolated from the late pupal stage of development. Recombinant DNA clones which mapped to 24 independent locations were identified. The clones containing the myosin light chain genes were identified from within this group. A cDNA library was generated from late pupal RNA. Clones homologous to each of the myosin light chains were identified and their cloned inserts were sequenced. Their classification as Drosophila myosin light chain genes was established by comparing the derived amino acid sequence to the protein sequence of the chicken myosin light chains. Each myosin light chain is single copy. The alkali myosin light chain maps to the chromosomal locus 98B. It hybrid selects RNA which may be translated in vitro to yeild several polypeptides of molecular weight 18,000 to 19,000 daltons. The gene for myosin light chain-2 maps to the chromosomal locus 99E and is shown to encode two proteins of apparent molecular weights 26,000 and 17,000 daltons. Each protein has a putative divalent cation binding domain.
" }, { "name": "Roach, Arthur Henry", "degree": "PhD", "year": "1985", "title": "The Genes for Myelin Basic Protein in Normal and Shiverer Mutant Mice", "advisor": "Hood, Leroy E.; Strumwasser, Felix", "url": "https://resolver.caltech.edu/CaltechTHESIS:01312019-172314851", "creators": [ { "name": { "family": "Roach", "given": "Arthur Henry" }, "id": "Roach-Arthur-Henry", "display_name": "Roach, Arthur Henry" } ], "advisors": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "advisor", "display_name": "Hood, Leroy E." }, { "name": { "family": "Strumwasser", "given": "Felix" }, "id": "Strumwasser-F", "role": "co-advisor", "display_name": "Strumwasser, Felix" } ], "committee": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "chair", "display_name": "Davidson, Eric H." }, { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "member", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Patterson", "given": "Paul H." }, "id": "Patterson-P-H", "role": "member", "display_name": "Patterson, Paul H." }, { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-B-J", "orcid": "0000-0003-3235-8130", "role": "member", "display_name": "Wold, Barbara J." }, { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "member", "display_name": "Hood, Leroy E." }, { "name": { "family": "Strumwasser", "given": "Felix" }, "id": "Strumwasser-F", "role": "member", "display_name": "Strumwasser, Felix" } ], "option_major": [ "biology" ], "doi": "10.7907/4d7s-zs52", "abstract": "A cDNA library was constructed from the brains of 18 day old rats, and was screened with a synthetic DNA probe to yield clones representing myelin basic protein (MBP). One 1.9 kb clone was sequenced and found to encode the 14 kd MBP. Using this clone as a hybridization probe, cosmid clones from a library of wild type mouse DNA were selected and characterized. One clone was shown to carry five exons which encode 14 kd MBP, distributed over a 32 kb region. A sixth exon was detected with a synthetic DNA probe, and was found to encode the 41 amino acids which distinguish 18.5 kd from 14 kd MBP. The 5' end ot the gene was mapped with S1 nuclease protection and primer extension experiments to a position 47 bp 5' of the initator codon for MBP synthesis. It was shown that the gene cloned is probably the only MBP gene in the mouse genome.
\r\n\r\nCloned DNAs were used to analyze the MBP gene and its expression in the myelin deficient mutant mouse shiverer. It was shown that a deletion has removed five out of six MBP exons, leaving only the 5'-most exon and 13 kb of the first intervening sequence. The deletion completely prevents expression of normal 2.1 kb MBP mRNAs, but a 16-fold lower number of transcripts are observed which initiate correctly at the 5' end of the first exon, are not correctly spliced, and are rarely polyadenylated. If translated, they would direct synthesis of a 61 amino acid peptide containing the first 56 amino acids of MBP. The MBP gene was mapped to mouse chromosome 18 by hybridization of MBP probes with DNA from Chinese hamster-mouse hybrid cell lines, showing it to be linked to the shiverer mutation. It is proposed that the partial deletion of the MBP gene is the primary lesion of the shiverer mutation.
" }, { "name": "Segall, Jeffrey Edward", "degree": "PhD", "year": "1985", "title": "Chemotaxis of Escherichia coli Studied Using Ionophoretic Stimulation", "advisor": "Berg, Howard C.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02132019-181420941", "creators": [ { "name": { "family": "Segall", "given": "Jeffrey Edward" }, "id": "Segall-Jeffrey-Edward", "display_name": "Segall, Jeffrey Edward" } ], "advisors": [ { "name": { "family": "Berg", "given": "Howard C." }, "id": "Berg-Howard-C", "role": "advisor", "display_name": "Berg, Howard C." } ], "committee": [ { "name": { "family": "Brokaw", "given": "Charles J." }, "id": "Brokaw-C-J", "role": "chair", "display_name": "Brokaw, Charles J." }, { "name": { "family": "Fender", "given": "Derek H." }, "id": "Fender-D-H", "role": "member", "display_name": "Fender, Derek H." }, { "name": { "family": "Hopfield", "given": "John J." }, "id": "Hopfield-J-J", "role": "member", "display_name": "Hopfield, John J." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Simon", "given": "Melvin I." }, "id": "Simon-M-I", "role": "member", "display_name": "Simon, Melvin I." }, { "name": { "family": "Berg", "given": "Howard C." }, "id": "Berg-Howard-C", "role": "member", "display_name": "Berg, Howard C." } ], "option_major": [ "biochem" ], "doi": "10.7907/6r2y-ba72", "abstract": "Chemotactic responses of the bacterium Escherichia coli were elicited by iontophoretic ejection of charged compounds from micropipettes. Responses were measured using cells tethered by a single flagellum and following the direction of rotation of the cell body (with cells viewed from above the surface to which the flagellum is attached) . Mean response latencies to addition of attractant or repellent were 0.1 to 0.2 seconds. Brief pulses of attractants and repellents were used to determine the impulse response for chemotaxis. In response to a brief pulse of attractant the counterclockwise (CCW) bias (fraction of time that the flagellar motor spent rotating CCW) increased rapidly to a peak, then fell below the prestimulus value, returning to baseline within 5 seconds -- the response was biphasic. Repellent impulse responses were similar but inverted. The attractant impulse response accurately predicted responses to step changes, linear ramp changes and sine wave changes in receptor occupancy. Mutants defective in the enzymes responsible for methylation (cheR) and demethylation (cheB), which do not adapt to attractant stimuli, gave monophasic impulse responses--only the initial peak was evident. Mutants defective in the cheZ gene product had responses that were slower than the wild-type by a factor of about 10. The responses of flagellar motors on filamentous cells also were studied. The reversals of two motors on the same cell were not correlated, but fluctuations in bias were correlated for motors less than l 0 microns apart. Responses of motors of filamentous cells to iontophoretic application of aspartate indicated that the internal chemotaxis signal travels about 2 microns in cells lacking the cheR and cheB gene products and about 6 microns in cells with a defective cheZ gene product. These data are consistent with a simple model involving destruction of a cellular chemotaxis signal by the cheZ gene product.
" }, { "name": "Sher, Beverly Taylor", "degree": "PhD", "year": "1985", "title": "Studies of Class I Genes in the Major Histocompatibility Complex of the BALB/c Mouse", "advisor": "Hood, Leroy E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01312019-165301208", "creators": [ { "name": { "family": "Sher", "given": "Beverly Taylor" }, "id": "Sher-Beverly-Taylor", "display_name": "Sher, Beverly Taylor" } ], "advisors": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "advisor", "display_name": "Hood, Leroy E." } ], "committee": [ { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "chair", "display_name": "Davidson, Norman R." }, { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "member", "display_name": "Lewis, Edward B." }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "member", "display_name": "Owen, Ray David" }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "member", "display_name": "Hood, Leroy E." } ], "option_major": [ "biology" ], "doi": "10.7907/4pj0-z147", "abstract": "This thesis contains the results of investigations into the structure and organization of Class I genes in the major histocompatibility complex of the BALB/c mouse.
\r\n\r\nIn the body of the thesis, the sequence of the BALB/c H-2Dd transplantation antigen gene is presented. This is the first complete sequence of an H-2Dd gene and is the only genomic sequence to be in full agreement with the available protein sequence. The H-2Dd gene sequence has been used to predict the protein sequence of the H-2Dd molecule, which has been compared to the protein sequences of other Class I molecules. The H-2Dd protein sequence is no more related to that of its closely linked partner, H-2Ld, than it is to the sequence of its presumptive allele, H-2Db, or to the sequence of the H-2Kb molecule, which is from not only another H-2 haplotype but another genetic subregion. The sequence differences between these transplantation antigens are spread throughout the molecules in a mosaic pattern that may have arisen,in part, from small gene conversion events. No obvious evidence of any recent gene conversion event affecting the H-2Dd gene was observed, however.
\r\n\r\nThree Class I genes have been cloned and mapped to the H-2D subregion in BALB/c. These include gene 16.1, whose product has not been identified; the H-2Dd gene; and the H-2Ld gene. There is serological evidence for the existence of additional H-2D-encoded transplantation antigen molecules in BALB/c, but no genes encoding these products have been identified. The sequence of the H-2Dd gene contains potential alternative splice sites in and around the exon encoding the first external domain. use of these splice sites could generate a transplantation antigen molecule with different serological determinants, and might help to resolve the discrepancy between the number of H-2D-subregion Class I genes and the number of serologically defined H-2D-subregion transplantation antigens.
\r\n\r\nThe appendices contain the results of a number of studies related to Class I gene organization and function. Appendix A contains the sequence of gene 27.1. This gene, also known as the Q8 gene, was identified as a Qa pseudogene based on the presence of termination codons in inappropriate locations in its sequence. Appendix B contains the sequence of the H-2Ld gene, which was the first transplantation antigen gene to be sequenced. Appendix C contains the results of DNA-mediated gene transfer experiments that Identified genomic clones containing the H-2Kd, H-2Ld, and H-2Dd transplantation antigen genes as well as Class I genes encoding the Qa2,3 molecule and two different TL differentiation antigen genes. Appendix D contains the results of calculations of protein sequence homology between different Class I molecules.
" }, { "name": "Cronin-Golomb, Alice Mary", "degree": "PhD", "year": "1984", "title": "Intrahemispheric Processing and Subcortical Transfer of Non-Verbal Information in Subjects with Complete Forebrain Commissurotomy", "advisor": "Sperry, Roger Wolcott", "url": "https://resolver.caltech.edu/CaltechTHESIS:10032018-103359828", "creators": [ { "name": { "family": "Cronin-Golomb", "given": "Alice Mary" }, "id": "Cronin-Golomb-Alice-Mary", "orcid": "0000-0001-5699-6204", "display_name": "Cronin-Golomb, Alice Mary" } ], "advisors": [ { "name": { "family": "Sperry", "given": "Roger Wolcott" }, "id": "Sperry-R-W", "role": "advisor", "display_name": "Sperry, Roger Wolcott" } ], "committee": [ { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "chair", "display_name": "Allman, John Morgan" }, { "name": { "family": "Hamilton", "given": "Charles R." }, "id": "Hamilton-Charles-R", "role": "member", "display_name": "Hamilton, Charles R." }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "member", "display_name": "Van Essen, David C." }, { "name": { "family": "Sperry", "given": "Roger Wolcott" }, "id": "Sperry-R-W", "role": "member", "display_name": "Sperry, Roger Wolcott" } ], "option_major": [ "biology" ], "doi": "10.7907/vt51-7x14", "abstract": "Subjects who had undergone complete surgical division of the forebrain commissures for treatment of intractable epilepsy were tested on a variety of cognitive and perceptual tasks. It was found that the right hemisphere performs as well as the left on a test of abstract concept comprehension when the stimulus materials are presented in a non-verbal format. In light of evidence of a selective right hemisphere deficiency for processing abstract words, this result is taken to imply a dissociation of language and cognition at a high level. A second experiment involved the nature of information which can cross subcortically between the cerebral hemispheres. With stimuli presented to opposite visual hemi-fields for prolonged durations, three commissurotomy subjects were able to make matches which convincingly demonstrated interhemispheric transfer and integration of cognitive information, including concrete and abstract concepts. Transfer between the hemispheres was equally successful in the two directions, though the pathway originating in the right and terminating in the left hemisphere may be more sensitive to some affective and semantic components of the stimulus. The information relayed subcortically is neither verbal nor imagic in nature, but appears to involve contextual or connotative associations of the stimulus. Implications for the evolution and development of non-verbal thought include the possible existence of a common bilateral cognitive system which permits interhemispheric communication of complex, if imprecise, associations that are distinct from the more specific verbal and visuospatial constructs of the left and right hemispheres, respectively. Finally, differences in the ability of the two hemispheres to perceive figure and background were described for four commissurotomy subjects. While the left hemisphere preferentially identified figures from briefly-presented picture compositions, the right hemisphere was equally adept at recognizing both figure and ground. The right hemisphere was also more sensitive to background influences on object perception, and was furthermore able to use \"natural\" gradient and perspective cues in evaluating an object's size and position in a field. In sum, the results demonstrate (1) the richness and complexity of non-verbal information and its place in human thought processes, and (2) the sophistication of the right hemisphere as a perceptual and cognitive system.
\r\n" }, { "name": "Crowley, Thomas Edward", "degree": "PhD", "year": "1984", "title": "Functions and Regulation of RNAs Transcribed from the Drosophila melanogaster 68C Puff", "advisor": "Meyerowitz, Elliot M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10302018-171502394", "creators": [ { "name": { "family": "Crowley", "given": "Thomas Edward" }, "id": "Crowley-Thomas-Edward", "display_name": "Crowley, Thomas Edward" } ], "advisors": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "advisor", "display_name": "Meyerowitz, Elliot M." } ], "committee": [ { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "chair", "display_name": "Davidson, Norman R." }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "member", "display_name": "Mitchell, Herschel K." }, { "name": { "family": "Parker", "given": "Carl Stevens" }, "id": "Parker-C-S", "orcid": "0000-0001-9795-4211", "role": "member", "display_name": "Parker, Carl Stevens" }, { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "member", "display_name": "Strauss, James H." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." } ], "option_major": [ "biology" ], "doi": "10.7907/7vtr-zc75", "abstract": "Puffs appear and disappear on Drosophila salivary gland polytene chromosomes at specific developmental timepoints or in response to external stimuli. This thesis is an analysis of the functions and regulation of three RNAs transcribed from the puff at 68C on the left arm of the third chromosome.
\r\n\r\nThe nucleotide sequence of the DNA encoding these RNAs was used to predict the physical and chemical properties expected of their protein products. Analysis of radiolabeled salivary glands revealed polypeptides having the characteristics predicted for the products of the 68C RNAs. Amino acid sequencing of these proteins showed that they are in fact encoded by the 68C RNAs. All three polypeptides were found to be part of the salivary gland glue: one is the previously described sgs-3, the others the newly identified glue proteins sgs-7 and sgs-8.
\r\n\r\nThe effect of the steroid hormone ecdysterone on the 68C RNAs was examined by culturing salivary glands in vitro in the presence or absence of the hormone. The presence of the steroid caused the RNAs to disappear more rapidly than they would in its absence. Pulse-labeling experiments demonstrated that the effect of ecdysterone is on an early step in RNA production, probably transcription. The effect on the 68C RNAs is very rapid, more rapid than puff regression. The three RNAs appear to be coordinately regulated.
\r\n\r\nThe expression of glue protein genes in a non-pupariating mutant strain of Drosophila has been studied. Although a puff is present at position 68C on the third chromosome in the mutants, the Sgs-3, Sgs-7 and Sgs-8 genes are not expressed. Pulse-labeling experiments indicate that the mutation affects transcription of these genes. Other researchers have mapped the non-pupariating mutation to position 2B on the X chromosome. It appears that a product of a gene at 2B or a product whose synthesis is induced by a gene at 2B is necessary for transcription of the 68C glue protein genes. This trans-acting regulatory element produces its effect by interacting with DNA sequences within or very close to the glue genes at 68C. These results along with transcription autoradiograms show that the puff at 68C is not caused by transcription of Sgs-3, Sgs-7, Sgs-8 or any\r\nother genes located within the puff region.
" }, { "name": "Eatock, Ruth Anne", "degree": "PhD", "year": "1984", "title": "Sensory Adaptation in Hair Cells of the Bullfrog's Sacculus", "advisor": "Hudspeth, A. James", "url": "https://resolver.caltech.edu/CaltechTHESIS:12072018-092224939", "creators": [ { "name": { "family": "Eatock", "given": "Ruth Anne" }, "id": "Eatock-Ruth-Anne", "orcid": "0000-0001-7547-2051", "display_name": "Eatock, Ruth Anne" } ], "advisors": [ { "name": { "family": "Hudspeth", "given": "A. James" }, "id": "Hudspeth-A-J", "role": "advisor", "display_name": "Hudspeth, A. James" } ], "committee": [ { "name": { "family": "Hudspeth", "given": "A. James" }, "id": "Hudspeth-A-J", "role": "chair", "display_name": "Hudspeth, A. James" }, { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "member", "display_name": "Van Essen, David C." }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "member", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Pine", "given": "Jerome" }, "id": "Pine-J", "role": "member", "display_name": "Pine, Jerome" } ], "option_major": [ "biology" ], "doi": "10.7907/6pam-hv78", "abstract": "Hair cells in the bullfrog's sacculus, a vestibular organ sensitive to linear acceleration, show sensory adaptation: the response to a constant stimulus peaks near the stimulus onset, then decays. This has been studied in two different preparations. In an excised in vitro preparation of the saccular sensory epithelium, intracellular responses of hair cells to step deflections of their hair bundles were recorded. The second set of experiments was conducted in vivo using steps of vertical linear acceleration as stimuli. The hair cell response was recorded extracellularly in the form of the saccular microphonic potential. Both the intracellular response to direct hair bundle deflection and the extracellular response to acceleration adapted to a steady-state value within the first 100 ms following the step onset. In both cases, the response decline was largely due to a shift in the operating range of the cells in the direction of the constant stimulus. This shift occurred without significant change in dynamic range or in sensitivity within the operating range. Thus the hair cells appear to respond to static stimuli by resetting the bias point of the operating range in the direction of the stimulus.
\r\n\r\nThe response of primary saccular neurons to acceleration steps also showed pronounced sensory adaptation. Comparison of the afferent activity and saccular microphonic potential suggests that adaptation of afferent responses to acceleration steps may be due largely to the adaptive operating range shift in the hair cell responses.
\r\n\r\nThe adaptation of saccular neurons to acceleration steps may be explained by the following simple model. The acceleration step causes displacement of the saccular otolith and deflection of the underlying hair bundles. The hair cells respond initially to the displacement, then adapt (as observed in vitro), and this information is faithfully translated postsynaptically into afferent spike rate. However, the possibility exists that the in vitro and in vivo adaptive shifts in operating range are not the same process. In vivo, one cannot distinguish an operating range shift within the hair cells from one due to mechanical adaptation of the stimulus to the hair bundles.
" }, { "name": "Goldberg, David Alan", "degree": "PhD", "year": "1984", "title": "Molecular Studies on the Alcohol Dehydrogenase Gene of Drosophila melanogaster", "advisor": "Meyerowitz, Elliot M.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10302018-173746907", "creators": [ { "name": { "family": "Goldberg", "given": "David Alan" }, "id": "Goldberg-David-Alan", "display_name": "Goldberg, David Alan" } ], "advisors": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "advisor", "display_name": "Meyerowitz, Elliot M." } ], "committee": [ { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "chair", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "member", "display_name": "Lewis, Edward B." }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "member", "display_name": "Mitchell, Herschel K." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Maniatis", "given": "Thomas P." }, "id": "Maniatis-T-P", "role": "member", "display_name": "Maniatis, Thomas P." } ], "option_major": [ "bioch" ], "doi": "10.7907/skx9-0t33", "abstract": "In this thesis, I describe the isolation of the alcohol dehydrogenase (Adh) gene of Drosophila melaogaster and some preliminary biochemical characterizations of the gene and its expression. The isolation of the Adh gene was accomplished by screening a bacteriophage \u03bb library containing inserts of Drosophila DNA with eDNA probe made from size selected mRNA. One clone which showed hybridization in the initial scr\u2022en was shown to contain Adh sequences by virtue of its lack of hybridization to Adh deficiency DNA, in situ hybridization, translation of ADH protein by mRNA selected by hybridization to the clone, and by partial DNA sequence analysis. Using the clone, approximately 35 kb of the Adh chromosomal region was isolated. This region was found to be composed largely of single-copy sequences, showed limited polymorphism between strains, and encoded only one RNA transcript prevalent in larvae and adults - the Adh mRNA. Two intervening sequences within the Adh coding region were demonstrated by S1 nuclease mapping.
\r\n\r\nIn order to identify sequences important in Adh expression, the cloned Adh gene was transformed into the Drosophila germ line by utilizing the hybrid dysgenesis P element vector of Spradling and Rubin. The correct developmental expression of the Adh gene was retained by the transformed gene, even though it had integrated into many locations. These results delimit the sequences and chromoscmal enviroiiilent necessary for correct developmental expression of the Adh gene. In addition, the 'transient' expression of cloned DNA in larvae and adults directly grown from injected embryos was investigated. In most instances, ADH activity was found only in tissues that normally express ADH, although low level of activity was observed in some cells which do not normally produce detectable levels of Adh. Together, these results form the basis for assay systems that may be combined with in vitro mutagenesis in order to determine in detail which sequences are necessary for correct developmental expression of the Adh gene.
" }, { "name": "Gordon, Herman J.", "degree": "PhD", "year": "1984", "title": "Postnatal Development of Motor Units in Rabbit and Rat Soleus Muscles", "advisor": "Van Essen, David C.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10032018-123356944", "creators": [ { "name": { "family": "Gordon", "given": "Herman J." }, "id": "Gordon-Herman-J", "display_name": "Gordon, Herman J." } ], "advisors": [ { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "advisor", "display_name": "Van Essen, David C." } ], "committee": [ { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "chair", "display_name": "Van Essen, David C." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "member", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Grinnell", "given": "Alan D." }, "id": "Grinnell-Alan-D", "role": "member", "display_name": "Grinnell, Alan D." } ], "option_major": [ "biology" ], "doi": "10.7907/3ecg-0z58", "abstract": "The development of motor unit properties in the soleus muscle of rabbits and rats was used to study the control of innervation and elimination of synapses from mammalian skeletal muscle fibers and the differentiation of those muscle fibers into various twitch types.
\r\n\r\n1. Motor unit twitches with distinctly different time courses were found in rabbit soleus muscle at a stage in development when all muscle fibers were polyinnervated. This observation implies that (1) muscle fibers have already begun their physiological differentiation into twitch types while still polyinnervated and (2) motor neurons of a specific type preferentially polyinnervate muscle fibers of a corresponding type.
\r\n\r\n2. It has recently been claimed that synapse elimination occurs preferentially among motor neurons from the more rostral of the two spinal roots contributing to the soleus muscle of the rat. Using an assay based on measurements of motor unit twitch tens ions, it was found, contrary to the previous claim, that synapses were lost to the same extent by motor neurons passing through all contributing spinal roots to both the rabbit and rat soleus muscles.
\r\n\r\n3. Two lines of evidence indicate that rabbit soleus motor neurons redistribute their terminals at a time after wholesale polyinnervation has been lost from the muscle. (1) Between 11 and 18 days and 5 weeks of age, the frequency of histochemically defined type I fibers increases from 30% to 65% while the incidence of physiologically defined slow motor units does not obviously change. (2) Over the same time period, the ratio of average slow twitch tension to average fast twitch tension quadruples after correction for changes 1n muscle fiber cross sectional area. I hypothesize that during this 3 week time window, slow twitch motor neurons take over end plates previously occupied by fast twitch motor terminals.
\r\n\r\n4. Activity has previously been shown to play a role in the overall rate of synapse elimination. I have conducted preliminary experiments to address whether a competition on active muscle fibers between terminals of active and tetrodotoxin-inactivated motor axons results in a preferential retention of active connections. With the paradigm used, there was at most a small bias favoring the survival of active synapses.
\r\n" }, { "name": "Katz, Lawrence Charles", "degree": "PhD", "year": "1984", "title": "Intrinsic Connectivity of Identified Projection Neurons in Cat Visual Cortex Brain Slices", "advisor": "Konishi, Masakazu", "url": "https://resolver.caltech.edu/CaltechTHESIS:11202018-101534170", "creators": [ { "name": { "family": "Katz", "given": "Lawrence Charles" }, "id": "Katz-Lawrence-Charles", "display_name": "Katz, Lawrence Charles" } ], "advisors": [ { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "advisor", "display_name": "Konishi, Masakazu" } ], "committee": [ { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "chair", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Heiligenberg", "given": "Walter F." }, "id": "Heiligenberg-Walter-F", "role": "member", "display_name": "Heiligenberg, Walter F." }, { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "member", "display_name": "Van Essen, David C." } ], "option_major": [ "neurobio" ], "doi": "10.7907/kahn-q578", "abstract": "The mammalian primary visual cortex is a structure of remarkable physiological and morphological heterogeneity. Despite quite intensive efforts, using a variety of approaches, the relationships between neuronal form and neuronal function have remained obscure. Most previous attempts proceeded on the assumption that a cell's physiological responses to particular stimuli were the best indication of its function. In this study a different assumption was made: that since different efferent targets of area 17 neurons were presumably engaged in different sorts of neural processing, area 17 cells that project to those targets should be engaged in different intrinsic circuits. This in turn might be reflected in distinctive patterns of intrinsic axons and dendrites. This hypothesis was tested by comparing the morphology of two groups of neurons within layer VI of cat area 17: those that project to the claustrum and those that project to the lateral geniculate nucleus. Since both projections occupy the same laminar position, and therefore have potential access to the same environmental and lamina-specific influences, this projection was an excellent system to examine the role of different efferent projections in defining neuronal form, not confounded by differences in laminar position.
\r\n\r\nThis study was carried out by retrogradely labeling, in vivo, one or the other of the projections with a newly developed fluorescent tracer, latex microspheres. Subsequently, in vitro brain slices were prepared from area 17, and the retrogradely labeled neurons were visualized, impaled, and intracellularly stained with a second fluorescent dye, lucifer yellow.
\r\n\r\nComparisons of the two efferent projection classes revealed nonoverlapping patterns of distributions of intrinsic axons and dendrites between the two groups, and a remarkable degree of homogeneity within each group. Particularly dramatic was the difference in intrinsic axons: claustrum projecting cells had long, horizontally directed collaterals restricted to layer VI, whereas LGN projecting neurons had few if any collaterals within layer VI, possessing instead thick, ascending collaterals which arborized in layer IV. Additionally, claustrum projecting cells had significantly fewer, yet more extensive, basal dendritic arms than LGN projecting cells. The apical dendrites of the two groups arborized within different overlying laminae, suggesting that the two classes receive different inputs. These differences in axons and dendrites demonstrate that the two cell classes participate in different intrinsic circuits within area 17.
\r\n\r\nIn addition to the two efferent projection classes, a considerable number of pyramidal cells lacking an efferent axon were observed. They resembled either one or the other of the projection classes, and may represent a substantial\r\npopulation of neurons that, during development, were unable to maintain an efferent projection.
\r\n\r\nThese results suggest that, independent of laminar differences, at least some of the cellular heterogeneity observed in cortex may be attributed to the different informational needs of various efferent targets.
\r\n" }, { "name": "Krouse, Mauri Eugene", "degree": "PhD", "year": "1984", "title": "Investigation of Competitive Antagonist Binding to the Nicotinic Acetylcholine Receptor Using Voltage-Jump and Light-Flash Techniques", "advisor": "Lester, Henry A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05282015-161004818", "creators": [ { "name": { "family": "Krouse", "given": "Mauri Eugene" }, "id": "Krouse-Mauri-Eugene", "display_name": "Krouse, Mauri Eugene" } ], "advisors": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "advisor", "display_name": "Lester, Henry A." } ], "committee": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "chair", "display_name": "Lester, Henry A." }, { "name": { "family": "Pine", "given": "Jerome" }, "id": "Pine-J", "role": "member", "display_name": "Pine, Jerome" }, { "name": { "family": "Chan", "given": "Sunney I." }, "id": "Chan-S-I", "orcid": "0000-0002-5348-2723", "role": "member", "display_name": "Chan, Sunney I." }, { "name": { "family": "Berg", "given": "Howard C." }, "id": "Berg-Howard-C", "role": "member", "display_name": "Berg, Howard C." }, { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "member", "display_name": "Van Essen, David C." } ], "option_major": [ "neurobio" ], "doi": "10.7907/wb48-ph26", "abstract": "1. The effect of 2,2\u2019-bis-[\u03b1-(trimethylammonium)methyl]azobenzene (2BQ), a photoisomerizable competitive antagonist, was studied at the nicotinic acetycholine receptor of Electrophorus electroplaques using voltage-jump and light-flash techniques.
\r\n\r\n2. 2BQ, at concentrations below 3 \u03bc\u039c, reduced the amplitude of voltage-jump relaxations but had little effect on the voltage-jump relaxation time constants under all experimental conditions. At higher concentrations and voltages more negative than -150 mV, 2BQ caused significant open channel blockade.
\r\n\r\n3. Dose-ratio studies showed that the cis and trans isomers of 2BQ have equilibrium binding constants (Ki) of .33 and 1.0 \u03bc\u039c, respectively. The binding constants determined for both isomers are independent of temperature, voltage, agonist concentration, and the nature of the agonist.
\r\n\r\n4. In a solution of predominantly cis-2BQ, visible-light flashes led to a net cis\u2192trans isomerization and caused an increase in the agonist-induced current. This increase had at least two exponential components; the larger amplitude component had the same time constant as a subsequent voltage-jump relaxation; the smaller amplitude component was investigated using ultraviolet light flashes.
\r\n\r\n5. In a solution of predominantly trans-2BQ, UV-light flashes led to a net trans\u2192cis isomerization and caused a net decrease in the agonist-induced current. This effect had at least two exponential components. The smaller and faster component was an increase in agonist-induced current and had a similar time constant to the voltage-jump relaxation. The larger component was a slow decrease in the agonist-induced current with rate constant approximately an order of magnitude less than that of the voltage-jump relaxation. This slow component provided a measure of the rate constant for dissociation of cis-2BQ (k_ = 60/s at 20\u00b0C). Simple modelling of the slope of the dose-rate curves yields an association rate constant of 1.6 x 108/M/s. This agrees with the association rate constant of 1.8 x 108/M/s estimated from the binding constant (Ki). The Q10 of the dissociation rate constant of cis-2BQ was 3.3 between 6\u00b0 and 20\u00b0C. The rate constants for association and dissociation of cis-28Q at receptors are\r\nindependent of voltage, agonist concentration, and the nature of the agonist.
\r\n\r\n6. We have measured the molecular rate constants of a competitive antagonist which has roughly the same Ki as d-tubocurarine but interacts more slowly with the receptor. This leads to the conclusion that curare itself has an association rate constant of 4 x 109/M/s or roughly as fast as possible for an encounter-limited reaction.
\r\n" }, { "name": "Mayne, Jeffrey Terrell", "degree": "PhD", "year": "1984", "title": "The Post-Translational Processing of Sindbis Virus Glycoproteins", "advisor": "Strauss, James H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:11282018-124040200", "creators": [ { "name": { "family": "Mayne", "given": "Jeffrey Terrell" }, "id": "Mayane-Jeffrry-Terrell", "display_name": "Mayne, Jeffrey Terrell" } ], "advisors": [ { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "advisor", "display_name": "Strauss, James H." } ], "committee": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "chair", "display_name": "Mitchell, Herschel K." }, { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "member", "display_name": "Hood, Leroy E." }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "member", "display_name": "Revel, Jean-Paul" }, { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "member", "display_name": "Strauss, James H." } ], "option_major": [ "bioch" ], "doi": "10.7907/jvbk-es85", "abstract": "A small glycoprotein (E3) was purified from the culture fluid of Sindbis virus infected chicken cells and shown to be produced from the cleavage of PE2 to produce E2. The N-terminal sequence of E3 is identical to that of PE2. The first 19 amino acids are hydrophobic and presumably serve as the signal sequence for PE2. This sequence is unusual in that it is not immediately cleaved from PE2 and is glycosylated at position 14. Labeling studies imply that the PE2 \u2192 E2 + E3 cleavage is not closely coupled to budding. E3 is cleaved and released into the culture fluid under conditions where no virions bud, and the kinetics of appearance of E3 in the culture fluid and E2 in virions are dissimilar. The maturation of E3 is discussed as it relates to the processing of cellular membrane glycoproteins.
\r\n\r\nHybridomas were selected by the fusion of NSI/1 myeloma cells with spleen cells from mice inoculated with Sindbis specific antigens. Ten stable hybridomas were obtained, seven producing E1-specific antibodies and three producing capsid-specific antibodies. The seven E1 specific antibodies were divided into two classes, which reacted with different E1 antigenic domains. The two classes of antibodies differed in several tested properties. Two E1 clones inhibited viral infectivity, and one of these precipitated E2 along with E1 in Triton-treated preparations. These properties are discussed with regard to the known relationships between the viral structural proteins.
\r\n\r\nThe tryptic glycopeptides of E1 and E2 grown in BHK or chick cells were purified and analyzed by N-terminal sequencing, pronase digestions and labeling with various radioactive sugars. We found that the glycosylation patterns for the two proteins were essentially identical in the two hosts. E2 contains exclusively complex chains attached to Asn196 and simple chains attached to Asn398. In E1, the Asn135 glycosylation site contained only complex chains, but the Asn245 site contained a mixture of simple and complex chains. A prediction as to the relative importance of the different glycosylation sites to protein function is offered.
" }, { "name": "Myers, Jay J.", "degree": "PhD", "year": "1984", "title": "Cognitive Transfer from Right to Left Hemisphere after Section of the Forebrain Commissures", "advisor": "Sperry, Roger Wolcott", "url": "https://resolver.caltech.edu/CaltechTHESIS:10302018-184155718", "creators": [ { "name": { "family": "Myers", "given": "Jay J." }, "id": "Myers-Jay-J", "display_name": "Myers, Jay J." } ], "advisors": [ { "name": { "family": "Sperry", "given": "Roger Wolcott" }, "id": "Sperry-R-W", "role": "advisor", "display_name": "Sperry, Roger Wolcott" } ], "committee": [ { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "chair", "display_name": "Allman, John Morgan" }, { "name": { "family": "Hamilton", "given": "Charles R." }, "id": "Hamilton-Charles-R", "role": "member", "display_name": "Hamilton, Charles R." }, { "name": { "family": "Thompson", "given": "Frederick B." }, "id": "Thompson-F-B", "role": "member", "display_name": "Thompson, Frederick B." }, { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "member", "display_name": "Van Essen, David C." }, { "name": { "family": "Sperry", "given": "Roger Wolcott" }, "id": "Sperry-R-W", "role": "member", "display_name": "Sperry, Roger Wolcott" } ], "option_major": [ "neurobio" ], "doi": "10.7907/agst-h905", "abstract": "Transfer of cognitive information from right to left hemisphere was examined in patients with complete surgical section of the forebrain commissures.
\r\n\r\nA simple new technique is described that allows lateralized presentation of visual input for prolonged viewing by a single hemisphere without attachments to the eye. This technique was applied in tests of the ability of two complete commissurotmy patients to name simple visual and tactual stimuli projected to the right hemisphere and to cross-compare bilateral input, in exception to characteristic disconnection effects. Special procedures and control tests were employed to determine the underlying mechanisms of such behaviors, and especially to assess the involvement of the left hemisphere. Three commissurotomy subjects were also tested for their ability to verbally describe pictures and printed nouns, corresponding to items associated with distinctive tastes and smells, presented for prolonged viewing in the left hemifield.
\r\n\r\nThe commissurotomy patients could sometimes name or cross-integrate the simple stimuli. Use of cognitive strategies and access to stimulus information by the left hemisphere was shown under these conditions. The subjects could not orally name more complex pictures and words. They could, however, provide relevant and appropriate verbal reports including evaluations, category and context cues and even distinct perceptual impressions and other specific associations but not the precise identity.
\r\n\r\nResults demonstrate that certain cognitive aspects of right hemisphere processing can transfer to the left hemisphere through brainstem channels. Verbalizations in response to stimuli presented in the left visual field and other recent exceptions to symptoms of disconnection may result from this subcortical communication. Other possibilities including oral naming by the right hemisphere cannot account for these results. The name or identity of stimuli is not conveyed by these interhemispheric transmissions but rather, less specific information that is more connotative or orientational in nature. Such transmissions are presumed to function also in normal cognitive processing. The findings provide further evidence for relatively high-level cognitive processing by the right hemisphere.
" }, { "name": "Block, Steven Michael", "degree": "PhD", "year": "1983", "title": "Chemotactic Responses of Tethered Bacteria", "advisor": "Berg, Howard C.", "url": "https://resolver.caltech.edu/CaltechTHESIS:09262018-173300466", "creators": [ { "name": { "family": "Block", "given": "Steven Michael" }, "id": "Block-Steven-Michael", "display_name": "Block, Steven Michael" } ], "advisors": [ { "name": { "family": "Berg", "given": "Howard C." }, "id": "Berg-Howard-C", "role": "advisor", "display_name": "Berg, Howard C." } ], "committee": [ { "name": { "family": "Berg", "given": "Howard C." }, "id": "Berg-Howard-C", "role": "chair", "display_name": "Berg, Howard C." }, { "name": { "family": "Hopfield", "given": "John J." }, "id": "Hopfield-J-J", "role": "member", "display_name": "Hopfield, John J." }, { "name": { "family": "Fender", "given": "Derek H." }, "id": "Fender-D-H", "role": "member", "display_name": "Fender, Derek H." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "member", "display_name": "Revel, Jean-Paul" } ], "option_major": [ "biology" ], "doi": "10.7907/2fzz-tb74", "abstract": "Escherichia coli swim in a three-dimensional random walk of alternating runs and tumbles, using their flagella for propulsion. When moving in a gradient of an attractant or repellent, they bias the walk in such a way as to migrate into a favorable region; this is a basis for chemotaxis. Bacteria may be tethered to a glass surface by means of a single flagellum. When tethered, cell bodies spin alternately clockwise (CW) and counterclockwise (CCW) under the influence of the rotary motor that drives the flagellum. The CCW state corresponds to the run mode and the CW state to the tumble mode. Tethered bacteria remain fixed in place, thereby providing an opportunity to study chemotactic behavior by direct manipulation of attractant or repellent concentration near the cells. Two experimental approaches have been used to exploit this opportunity. In the first, a mixing device that provides programmable concentration changes was used to stimulate tethered cells with exponential temporal gradients or exponentiated sine waves of the attractant \u03b1-methyl-D,L-aspartate. Such changes cause chemoreceptor occupancy to be changed linearly or sinusoidally, respectively. Exponential temporal gradients (both positive and negative) were found to shift the rotational bias (defined as the fraction of time spent spinning CCW) by a fixed amount related to the steepness of the gradient. The bias shifts produced indicate that cells are exquisitely sensitive to small changes in chemoreceptor occupancy. Distributions of CW and CCW intervals remained exponential during such gradients. This result is inconsistent with a response regulator model in which rotational transitions are associated with level-crossings of a fluctuating, hypothetical intermediate. It is consistent with a model in which transitions occur at random between rotational states, the transition probabilities being governed by chemotactic signals. In the second approach, short bursts of an attractant or repellent were delivered iontophoretically, producing an impulse response in the tethered bacteria. Properties of the impulse response show both adaptive and integrative behavior, and imply that cells respond maximally to changes in concentration which occur over times comparable with the length of a run. The impulse response can be used to predict the behavior of cells towards an arbitrary stimulus in the linear domain. Impulse responses from a series of chemotaxis mutants showed that some were defective in adaptation but not excitation; others were defective in both. Taken together, the experiments provide information about the spectral response of bacteria to concentration changes with frequencies ranging from 10-3 Hz up to almost 10 Hz. Both sets of data are consistent with the notion of a cellular \"bias regulator\" signal that sets the transition probabilities between two states; one representing CCW and the other representing CW rotation.
" }, { "name": "Crews, Stephen Thomas", "degree": "PhD", "year": "1983", "title": "The Structure of Mammalian Genes: (1) Antibody Heavy Chain Variable Region Genes: Organization, Diversity, and Somatic Mutation. (2) Structure and Transcription of the DNA Encompassing the Origin of Replication of Human Mitochondrial DNA", "advisor": "Hood, Leroy E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08302019-152439482", "creators": [ { "name": { "family": "Crews", "given": "Stephen Thomas" }, "id": "Crews-Stephen-Thomas", "orcid": "0000-0002-1432-401X", "display_name": "Crews, Stephen Thomas" } ], "advisors": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "advisor", "display_name": "Hood, Leroy E." } ], "committee": [ { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "chair", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "member", "display_name": "Owen, Ray David" }, { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "member", "display_name": "Strauss, James H." }, { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "member", "display_name": "Hood, Leroy E." } ], "option_major": [ "biology" ], "doi": "10.7907/4dgr-za66", "abstract": "This thesis describes two experimental systems utilized to study mammalian gene structure and expression: (1) antibody heavy chain variable region genes and (2) mitochondrial DNA.
\r\n\r\nIn order to study the organization and structure of antibody genes and the relative germline and somatic contributions towards antibody diversity, we have analyzed the germline genes encoding the murine immune response to phosphorylcholine. Molecular cloning studies were undertaken and conclusively show that there is only one germline VH gene segment encoding the immune response to phosphorylcholine. Protein sequencing work on monoclonal antibodies that bind phosphorylcholine reveals many different protein sequences related to one predominant sequence. We are able to conclude that these variant sequences are the result of somatic diversification operating on one germline gene segment. We are further able to show that this diversification is mutational and not recombinational. Finally, somatic mutation is correlated with the class of the antibody; IgG and IgA antibodies undergo somatic mutation, IgM antibodies do not.
\r\n\r\nWe have isolated and sequenced a family of four closely related VH gene segments designated V1, V3, V11 and V13. Their function varies: V1 encodes the immune response to phosphorylcholine, V3 is a pseudogene, V11 encodes the immune response to influenza hemagglutinin, and V13 has an unknown function but is not obviously a pseudogene. Structural analysis of recombinant clones containing this family of related VH gene segments and other VH gene segments reveals several important points about the organization of VH gene segments. First, closely related VH gene segments can be clustered together within the VH gene locus. Second, the spacing distance between adjacent VH gene segments is variable; it may be as short as 5 kb and greater than 30 kb. Finally, the average spacing distance between VH gene segments is large, at least 23 kb. Assuming a minimum of 200 germline VH gene segments, the size of the VH gene locus may be greater than 5 million base pairs.
\r\n\r\nThe human mitochondrial genome is the second system that has been chosen to study gene structure and expression, and to accomplish this, we have applied both DNA and RNA sequencing technologies. We sequenced the DNA encompassing the origin of DNA replication and then localized the origin at the nucleotide level. The human mitochondrial origin of DNA replication shares structural characteristics with other known origins of DNA replication; in particular, the presence of extensive secondary structure in the form of a stem-loop structure. In order to precisely localize mitochondrial transcripts to the DNA, we developed techniques that allowed the isolation and sequencing of the 5'-ends of mitochondrial transcripts. This technology was utilized to precisely localize the 5'-end of the mitocohndrial 12S rRNA species 457 nucleotide pairs 5'-to the origin of DNA replication. Analysis of the DNA sequence in this region revealed a phenylalanine tRNA gene whose 3'-end was joined end-to-end with the 5'-end of the 12S rRNA. This analysis first demonstrated the extreme enconomy of genetic material in mammalian mitochondrial DNA.
" }, { "name": "Ellison, Jay William", "degree": "PhD", "year": "1983", "title": "Structure and Evolution of Human Immunoglobulin C\u03b3 Genes", "advisor": "Hood, Leroy E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:09032019-120820356", "creators": [ { "name": { "family": "Ellison", "given": "Jay William" }, "id": "Ellison-Jay-William", "display_name": "Ellison, Jay William" } ], "advisors": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "advisor", "display_name": "Hood, Leroy E." } ], "committee": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "chair", "display_name": "Hood, Leroy E." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Lazarides", "given": "Elias" }, "id": "Lazarides-E", "role": "member", "display_name": "Lazarides, Elias" }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." } ], "option_major": [ "biology" ], "doi": "10.7907/9crt-qq78", "abstract": "In order to learn about the evolution of the human immunoglobulin C\u03b3 gene family, the structural features of individual C\u03b3 genes were examined. The complete nucleotide sequences were determined for three members of the gene family-the C\u03b31, C\u03b32, and C\u03b34 genes. A comparison of these sequences with those of the three reported mouse C\u03b3 genes (C\u03b31, C\u03b32a, C\u03b32b) fails to reveal any pairs of corresponding genes in the two species. Moreover, the sequence homology shared by human C\u03b3 genes in both coding and noncoding regions (about 95%) is significantly greater than that seen within the mouse C\u03b3 family (about 70-80%). The presumably neutral mutations accumulated in the noncoding regions of the human genes have been used to estimate that approximately 6-8 million years have elapsed since the divergence of these genes from a common ancestral sequence. This divergence is considerably more recent than inferred for the mouse C\u03b3 genes, and suggests that gene duplication or gene correction events have occurred more recently in humans than in mice.
\r\n\r\nIn contrast to the CH domain exons and adjacent noncoding regions, the hinge exons of human C\u03b3 genes are quite divergent both in length and sequence. This coding sequence variability is seen to extend into the regions of CH domains which border the hinge in the polypeptide chain. This divergence is interpreted as being the result of natural selection for particular hinge structures in the IgG subclasses. The implication is that these polypeptide regions are important for immunologic effector functions carried out by IgG molecules.
\r\n\r\nThe arrangement of the C\u03b32 and C\u03b34 genes in human chromosomal DNA has been determined to be 5'-C\u03b32-17 kilobase pairs-C\u03b34-3'. The genetic processes generating hybrid IgG molecules from these two genes are discussed, along with the relationship of these processes to gene duplication and gene correction.
" }, { "name": "Fryxell, Karl Joseph", "degree": "PhD", "year": "1983", "title": "Biochemical and Genetic Studies of Peripheral Myelination in Normal Development and in the Mouse Mutant Trembler", "advisor": "Revel, Jean-Paul", "url": "https://resolver.caltech.edu/CaltechTHESIS:10112019-091636747", "creators": [ { "name": { "family": "Fryxell", "given": "Karl Joseph" }, "id": "Fryxell-Karl-Joseph", "orcid": "0000-0002-2975-7897", "display_name": "Fryxell, Karl Joseph" } ], "advisors": [ { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "advisor", "display_name": "Revel, Jean-Paul" } ], "committee": [ { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "chair", "display_name": "Revel, Jean-Paul" }, { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "member", "display_name": "Hood, Leroy E." }, { "name": { "family": "Hudspeth", "given": "A. James" }, "id": "Hudspeth-A-J", "role": "member", "display_name": "Hudspeth, A. James" }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "member", "display_name": "Owen, Ray David" } ], "option_major": [ "biology" ], "doi": "10.7907/eahp-bx93", "abstract": "I developed a radioimmunoassay for P0, the major peripheral myelin protein, and adapted an existing radioimmunoassay for myelin basic protein. Results from these assays showed that Schwann cells do not make either protein before myelination begins, and accumulate P0 and myelin basic protein with the same time course during development. Schwann cells cultured in the absence of neurons do not express detectable levels of either protein, but they do continue to synthesize sulfatide, a myelin sulfolipid, apparently indefinitely, as shown by biosynthetic labeling.
\r\n\r\nThe trembler (Tr/+) mouse mutant has a pronounced reduction in peripheral myelin, while central myelin and peripheral unmyelinated nerves appear normal. This myelin deficiency is caused by an autonomous Schwann cell defect. Since the Trembler phenotype is expressed in heterozygotes, most previous studies were confined to Tr/+ mice. Using two alleles, I show here that the six possible genotypes can be ordered as follows: +/+ > Trj/+ > Tr/+ > Trj/Trj > Trj/Tr > Tr/Tr, based on myelin basic protein radioimmunoassays of sciatic nerve extracts. The amount of compact myelin in each genotype, as shown by electron microscopy, is in good agreement with the radioimmunoassay results, with two important provisos. First, Tr/Tr mice have 1% of wild-type myelin basic protein levels but essentially no compact myelin. Secondly, the first steps in the above genetic series reduce both the average myelin sheath area and the number of myelin sheaths by similar amounts, while the last steps primarily reduce the number of myelin sheaths. These results suggest that, if the activity of the Tr+ gene product is reduced below a certain point, myelin protein synthesis can be induced without forming a myelin sheath. Visualization of P0 by indirect immunofluorescence shows that Schwann cells without compact myelin express much lower levels of P0 than adjacent Schwann cells, associated with the same axon, that do form compact myelin. Finally, although Trembler Schwann cells proliferate excessively in vivo, their proliferation is not affected by Tr gene dose. In vitro Trj/Trj Schwann cell proliferation is apparently normal. Thus, it is likely that the function of the Tr+ gene product is not directly concerned with Schwann cell proliferation.
" }, { "name": "Gomer, Richard Hans", "degree": "PhD", "year": "1983", "title": "The Role of Filamin in the Morphogenesis of the Skeletal Muscle Sarcomere", "advisor": "Lazarides, Elias", "url": "https://resolver.caltech.edu/CaltechETD:etd-12212004-095323", "creators": [ { "name": { "family": "Gomer", "given": "Richard Hans" }, "id": "Gomer-Richard-Hans", "orcid": "0000-0003-2361-4307", "display_name": "Gomer, Richard Hans" } ], "advisors": [ { "name": { "family": "Lazarides", "given": "Elias" }, "id": "Lazarides-E", "role": "advisor", "display_name": "Lazarides, Elias" } ], "committee": [ { "name": { "family": "Lazarides", "given": "Elias" }, "id": "Lazarides-E", "role": "chair", "display_name": "Lazarides, Elias" }, { "name": { "family": "Brokaw", "given": "Charles J." }, "id": "Brokaw-C-J", "role": "member", "display_name": "Brokaw, Charles J." }, { "name": { "family": "Hudspeth", "given": "A. James" }, "id": "Hudspeth-A-J", "role": "member", "display_name": "Hudspeth, A. James" }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "member", "display_name": "Revel, Jean-Paul" } ], "option_major": [ "biology" ], "doi": "10.7907/97M8-B306", "abstract": "During chicken skeletal myogenesis in tissue culture, filamin is found on stress fibers in myoblasts and early myotubes. Approximately one day after fusion and shortly before \u03b1-actinin transits to Z lines, filamin disappears from the cells. The disappearance of filamin is correlated with a cessation of its synthesis. Approximately six days after fusion, filamin reappears at the Z lines of myogenic cells, shortly before desmin and vimentin transit to the Z line. In adult muscle, filamin is found at the periphery of the Z disk, along with desmin and vimentin. Peptide map analysis of the various filamins shows that gizzard and fibroblast filamins are identical while myoblast filamin is quite similar to these two filamins. Cultured myotube and adult myofibril filamins are virtually identical to each other and are quite different polypeptides when compared to gizzard, fibroblast and myoblast filamins. Analysis of terminally differentiated slow and fast muscle shows that both muscle types contain identical, skeletal muscle type filamins although in the slow muscle, filamin is distributed additionally on the I band. The molar filamin to actin ratio is 1:25 in gizzard and fibroblast, 1:54 in myoblasts, 1:820 in fast skeletal myofibrils and 1:82 in slow skeletal myofibrils.
\r\n\r\nThese results offer several new insights into eucaryotic molecular morphogenesis. From the disappearance of filamin during myogenesis, we see that a morphogenetic process may involve the temporary removal of a family of proteins. The different distributions of identical filamin polypeptides in slow and fast muscle indicates that filamin may be synthesized at different times and rates in the two myogenic processes. It appears that, at least in the case of the skeletal muscle sarcomere, temporal control of protein synthesis may be an important part of eucaryotic molecular morphogenesis.
" }, { "name": "Kronenberg, Mitchell", "degree": "PhD", "year": "1983", "title": "Gene Expression in B and T Lymphocytes: (1) Evolution of Rat C\u03ba Alleles (2) The T-cell Receptor Problem", "advisor": "Hood, Leroy E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10182019-143201215", "creators": [ { "name": { "family": "Kronenberg", "given": "Mitchell" }, "id": "Kronenberg-Mitchell", "orcid": "0000-0001-6318-6445", "display_name": "Kronenberg, Mitchell" } ], "advisors": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "advisor", "display_name": "Hood, Leroy E." } ], "committee": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "chair", "display_name": "Hood, Leroy E." }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "member", "display_name": "Owen, Ray David" }, { "name": { "family": "Rothenberg", "given": "Ellen V." }, "id": "Rothenberg-E-V", "orcid": "0000-0002-3901-347X", "role": "member", "display_name": "Rothenberg, Ellen V." } ], "option_major": [ "molbio" ], "doi": "10.7907/p24z-a733", "abstract": "The amino acid sequence of the two kappa chain constant region allotypes found in inbred rat strains indicated that these alleles are very different and therefore may have had an unusual evolutionary history. To understand the evolution of these genes, serologic tests were performed to determine if inbred rats express latent or unexpected C\u03ba alleles. They apparently do not do so. Wild Norway rats were tested, and it was found that the laboratory strains do not represent a subset of the rat C\u03ba polymorphism. Further tests indicated that only one of the two serologic specificities could be found in related rodent species.
\r\n\r\nThe structure of the T cell antigen-binding receptor is a major controversial issue in immunology. It has been asserted that the T cell antigen-receptor is homologous to immunoglobulins, and one popular theory contends that VH genes are responsible for the specificity of the receptor. We tested these theories by hybridizing immunoglobulin DNA probes to RNA and DNA from cloned T cells. First, we determined that the C\u03bb, J\u03ba, C\u03ba, JH, C\u00b5 and C\u03b1 genes and the sequences involved in heavy chain class switching are not rearranged in a T helper, a cytotoxic T cell and a T lymphoma. These cells also do not transcribe C\u03ba, C\u03bb, JH, C\u00b5 and C\u03b1 RNA. Second, a cDNA clone encoding heavy chain variable region characteristic of most B cells which respond to the antigen GAT was isolated and sequenced. Poly(A)+ RNA was prepared from 12 cloned T lymphocytes specific for GAT. While six of these T cells display antigenic determinants present on immunoglobulins that bind GAT, none of them contained a transcript homologous to the cDNA probe. Finally, using a random primer, large cDNA libraries (105-106 colonies) were constructed from three T-cell hybridomas. These libraries were screened by two separate, well-characterized methods which should permit the detection of all or most VH gene segments. No VH cDNA colonies were found by these methods. Therefore immunoglobulin gene segments are not likely to be part of the T cell antigen receptor.
\r\n\r\nThe I-J serologic specificity has been reported to be present on T cell-derived antigen-binding molecules. Cosmid clones have been previously obtained containing all the sequences between the I-A and I-E subregions of the murine major histocompatibility complex, where I-J has been genetically mapped. The putative I-J DNA does not, however, hybridize to RNA from I-J positive suppressor T cells. Also, suppressor T lymphocytes do not rearrange this DNA. Therefore the I-J coding sequences must map elsewhere.
" }, { "name": "Kuppermann, Baruch Davidson", "degree": "PhD", "year": "1983", "title": "Studies on the Role of Afferent Activity in the Visual Pathways of the Cat: I. Mechanisms Involved in the Control of Geniculate Cell Size. II. Control of the Critical Period After Dark-Rearing", "advisor": "Van Essen, David C.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10182019-151127683", "creators": [ { "name": { "family": "Kuppermann", "given": "Baruch Davidson" }, "id": "Kuppermann-Baruch-Davidson", "orcid": "0000-0002-8380-8464", "display_name": "Kuppermann, Baruch Davidson" } ], "advisors": [ { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "advisor", "display_name": "Van Essen, David C." } ], "committee": [ { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "chair", "display_name": "Van Essen, David C." }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Fender", "given": "Derek H." }, "id": "Fender-D-H", "role": "member", "display_name": "Fender, Derek H." }, { "name": { "family": "Hudspeth", "given": "A. James" }, "id": "Hudspeth-A-J", "role": "member", "display_name": "Hudspeth, A. James" }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" } ], "option_major": [ "biology" ], "doi": "10.7907/8snk-xj55", "abstract": "Two studies were conducted examining the role of afferent activity on synaptic connectivity in the cat's visual pathways. The first study investigated the effects of retinal inactivity on cell size in the lateral geniculate nucleus (LGN). A complete retinal blockade was produced in one eye of 7-week-old kittens by intravitreal injections of tetrodotoxin (TTX). Within one week, cell shrinkage of ~20% in laminae with input from the inactive eye was observed in the binocular and monocular segments of the LGN. Cell growth of 10% was observed in active laminae only in the binocular segment. Adult cats subjected to one week of monocular TTX treatment and kittens placed in the dark during the treatment period underwent uniform changes in cell size of ~20% throughout the binocular and monocular segments of the LGN. The cell size changes in the monocular segment of the kittens left in the light, and throughout the LGN of the kittens placed in the dark and of the adult cats were not indicative of a competitive response to differential activity from the two eyes. The results suggest a strong control of LGN cell size by afferent (retinal) activity.
\r\n\r\nIn the second study, cats were reared in the dark from birth until adulthood, then subjected to various visual exposure paradigms to test their residual cortical plasticity. Animals allowed two weeks of binocular visual exposure after dark-rearing, then monocularly lid-sutured underwent changes in cortical ocular dominance in response to the monocular deprivation. Animals which were monocularly deprived immediately upon removal from the dark and 25 days later reverse-sutured for 4-6 months (closure of initially open eye, opening of previously sutured eye) underwent shifts in ocular dominance towards the experienced eye both after the initial monocular deprivation and again in response to reverse-suturing. The LGN of dark-reared/monocularly deprived animals did not exhibit any morphological abnormalities. Depletion of cortical norepinephrine reduced but did not abolish the sensitivity of the dark-reared cortex to monocular deprivation. Visual exposure for four weeks after birth, followed by extended dark-rearing did not halt the subsequent prolongation of cortical plasticity in the dark.
" }, { "name": "Lemke, Greg Erwin", "degree": "PhD", "year": "1983", "title": "Identification and Characterization of Glial Growth Factor", "advisor": "Kennedy, Mary B.; Brockes, Jeremy P.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10182019-171906142", "creators": [ { "name": { "family": "Lemke", "given": "Greg Erwin" }, "id": "Lemke-Greg-Erwin", "display_name": "Lemke, Greg Erwin" } ], "advisors": [ { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "advisor", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Brockes", "given": "Jeremy P." }, "id": "Brockes-Jeremy-P", "orcid": "0000-0002-3395-5159", "role": "co-advisor", "display_name": "Brockes, Jeremy P." } ], "committee": [ { "name": { "family": "Brockes", "given": "Jeremy P." }, "id": "Brockes-Jeremy-P", "orcid": "0000-0002-3395-5159", "role": "chair", "display_name": "Brockes, Jeremy P." }, { "name": { "family": "Hudspeth", "given": "A. James" }, "id": "Hudspeth-A-J", "role": "member", "display_name": "Hudspeth, A. James" }, { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "member", "display_name": "Hood, Leroy E." }, { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "member", "display_name": "Revel, Jean-Paul" }, { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "member", "display_name": "Kennedy, Mary B." } ], "option_major": [ "biology" ], "doi": "10.7907/774r-7520", "abstract": "A combination of biochemical, cell biological and immunological techniques have been employed to identify a novel and potent polypeptide mitogen of the brain and pituitary. This molecule, named glial growth factor (GGF), stimulates DNA synthesis and cell division in cultured rat Schwann cells, astrocytes, and fibroblasts.
\r\n\r\nThree independent lines of evidence indicate that GGF activity resides in a basic protein of molecular weight 3.1 x 104. (a) When partially purified preparations are analyzed by native gel electrophoresis at pH 4.5, mitogenic activity migrates with a protein of this molecular weight, as revealed by bioassay coupled with a second dimension of SDS gel electrophoresis. (b) A set of monoclonal antibodies which deplete growth factor activity from heterogeneous solutions specifically recognize a 31,000 dalton protein antigen, as determined by gel immunoautoradiography. (c) GGF activity is recovered at a molecular weight of 3.1 x 104 after denaturing polyacrylamide gel electrophoresis in SDS.
\r\n\r\nThree large-scale purifications of GGF, employing a combination of column chromatography steps and preparative electrophoreses, are described. The molecule has been purified to apparent homogeneity from anterior lobes of the bovine pituitary.
\r\n\r\nThrough the use of nucleic acid precursor incorporation assays, GGF has been shown to be markedly mitogenic for rat Schwann cells, astrocytes and fibroblasts, but inactive when assayed on oligodendrocytes or microglia. Electrophoretic analyses suggest that all responsive cell types are stimulated by a single (the same) molecular species. GGF is the only defined mitogen to which rat Schwann cells respond.
\r\n\r\nGlial growth factor from bovine brain has been found to be indistinguishable from bovine pituitary GGF, as determined by biochemical, immunological and bioactivity criteria. GGF is non-uniformly distributed among bovine brain regions. It is present in brain extracts prepared from a wide variety of vertebrate species.
\r\n\r\nPurified human platelet-derived growth factor (PDGF) shares many important properties with GGF. PDGF has been shown to be unable to significantly stimulate the division of rat Schwann cells, however, and therefore appears to be distinct.
\r\n\r\nObservations made in vitro suggest several possible biological roles for GGF in vivo. These are discussed.
\r\n" }, { "name": "McCasland, James Stacy", "degree": "PhD", "year": "1983", "title": "Neuronal Control of Bird Song Production", "advisor": "Konishi, Masakazu", "url": "https://resolver.caltech.edu/CaltechTHESIS:10212019-145504230", "creators": [ { "name": { "family": "McCasland", "given": "James Stacy" }, "id": "McCasland-James-Stacy", "display_name": "McCasland, James Stacy" } ], "advisors": [ { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "advisor", "display_name": "Konishi, Masakazu" } ], "committee": [ { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "chair", "display_name": "Allman, John Morgan" }, { "name": { "family": "Arnold", "given": "Arthur P." }, "id": "Arnold-Arthur-P", "role": "member", "display_name": "Arnold, Arthur P." }, { "name": { "family": "Hudspeth", "given": "A. James" }, "id": "Hudspeth-A-J", "role": "member", "display_name": "Hudspeth, A. James" }, { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "member", "display_name": "Van Essen, David C." }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" } ], "option_major": [ "neurobio" ], "doi": "10.7907/z6h5-1n28", "abstract": "Male songbirds sing to establish species and individual identities, to maintain territories, and to stimulate reproductive behavior in conspecific females. The ability to produce a stereotyped song is therefore necessary for reproduction. In many species the patterns of song are learned by young birds from adults in a process involving two stages - an auditory phase involving storage of a song model, and a sensorimotor phase in which the bird learns to reproduce the model by using auditory feedback. Lesion results (Nottebohm et al., 1976) demonstrated that at least three discrete nuclei -- HVc (Hyperstriatum ventrale, pars caudale), RA (n. Robustus Archistriatalis), and nXIIts (n. Hypoglossus, pars tracheosyringealis) -- are indispensable to normal song production. These findings opened the way to study of a discrete vertebrate neural system which, uniquely, mediates production of an acquired yet stereotyped behavior.
\r\n\r\nHowever, the functional specializations of these nuclei cannot be discerned through lesion studies. For this reason I developed techniques for examining directly the neural correlates of song production -- by making neuronal recordings from the freely-behaving, singing bird. From my studies I can draw the following generalizations about the relative roles of vocal control nuclei: (1) the telencephalic nucleus NIf (Nucleus Interfacialis, of Nottebohm, 1980), which provides an input to HVc and is anatomically the \"highest\" nucleus in the descending motor pathway, is uniquely placed among vocal control nuclei to be a generator of timing cues for song; (2) consistent with the unidirectional and serial connections between nuclei of the descending tract, NIf, HVc, RA, and nXIIts are activated sequentially prior to sound onset; (3) single-unit recordings demonstrate that there are neurons specialized for production of specific song elements; (4) a re-examination of hemispheric dominance in song control shows that both hemispheres normally make similar contributions to all song elements; and (5) at least two types of inhibitory interactions between auditory and motor activities can be observed in the behaving bird.
\r\n\r\nThe source of timing cues for song. Previous lesion and neuroanatomical studies have implicated eight brain nuclei in the control of song. I recorded neural activity from seven of the eight nuclei, in order to assess the presence and patterning of song-related activity, and to localize the site or sites where timing cues for song are generated. I confirmed the suggestion from lesion studies that the serially connected nuclei HVc, RA, and nXIIts each produce song-correlated neural activity and showed quantitatively that these nuclei are activated sequentially when the bird vocalizes. I further demonstrated that many multiple-unit recordings from these areas show a clear modulation of activity pattern which corresponds to the temporal pattern of song. This finding implicates these nuclei in the genesis or transmission of timing cues for song. I then made recordings from Nucleus Interfacialis (NIf), which provides an input to HVc, and found a similar song-related pattern of activity. The pre-sound latency of recordings from NIf was longer than that of comparable recordings from HVc, suggesting that NIf provides timing information to HVc. To test this interpretation, I sectioned the pathway from NIf to HVc; postoperatively, these subjects were unable to produce stereotyped song. A sham operation involving the same amount of tissue damage but sparing NIf had no effect on song. HRP studies show only one input to NIf (with the possible exception of the auditory nucleus Field L), from nucleus Uva of the thalamus. Recordings from this nucleus showed no changes in activity during vocalization in the adult, and bilateral lesions of this nucleus had no effect on song. To test for other inputs to NIf which might not transport HRP, I made a series of recordings from eight sites in the vicinity of NIf. None of these recordings showed song-related activity. The combination of three findings -- song-related patterning of activity in NIf, the necessity of NIf for normal song patterning, and the absence of song-related activity in inputs to NIf -- imply that NIf is a source of timing cues for song, and is the only song control nucleus to which this statement can be unequivocally applied. The fact that elimination of input to NIf has no effect on song suggests that NIf produces a learned central motor program for song.
\r\n\r\nRecordings made from two other putative song control nuclei, MAN (Magnocellular nucleus of the Anterior Neostriatum) and area X, showed no changes in activity during song or other vocalizations. Transection of the pathways linking these two nuclei to HVc had no effect on song. Thus my recording experiments demonstrate that lesion effects provide a better indication of the necessity of a nucleus for adult song production than do studies of anatomical connections. Whether Uva, MAN, or area X plays a crucial role in song development remains to be determined.
\r\n\r\nSingle units with specialized roles in song production. Elucidation of the neural mechanisms of song control ultimately requires an analysis at the single-unit level: the clues to the interactions involved may be masked in multi-unit recordings. Accordingly, I have developed a new technique for recording from single neurons in song system nuclei of the freely-behaving, singing mockingbird. The method employs an X-Y microdrive which, when chronically implanted, is sufficiently stable so that units can be isolated and held for periods of up to several hours.
\r\n\r\nMy recordings from mockingbird HVc revealed single units with several classes of specialized roles in song production. Many cells exhibit premotor activity for all song syllables, and do not respond to those same syllables presented as auditory stimuli. Some of these cells show long-latency (e.g., 500 msec) \"anticipatory\" activity at the initiation of song and between syllables of a long song bout, thus demonstrating a role for HVc beyond the purely motor aspects of sound production. A few cells show more selective premotor activity, producing highly stereotyped bursts of spikes for only a few syllables. The distinctions between sounds for which these cells do or do not show activity may be quite subtle, suggesting that HVc units may encode subtle variations in sound production. This interpretation is supported by the high degree of temporal specificity of unit firing with respect to timing of the syllable.
\r\n\r\nA re-examination of hemispheric dominance in song control. Series of studies by Nottebohm et al. demonstrated that left-side lesions in the song system of several species inflict much more severe damage to song than comparable right-side lesions. These studies led to the hypothesis that the left hemisphere plays a dominant role in song production. My neural recordings, however, led to a completely different interpretation of the lesion results. Recordings from the left and right hypoglossal nerves innervating the syrinx invariably showed very similar activity patterns for any given vocalization. These patterns consist of neural bursts and silent periods of different durations, with each song syllable represented by a unique neural homologue. Because the hypoglossal nerve represents the \"final common pathway\" for song control, the neural activity it transmits must convey coded commands for song production. To the extent that my recordings reveal these commands, it appears that both right and left nerves are transmitting the same messages to their respective syringeal halves. Section of right or left hypoglossal nerve in a subject from which nerve recordings were also made led to the classical behavioral deficit, including complete disappearance of certain syllables, but these behavioral deficits were uncorrelated with any consistent pattern differences in the two nerves. Recordings from right and left HVc of the same bird also showed similar activity patterns for a given song element. These results are incompatible with any all-or-nothing mechanism of lateralization in the song control system. However, they are consistent with the absence of hemispheric anatomical asymmetries in song control nuclei.
\r\n\r\nThe critical feature of the qualitative dominance theory is the independent contribution of different sets of sounds by the two sound sources in the syrinx -- the right and left internal tympaniform membranes. By blocking airflow through the right or left bronchus, I was able to eliminate the function of one membrane and observe the set of song syllables produced by the bird with only the other membrane. With this bronchus-plugging technique, I found that the right syringeal half alone is sufficient to produce easily recognizable counterparts to most syllables normally produced by both syringeal halves. This result contradicts the conclusion from nerve-section experiments that most sounds are contributed by the left side only, but is completely consistent with the nerve recordings which indicate bilateral participation in sound production. Taken together, these results indicate that both hemispheres and syringeal halves make similar contributions to the production of all song elements.
\r\n\r\nInteraction between auditory and motor activities in an avian song control nucleus. Intracellular recordings from anesthetized birds have shown that many neurons in HVc respond to auditory stimuli. I confirmed this result in multi-unit recordings from awake behaving birds, and further demonstrated responses of HVc neurons to playback of the bird's own song. The functional significance of these responses is not yet clear, but behavioral studies show that auditory feedback plays a crucial role in the development of normal song. I showed that the song-correlated temporal pattern of neural activity persists even in the deaf bird. Furthermore, in the normal bird the activity pattern correlated with production of certain song elements can be clearly distinguished from the pattern of auditory responses to the same song elements. This result implies that an interaction occurs in HVc of the singing bird between motor and auditory activity. Through experiments involving playback of sound while the bird is singing, I showed that the interaction consists of motor inhibition of auditory activity in HVc and that this inhibition decays slowly over a period of seconds after the song terminates.
\r\n\r\nThe time-locking of pre-motor activity in HVc to song elements survives the loss of auditory feedback by deafening even though auditory inputs to HVc produce clear responses to sounds heard by the normal quiescent bird. Normal songs are produced by deafened adults in some species. These findings suggest the possibility of a learned central motor program for song, functioning independently of sensory input in the adult. Because the bird must make use of auditory feedback to develop normal song, the autonomy of the motor program would have to be acquired during ontogeny. Recordings from Field L neurons in the singing bird show auditory responses to song elements which persist during singing, suggesting that the sensorimotor interaction I observed occurs within HVc. If so, it is likely that the motor inhibition of auditory inputs to HVc plays a role in song development or song maintenance. If present throughout development of song, this inhibition would appear to serve the paradoxical function of rendering these inputs inaccessible for guidance of motor learning, unless such guidance does not occur within HVc or involves nonspiking interaction between or within cells. These considerations thus impose constraints on possible neural mechanisms of song control.
\r\n\r\nIn single-unit recordings I have confirmed the expectation from multi-unit data that at least some auditorily responsive neurons are inhibited during singing, thus demonstrating a type of sensorimotor interaction at the single cell level. One cell in mockingbird HVc was inhibited by playback of all syllables of the bird's own song, and showed premotor activity for some syllables. Another cell which was inhibited by playback showed specific motor activity, firing for only one of two very similar song syllables. While the functional significance of these interactions is unknown, it will clearly be of great theoretical importance to know whether other types of sensorimotor interaction are exhibited by single cells in the vocal control system. Such cells would be likely candidates for specialized roles in song learning or song maintenance.
" }, { "name": "Nicholson, Bruce John", "degree": "PhD", "year": "1983", "title": "Biochemistry and Diversity of the Gap Junction Protein: A Study of Liver, Heart and Lens", "advisor": "Revel, Jean-Paul", "url": "https://resolver.caltech.edu/CaltechTHESIS:10222019-124810283", "creators": [ { "name": { "family": "Nicholson", "given": "Bruce John" }, "id": "Nicholson-Bruce-John", "orcid": "0000-0003-1649-7173", "display_name": "Nicholson, Bruce John" } ], "advisors": [ { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "advisor", "display_name": "Revel, Jean-Paul" } ], "committee": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "chair", "display_name": "Hood, Leroy E." }, { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "member", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "member", "display_name": "Mitchell, Herschel K." }, { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "member", "display_name": "Strauss, James H." }, { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "member", "display_name": "Revel, Jean-Paul" } ], "option_major": [ "biology" ], "doi": "10.7907/bhjv-8053", "abstract": "Fractions highly enriched for gap junctions by morphological criteria have been isolated from rat liver, heart and eye lens, although some question exists as to the nature of the structures from lens. The junctions from each tissue are comprised of a single major protein of Mr 28,000 in the liver, Mr 30,000 in the heart, and Mr 26,000 (MIP 26) in the lens. The polypeptide profile of the liver fraction is complicated by endogenous proteolysis and aggregation in SDS of the gap junction protein and the presence of about 20% non-junctional material. Heart and lens junction proteins are also found to aggregate in SDS, while endogenous proteolysis typically reduces the cardiac gap junction protein to Mr 28,000 during isolation.
\r\n\r\nComparisons of two-dimensional peptide maps of the junctional proteins from these tissues, and the use, where necessary, of a third dimension of resolution (HPLC), demonstrates the three proteins to be very different in terms of their primary structures. The protein of each tissue, however, seems well conserved between mammalian species. For liver and lens, this finding has been confirmed in amino acid analyses and partial NH2-terminal sequences (to 58 and 33 residues, respectively). Cleavage products of these two proteins have also been produced to allow further sequence analysis in the future. In spite of the differences in primary structure, some conservation of the tertiary structures of these proteins is suggested by proteolysis of intact junctions (likely restricted to the cytoplasmic surfaces). Liver and heart gap junction proteins are reduced by trypsin to two fragments of Mr 10,000, while a single Mr 21,000 fragment is produced from lens MIP 26. Sequence analysis (liver and lens only) indicates that most of the protein removed by tryptic hydrolysis is from the carboxy-terminus, although an additional loop of 4,000 daltons is excised from the center of the liver polypeptide and five residues are lost from the NH2-terminus of the lens protein.
\r\n\r\nThe extent and possible significance of this surprising tissue specificity of the gap junction protein are discussed in the light of these findings.
" }, { "name": "Snyder, Michael Paul", "degree": "PhD", "year": "1983", "title": "Organization and Expression of a Cluster of Drosophila Cuticle Genes", "advisor": "Davidson, Eric H.; Davidson, Norman R.", "url": "https://resolver.caltech.edu/CaltechTHESIS:11042019-115006064", "creators": [ { "name": { "family": "Snyder", "given": "Michael Paul" }, "id": "Snyder-Michael-Paul", "orcid": "0000-0003-0784-7987", "display_name": "Snyder, Michael Paul" } ], "advisors": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "advisor", "display_name": "Davidson, Eric H." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "co-advisor", "display_name": "Davidson, Norman R." } ], "committee": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "chair", "display_name": "Hood, Leroy E." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Parker", "given": "Carl Stevens" }, "id": "Parker-C-S", "orcid": "0000-0001-9795-4211", "role": "member", "display_name": "Parker, Carl Stevens" }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "display_name": "Davidson, Norman R." } ], "option_major": [ "biology" ], "doi": "10.7907/mfee-te69", "abstract": "A 50 kb DNA segment of the Drosophila genome has been cloned and characterized. This segment lies at chromosomal location 44D and contains two small gene families. One family is comprised of four related cuticle genes clustered within 7.9 kb of DNA. The four genes encode four of the five major third instar larval cuticle proteins. These cuticle genes are coordinately expressed in the integument of third instar larvae, and they are not abundantly expressed in other developmental stages. A fifth cuticle-like gene lies within this gene cluster. It is judged to be a pseudogene, because several features of its structure and the absence of transcripts suggest that it is nonfunctional. Sequence comparisons indicate it arose by an unequal crossing over event involving two closely related and adjacent cuticle genes.
\r\n\r\nEleven kb away from the cuticle gene cluster lies another gene family. This family is comprised of three genes that are 55-60% homologous in DNA sequence and clustered within 8 kb of DNA. The three genes are expressed together in larval stages and adults but show a different pattern of developmental expression from the third instar larval cuticle protein genes. Thus two small gene families can lie adjacent on the chromosome and exhibit different patterns of developmental expression, even though individual genes within a clustered family are coordinately expressed.
\r\n\r\nAdditionally, a Drosophila strain has been studied which fails to synthesize one of the cuticle proteins. A molecular characterization of this strain is reported, which includes the finding of a transposable element in the promoter region of the unexpressed gene.
" }, { "name": "Wang, Chung", "degree": "PhD", "year": "1983", "title": "Induction and Methylation of Heat Shock Proteins in Cultured Vertebrate Cells", "advisor": "Lazarides, Elias", "url": "https://resolver.caltech.edu/CaltechTHESIS:10292019-140253277", "creators": [ { "name": { "family": "Wang", "given": "Chung" }, "id": "Wang-Chung", "display_name": "Wang, Chung" } ], "advisors": [ { "name": { "family": "Lazarides", "given": "Elias" }, "id": "Lazarides-E", "role": "advisor", "display_name": "Lazarides, Elias" } ], "committee": [ { "name": { "family": "Lazarides", "given": "Elias" }, "id": "Lazarides-E", "role": "chair", "display_name": "Lazarides, Elias" }, { "name": { "family": "Brokaw", "given": "Charles J." }, "id": "Brokaw-C-J", "role": "member", "display_name": "Brokaw, Charles J." }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "member", "display_name": "Revel, Jean-Paul" }, { "name": { "family": "Clarke", "given": "Steven G." }, "id": "Clarke-Steven-G.", "role": "member", "display_name": "Clarke, Steven G." } ], "option_major": [ "biology" ], "doi": "10.7907/mpys-2s35", "abstract": "When vertebrate cells are exposed to sodium arsenite, they respond by increased synthesis of a small number of polypeptides similar to the heat shock proteins, which are induced by exposure to 40-45\u00b0C. The 70,000 dalton inducible protein (hsp70) is the most commonly induced species and this protein is methylated at both lysyl and arginyl residues. In chicken fibroblasts, the hsp70 is composed of two major distinct isoelectric variants, as well as several minor components. The more acidic one (hsp70A; pI 5.6) is highly conserved and found in all vertebrate tissues and cultured cells examined, while the more basic one (hsp70B; pI 6.0) at present has only been found in avian cells.
\r\n\r\nThe hsp70 is a prominent cytoplasmic constituent under normal growth conditions. In chicken fibroblasts, the induction by arsenite not only increases the synthesis of hsp70 but also results in an accumulation of this protein. In contrast, the induction in 3T3 and SR-RSV 3T3 cells results in increased synthesis, but no accumulation of this polypeptide.
\r\n\r\nIn chicken cells, \u03b5-N-trimethyl-lysine has been identified as the major component of methyl-lysine species in hsp70, but the contribution of \u03b5-N-monomethyl-lysine and \u03b5-N-dimethyl-lysine is evident. The methyl-arginine of hsp70 is exclusively NG-monomethyl-arginine; these methylations appear to be stoichiometric. Furthermore, these methylations can be modulated by arsenite. In particular, in hsp70A the amount of \u03b5-N-trimethyl-lysine decreases and the \u03b5-N-dimethyl-lysine significantly increases, while in hsp70B the quantity of NG-monomethyl-arginine is reduce fivefold in the presence of sodium arsenite. In 3T3 and SR-RSV 3T3 cells, \u03b5-N-trimethyl-lysine appears to be the only methylated lysine species; both NG-monomethyl-arginine and NG,NG-dimethyl-arginine have been identified as the methylated arginyl residues. Similar to the homologous polypeptide in chicken fibroblasts, the level of arginyl methylation of hsp70 of 3T3 cells can be reduced in the presence of arsenite. This arsenite-induced reduction in arginyl methylation of hsp70 appears to be restricted to the polypeptides synthesized during the arsenite incubation. However, the arginyl methylation level of hsp70 of SR-RSV 3T3 is constitutively lower than their untransformed counterpart (3T3 cells), and the methylation cannot be reduced further by arsenite. In addition, the level of lysyl methylation of hsp70 remains the same after arsenite treatment and transformation. Since the basic amino acid methylation in total cellular proteins remains unchanged after arsenite incubation and Rous sarcoma virus transformation, the reduction in arginyl methylation of hsp70 in chicken fibroblasts and 3T3 cells by these treatments appears to be specific.
" }, { "name": "Gard, David Lynn", "degree": "PhD", "year": "1982", "title": "Intermediate Filaments and Myogenesis in vitro", "advisor": "Lazarides, Elias", "url": "https://resolver.caltech.edu/CaltechTHESIS:05142018-174410594", "creators": [ { "name": { "family": "Gard", "given": "David Lynn" }, "id": "Gard-David-Lynn", "display_name": "Gard, David Lynn" } ], "advisors": [ { "name": { "family": "Lazarides", "given": "Elias" }, "id": "Lazarides-E", "role": "advisor", "display_name": "Lazarides, Elias" } ], "committee": [ { "name": { "family": "Lazarides", "given": "Elias" }, "id": "Lazarides-E", "role": "chair", "display_name": "Lazarides, Elias" }, { "name": { "family": "Brokaw", "given": "Charles J." }, "id": "Brokaw-C-J", "role": "member", "display_name": "Brokaw, Charles J." }, { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "member", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "member", "display_name": "Mitchell, Herschel K." }, { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "member", "display_name": "Revel, Jean-Paul" } ], "option_major": [ "biology" ], "doi": "10.7907/e5gk-4c77", "abstract": "This thesis describes my investigations into the composition and function of intermediate filaments (IF) during myogenesis in vitro. I have found that avian embryonic myotubes cultured in vitro contain two intermediate filament subunits, desmin and vimentin. Prior to myoblast fusion vimentin is the sole IF subunit protein detectable by electrophoretic and immunological techniques. The onset of desmin synthesis and its cytoplasmic accumulation appear to coincide with fusion of myoblasts into multinucleate myotubes. Immunofluorescence reveals dense networks of desmin- and vimentin-containing filaments in the sarcoplasm of immature myotubes; however, late in myogenesis antisera to both desmin and vimentin are observed to stain the Z-lines of myofibrils. Double immunofluorescence microscopy using antisera to \u03b1-actinin and desmin revealed that this association occurs after the assembly of \u03b1-actinin into Z-lines, at a time when individual myofibrils are being organized into bundles. Phosphorylation of intermediate filament proteins in muscle has been previously reported (O'Connor et al., Proc. Natl. Acad. Sci. U.S.A. 76: 819-823, 1979). Using two-dimensional tryptic analysis I have found that desmin from embryonic myotubes is phosphorylated at multiple sites which correspond to sites phosphorylated by cAMP-dependent protein kinase in vitro. I have observed phosphorylation of desmin and vimentin in intact myotubes at all stages of myogenesis. However, treatment of mature (7 day and older) myotubes with 8-BrcAMP or isoproterenol results in a specific 2-3 fold increase in phosphorylation of these proteins, with a corresponding increase in 32PO4 incorporation into one major phosphopeptide of desmin. Preliminary evidence indicates that treatment of 6-8 day myotubes with 8-BrcAMP or isoproterenol significantly inhibits the transition of intermediate filaments to the Z-line which occurs during normal myogenesis. These observations suggest that intermediate filaments containing desmin and vimentin are responsible for the organization of skeletal muscle myofibrils into an integral contractile machinery, and that cAMP-dependent phosphorylation of desmin and vimentin plays an important role in the regulation of this function.
" }, { "name": "Granger, Bruce Leslie", "degree": "PhD", "year": "1982", "title": "Composition and Function of Intermediate Filaments in Avian Muscle Cells and Erythrocytes", "advisor": "Lazarides, Elias", "url": "https://resolver.caltech.edu/CaltechTHESIS:05152018-105719665", "creators": [ { "name": { "family": "Granger", "given": "Bruce Leslie" }, "id": "Granger-Bruce-Leslie", "display_name": "Granger, Bruce Leslie" } ], "advisors": [ { "name": { "family": "Lazarides", "given": "Elias" }, "id": "Lazarides-E", "role": "advisor", "display_name": "Lazarides, Elias" } ], "committee": [ { "name": { "family": "Lazarides", "given": "Elias" }, "id": "Lazarides-E", "role": "chair", "display_name": "Lazarides, Elias" }, { "name": { "family": "Brockes", "given": "Jeremy P." }, "id": "Brockes-Jeremy-P", "orcid": "0000-0002-3395-5159", "role": "member", "display_name": "Brockes, Jeremy P." }, { "name": { "family": "Brokaw", "given": "Charles J." }, "id": "Brokaw-C-J", "role": "member", "display_name": "Brokaw, Charles J." }, { "name": { "family": "Berg", "given": "Howard C." }, "id": "Berg-Howard-C", "role": "member", "display_name": "Berg, Howard C." }, { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "member", "display_name": "Revel, Jean-Paul" } ], "option_major": [ "biology" ], "doi": "10.7907/z9x1-xs25", "abstract": "Intermediate filaments comprise a family of morphologically similar cytoplasmic structures whose individual subunits are biochemically and immunologically distinguishable, and largely cell-type specific. This thesis is an investigation of the intermediate filaments in avian smooth and skeletal muscle, and avian erythrocytes.
\r\n\r\nConditions have been found under which sheets of interconnected Z-discs can be generated from skeletal muscle. The stability of these sheets demonstrates that the Z-discs of adjacent myofibrils are firmly linked to one another. Desmin, the intermediate filament subunit characteristic of muscle, encircles each Z-disc and thereby forms an insoluble, two-dimensional scaffold at right angles to the fiber axis within each Z-plane. Desmin may thus be responsible for maintaining the cross-striated appearance of skeletal muscle fibers, and may function to mechanically integrate the contractile actions of their constituent myofibrils. Vimentin, the intermediate filament subunit characteristic of mesenchymal cells, coexists with desmin at the periphery of the Z-disc, and demonstrates that a terminally differentiated cell can possess more than one class of intermediate filament subunit.
\r\n\r\nA 230,000 dalton polypeptide, named synemin, copurifies with desmin from smooth muscle. It coexists and colocalizes with desmin and vimentin in skeletal muscle at all stages of differentiation, from fusing myoblasts to mature fibers. Nondenaturing conditions under which synemin can be separated from desmin and vimentin have not been found.
\r\n\r\nDetection of synemin in avian erythrocytes has led to the realization that this cell might be a relatively simple model system for the study of intermediate filaments. A fraction of the intermediate filaments in avian erythrocytes is stably associated with the plasma membrane, but can be selectively removed from it with water; this results in preparations consisting predominantly of vimentin and synemin. Immunoelectron microscopy reveals that vimentin forms the bulk of the core filament in these cells, and synemin exists at regular intervals along this core. The axial periodicity of synemin appears to change during erythropoiesis, perhaps in accordance with some structural or functional change in the filaments. Synemin appears to crosslink the filaments through self-association, and may thus regulate the rigidity or dispersion of the intermediate filament network in erythrocytes as well as in muscle cells.
" }, { "name": "Green, Steven Haym", "degree": "PhD", "year": "1982", "title": "Genetic Studies of Neuronal Development in Drosophila melanogaster", "advisor": "Lewis, Edward B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05162018-092739245", "creators": [ { "name": { "family": "Green", "given": "Steven Haym" }, "id": "Green-Steven-Haym", "display_name": "Green, Steven Haym" } ], "advisors": [ { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "advisor", "display_name": "Lewis, Edward B." } ], "committee": [ { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "chair", "display_name": "Lewis, Edward B." }, { "name": { "family": "Konopka", "given": "Ronald J." }, "id": "Konopka-Ronald-J", "role": "member", "display_name": "Konopka, Ronald J." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "member", "display_name": "Revel, Jean-Paul" }, { "name": { "family": "Strumwasser", "given": "Felix" }, "id": "Strumwasser-F", "role": "member", "display_name": "Strumwasser, Felix" } ], "option_major": [ "biology" ], "doi": "10.7907/3h8z-9176", "abstract": "The projections into the central nervous system (CNS) of several wild-type and genetically ectopic sensory structures were studied by cobalt filling or silver staining and compared for the purpose of determining what factors guide the growth of the sensory axons. The head bristles all arborize in a similar fashion in the subesophageal ganglion although they reach the target by three different routes depending on their position on the head. This arborization is L-shaped with a longitudinal branch and a medially directed branch that crosses the midline. The antennal projection consists of an olfactory lobe component, organized into glomeruli, and an antennal mechanosensory component which can be further subdivided into three branches, the anteriormost of which is identical to the head bristle projection. The tarsi all have similar U-shaped projections into their segment's neuromere with no ascending, descending or contralateral branches.
\r\n\r\nAxons of ectopic thoracic bristles on the head may enter the brain or the optic lobes. The routes into the brain taken by the ectopic bristles were initially like those of the normal head bristles but were followed for greater or lesser distances and the region of the subesophageal ganglion that is the target of the head bristles was seldom reached. The terminal arborizations of the ectopic bristle axons were generally irregular regardless of where they were: in the subesophageal ganglion, brain or optic lobes. They resembled neither their normal arborizations in the ventral ganglion nor those of the local head sensilla in the brain.
\r\n\r\nAxons from antennal legs have a pattern of projection grossly similar to that of wild-type antennae in that the same regions of neuropil were innervated. The non-olfactory lobe components of the antennal leg projection were like those of the antenna. However, the arborization in the olfactory lobe was chaotic and there were adventitious projections from the lobe into adjacent neuropil, particularly the subesophageal ganglion. Some elements of these adventitious projections in the subesophageal ganglion were found consistently in almost every preparation. No element of the projection resembled the leg projection in the ventral ganglion.
\r\n\r\nThe axons of ectopic sensilla can reach a normal target if the distance to it from the new location is sufficiently small: axons from abdominal legs in bxd mutants terminate in normal metathoracic leg sensory neuropil and the axons of antennae misplaced as a result of the mutation ant can enter normal antennal targets.
\r\n\r\nIn summary, axons of ectopic sensilla can't reach their normal targets if they enter the CNS far from those targets which suggests that there are no long range cues for guidance of sensory axons. In the \"foreign\" part of the CNS the axons of ectopic sensilla do not make projections that resemble their normal ones. They initially take routes characteristic of sensilla in their new location but do not follow them consistently. The exception, antennal leg mechanosensory projections, is likely to be a result of a homology between antennal and leg mechanosensory sensilla. These results suggest the following: insect sensory neurons reach their targets mainly by following local and not long-range cues. The growth of these axons is constrained to specific tracts and it is by these that they are guided over long distances to their targets. Tracts recognized by the axon can be recognized at any point and, as the present study shows, this recognition is required not only at the point of entry but continuously, all along the tract, for guidance of the axon. Guidance by the tract appears to depend on an affinity between the axon and the tract that may also exist between axons and tracts of their segmental or functional homologues. Since axons in foreign neuropil have irregular arborizations characteristic neither of their normal ones nor of those of the local sensilla, the arborization pattern is not a result of an internal branching program alone nor of the axon's milieu directing the branching but must depend on a specific interaction between the axon and its target.
\r\n\r\nThe leg motorneurons were identified and described after HRP backfilling from cut legs. The pattern of their positions differs from segment to segment. The bithorax mutations transform the metathoracic pattern into a mesothoracic pattern, paralleling their effect on the epidermis.
" }, { "name": "Jennings, Kent Richard", "degree": "PhD", "year": "1982", "title": "Studies of Excitability in a Model Peptidergic System: The Roles of Cyclic AMP, Protein Phosphorylation and Serotonin During Afterdischarge in the Bag Cell Neurons of Aplysia californica", "advisor": "Strumwasser, Felix", "url": "https://resolver.caltech.edu/CaltechTHESIS:05152018-142224321", "creators": [ { "name": { "family": "Jennings", "given": "Kent Richard" }, "id": "Jennings-Kent-Richard", "display_name": "Jennings, Kent Richard" } ], "advisors": [ { "name": { "family": "Strumwasser", "given": "Felix" }, "id": "Strumwasser-F", "role": "advisor", "display_name": "Strumwasser, Felix" } ], "committee": [ { "name": { "family": "Strumwasser", "given": "Felix" }, "id": "Strumwasser-F", "role": "chair", "display_name": "Strumwasser, Felix" }, { "name": { "family": "Brockes", "given": "Jeremy P." }, "id": "Brockes-Jeremy-P", "orcid": "0000-0002-3395-5159", "role": "member", "display_name": "Brockes, Jeremy P." }, { "name": { "family": "Hudspeth", "given": "A. James" }, "id": "Hudspeth-A-J", "role": "member", "display_name": "Hudspeth, A. James" }, { "name": { "family": "Konopka", "given": "Ronald J." }, "id": "Konopka-Ronald-J", "role": "member", "display_name": "Konopka, Ronald J." }, { "name": { "family": "Lazarides", "given": "Elias" }, "id": "Lazarides-E", "role": "member", "display_name": "Lazarides, Elias" } ], "option_major": [ "biology" ], "doi": "10.7907/8k0z-ft54", "abstract": "The polypeptide hormone-secreting bag cell neurons from the abdominal ganglion of Aplysia can be induced to fire repetitively when triggered by a brief electrical stimulus to the afferent pathway. This thesis investigates the mechanism of this afterdischarge by employing biochemical, pharmacological and electrophysiological approaches.
\r\n\r\nThe description of bag cell afterdischarge, its modulation by the transmitters serotonin and dopamine and evidence for the role of cyclic AMP in the genesis of afterdischarge is presented in Chapter 1. Bag cell afterdischarge is shown to be inhibited by the application of serotonin and lengthened by the application of dopamine or the methylxanthine phosphodiesterase inhibitors. Cyclic AMP undergoes a 2-3 fold increase in the bag cell clusters during an electrically-stimulated afterdischarge but not in matched controls where equivalent electrical stimulation did not elicit afterdischarge. As further evidence for a role for cyclic AMP in the genesis of afterdischarge, afterdischarges were obtained in unstimulated preparations by the extracellular application of the cyclic AMP analogues, 8-benzylthio-cyclic AMP and 8-methylthio-cyclic AMP.
\r\n\r\nChapter 2 describes protein phosphorylation in bag cell tissues under a number of different conditions. The presence of an endogenous, cyclic AMP-dependent protein kinase activity is demonstrated in crude membranes prepared from bag cells and the substrate specificity for this activity is shown to be similar to that of protein kinase catalytic subunit prepared from bovine heart. Increases in phosphorylation of a 33,000 dalton and 21,000 dalton phosphoprotein are shown to occur during electrically-stimulated afterdischarge in bag cells. The 21,000 dalton substrate is shown to be apparently specific to bag cell tissues and an amino acid composition and partial amino acid sequence of this protein is presented.
\r\n\r\nChapter 3 presents evidence that serotonin, within the physiological range reported by other workers for Aplysia (0.1-1.0 \u03bcM) brings about a rapid inhibition of an ongoing afterdischarge. This inhibition is antagonized by the stereospecific blocker of serotonin action, D-butaclamol but not its inactive isomer, L-butaclamol. Serotonergic inhibition is shown to be associated with decreased bag cell action potential duration and height and an increased threshold to spike generation. Evidence is presented that the second, calcium-dependent phase of bag cell afterdischarge is most sensitive to the action of the transmitter and that the potassium channel blocker, tetraethylammonium can overcome serotonin's inhibitory effect. This raises the possibility that serotonin may cause inhibition of bag cell afterdischarge by increasing potassium conductance. The possible functional role of serotonin inhibition of egg-laying is discussed.
" }, { "name": "Maunsell, John Henry Richard", "degree": "PhD", "year": "1982", "title": "Functional Organization and Connections of the Middle Temporal Visual Area in the Macaque Monkey", "advisor": "Van Essen, David C.", "url": "https://resolver.caltech.edu/CaltechTHESIS:07122018-165408194", "creators": [ { "name": { "family": "Maunsell", "given": "John Henry Richard" }, "id": "Maunsell-John-Henry-Richard", "display_name": "Maunsell, John Henry Richard" } ], "advisors": [ { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "advisor", "display_name": "Van Essen, David C." } ], "committee": [ { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "chair", "display_name": "Allman, John Morgan" }, { "name": { "family": "Hamilton", "given": "Charles R." }, "id": "Hamilton-Charles-R", "role": "member", "display_name": "Hamilton, Charles R." }, { "name": { "family": "Hudspeth", "given": "A. James" }, "id": "Hudspeth-A-J", "role": "member", "display_name": "Hudspeth, A. James" }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "member", "display_name": "Van Essen, David C." } ], "option_major": [ "biology" ], "doi": "10.7907/zmmw-v278", "abstract": "A variety of anatomical and physiological criteria have shown that the extrastriate visual cortex of the macaque monkey is subdivided into many distinct areas. There is evidence to suggest that there is functional specialization among these areas. Previous studies have shown that the middle temporal visual area (MT) contains a high proportion of cells which are selective for the direction of movement of visual stimuli, and yet relatively non-selective for stimulus color or form. The experiments reported here examined the response properties in MT in greater detail, and demonstrated the inputs and outputs of the area by means of anatomical tracers.
\r\n\r\nA computer-driven stimulator was used to examine quantitatively the responses of 163 single units from five anesthetized and paralyzed Macaca fascicularis. The experiments included tests of selectivity for stimulus direction, speed, orientation and disparity. Cells were also tested with stimuli which simulated trajectories with components of motion toward or away from the animal. The results show that in addition to direction selectivity, many cells in MT are sharply tuned for stimulus speed and disparity. This suggests that neurons in MT are well adapted for the analysis of motion in three-dimensional space.
\r\n\r\nHorseradish peroxidase and 3H-proline were injected into MT in three animals to demonstrate its anatomical inputs and outputs. Connections were seen with a large number of subcortical and cortical areas. In addition, connections with MT provide evidence contributing to the identification of two previously unrecognized cortical areas, which we have designated the medial superior temporal area (MST) and the ventral intraparietal area (VIP). The cortical layers in which projections originate and terminate are shown to provide objective anatomical criteria for assigning most cortical visual areas to a hierarchical order.
" }, { "name": "Orr, Dominic Ping-Yim", "degree": "PhD", "year": "1982", "title": "Behavioral Neurogenetic Studies of a Circadian Clock in Drosophila melanogaster", "advisor": "Strumwasser, Felix; Konopka, Ronald J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10222019-143344602", "creators": [ { "name": { "family": "Orr", "given": "Dominic Ping-Yim" }, "id": "Orr-Dominic-Ping-Yim", "display_name": "Orr, Dominic Ping-Yim" } ], "advisors": [ { "name": { "family": "Strumwasser", "given": "Felix" }, "id": "Strumwasser-F", "role": "advisor", "display_name": "Strumwasser, Felix" }, { "name": { "family": "Konopka", "given": "Ronald J." }, "id": "Konopka-Ronald-J", "role": "co-advisor", "display_name": "Konopka, Ronald J." } ], "committee": [ { "name": { "family": "Brokaw", "given": "Charles J." }, "id": "Brokaw-C-J", "role": "chair", "display_name": "Brokaw, Charles J." }, { "name": { "family": "Konopka", "given": "Ronald J." }, "id": "Konopka-Ronald-J", "role": "member", "display_name": "Konopka, Ronald J." }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Hopfield", "given": "John J." }, "id": "Hopfield-J-J", "role": "member", "display_name": "Hopfield, John J." }, { "name": { "family": "Strumwasser", "given": "Felix" }, "id": "Strumwasser-F", "role": "member", "display_name": "Strumwasser, Felix" } ], "option_major": [ "biology" ], "doi": "10.7907/wp4e-5054", "abstract": "The circadian clock controlling the locomotor activity of the adult fruitfly, Drosophila melanogaster, is studied in one wild-type and five clock mutant strains. Locomotive activity of individual flies are monitored using arrays of infra-red beams and detectors. It is found that the temperature compensation mechanism is intact in the mutants And and ClkK06, is slightly defective in the mutant pers and is grossly defective in the mutants perl1 and perl2. In the pers and perl1 mutants, this defect is enhanced when both eyes and major parts of both optic lobes are eliminated by a genetic mutation (sine oculus). The inter-individual variation of periods in a strain is found to increase much more than linearly with the average period of the same strain. The interaction between the And and the per loci and that between the And and ClkK06 loci are found to be either very weak or non-existent (effects of mutations additive), whereas the interactions among the various alleles in the per locus are found to be strong (effects of mutations non-additive).
\r\n\r\nTen 'Phase Resetting Curves' (PRC) obtained with saturating light pulses for six strains of flies at various temperatures are presented. All the ten cases exhibit basically 'type-1' resetting behavior (average slope = 1). Comparisons of the PRC's for pers, perl1 and wild-type at 17\u00b0C suggest that the mutations pers and perl1 change the period of the circadian clock by differentially shortening and lengthening, respectively, the duration of the 'subjective day' phase of the oscillation. Comparisons between the PRC's for pers at 17\u00b0C, 22\u00b0C, and 25\u00b0C and comparison between the wild-type PRC's at 17\u00b0C and 22\u00b0C do not reveal major changes in the temporal structure of these two circadian clocks over the stated temperature ranges.
\r\n\r\nThe responses of one wild-type and five mutant circadian clocks to sustained dim light of the range 5 x 10-4 lux to 50 lux at 22\u00b0C are studied. In each strain, a critical 'window' of light intensity is found within which a variety of unstable clock features, including arrhythmia, are observed. The light intensity at which this critical window occurs in each of the mutant is 5 to 10 times lower than that in the wild-type. Responses from a ERG-defective mutant (norpA) are found to be qualitatively, but not quantitatively, similar to that of the wild-type. Responses from an eyeless and ocelli-less mutant (sine oculus) indicate that both period changes and arrhythmicity can be elicited by light in the absence of the compound eyes and ocelli. However, the sharp dependence of the occurences of these phenomena on light intensity is lost in this mutant.
\r\n\r\nArguments are presented to suggest that none of the four mutations -- And, ClkK06, pers, and perl1 -- cause changes of period by mimicking the effects of tonic light on the Drosophila circadian system.
\r\n\r\nThe phase resetting curves (PRC) and the dim light responses described above are found to be incompatible with a particular model of the Velocity Response Curve (VRC) theory to inter-relate the phasic to tonic effects of light, in which the tonic effect of light is assumed to be the result of a summation of the effects of a contiguous series of single Light pulses, taking into account adaptation.
" }, { "name": "Ou, Jing-hsiung James", "degree": "PhD", "year": "1982", "title": "Structure and Replication of Alphavirus RNAs", "advisor": "Strauss, James H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05092018-121105492", "creators": [ { "name": { "family": "Ou", "given": "Jing-hsiung James" }, "id": "Ou-Jing-hsiung-James", "display_name": "Ou, Jing-hsiung James" } ], "advisors": [ { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "advisor", "display_name": "Strauss, James H." } ], "committee": [ { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "chair", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "member", "display_name": "Mitchell, Herschel K." }, { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "member", "display_name": "Strauss, James H." } ], "option_major": [ "biology" ], "doi": "10.7907/2sr4-6m60", "abstract": "Both ends of the alphavirus genomic RNA are potentially important in its replication. The region preceding and including the 5'-end of the subgenomic 26S RNA in genomic RNA might also be involved in 26S RNA transcription. Sequences of these regions of up to 10 alphaviruses were determined by using strategies including enzymatic, chain-termination and cDNA sequencing methods.
\r\n\r\nComparison of the nucleotide sequences reveals three highly conserved sequences. The first conserved sequence is 19 nucleotides in length and is located at the extreme 3'-end next to the poly(A) tail. The second conserved sequence, which is 21 nucleotides in length, precedes the 5'-end of 26S RNA and includes the first two nucleotides of it. The third conserved sequence is 51 nucleotides in length and is located at a position of about 130 to 150 nucleotides from the 5'-end, depending on the virus. The last conserved sequence in all alphaviruses examined is capable of forming two stable hairpin structures and could also base-pair stably with the 3'-terminal sequences to cyclize genomic RNAs. Besides these three conserved sequences, a highly conserved stem and loop structure could also be formed at the extreme 5'-end of genomic RNA.
\r\n\r\nDefective interfering (DI) RNAs of alphaviruses are mutated genomic RNAs which often contain deleted, repeated and translocated sequences, but yet retain all elements essential for their replication. By studying the sequence organization of alphavirus DI RNAs, and the 3'-terminal sequences of the genomic RNAs of two alphavirus variants and their replication, the importance of these conserved sequences and secondary structures in alphavirus replication are discussed.
\r\n\r\nBoth the 3'- and 5'-terminal sequences of several alphavirus 26S RNAs were also determined. Results show that 26S and genomic RNAs are coterminal. Together with the results previously published, the total length of the 26S RNAs of two alphaviruses, Sindbis virus and Semliki Forest virus, were determined to be 4102 and 4074 nucleotides, respectively.
\r\n\r\nThe NH2- and COOH-terminal sequences of the precursors of nonstructural proteins (translated from genomic RNA) and structural proteins (translated from 26S RNA) of several alphaviruses were deduced from the nucleotide sequences determined. The initiation codons of most alphavirus genomic and 26S RNAs are preceded by the sequence CANN. To determine the importance of these tetranucleotides, their sequences in 65 eucaryotic mRNAs were surveyed. Results show that the sequence distribution of these tetranucleotides are non-random and they might be involved in initiation of translation.
\r\n\r\nThe 3'-noncoding regions of alphavirus genomic RNAs contain AU rich sequences. Sequence organization in the 3'-noncoding regions is similar to those in alphavirus DI RNAs. Mechanisms for the generation of these sequence rearrangements are discussed.
" }, { "name": "Petersen, Steven Elery", "degree": "PhD", "year": "1982", "title": "Visual Response Properties of Neurons in Extrastriate Cortex of the Owl Monkey", "advisor": "Allman, John Morgan", "url": "https://resolver.caltech.edu/CaltechTHESIS:05142018-121737381", "creators": [ { "name": { "family": "Petersen", "given": "Steven Elery" }, "id": "Petersen-Steven-Elery", "display_name": "Petersen, Steven Elery" } ], "advisors": [ { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "advisor", "display_name": "Allman, John Morgan" } ], "committee": [ { "name": { "family": "Sperry", "given": "Roger Wolcott" }, "id": "Sperry-R-W", "role": "chair", "display_name": "Sperry, Roger Wolcott" }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "member", "display_name": "Van Essen, David C." }, { "name": { "family": "Hamilton", "given": "Charles R." }, "id": "Hamilton-Charles-R", "role": "member", "display_name": "Hamilton, Charles R." }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" } ], "option_major": [ "biology" ], "doi": "10.7907/j0a4-ne77", "abstract": "The neurophysiological response properties of single neurons were studied quantitatively in four extrastriate areas of the owl monkey: the medial (M), dorsomedial (DM), dorsolateral (DL), and the middle temporal (MT) areas. Directionality was computed by comparing the responses to stimuli moved in the optimal and opposing directions; MT cells had much higher directionality to moving bars than cells in the other areas. Cells in all four areas were sharply tuned to the orientation of stationary flashed bars. Tuning for moving bars was broader than for flashed bars; DM cells were more sharply tuned to moving bars than were cells in the other areas. Tuning was broader to spots than to bars, while directionality was relatively unaffected. A moving array of random dots was the best stimulus for many MT neurons. Random dot stimuli were also effective in M, but evoked weak or no response from DM and DL cells. Extrastriate receptive fields were much larger than striate receptive fields. Eccentricity was correlated with receptive field size, but was uncorrelated with other variables.
\r\n\r\nNeurons in these four areas were tested for their selectivity to the spatial dimensions, the length and width, of visual stimuli. Cells in DL were much more selective for the spatial dimension than were cells in the other areas. The dimensional selectivity of DL cells is independent of the amount or sign of contrast in the receptive field, and the position of the stimulus within the receptive field. The optimal lengths and widths of visual stimuli are specified independently, and have a wide range of optimal dimensions from 1 to 30\u00b0 in length, and from 0.25 to 7\u00b0 in width.
\r\n\r\nSince many of the neurons of MT show strong directionality, it has been hypothesized that MT contributes to the perception of motion. A well-known aspect of motion perception is the phenomenon of direction-specific adaptation. We tested the neurons of MT for changes in responsiveness due to prolonged adaptation to stimuli moving in various directions. For directional cells, the response to a bar was suppressed following adaptation in the best direction, and enhanced following adaptation in the opposite direction, when compared to the response to a bar following a period of stationary stimulation. For nondirection cells, the effects were much weaker, or absent.
\r\n\r\nThese results support the notion of a localization of function among the various extrastriate areas.
" }, { "name": "Posakony, James William", "degree": "PhD", "year": "1982", "title": "Studies of the Organization and Expression of Individual Repetitive Sequence Families of the Sea Urchin Genome", "advisor": "Davidson, Eric H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:07132018-112110004", "creators": [ { "name": { "family": "Posakony", "given": "James William" }, "id": "Posakony-James-William", "display_name": "Posakony, James William" } ], "advisors": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "advisor", "display_name": "Davidson, Eric H." } ], "committee": [ { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "chair", "display_name": "Davidson, Norman R." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "member", "display_name": "Owen, Ray David" }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." } ], "option_major": [ "biodev" ], "doi": "10.7907/4e58-pq58", "abstract": "Individual repetitive DNA sequence families of the sea urchin Strongylocentrotus purpuratus were investigated with regard to their genomic organization, the internal structure of their members, and the structural and developmental characteristics of their RN A transcripts.
\r\n\r\nAnalysis by gel blot hybridization and reassociation kinetics of cloned genomic DNA fragments containing members of three specific repeat families reveals a different pattern of organization in each case. One family is organized into long regions of repeated DNA, usually containing several members of the family in a tandem or clustered arrangement. A second family exists as long repeated elements occurring only once in a local genomic region. The third family consists of short repetitive sequence elements which are generally flanked on either side by single-copy sequences.
\r\n\r\nThe internal structure of eight cloned repetitive sequence elements was examined by determination of their nucleotide sequences. The lack of sequence homology among the eight elements indicates that they are representative of distinct repeat families. For the most part they consist of complex sequence internally, with a minor fraction of the length of five of the eight occupied by direct or inverse sequence repetitions. Six of the eight sequences are not translatable. Comparison of the nucleotide sequences of three different members of the same repeat family reveals that they are not simply colinear sequence variants, but that they differ in the presence and/or arrangement of small sequence subelements.
\r\n\r\nHybridization with cloned repetitive sequence elements was used to demonstrate that the level of representation of specific repeat sequences is quantitatively similar in the egg RNA of two sea urchin species, S. purpuratus and S. franciscanus.
\r\n\r\nEgg and embryo polyadenylated RNAs bearing specific repetitive sequences were analyzed by cDNA cloning, DNA and RNA gel blot hybridization, and DNA sequencing. It was found that the two complements of a given repeat are carried on different sets of polyadenylated transcripts, which are generally quite long (> 3 kilobases, with an estimated number average length of 5-6 kilobases). Within these transcripts, specific short repetitive sequence elements are found interspersed either with single-copy sequences or with other repeat sequences. It is demonstrated by sequencing that one such repeat-containing region is not translatable. The sets of polyadenylated transcripts deriving from several individual repeat families undergo substantial quantitative and probably qualitative modulation during early sea urchin development. Analysis of specific transcripts with single-copy probes from repeat-containing cDNA clones indicates that the embryo genome is transcribed to produce at least some of the same interspersed RNAs as are stored in the oocyte during oogenesis. Finally, the transcripts bearing specific repeat sequences in the polyadenylated egg RNA of two related sea urchin species were found to be qualitatively dissimilar.
" }, { "name": "Reyes, Antonio Arevalo", "degree": "PhD", "year": "1982", "title": "Application of Synthetic Oligonucleotides in the Isolation of Murine Transplantation Antigen cDNA Clones", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05162018-154934919", "creators": [ { "name": { "family": "Reyes", "given": "Antonio Arevalo" }, "id": "Reyes-Antonio-Arevalo", "display_name": "Reyes, Antonio Arevalo" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "chair", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "member", "display_name": "Hood, Leroy E." }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "member", "display_name": "Mitchell, Herschel K." }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "member", "display_name": "Owen, Ray David" }, { "name": { "family": "Wallace", "given": "R. Bruce" }, "id": "Wallace-R-Bruce", "role": "member", "display_name": "Wallace, R. Bruce" } ], "option_major": [ "biology" ], "doi": "10.7907/hmha-5v32", "abstract": "Cytoplasmic poly(A)+ RNA was extracted from the murine thymoma cell line EL4 (b haplotype) and used to construct a library of cloned cDNA in pBR322. Initially, 30,000 colonies were screened with a mixture of eight hexadecanucleotides representing all possible coding sequences for residues 51-56 of H-2Kb. The only clone isolated, pH2K01, contains the coding sequence for residues 50 through 91 of H-2Kb, followed by a Glu codon and a termination codon. It is speculated that the mRNA from which pH2K01 was derived and H-2Kb mRNA have a common precursor but differ in the manner of post-transcriptional splicing.
\r\n\r\nA 133-nucleotide probe containing most of the coding sequence of pH2K01 was constructed and used to screen the remainder of the library. Two clones were isolated. pH202 encodes H-2Kb from residue 66 through the carboxy-terminus and includes 386 nucleotides of 3'-untranslated sequence. pH203 codes for H-2Db, from residue 82 through the carboxy-terminus, together with 476 nucleotides of 3'-untranslated sequence.
\r\n\r\nH-2Kb and H-2Db share sequence homologies of 83% and 91% at the protein and nucleotide levels, respectively. The cytoplasmic region of the molecule proximal to the membrane is identical in both antigens. The next most conserved region is the third external domain.
\r\n\r\nThe H-2Kb molecule is 10 amino acid residues longer than the H-2Db molecule. Analysis of 3'-end coding sequences of pH202, pH203 and other H-2 clones reported in the literature suggests that the difference in length could be the result of different splicing patterns of the mRNAs.
" }, { "name": "Sarmiento, Loveriza A.", "degree": "PhD", "year": "1982", "title": "Developmental Regulation in Drosophila melanogaster", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05162018-171212203", "creators": [ { "name": { "family": "Sarmiento", "given": "Loveriza A." }, "id": "Sarmiento-Loveriza-A", "display_name": "Sarmiento, Loveriza A." } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "chair", "display_name": "Mitchell, Herschel K." }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "member", "display_name": "Owen, Ray David" }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "member", "display_name": "Strauss, James H." }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." } ], "option_major": [ "molbio" ], "doi": "10.7907/an7n-mz78", "abstract": "An examination by time lapse movie of the process of pupation in Drosophila melanogaster shows a series of muscular contractions to effect breakage of the tracheae and subsequent head invagination.
\r\n\r\nIn order to gain an understanding of the biochemical changes during pre-pupal development, salivary glands from animals in the late larval to the pupal stages were pulse-labeled with methionine or cysteine and the patterns of the proteins were analyzed on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Many of the proteins detectable by Coomassie are generally present in all stages from late larvae to pupae. However, autoradiographs of the same gel show rapid rates of synthesis in the prepupal stages especially for a set of low molecular weight proteins (3K-12K). These small polypeptides show a dramatic reduction in their rates of synthesis at the time of pupation.
\r\n\r\nAnalyses of proteins from the salivary glands of animals injected with labeled methionine and cysteine show a general maintenance of most, if not all, of the proteins except for the low molecular weight components which diminish drastically at pupation. Results also suggest a transport of these small polypeptides from the salivary glands to the fluid between the pupal case and the prepupal cuticle (pupation fluid). These low molecular weight proteins are very basic and each component actually consists of several sub-components.
\r\n\r\nAnother aspect of development that was investigated concerns the regulation of bristle and hair formation. Heat treatment (40.2\u00b0C for 40 min) of pupae at different stages resulted in the production of four separate phenocopies designated as angle bristle, smooth bristle, multiple hairs and spear bristle. Each phenocopy is induced at a specific time when the cell is in a susceptible state and the sensitive period lasts for less than two hours.
\r\n\r\nWhen animals are heat treated under conditions which do not turn off the protein synthesis in progress but which induce the heat shock proteins, this results in increased survival and protection against phenocopy production. Comparison of the resumption of protein synthesis to that of RNA synthesis suggests the storage of mRNA as a factor in the protection against phenocopy induction.
" }, { "name": "Shotwell, Sandra Lee", "degree": "PhD", "year": "1982", "title": "A Biochemical and Genetic Analysis of the Cyclic AMP Phosphodiesterase Defect in Dunce, a Memory Mutant of Drosophila", "advisor": "Benzer, Seymour", "url": "https://resolver.caltech.edu/CaltechTHESIS:05162018-173145200", "creators": [ { "name": { "family": "Shotwell", "given": "Sandra Lee" }, "id": "Shotwell-Sandra-Lee", "display_name": "Shotwell, Sandra Lee" } ], "advisors": [ { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "advisor", "display_name": "Benzer, Seymour" } ], "committee": [ { "name": { "family": "Brockes", "given": "Jeremy P." }, "id": "Brockes-Jeremy-P", "orcid": "0000-0002-3395-5159", "role": "chair", "display_name": "Brockes, Jeremy P." }, { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "member", "display_name": "Benzer, Seymour" }, { "name": { "family": "Hudspeth", "given": "A. James" }, "id": "Hudspeth-A-J", "role": "member", "display_name": "Hudspeth, A. James" }, { "name": { "family": "Kennedy", "given": "Mary B." }, "id": "Kennedy-M-B", "orcid": "0000-0003-1369-0525", "role": "member", "display_name": "Kennedy, Mary B." }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "member", "display_name": "Mitchell, Herschel K." }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "member", "display_name": "Owen, Ray David" } ], "option_major": [ "biology" ], "doi": "10.7907/67ye-6j88", "abstract": "Drosophila can learn in several associative conditioning paradigms. Flies carrying the mutation dunce were selected for their poor performance in one such task, a negative reinforcement olfactory conditioning paradigm (Dudai et al., 1976). dunce flies express two other mutant phenotypes, female sterility, and reduced activity for one of the two cyclic AMP phosphodiesterases present in normal flies, PDE II (Byers et al., 1981) . . The experiments described below indicate that the normal dunce gene (dunce+) probably codes for PDE II itself, rather than for a regulator that affects PDE II and possibly other activities.
\r\n\r\nA micro-assay technique is described that allows the separate measurement of PDE I and PDE II when both are present in mixture. PDE II is shown to occur at high specific activity in the nervous system, which is consistent with a role for this enzyme in neuronal function. The phenotype of female sterility associated with dunce mutants can be suppressed by any of three suppressor mutations. These do not suppress the other two phenotypes of reduced PDE II activity and poor learning, indicating that these phenotypes are closer to the primary defect associated with dunce mutants. Reduced PDE II activity correlates with poor learning in dunce flies in all three developmental stages that were tested (first and third instar larvae, and adults), as well as in response to genetic modifications of dunce gene activity. The results of several biochemical and genetic experiments fail to reveal any abnormal regulation of PDE II activity in dunce flies. In Drosophila, as a rule, the activity level of an enzyme correlates linearly with the activity of the enzyme's structural gene. The specific activity of PDE II is shown to correlate in a one to one fashion with the level of normal dunce gene activity at five different doses of dunce+.
\r\n\r\nTaken as a whole, these experiments provide strong support for the hypothesis that PDE II represents the primary product of the dunce gene, indicating a role for this enzyme in the learning of Drosophila.
" }, { "name": "Smith, Randall Forrest", "degree": "PhD", "year": "1982", "title": "Genetic Analysis of the Circadian Clock System of Drosophila melanogaster", "advisor": "Konopka, Ronald J.; Owen, Ray David", "url": "https://resolver.caltech.edu/CaltechTHESIS:05172018-103149078", "creators": [ { "name": { "family": "Smith", "given": "Randall Forrest" }, "id": "Smith-Randall-Forrest", "display_name": "Smith, Randall Forrest" } ], "advisors": [ { "name": { "family": "Konopka", "given": "Ronald J." }, "id": "Konopka-Ronald-J", "role": "advisor", "display_name": "Konopka, Ronald J." }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "co-advisor", "display_name": "Owen, Ray David" } ], "committee": [ { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "chair", "display_name": "Owen, Ray David" }, { "name": { "family": "Konopka", "given": "Ronald J." }, "id": "Konopka-Ronald-J", "role": "member", "display_name": "Konopka, Ronald J." }, { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "member", "display_name": "Lewis, Edward B." }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "member", "display_name": "Mitchell, Herschel K." }, { "name": { "family": "Strumwasser", "given": "Felix" }, "id": "Strumwasser-F", "role": "member", "display_name": "Strumwasser, Felix" } ], "option_major": [ "biology" ], "doi": "10.7907/9rwg-p020", "abstract": "The circadian rhythm phenotypes of eight chromosome aberrations with a breakpoint in the region of the per locus (3B1-2) of Drosophila melanogaster have been analyzed. Two duplications and five deficiencies with a 3B1-2 breakpoint produce either a wild-type (approx. 24-h period) or an arrhythmic clock phenotype while one translocation with a 3B1-2 breakpoint, T(1;4)JC43, produces locomotor-activity rhythms with either very-long periods (31-39 hr), rhythms that grade into arrhythmicity, or completely arrhythmic phenotypes. The clock phenotypes of 3B1-2 chromosome aberrations suggest that arrhythmicity results from the total lack of per function while long-period phenotypes result from a reduction, but not complete elimination, of per activity. An extensive complementation analysis of 3B1-2 chromosome aberrations and per mutant alleles provided no compelling evidence for genetic complexity at the per locus. This is in contrast to the report of Young and Judd (1978). Analysis of both the locomotor-activity and eclosion phenotypes of 3B1-2 chromosome aberrations did not uncover differences in the genetic control of these two rhythms.
\r\n\r\nThe normal 24-h period of the circadian rhythms of locomotor activity and eclosion of Drosophila is shown to be altered by changes in per gene dosage. Females with only one dose of per+ or pers (the 19-h short-period mutant allele) or perl (the 29-h long-period mutant allele) have periods which are about 1-2 h longer than the corresponding females with 2 doses. Females with 3 doses of per+ and males with 2 doses of per+ or pers have periods which are 1/2 to 1 h shorter than the corresponding individuals without the extra dose. Males with three per+ doses have periods which are about 1.5 h shorter than wild-type males; additional per+ doses do not shorten period further. The observation that decreased per dosage lengthens period while increased dosage shortens period suggests that the long- and short-period mutations alter period by respectively decreasing and increasing per gene or gene product activity. The per+ dosage results and the complementation behavior of pers indicate that the hypermorphic phenotype of pers results from increased activity of the pers gene product rather than an overproduction of per+ product. This is the first report of such a mutant action in Drosophila.
\r\n\r\nBy screening mutagenized sex-linked and autosomal stocks for ones in which the normal period or phase of the circadian rhythm of eclosion (adult emergence) has been altered, a new X-linked clock mutant has been isolated which lengthens the normal 24-h period of both the the eclosion and adult locomotor-activity rhythms to about 25.5 h. This mutant, which we have named Andante (And), is not an allele of the per locus; recombination and deficiency mapping has placed the Andante locus at a separate site between polytene chromosome bands 10E2 and 10F1 (tentatively at 10E3, just proximal to the m-dy complex at 10E2-3). Andante, like all of the per mutant alleles, has a semi-dominant effect on period. The eclosion rhythm of Andante, like wild-type, has a low-amplitude (Type 1) phase-resetting response to light pulses, but compared to wild-type the Andante phase-resetting curve (PRC) is lengthened by 1-2 h per cycle.
" }, { "name": "Aston-Jones, Gary", "degree": "PhD", "year": "1981", "title": "The Behavioral Physiology of Locus Coeruleus Neurons", "advisor": "Olds, James; Bloom, Floyd E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04082026-180424449", "creators": [ { "name": { "family": "Aston-Jones", "given": "Gary" }, "id": "Aston-Jones-Gary", "orcid": "0000-0002-5034-3816", "display_name": "Aston-Jones, Gary" } ], "advisors": [ { "name": { "family": "Olds", "given": "James" }, "id": "Olds-J", "role": "advisor", "display_name": "Olds, James" }, { "name": { "family": "Bloom", "given": "Floyd E." }, "id": "Blom-Floyd-E", "role": "co-advisor", "display_name": "Bloom, Floyd E." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "neurobio" ], "doi": "10.7907/rpgq-v891", "abstract": "Factors controlling discharge of known norepinephrine-containing locus\r\ncoeruleus (NE-LC) neurons were studied in unanesthetized behaving rats, and\r\nthese neurons' efferent impulse conduction properties were examined in\r\nanesthetized rats. Single-unit (SU) and multiple-unit (MU) extracellular\r\nrecordings in unanesthetized preparations demonstrated the following: (1)\r\nTonic discharge co-varied with stages of the sleep-waking cycle (S-WC),\r\nbeing highest during waking (W), lower during slow-wave sleep (SWS), and\r\nvirtually absent during paradoxical sleep (PS). (2) Altered discharge\r\npredictably anticipated S-WC stages as well as phasic cortical activity\r\nsuch as spindles during SWS. (3) Discharge was reduced within active\r\nwaking during grooming and sweet water consumption. (4) Bursts of impulses\r\naccompanied spontaneous or sensory-evoked interruptions of sleep, grooming,\r\nconsumption, or other such ongoing behaviors. (5) Discharge was not linked\r\nto movement per se. (6) Field potentials (FPs) occurred spontaneously in\r\nNE-LC recordings, temporally synchronized with bursts of unit activity from\r\nthe same electrodes during Wand SWS, but at highest rates during PS, when\r\ndischarge was virtually absent. (7) Short-latency (15-50 msec), transient,\r\nbiphasic unit responses and synchronous FPs were predictably evoked by\r\nnon-noxious auditory, visual and somatosensory stimuli; individual\r\nrecordings typically exhibited similar response patterns for each sensory\r\nmodality. (8) The magnitudes of sensory-evoked response varied as a\r\nfunction of vigilance, such that largest responses occurred for stimuli\r\nwhich awakened animals and least responsiveness was exhibited during\r\nuninterrupted sleep. (9) Sensory responsiveness also decreased during\r\ngrooming and sweet water consumption. (10) Transiently reduced discharge\r\noccurred in response to gustatory stimulation accompanying voluntary\r\nconsumption of sweet water. (11) SU and MU recordings throughout the\r\nnucleus yielded remarkably homogeneous results. (12) Robust phasic\r\ndischarge was markedly synchronized among neurons in MU populations.
\r\n\r\nSU recordings of spontaneous and antidromic NE-LC impulse activity in\r\nanesthetized rats indicated the following: (1) Impulse conduction velocity\r\nfluctuated as a function of basal conduction latency, impulse rate, and\r\nnumber of impulses in, a train of activity. (2) Impulse conduction velocity\r\nincreased briefly, then exhibited a more pronounced, gradual decrease\r\nduring the same train of activity. (3) Large increases in conduction\r\nlatency occurred during low-frequency trains of impulse activity. (4)\r\nCalculations indicated that these axons may modulate their own impulse flow\r\nas a result of ion fluxes associated with spike propagation.
\r\n\r\nThese results are interpreted in light of previous data on the\r\npostsynaptic physiology of norepinephrine to indicate that robust activity\r\nin the NE-LC system may participate in terminating CNS and behavioral\r\nprocesses which have minimal value in coping with phasic external events,\r\nand simultaneously enhance activity within systems primarily concerned with\r\nsuch immediate responses. Conversely, low levels of spontaneous or\r\nsensory-evoked NE-LC discharge may enable tonic, endogenously generated\r\nvegetative behaviors to proceed. In this way, the NE-LC system may bias\r\nglobal behavioral orientation between the external and internal\r\nenvironments.
" }, { "name": "Bell, John Richard", "degree": "PhD", "year": "1981", "title": "The Isolation and Partial Biochemical Analysis of Sindbis Virus Proteins", "advisor": "Strauss, James H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06022015-104921135", "creators": [ { "name": { "family": "Bell", "given": "John Richard" }, "id": "Bell-John-Richard", "display_name": "Bell, John Richard" } ], "advisors": [ { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "advisor", "display_name": "Strauss, James H." } ], "committee": [ { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "chair", "display_name": "Strauss, James H." }, { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "member", "display_name": "Hood, Leroy E." }, { "name": { "family": "Lazarides", "given": "Elias" }, "id": "Lazarides-E", "role": "member", "display_name": "Lazarides, Elias" }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "member", "display_name": "Mitchell, Herschel K." }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "member", "display_name": "Owen, Ray David" } ], "option_major": [ "bioch" ], "doi": "10.7907/w84z-ee41", "abstract": "Recently, the amino acid sequences have been reported for several proteins, including the envelope glycoproteins of Sindbis virus, which all probably span the plasma membrane with a common topology: a large N-terminal, extracellular portion, a short region buried in the bilayer, and a short C-terminal intracellular segment. The regions of these proteins buried in the bilayer correspond to portions of the protein sequences which contain a stretch of hydrophobic amino acids and which have other common characteristics, as discussed. Reasons are also described for uncertainty, in some proteins more than others, as to the precise location of some parts of the sequence relative to the membrane.
\r\n\r\nThe signal hypothesis for the transmembrane translocation of proteins is briefly described and its general applicability is reviewed. There are many proteins whose translocation is accurately described by this hypothesis, but some proteins are translocated in a different manner.
\r\n\r\nThe transmembraneous glycoproteins E1 and E2 of Sindbis virus, as well as the only other virion protein, the capsid protein, were purified in amounts sufficient for biochemical analysis using sensitive techniques. The amino acid composition of each protein was determined, and extensive N-terminal sequences were obtained for E1 and E2. By these techniques E1 and E2 are indistinguishable from most water soluble proteins, as they do not contain an obvious excess of hydrophobic amino acids in their N-terminal regions or in the intact molecule.
\r\n\r\nThe capsid protein was found to be blocked, and so its N-terminus could not be sequenced by the usual methods. However, with the use of a special labeling technique, it was possible to incorporate tritiated acetate into the N-terminus of the protein with good specificity, which was useful in the purification of peptides from which the first amino acids in the N-terminal sequence could be identified.
\r\n\r\nNanomole amounts of PE2, the intracellular precursor of E2, were purified by an immuno-affinity technique, and its N-terminus was analyzed. Together with other work, these results showed that PE2 is not synthesized with an N-terminal extension, and the signal sequence for translocation is probably the N-terminal amino acid sequence of the protein. This N-terminus was found to be 80-90% blocked, also by Nacetylation, and this acetylation did not affect its function as a signal sequence. The putative signal sequence was also found to contain a glycosylated asparagine residue, but the inhibition of this glycosylation did not lead to the cleavage of the sequence.
" }, { "name": "Chiu, Arlene Yuen-Chin", "degree": "PhD", "year": "1981", "title": "Biochemical and Immunohistochemical Studies of the Egg-Laying Hormone of Aplysia californica: Purification, Primary Structure, Neurosecretion and Morphological Distribution", "advisor": "Strumwasser, Felix", "url": "https://resolver.caltech.edu/CaltechETD:etd-09272005-141028", "creators": [ { "name": { "family": "Chiu", "given": "Arlene Yuen-Chin" }, "id": "Chiu-Arlene-Yuen-Chin", "display_name": "Chiu, Arlene Yuen-Chin" } ], "advisors": [ { "name": { "family": "Strumwasser", "given": "Felix" }, "id": "Strumwasser-F", "role": "advisor", "display_name": "Strumwasser, Felix" } ], "committee": [ { "name": { "family": "Strumwasser", "given": "Felix" }, "id": "Strumwasser-F", "role": "chair", "display_name": "Strumwasser, Felix" }, { "name": { "family": "Hudspeth", "given": "A. James" }, "id": "Hudspeth-A-J", "role": "member", "display_name": "Hudspeth, A. James" }, { "name": { "family": "Lazarides", "given": "Elias" }, "id": "Lazarides-E", "role": "member", "display_name": "Lazarides, Elias" }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "member", "display_name": "Mitchell, Herschel K." }, { "name": { "family": "Brockes", "given": "Jeremy P." }, "id": "Brockes-Jeremy-P", "orcid": "0000-0002-3395-5159", "role": "member", "display_name": "Brockes, Jeremy P." } ], "option_major": [ "biology" ], "doi": "10.7907/50RT-EZ43", "abstract": "Egg-laying behavior in Aplysia californica can be triggered by the introduction of a neuropeptide, the Egg-Laying Hormone (ELH) into the circulation. ELH is synthesized by the neurosecretory bag cells of the abdominal ganglion and released when these neurons are induced to fire repetitively. In this thesis, biochemical and immunohistoehemical techniques have been employed to study the primary structure, release and distribution of ELH in the nervous system of Aplysia.
\r\n\r\nThe purification of ELH to homogeneity from extracts of bag cell clusters, and the analysis of its primary structure are discussed in Chapter 1. A 100-fold enrichment of bioactive material was obtained by cation exchange chromatography (Sephadex SP C25) followed by gel filtration (BioRad P-6). This purified material was determined to be homogeneous by four lines of analysis: (i) SDS polyacrylamide gel electrophoresis, (ii) isoelectric focussing, (iii) microsequence analysis, and (iv) comparison of the amino acid compositions from acid hydrolysis and from microsequence data. ELH is a 36 amino acid, basic peptide with a calculated molecular weight of 4385 and an apparent isoelectric point of 9.0-9.2. Its amino acid sequence was determined as:
\r\n\r\nH-lle-Ser-Ile-Asn-Gln-Asp-Leu-Lys-Ala-Ile-Thr-Asp-Met-Leu-Leu-Thr-Glu-Gln-lleArg-Glu-Arg-Gln-Arg-Tyr-Leu-Ala-Asp-Leu-Arg-Gln-Arg-Leu-Leu-Glu-Lys-OH
\r\n\r\nChapter 2 demonstrates the release of ELH, identified by molecular weight, pI and bioactivity, when bag cell clusters afterdischarge in vitro. During such synchronous and prolonged electrical activity, bag cell clusters, which have been pulsed in 35S-Met, secrete at least four labeled presumed peptides of different molecular weights. One of these comigrates with 3H-Leu labeled, purified ELH on gel filtration chromatography and causes egg laying when injected into a test animal. This material also comigrates with 3H-ELH on isoelectrifocussing gels. A second released peptide has a molecular weight of approximately 5-6 K and a pI of 4.8; its function, and the functions of the other released molecules, are unknown.
\r\n\r\nIn order to study the distribution of ELH in the nervous system of Aplysia, antibodies were generated against the purified neuropeptide, coupled to a carrier molecule, thyroglobulin (Tg). Immune sera, enriched for anti-ELH antibodies by passage through an affinity column to remove antibodies which bound to Tg, was used for localizing ELH-like immunoreactivity in frozen sections of Aplysia ganglia. These results were discussed in Chapters 3 and 4.
\r\n\r\nWhen sections of the abdominal ganglion were stained by the PAP method for ELH, all neurons within the bag cell clusters were found to be immunoreactive. Except for occasionally displaced bag cells, all neurons within the ganglion remained unreactive, reflecting the specificity of the antiserum. Immunopositive processes from bag cells proliferate in the vascularized connective tissue capsule which serves as a neurohemal organ facilitating release of neurohormones. Some processes form a spiralling cuff around the nerve trunks of the pleuro-visceral connective and the vulvar nerves; others invade the ganglion in association with connective tissue septa which form partitions between groups of neurons. Immunoreactive fibers with varicosities are also found within the neuropile and the commissure between the two hemiganglia. This light microscopic visualization of the bag cell neuroendocrine system provides morphological support for the model of local hormone action of ELH upon other neurons in the abdominal ganglion. The immunoreactivity of all neurons within the clusters provides the strongest evidence to date of the homogeneity of the bag cell population.
\r\n\r\nAntibodies generated against ELH from A. californica selectively stained the bag cell systems of three other species of Aplysia - A. braziliana, A. vaccaria and A. dactylomela - which also share cross bioactivity. It is likely that receptor binding sites and antigenic determinants are conserved in their ELHs.
\r\n\r\nThe fourth chapter describes the organization of cells and fiber tracts with ELH-like immunoreactivity, endogenous to the head ganglia. Each pleural ganglion has 1-5 immunopositive somata which are strikingly similar to bag cells in cell and nuclear sizes, process morphology and location. These similarities, coupled with the close developmental association of the pleural and abdominal ganglia, suggest a common heritage for both populations of ELH+ cells.
\r\n\r\nThe ELH immunoreactive system in the cerebral ganglion consists of two laterally located clusters of small cells on the dorsal surface of the ganglion and extensive fiber tracts throughout the neuropile. The nature of immunoreactive molecules and the function of these systems within the cerebral and pleural ganglia are unknown. However, perfusion of ELH is known to induce long-term changes in the electrical activity of head ganglia neurons in vitro, and some of these changes may be linked to the suppression of feeding and locomotion during egg laying. The presence of these immunopositive systems in the pleural and cerebral ganglia raises the possibility that ELH target neurons in head ganglia may respond to local sources of ELH or ELH-like molecules.
" }, { "name": "Davis, Mark Morris", "degree": "PhD", "year": "1981", "title": "Programmed DNA Rearrangements During Differentiation: Immunoglobulin Class Switching", "advisor": "Hood, Leroy E.; Lewis, Edward B.", "url": "https://resolver.caltech.edu/CaltechETD:etd-04292005-082735", "creators": [ { "name": { "family": "Davis", "given": "Mark Morris" }, "id": "Davis-Mark-Morris", "orcid": "0000-0001-6868-657X", "display_name": "Davis, Mark Morris" } ], "advisors": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "advisor", "display_name": "Hood, Leroy E." }, { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "advisor", "display_name": "Lewis, Edward B." } ], "committee": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "chair", "display_name": "Hood, Leroy E." }, { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "member", "display_name": "Lewis, Edward B." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Delbruck", "given": "Max" }, "id": "Delbr\u00fcck-M", "role": "member", "display_name": "Delbruck, Max" }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Maniatis", "given": "Thomas P." }, "id": "Maniatis-T-P", "role": "member", "display_name": "Maniatis, Thomas P." } ], "option_major": [ "molbio" ], "doi": "10.7907/KFJS-G857", "abstract": "The events of B-lymphocyte differentiation can be reconstructed in part\r\nthrough an analysis of the organization of heavy-chain genes isolated from B-cell\r\ntumors (myelomas). A mouse immunoglobulin alpha heavy-chain gene is shown to be\r\ncomposed of at least three non-contiguous segments of germline DNA -- a VH gene\r\nsegment, a JH gene segment adjacent to the C\u03bc coding region, and a C\u03b1 gene\r\nsegment. These gene segments are joined together by two distinct types of DNA\r\nrearrangements: variable region formation and immunoglobulin class switching.\r\nThree examples of IgM \u2192 IgA elass switching were examined and in each case a\r\ndifferent site adjacent to C\u03bc and a different site adjacent to C\u03b1 were joined together\r\nin the process of switching. Two of the three C\u03bc sites shared significant homology to\r\neach other (15/25 nucleotides) and all three of C\u03b1 sites were highly homologous (22/30\r\nnucleotides). We believe these sequences serve as recognition sites for class\r\nswitching. Furthermore, the lack of homology between the C\u03b1 consensus sequence\r\nand sequences reported for C\u03b31 and c\u03b32b recombination sites suggests that this\r\nprocess is mediated by class-specific recognition sequences and, presumably, class-specific\r\nregulatory mechanisms. A number of predictions and possible explanations of\r\nimmune phenomena result from this observation. Apparently nonproductive DNA\r\nrearrangements, occurring in the same tumor lines, seem also to utilize some of the\r\n_same regulatory apparati. In addition, it appears that in one example, MClOl, class\r\nswitching has progressed from C\u03bc \u2192 C\u03b1 \u2192 C\u03b31. This switching pathway presents\r\ndifficulties for the simple deletional model of CH switching." }, { "name": "Johnson, Deanna Ojala", "degree": "PhD", "year": "1981", "title": "Transcription of the HeLa Cell Mitochondrial Genome", "advisor": "Attardi, Giuseppe", "url": "https://resolver.caltech.edu/CaltechTHESIS:10272025-221626339", "creators": [ { "name": { "family": "Johnson", "given": "Deanna Ojala" }, "id": "Johnson-Deanna-Ojala", "display_name": "Johnson, Deanna Ojala" } ], "advisors": [ { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "advisor", "display_name": "Attardi, Giuseppe" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "molbio" ], "doi": "10.7907/5fvg-t302", "abstract": "The HeLa cell mitochondrial genome has been shown to encode, in addition\r\nto a number of tRNAs, two ribosomal RNAs and at least 17 discrete poly(A)-containing\r\nRNA species. A variety of techniques, including Southern blots, Northern blots\r\nand Berk and Sharp analyses, were utilized to orient the ribosomal and polyadenylated\r\nspecies with respect to a detailed restriction map. Thus, nineteen species were\r\nsuccessfully localized to within an accuracy of about 30 to 40 nucleotides, thereby\r\nallowing a number of interesting observations to be made concerning the process\r\nof transcription of this genome. First, both the ribosomal RNAs and polyadenylated\r\nRNA components are transcribed colinearly; no intervening sequences exist. This\r\nis in dramatic contrast to the situation observed in mitochondrial DNA of some\r\nstrains of yeast. Second, there exists no overlapping of the mature polyadenylated\r\nspecies, the ribosomal RNAs and the tRNAs (within the resolution described) and,\r\nwith the exception of the D-loop region, the mitochondrial DNA sequences appear\r\nto be completely utilized for the synthesis of the transcripts. Third, a comparison\r\nof the positions of the DNA sequences which encode the polyadenylated transcripts\r\nwith respect to those encoding the tRNAs shows that the majority of those transcripts\r\nare flanked at both 5'- and 3'-ends by a tRNA.
\r\n\r\nDirect sequencing analyses have been used to investigate the precise relationship\r\nof the 5'- and 3'-ends of many of the polyadenylated transcripts (which are\r\npresumptive mRNAs) with respect to the ends of the tRNAs. Results obtained have\r\ndemonstrated that those species which have been shown to be flanked by a tRNA\r\ngene at their 5'-end begin immediately following the tRNA 3'-terminal nucleotide.\r\nCorrespondingly, the 3'-terminal nucleotide of seven of these RNA transcripts has\r\nbeen found to be immediately juxtaposed to the 5'-terminal nucleotide of the flanking\r\ntRNA. In one case, the 3'-terminal nucleotide is adjacent to the 5'-terminal nucleotide\r\nof the following mRNA. Moreover, an additional striking feature of these RNAs\r\nis that, with one possible exception, they either lack a 5'-end noncoding stretch,\r\nor contain an abbreviated version of it. Thus six species begin directly with AUG\r\nor A UA (which codes for methionine in human mitochondria), while three species\r\ncontain those triplets from within one to eight nucleotides of the 5'-end.
\r\n\r\nA model is proposed whereby structural features or sequences of tRNA\r\nspecific transcripts play a role in the processing events which form the mature species.\r\nSpecifically, the tRNA would provide a signal for a precise endonucleolytic cleavage\r\nevent which would generate the mature species from a larger precursor. The results\r\npresented in this thesis, in correspondence with sequencing data (B. Barrell and F.\r\nSanger, personal communication}, describe a transcriptional system characterized\r\nby simplicity, efficiency and economy.
" }, { "name": "Johnson, Nelson Daniell", "degree": "PhD", "year": "1981", "title": "An Analysis of Patterns of Diversity in Antibodies with Defined Specificity", "advisor": "Hood, Leroy E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05082018-140634895", "creators": [ { "name": { "family": "Johnson", "given": "Nelson Daniell" }, "id": "Johnson-Nelson-Daniell", "display_name": "Johnson, Nelson Daniell" } ], "advisors": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "advisor", "display_name": "Hood, Leroy E." } ], "committee": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "chair", "display_name": "Hood, Leroy E." }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "member", "display_name": "Owen, Ray David" }, { "name": { "family": "Richards", "given": "John H." }, "id": "Richards-J-H", "role": "member", "display_name": "Richards, John H." }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Maniatis", "given": "Thomas P." }, "id": "Maniatis-T-P", "role": "member", "display_name": "Maniatis, Thomas P." } ], "option_major": [ "bioch" ], "doi": "10.7907/1wj1-r628", "abstract": "Antibodies can recognize a large number of molecular determinants (antigens) because of the diversity present in antibody combining sites. This diversity resides in regions of extensive amino acid variability termed variable (V) regions. Variable region diversity is encoded in multiple germline variable region genes and can also arise from somatic modification of these genes. An important class of somatic modifications is the rearrangement of gene segments to form complete variable region genes. In this way complete VL genes arise from the joining of VL and JL gene segments while VH genes arise from VH, D, and JH gene segment joining.
\r\n\r\nStudies of the V region protein sequences of hybridoma and myeloma immunoglobulins which bind phosphorylcholine show that IgM antibody V regions are considerably less diverse than IgG and IgA V regions. A comparison of protein sequence data with experiments on germline DNA suggests that at least some V segment diversity in IgG and IgA antibodies is the result of somatic mutations. D segments from phosphorylcholine-binding IgM antibodies as well as from IgG and IgA antibodies show extensive amino acid interchanges and size differences. In addition, diversity in the antibody response to phosphorylcholine is generated by associating a single VH region with at least two different VL regions.
\r\n\r\nThe complete sequences of the V L and V H regions from two antibodies binding \u03b2 (2 \u2192 1) levan have also been determined. A comparison of these sequences to protein sequence data from other \u03b2 (2 \u2192 1)) levan-binding proteins and to a germline DNA sequence suggests that the levan-binding proteins may arise from multiple germline genes differing at the protein level by only a few amino acids. Unlike the D segments of the phosphorylcholine binding proteins, the levan-binding immunoglobulin D segments show very little diversity. In addition, the protein sequences of levan-binding immunoglobulins can be compared to published V region idiotype and antigen binding studies. These comparisons show that idiotypes may focus on certain sections of antibody V regions, and hence be of limited value as a probe of antibody V region fine structure.
" }, { "name": "Lacy, Elizabeth Hardy", "degree": "PhD", "year": "1981", "title": "The Structure, Expression and Chromosomal Arrangement of Rabbit \u03b2 Globin Genes", "advisor": "Maniatis, Thomas P.", "url": "https://resolver.caltech.edu/CaltechTHESIS:09212021-231109296", "creators": [ { "name": { "family": "Lacy", "given": "Elizabeth Hardy" }, "id": "Lacy-Elizabeth-Hardy", "orcid": "0000-0001-5457-0728", "display_name": "Lacy, Elizabeth Hardy" } ], "advisors": [ { "name": { "family": "Maniatis", "given": "Thomas P." }, "id": "Maniatis-T", "orcid": "0000-0002-2722-8633", "role": "advisor", "display_name": "Maniatis, Thomas P." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/z3xt-k222", "abstract": "Rabbits contain at least three \u03b2-like and two \u03b1-like globin polypeptides which are differentially synthesized during embryonic development. The structure, expression and chromosomal arrangement of the gene family encoding the \u03b2-like polypeptides is described.
\r\n\r\nA novel procedure was used to isolate the rabbit \u03b2 globin gene family which does not require the partial purification of single copy genes. Large random rabbit genomic DNA fragments were joined to phage lambda vectors by using synthetic DNA linkers. The resulting recombinant lambda-rabbit DNA molecules were packaged in vitro into viable phage particles and amplified to produce a permanent library of rabbit genomic sequences. The library was screened using cloned globin cDNA probes and an in situ plaque hybridization procedure.
\r\n\r\nFrom a screen of the rabbit library, nine clones were isolated which contain four different \u03b2-like gene sequences (\u03b21, \u03b22, \u03b23 and \u03b24). Restriction enzyme mapping and blot-hybridization studies indicated that the nine clones contain overlapping restriction fragments, which together encompass 44 kilobase pairs (kb) of contiguous rabbit chromosomal DNA. Therefore, the four rabbit \u03b2-like globin genes are physically linked. In addition, all four genes are transcribed off the same strand of DNA in the orientation 5'-\u03b24-\u03b23-\u03b22-\u03b21-3'. A combination of blot-hybridization, R-looping, and DNA sequencing experiments demonstrated the presence of a large intervening sequence in all four genes and a second, smaller intervening sequence in \u03b21, \u03b22 and \u03b24.
\r\n\r\nDetermination of the nucleotide sequence of \u03b21 showed that this gene codes for the second type of two common codominant alleles encoding the rabbit adult \u03b2 globin chain. A presumptive 1450 nt polyadenylated precursor to \u03b21 mRNA was detected in adult bone marrow. Mature mRNA transcripts from genes \u03b23 and \u03b24 were found in rabbit embryonic erythroid cells, thereby identifying \u03b23 and \u03b24 as embryonic and/or fetal \u03b2-like glob in genes. RNA blotting experiments confirmed that genes \u03b21, \u03b23 and \u03b24 are differentially expressed during development.
\r\n\r\nNo \u03b22 transcripts were identified in anemic adult bone marrow or reticulocytes or in 12-day rabbit embryos. A DNA sequence analysis demonstrated that \u03b22 cannot code for a functional \u03b2 globin polypeptide and is therefore a globin pseudogene.
\r\n\r\nThe mRNA expression of \u03b21 was analyzed in a heterologous host cell using DNA-mediated transformations. Thymidine kinase (tk) minus mouse L cells were cotransformed with a lambda clone containing a chromosomal copy of the rabbit adult \u03b2 globin gene, \u03b21, and the herpes virus tk gene. The tk+ transformants containing copies of the rabbit gene were analyzed for rabbit \u03b2 globin transcripts. One transformant was found to contain 5 copies per cell of a cytoplasmic 9S polyadenylated rabbit \u03b2 globin transcript. Berk-Sharp S1 experiments demonstrated that both intervening sequences are spliced out precisely from the rabbit 9S \u03b2 globin transcripts in the transformed mouse L cells. This result indicates that RNA splicing mechanisms are not species-specific. However, the 5' termini do not contain 48 \u00b1 5 nucleotides present in the \u03b2 globin mRNA of rabbit reticulocytes. Therefore, the initiation and/or 5' processing of rabbit \u03b2 globin nuclear RNA precursors are not identical in transformed mouse fibroblasts and rabbit reticulocytes.
" }, { "name": "Lauer, Joyce Ellen", "degree": "PhD", "year": "1981", "title": "Molecular Cloning of the Human \u03b1-Globin Gene Family", "advisor": "Maniatis, Thomas P.", "url": "https://resolver.caltech.edu/CaltechTHESIS:09082025-201021359", "creators": [ { "name": { "family": "Lauer", "given": "Joyce Ellen" }, "id": "Lauer-Joyce-Ellen", "display_name": "Lauer, Joyce Ellen" } ], "advisors": [ { "name": { "family": "Maniatis", "given": "Thomas P." }, "id": "Maniatis-T", "orcid": "0009-0009-4304-1391", "role": "advisor", "display_name": "Maniatis, Thomas P." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/tdac-ny02", "abstract": "During human development a series of \u03b1-like and \u03b2-like subunits of hemoglobin\r\nare produced. Five \u03b2-like polypeptides, embryonic (\u0190), fetal (G\u03b3, A\u03b3) and adult\r\n(\u03b4, \u03b2), and two \u03b1-like polypeptides, embryonic (z;) and adult (\u03b1), have been identified.\r\nA structural analysis of the gene family encoding the human \u03b1-like globins is described.
\r\n\r\nAn improved gene isolation procedure was developed. This procedure makes\r\nit possible to isolate genes and their flanking sequences, and thus to directly determine\r\nthe linkage relationship between genes, without requiring partial purification of genes.\r\nLarge random genomic DNA fragments resulting from physical shear of DNA were\r\njoined to phage lambda vectors using synthetic DNA linkers. The resulting\r\nrecombinant DNA molecules were packaged in vitro into viable phage to yield a\r\ncollection of cloned overlapping DNA fragments which were then amplified to produce\r\na permanent library of genomic DNA sequences. This library can be screened\r\nrepeatedly using nucleic acid probes and an in situ plaque hybridization procedure in\r\norder to isolate many different genes.
\r\n\r\nClones containing the duplicated human \u03b1-globin genes (a1 and a2) were\r\nisolated from a library of human DNA. Also present on these clones are an \u03b1-like\r\npseudogene (\u03c8\u03b11) and an embryonic \u03b1-like gene (\u03c21) which were identified by blot\r\nhybridization experiments and DNA sequence analysis. Genomic blotting using the \u03c21\r\ncoding sequence as probe identified a second embryonic gene (\u03c22). The \u03c22 gene was\r\nisolated by cloning a DNA fragment which overlaps the clones containing the other \u03b1-like\r\ngenes. All five genes are transcribed from the same DNA strand and are arranged\r\nin the order 5'-\u03c22-\u03c21\u03c8\u03b11-\u03b12-\u03b11-3'.
\r\n\r\nComparison of \u03b11 and \u03b12 by restriction mapping and heteroduplex analysis of\r\nDNA fragments containing al or \u03b12 plus 5' flanking sequences demonstrated that each\r\ngene is located within an approximately 4 kb region of homology interrupted by two\r\nshort regions of nonhomology. The association of these large blocks of homology with\r\ngenes which are thought to have duplicated long ago suggests the existence of a\r\nmechanism for sequence matching.
\r\n\r\nTwo types of deletions invariably occur during propagation of clones\r\ncontaining \u03b11 and \u03b12. The breakpoints of these two types of deletions are located\r\nwithin the two blocks of al-a2 homology. The positions and the precise lengths of\r\nthese deletions indicate that deletion occurs by homologous but unequal crossing-over\r\nbetween corresponding regions of al and a2. The lengths and positions of these\r\ndeletions are indistinguishable from those of the two types of deletions which are\r\nassociated with \u03b1-thalassemia 2, suggesting that this common genetic disease results\r\nfrom homologous but unequal crossing-over between regions within and/or surrounding\r\nthe adult \u03b1-globin genes.
" }, { "name": "Rice, Charles Moen, III", "degree": "PhD", "year": "1981", "title": "Studies on the Structural Proteins of Sindbis Virus", "advisor": "Strauss, James H.", "url": "https://resolver.caltech.edu/CaltechETD:etd-10112005-110000", "creators": [ { "name": { "family": "Rice", "given": "Charles Moen, III" }, "id": "Rice-Charles-Moen-III", "orcid": "0000-0003-3087-8079", "display_name": "Rice, Charles Moen, III" } ], "advisors": [ { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "advisor", "display_name": "Strauss, James H." } ], "committee": [ { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "chair", "display_name": "Davidson, Norman R." }, { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "member", "display_name": "Hood, Leroy E." }, { "name": { "family": "Hudspeth", "given": "A. James" }, "id": "Hudspeth-A-J", "role": "member", "display_name": "Hudspeth, A. James" }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "member", "display_name": "Mitchell, Herschel K." }, { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "member", "display_name": "Strauss, James H." }, { "name": { "family": "Maniatis", "given": "Thomas P." }, "id": "Maniatis-T-P", "role": "member", "display_name": "Maniatis, Thomas P." } ], "option_major": [ "bioch" ], "doi": "10.7907/9aan-kg88", "abstract": "Conditions are described for the synthesis of long cDNA transcripts of Sindbis virus 26S and 49S RNA in high yield. This single-stranded cDNA could be cut with Type II restriction endonucleases including Hae III, Hha I, Rsa I, or Taq I to give reproducible patterns of discrete, virus-specific fragments which were suitable for subsequent end-labeling and direct sequence analysis. Using these methods, the strategy used for obtaining nearly the entire 26S RNA sequence from cDNA synthesized in vitro is presented. The 26S RNA is approximately 4.2 kb in length, and from the AUG codon initiating synthesis of the capsid protein, contains an open reading frame for 3735 nucleotides. From this sequence, the amino acid sequences of the encoded virus structural proteins, which include a basic capsid protein and two integral membrane glycoproteins (El and E2), as well as the sequences of two nonstructural polypeptides have been deduced. Features of the primary structure of these proteins and the proteolytic cleavage sites involved in their processing are discussed.
\r\n\r\nThe orientation of the virion glycoproteins with respect to the lipid bilayer was studied by digesting intact Sindbis virus with \u03b1-chymotripsin. A single membrane-associated peptide is produced from each of the two virion glycoproteins. These peptides contain covalently attached palmitic acid, are rich in hydrophobic amino acids and are located at the extreme COOH-terminal end of each glycoprotein. Both peptides contain uninterrupted sequences of uncharged amino acids of sufficient length to span the lipid bilayer, and it is suggested that they serve to anchor the viral glycoproteins in the membrane. The properties of these and other well-characterized transmembrane segments are discussed.
\r\n\r\nSpecific antisera to each of the virus structural proteins was produced and used to study the association of the virion glycoproteins and their precursors. E1 and E2 could be cross-linked into heterodimers using bifunctional amino-reactive imidates. This association is present both in intact virions and infected cells and is stable after solubilization of the virion envelope by Triton X-100. Cross-linking data of pulse-labeled monolayers and cells infected with ts mutants are summarized. These data suggest that PE2 (the precursor to E2) and E2 are in different conformations with respect to E1, and that the glycoprotein precursors synthesized at elevated temperatures have an increased tendency to undergo intracellular aggregation.
" }, { "name": "Sargent, Thomas Dean", "degree": "PhD", "year": "1981", "title": "The Rat Serum Albumin Gene", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechTHESIS:02172017-094958964", "creators": [ { "name": { "family": "Sargent", "given": "Thomas Dean" }, "id": "Sargent-Thomas-Dean", "display_name": "Sargent, Thomas Dean" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "chair", "display_name": "Bonner, James Frederick" }, { "name": { "family": "Meyerowitz", "given": "Elliot M." }, "id": "Meyerowitz-E-M", "orcid": "0000-0003-4798-5153", "role": "member", "display_name": "Meyerowitz, Elliot M." }, { "name": { "family": "Lazarides", "given": "Elias" }, "id": "Lazarides-E", "role": "member", "display_name": "Lazarides, Elias" }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "display_name": "Campbell, Judith L." } ], "option_major": [ "bioch" ], "doi": "10.7907/22x8-g069", "abstract": "The messenger RNA's that encode the Proteins rat serum albumin (RSA) and rat alphafetoprotein (RAFP) have been purified to virtual homogeneity by a combination of immunoprecipitation of polysomes and other physical isolation methods. Radioactive cDNA copies of these mRNA's have been prepared and used to monitor the changes in abundance of RSA- and RAFP-synthesizing polysomes in neonatal rat liver and in Morris hepatoms 7777, a rat liver tumor. These cDNA probes have also been used to determine the stability and reiteration frequency of their respective genes in various rat tissues.
\r\n\r\nThe RSA mRNA sequence has been converted into a series of bacterial plasmids by recombinant DNA methodology, and the RSA gene has been isolated from a library of recombinant bateriophage. Restriction endonuclease site mapping, R-loop mapping, \"Southern\" blot and extensive nucleotide sequence determination have been employed to elucidate the organization of the cloned sequences.
\r\n\r\nThe rat serum albumin gene has been found to be interrupted at fourteen locations by introns. The fifteen exons are spread over approximately 15,000 nucleotides of contiguous chromosomal DNA. The evolutionary history of albumin has been deduced by analysis of the patterns of internal periodic homology in this gene. Albumin apparently evolved by a series of at least three intragenic duplications followed by accumulation of many point mutations and small deletions. These events probably occurred over 300 million years ago. The rat alphafetoprotein gene has been shown to be related to the rat serum albumin gene by a common ancestor gene, and thus two (or possibly more) highly complex genes with many exons and many protein domains have evolved by duplication mechanisms. That this might be an important general source of the complexity of eukaryotic genomes is discussed.
" }, { "name": "Schilling, James Walter, Jr.", "degree": "PhD", "year": "1981", "title": "Antibody Diversity", "advisor": "Hood, Leroy E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03292017-102353875", "creators": [ { "name": { "family": "Schilling", "given": "James Walter, Jr." }, "id": "Schilling-James-Walter", "display_name": "Schilling, James Walter, Jr." } ], "advisors": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "advisor", "display_name": "Hood, Leroy E." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "molbio" ], "doi": "10.7907/npny-et15", "abstract": "Vertebrate organisms possess a large and diverse repertoire of\r\nantibody variable regions. A number of different genetic mechanisms\r\nhave been proposed to account for immunoglobulin variable (V) region\r\ndiversity, including multiple germline genes, somatic mutation,\r\nsomatic recombination, and multiple small gene segments which are\r\njoined to form a complete variable region gene segment. Analyses of\r\nvariable region amino acid sequences demonstrate the relative contribution\r\nof each of these mechanisms to antibody diversity.
\r\n\r\n\r\nTwenty-four VK21 chains have been examined. They suggest that the\r\nkappa chain variable region is encoded in two separate gene segments:\r\nVK and JK which are rearranged and joined during B cell differentiation.\r\nDiversification of the N terminus of the JK segment occurs as a consequence of VK-JK joining and has been explained by a site-specific\r\nrecombination model. The amino acid sequence data are consistent with\r\nthe existence of a minimum of six VK and five JK germline gene segments.\r\nPossible cases of somatic mutation are also observed. These conclusions\r\nare supported by nucleic acid sequence analyses performed by\r\nothers.
\r\n\r\n\r\nComplete variable region amino acid sequences have been determined\r\nfor twenty-one heavy chains from dextran binding antibodies. These\r\nsequences suggest that the heavy chain variable region is encoded by\r\nthree gene segments: VH, D, and JH. Nucleic acid sequence analyses\r\nare consistent with this conclusion. The existence of a minimum of\r\ntwo VH and four JH germline gene segments is suggested by these\r\nsequences. Possible examples of somatic mutation of VH and JH gene\r\nsegments have also been found. Diversification of the N-terminal residue\r\nof the JH segment may occur as a consequence of D-JH joining by a\r\nmechanism analogous to that observed in kappa chains. Although comprised\r\nof only two residues, the D segment is the most diverse portion\r\nof dextran binding heavy chains.
\r\n\r\n\r\nCombinatorial joining of VK and JK gene segments and VH, D, and\r\nJH gene segments contributes significantly to antibody diversity.
\r\n\r\n\r\nPrecise molecular locations of idiotypic determinants can be\r\nestablished in the dextran heavy chains. A cross-reactive idiotypic\r\ndeterminant (IdX) is located in the second hypervariable region of the\r\nVH segment. Individual idiotypic determinants (IdIs) correspond to\r\nparticular D segments.
\r\n\r\n\r\n\r\n\r\n" }, { "name": "Asai, David John", "degree": "PhD", "year": "1980", "title": "Immunological Approaches to Flagellar Movement", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechETD:etd-08232006-093449", "creators": [ { "name": { "family": "Asai", "given": "David John" }, "id": "Asai-David-John", "display_name": "Asai, David John" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/NY0R-TB57", "abstract": "This thesis summarizes attempts in our laboratory to use antibodies to study flagellar motility. After preliminary work on the effects of antibodies to intact dynein 1 and a tryptic fragment of dynein 1, a thorough study of antibodies against outer doublet tubulin has been made.\r\n\r\nBoth anti-dynein 1 and anti-fragment 1A inhibit movement-coupled ATP dephosphorylation and the movement of reactivated Strongylocentrotus purpuratus spermatozoa. Anti-fragment 1A, which also inhibits the ATPase activity of isolated dynein, inhibits the frequency and bend angle of the flagella. Anti-dynein 1 has a relatively smaller effect on beat frequency and is inhibitory of bend angle. These results suggest that binding to dynein is not, by itself, sufficient to strongly inhibit movement and that a more specific interference with ATPase activity produces more severe effects on movement.\r\n\r\nAntibodies to tubulin were purified from rabbit sera by tubulin-affinity chromatography. Four induced anti-tubulins from immune sera and four spontaneous anti-tubulins from the corresponding preimmune sera were isolated and characterized. Both induced and spontaneous anti-tubulins are of the IgG class of antibody. Both are specific for tubulin when presented with a crude mixture of axonemal proteins. Only induced anti-tubulin precipitates tubulin in immunodiffusion assays. Both anti-tubulins as well as their monovalent Fab fragments bind to sea urchin axonemes in a radiobinding assay. Both stain demembranated sperm flagella in indirect immunofluorescence but only induced anti-tubulin decorates chick fibroblast cytoplasmic microtubules. I conclude that the affinity-purified antibodies are specific for tubulin and are able to bind sea urchin sperm flagella.\r\n\r\nInduced anti-tubulins from four different immunizations all specifically reduce bend angle and symmetry of reactivated flagella without affecting beat frequency. At identical concentrations, spontaneous anti-tubulins, and immune and preimmune Fab fragments have no effect on any of the parameters of flagellar movement. The ATP-mediated disintegration of elastase-digested axonemes is not prevented by concentrations of induced anti-tubulin which are more than sufficient to paralyze reactivated spermatozoa, but the ATP concentration threshold for sliding is raised. Under similar conditions, anti-fragment 1A has been shown to completely inhibit microtubule sliding. Induced anti-tubulin behaves very similarly to CO2 in its movement-inhibitory activity.\r\n\r\nThese three antibodies all inhibit the bend angle of the reactivated flagella; they represent the first reports of amplitude inhibition by reagents known to be specific for the components responsible for active sliding. The specific bend angle inhibition and lack of effect on sliding by induced anti-tubulin is consistent with a microtubule conformational control of cross-bridge activity as a mechanism controlling the bending of the flagellum. The induced anti-tubulin effect is the first report of an antibody to tubulin having an inhibitory activity on microtubule-associated movement." }, { "name": "Byers, Andrew Duncan", "degree": "PhD", "year": "1980", "title": "Studies on Learning and Cyclic AMP Phosphodiesterase of the Dunce Mutant of Drosophila melanogaster", "advisor": "Benzer, Seymour", "url": "https://resolver.caltech.edu/CaltechTHESIS:03102026-174727484", "creators": [ { "name": { "family": "Byers", "given": "Andrew Duncan" }, "id": "Byers-Andrew-Duncan", "display_name": "Byers, Andrew Duncan" } ], "advisors": [ { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "advisor", "display_name": "Benzer, Seymour" } ], "committee": [ { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "chair", "display_name": "Benzer, Seymour" }, { "name": { "family": "Hudspeth", "given": "A. James" }, "id": "Hudspeth-A-J", "role": "member", "display_name": "Hudspeth, A. James" }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Konopka", "given": "Ronald J." }, "id": "Konopka-Ronald-J", "role": "member", "display_name": "Konopka, Ronald J." }, { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "member", "display_name": "Lewis, Edward B." }, { "name": { "family": "Olds", "given": "James" }, "id": "Olds-J", "role": "member", "display_name": "Olds, James" } ], "option_major": [ "biology" ], "doi": "10.7907/k8cd-wk36", "abstract": "Normal Drosophila learn to avoid an odorant associated with electric\r\nshock. This dissertation describes the isolation of two x-linked\r\nmutants that fail to display this learning, in spite of being able to\r\nsense the odorants and electric shock. The mutants are proved to be\r\nalleles of one gene, and are named dunce1 and dunce2. The finding that\r\ndunce2 females are sterile led to the discovery that the dunce gene was\r\nindependently known in two other laboratories.
\r\n\r\nKiger, Davis, and Golanty of the University of California at Davis\r\nfind (one) that normal Drosophila have two forms (I and II) of soluble\r\ncyclic AMP phosphodiesterase, differing in molecular weight and other\r\nproperties; (two) that a gene appearing to control form II is included\r\nwithin chromosomal bands 3D3 and 3D4; and (three) that females with\r\nhomozygous deficiency of these two bands are sterile. Mohler of the\r\nUniversity of Iowa isolated 225 x-linked female-sterile mutants. Among\r\nthese Davis and Kiger found two that map within 3D3 and 3D4 and lack\r\nthe form II of soluble cyclic AMP phosphodiesterase.
\r\n\r\nThe two mutants of Mohler learn poorly. They do not complement\r\ndunce1 or dunce2 in learning nor dunce2 in fertility, and therefore are\r\nalleles of the dunce gene. Mapping of learning with genetic deficiencies\r\nand duplications places dunce1 and dunce2 within region 3D3 and 3D4.\r\nFemales with homozygous deficiency of five adjacent bands, including\r\nthese two, are viable and vigorous, but learn poorly and are sterile.
\r\n\r\nThe soluble cyclic AMP phosphodiesterase II is inactive or absent\r\nin homogenates of dunce1 and dunce2 flies. In addition, cyclic AMP\r\nlevels are elevated in the dunce mutants, as expected if cyclic AMP\r\nphosphodiesterase activity is deficient.
\r\n\r\nIt is concluded that the dunce gene of normal Drosophila has functions\r\nin olfactory learning performance, reproduction, and cyclic AMP metabolism.\r\nRecent work with other organisms has suggested that cyclic AMP\r\nmay be intimately involved in mechanisms of learning; the dunce mutant\r\nshould prove helpful in testing this.
" }, { "name": "Corey, David Paul", "degree": "PhD", "year": "1980", "title": "A Biophysical Approach to Sensory Transduction by Vertebrate Hair Cells", "advisor": "Hudspeth, A. James", "url": "https://resolver.caltech.edu/CaltechTHESIS:01252017-090732325", "creators": [ { "name": { "family": "Corey", "given": "David Paul" }, "id": "Corey-David-Paul", "display_name": "Corey, David Paul" } ], "advisors": [ { "name": { "family": "Hudspeth", "given": "A. James" }, "id": "Hudspeth-A-J", "role": "advisor", "display_name": "Hudspeth, A. James" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "neurobio" ], "doi": "10.7907/42z7-s748", "abstract": "An in vitro preparation of the bullfrog sacculus was developed, in which the\r\nmacular epithelium was arranged as a septum between two chambers. Hair cells are\r\nstimulated by moving the overlying otolithic membrane with a glass stimulus probe;\r\nthe response is recorded as the transepithelial current with the epithelium voltage-clamped.\r\nThe preparation complements the intracellular studies of individual hair\r\ncells under way in this laboratory, and has the advantages of lower electrical noise,\r\nhigher temporal resolution, greater stability over time, and separate control of\r\nsolutions bathing apical and basal surfaces of the epithelium. Its major disadvantage\r\nis that stimulation and recording methods are somewhat less direct. Four projects\r\nwere carried out with this preparation:
\r\n\r\nThe generation of the transepithelial \"microphonic\" current was analyzed in\r\nterms of the passive electrical properties of the epithelium and time- and voltage-dependent\r\nconductances of the hair cell membrane. The summed receptor currents\r\nare modified by the change in membrane potential of hair cells, by a voltage-dependent\r\npotassium conductance, and by an adaptive shift of the responsive range of\r\nhair bundles. A mathematical model that includes these effects predicts the observed\r\nwaveform of the microphonic current.
\r\n\r\nThe ionic selectivity of the transduction channel was studied with this\r\npreparation and with intracellular voltage-clamping of individual hair cells. The\r\ntransduction channel is permeable to all alkali cations, to at least some of the\r\ndivalent alkaline earth metals, and to many small organic cations. The permeability\r\nto anions is not clear. The channel constitutes a large, water-filled pore, of at least\r\n0.65 nm diameter, which appears to contain a binding site for permeant organic\r\ncations. While a good fit to the current-voltage relation in Na+ saline can be\r\nobtained with a simple model that includes two energy barriers to permeation near\r\nthe middle of the membrane, the distribution of barriers is not known with any\r\nconfidence.
\r\n\r\nThe hair cell response saturates if the mechanically sensitive hair bundle is\r\ndisplaced by more than about 0.4 \u03bcm. An adaptation, which follows such saturation\r\nand had been seen intracellularly, was shown to be an adaptive shift of the responsive\r\nrange such as to restore the sensitivity. The shape of the response curve is unchanged\r\nby adaptation. The adaptation constitutes a relaxation of the link between hair\r\nbundle displacement and bias on the transduction element. The position and shape of\r\nthe response curve is changed by intracellular Ca++ and/or pH, and the rate of the\r\nshift is increased by increasing the Ca++ concentration.
\r\n\r\nFollowing a step displacement of the otolithic membrane, the microphonic\r\ncurrent approaches a new equilibrium value over several tens of microseconds. The\r\nlatency of the response is less than 40 \u03bcs at 22\u00b0C. The kinetics of the approach to\r\nequilibrium are slower at lower temperatures, and depend on the position of the hair\r\nbundle. A three-state model for transduction channel gating is presented that\r\nsupposes that changing the position of the hair bundle directly and continuously\r\nchanges the free energies of states of the channel. The model can quantitatively\r\npredict the observed kinetics of the current.
\r\n\r\n" }, { "name": "Costantini, Franklin David", "degree": "PhD", "year": "1980", "title": "Studies of Repetitive Sequence Transcripts in the Sea Urchin", "advisor": "Davidson, Eric H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01252017-095417305", "creators": [ { "name": { "family": "Costantini", "given": "Franklin David" }, "id": "Costantini-Franklin-David", "display_name": "Costantini, Franklin David" } ], "advisors": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "advisor", "display_name": "Davidson, Eric H." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biodev" ], "doi": "10.7907/p0b3-e643", "abstract": "The nature of transcripts from repetitive DNA sequences in the sea urchin,\r\nStrongylocentrotus purpuratus, is investigated. Hybridization experiments utilizing\r\nindividual cloned repeat sequences, as well as fractions of total repetitive DNA,\r\nindicate that the expression of repeat sequences in RNA is specifically regulated\r\nin development. A different set of repeat families is highly represented in each\r\nof three RNA populations examined, the nuclear RNAs of gastrula stage embryos\r\nand adult intestine tissue, and the total RNA of eggs. Essentially all the genomic\r\nrepeat families are represented in each RNA, but the prevalence of transcripts\r\nrepresenting different repeat families can vary by more than two orders of magnitude\r\nin a given RNA. Both complementary strands of most repeat families are represented\r\nat similar levels, raising the possibility that RNA-RNA repeat duplex formation\r\noccurs in the cell. Two cloned repeat sequences examined were both found primarily\r\non large transcripts in the nuclear RNA, and many of the nuclear repeat transcripts\r\nare believed to occur on long interspersed RNA molecules.
\r\n\r\nSeveral lines of evidence indicate that most repeat sequences in the egg\r\nRNA are contained on transcripts with the properties of maternal messenger RNA.\r\nA large fraction of the repeat-containing transcripts are polyadenylated. Most of\r\nthe repeats are found on long transcripts, while in the genome, these repeats are\r\nshort and interspersed with single-copy sequences. The repeat-containing RNAs\r\nare isolated and directly shown to consist of short repeats linked to longer single-copy\r\nsequences. These interspersed egg RNAs are shown to include nearly all of\r\nthe diverse single-copy sequences of total egg RNA, most of which are believed\r\nto be message sequences. Several implications of these findings are discussed.\r\nParticularly interesting is the conclusion that the single-copy maternal message\r\nsequences must be associated primarily with a restricted group of the diverse\r\ngenomic repeat families. The message sequences thus fall into several hundred\r\nsets, each containing transcripts from a different repeat family.
" }, { "name": "Early, Philip Warren", "degree": "PhD", "year": "1980", "title": "Mouse Immunoglobulin Heavy Chain Gene Organization and Rearrangement: Genetic Bases for Antibody Diversity and Regulated Expression", "advisor": "Davidson, Norman R.; Hood, Leroy E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04062026-221212098", "creators": [ { "name": { "family": "Early", "given": "Philip Warren" }, "id": "Early-Philip-Warren", "display_name": "Early, Philip Warren" } ], "advisors": [ { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "advisor", "display_name": "Davidson, Norman R." }, { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "co-advisor", "display_name": "Hood, Leroy E." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/c5ya-yy20", "abstract": "Immunoglobulin heavy chains each display one of a wide range of diverse antigenbinding\r\nvariable regions. At least one class of immunoglobulin, IgM, contains heavy chains\r\nwhich exist as two forms, either bound to the outside of a cell membrane or linked by\r\ndisulfide bonds in secreted antibodies. I have used recombinant DNA techniques to isolate\r\nand determine the nucleotide sequences of genes encoding mouse immunoglobulin heavy\r\nchains. This has enabled me to examine genetic bases for the diversity of heavy chain\r\nvariable regions and for the synthesis of membrane-bound and secreted forms of IgM\r\nheavy chains.
\r\n\r\nI found that genes encoding heavy chain variable regions are created somatically\r\nby joining three segments of DNA: VH, D, and JH The JH gene segments are closely\r\nlinked to the IgM heavy chain constant region gene in germline DNA, where they are\r\nwidely separated from VH gene segments. In an immunoglobulin-producing cell, one VH\r\nand one JH gene segment are joined, together with a D sequence which is probably also\r\na germline gene segment, to form the expressed heavy chain variable region gene. Both\r\ncombinatorial association of gene segments and variations in the exact sites of DNA\r\njoining between gene segments can contribute to heavy chain variable region diversity.\r\nBased on observations of certain conserved nucleotides and spacer sequences adjacent\r\nto unrearranged immunoglobulin gene segments, I propose a mechanism for variable\r\nregion gene rearrangement during differentiation.
\r\n\r\nSecreted and membrane-bound forms of IgM heavy chains were found to be\r\nencoded by separate mRNAs transcribed from the same gene. These mRNAs differ only\r\nat their 3' ends, where one encodes a 20 amino acid secretory C-terminal segment, and\r\nthe other encodes a 41 amino acid transmembrane C-terminal segment. Synthesis of\r\nthe two forms of lgM heavy chain mRNA appears to be developmentally regulated by\r\ncontrolling the site of 3' terminal polyadenylation. The site of polyadenylation defines\r\nthe lengths of RNA transcripts and thereby determines which of two alternative RNA\r\nsplicing patterns will be followed, leading to mRNAs encoding either the secreted or\r\nmembrane-bound forms of IgM heavy chains.
" }, { "name": "Frelinger, John Gregory", "degree": "PhD", "year": "1980", "title": "Studies on the Major Histocompatibility Complex of the Mouse and Rat", "advisor": "Hood, Leroy E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03122026-170817040", "creators": [ { "name": { "family": "Frelinger", "given": "John Gregory" }, "id": "Frelinger-John-Gregory", "display_name": "Frelinger, John Gregory" } ], "advisors": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "advisor", "display_name": "Hood, Leroy E." } ], "committee": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "chair", "display_name": "Hood, Leroy E." }, { "name": { "family": "Brockes", "given": "Jeremy P." }, "id": "Brockes-Jeremy-P", "orcid": "0000-0002-3395-5159", "role": "member", "display_name": "Brockes, Jeremy P." }, { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "member", "display_name": "Lewis, Edward B." }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "member", "display_name": "Mitchell, Herschel K." }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "member", "display_name": "Owen, Ray David" } ], "option_major": [ "bioch" ], "doi": "10.7907/d8rx-2d85", "abstract": "This thesis contains investigations into the Major Histocompatibility\r\nComplex (MHC) of two closely related species, rats and mice. In particular, I\r\nhave concentrated on molecules encoded in one part of the MHC, the I region\r\nin mice, and its equivalent in rats. The first part of the thesis consists of experiments\r\ndealing with the expression of molecules encoded by genes in the I region,\r\nthe Ia molecules. Ia molecules are expressed on immune related cells and surprisingly\r\non epidermal cells.
\r\n\r\nSeveral conclusions can be drawn from my studies on these epidermal\r\nIa molecules. The Ia molecules isolated from radiolabeled detergent solubilized\r\nepidermal cell extracts are not contributed by contaminating lymphocytes. The\r\nIa molecules from epidermal cell extracts are identical to their counterparts isolated\r\nfrom spleen cells by both sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis\r\nand high pressure liquid chromatography tryptic peptide map analyses.\r\nThe Ia molecules are synthesized by a non-T and non-B cell bone-marrow-derived\r\ncell. This cell is probably the macrophage-like Langerhans cell. This work supports\r\nthe theory that Ia molecules are involved in the immune response and are present\r\nonly on immune related cells.
\r\n\r\nThe second part of this thesis deals with the Class II (Ia-like) molecules\r\nencoded by the rat equivalent of the I region. Two Class II molecules can be immunoprecipitated\r\nusing cross-reactive mouse anti-la sera. These reagents will be extremely\r\nuseful in the further elucidation of the rat MHC.
" }, { "name": "Gaston, Karen Elizabeth", "degree": "PhD", "year": "1980", "title": "Behavioral Studies on Learning and Interocular Transfer in the Domestic Chick", "advisor": "Sperry, Roger Wolcott", "url": "https://resolver.caltech.edu/CaltechTHESIS:02272026-182254777", "creators": [ { "name": { "family": "Gaston", "given": "Karen Elizabeth" }, "id": "Gaston-Karen-Elizabeth", "display_name": "Gaston, Karen Elizabeth" } ], "advisors": [ { "name": { "family": "Sperry", "given": "Roger Wolcott" }, "id": "Sperry-R-W", "role": "advisor", "display_name": "Sperry, Roger Wolcott" } ], "committee": [ { "name": { "family": "Sperry", "given": "Roger Wolcott" }, "id": "Sperry-R-W", "role": "chair", "display_name": "Sperry, Roger Wolcott" }, { "name": { "family": "Hamilton", "given": "Charles R." }, "id": "Hamilton-Charles-R", "role": "member", "display_name": "Hamilton, Charles R." }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Konopka", "given": "Ronald J." }, "id": "Konopka-Ronald-J", "role": "member", "display_name": "Konopka, Ronald J." }, { "name": { "family": "Cherkin", "given": "Arthur" }, "id": "Cherkin-Arthur", "role": "member", "display_name": "Cherkin, Arthur" } ], "option_major": [ "biology" ], "doi": "10.7907/0znp-t143", "abstract": "Acquisition and interocular transfer of two kinds of visual learning, a\r\npattern discrimination task and an illness-induced aversion to a colored food,\r\nwere studied in young domestic chicks.
\r\n\r\nThe experiment reported in Chapter I was designed to assess interocular\r\ntransfer of a monocularly acquired operant pattern discrimination task reinforced\r\nby heat, and to determine whether the extent of transfer might vary with age\r\nand possible maturation of participating interhemispheric connections. The results\r\ndemonstrated that pattern discrimination learning failed to transfer from trained\r\nto untrained eye in intact chicks up to 16 days post-hatch, indicating that the\r\nmonocular learning was stored in the form of a unilateral engram which was not\r\navailable to the untrained hemisphere. This finding illustrates the extent to which\r\nthe two halves of the chick brain are separately organized and able to function\r\nindependently under certain conditions.
\r\n\r\nThe series of experiments reported in Chapters II, III, and IV examined\r\nacquisition and interocular/interhemispheric transfer of a conditioned aversion\r\nto a colored food. It was found that 10-day-old chicks learned to avoid drinking\r\na novel colored sucrose solution if their first experience with it was followed\r\nby illness induced by intraperitoneal injection of LiCl. Non-illness control subjects\r\ndid not show an aversion. When novel taste and color were both present, the aversion\r\nwas based on the visual (color) cue and not on taste. When color was eliminated,\r\nso that taste was the only novel cue available, then conditioning failed. Interocular\r\ntransfer of this learning was evaluated by conditioning chicks with one eye closed\r\nand testing for an aversion to the colored sucrose with either the trained or the\r\nuntrained eye open. The results showed that chicks avoided drinking the colored\r\nsucrose regardless of which eye was open during testing, indicating good interocular\r\ntransfer of the monocularly acquired aversion.
\r\n\r\nTo investigate further the roles of visual and gustatory cues in acquisition\r\nand transfer of the conditioned aversion, chicks were trained monocularly with\r\nnovel color combined with either novel or familiar sucrose taste. They were then\r\ntested, with either the trained or the untrained eye open, for learned aversions\r\nto colored or uncolored sucrose. Chicks tested with the trained eye showed an\r\naversion to the colored, but not the uncolored, liquid regardless of whether the\r\ntaste was novel or familiar during training. This result confirmed that the aversion\r\nwas mediated by the visual (color) cue and demonstrated that novel taste was\r\nnot required for acquisition of the visual aversion. In contrast, chicks tested with\r\nthe untrained eye open showed an aversion to either colored or uncolored sucrose,\r\nbut in each case only if the taste was novel during training. The avoidance of\r\nuncolored sucrose was clearly a taste aversion acquired only by the so-called\r\n\"untrained\" hemisphere which was deprived of primary visual input during the\r\ntraining session. These findings demonstrated that interocular/interhemispheric\r\ntransfer of the visual aversion failed in chicks trained with familiar taste; they\r\ndid not, however, rule out the occurrence of transfer when novel taste was present.
\r\n\r\nTaken together, the results of this series of experiments on conditioned\r\nfood aversions in chicks indicate that, while the visual system was normally\r\ndominant, eliminating primary visual input to half the brain somehow enhanced\r\nthe effective significance of taste information and enabled that hemisphere to\r\nassociate novel taste with the subsequent illness experience. It seems, then, that\r\nunder certain conditions the two halves of the chick brain are capable of independent\r\nand concurrent avoidance learning based on different types of sensory information.
" }, { "name": "Gelfand, Robert Allen", "degree": "PhD", "year": "1980", "title": "Studies on RNA Metabolism in HeLa Mitochondria", "advisor": "Unknown, Unknown", "url": "https://resolver.caltech.edu/CaltechETD:etd-08042006-124851", "creators": [ { "name": { "family": "Gelfand", "given": "Robert Allen" }, "id": "Gelfand-Robert-Allen", "display_name": "Gelfand, Robert Allen" } ], "advisors": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/rjx2-w897", "abstract": "The metabolism of mitochondrial ribonucleic acid was studied in the human cell line HeLa. Studies of labeling kinetics show that polyadenylated mitochondrial RNA species are turned over with half-lives ranging from a few minutes to about two hours.\r\n\r\nSeveral giant RNA species transcribed from the mitochondrial DNA light strand were studied. They range in size from approximately 25% to 60% of the length of the mitochondrial genome and overlap in their mapping locations.\r\n\r\nThe kinetics of labeling of mitochondrial polyadenylic acid not covalently linked to larger RNA molecules was studied. This \"free\" polyadenylic acid is not derived from the breakdown of larger polyadenylated RNA molecules and may be a precursor to polyadenylic acid covalently linked to messenger RNA molecules." }, { "name": "Gurney, Mark E.", "degree": "PhD", "year": "1980", "title": "Sexual Differentiation of Brain and Behavior in the Zebra Finch (Poephila guttata): a Cellular Analysis", "advisor": "Konishi, Masakazu", "url": "https://resolver.caltech.edu/CaltechTHESIS:04072026-204901171", "creators": [ { "name": { "family": "Gurney", "given": "Mark E." }, "id": "Gurney-Mark-E", "orcid": "0000-0003-4901-3083", "display_name": "Gurney, Mark E." } ], "advisors": [ { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "advisor", "display_name": "Konishi, Masakazu" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/5x7d-5114", "abstract": "Male zebra finches sing a brief song phrase to the female during courtship.\r\ncastration of an adult male reduces the bird's frequency of singing; testosterone\r\nreplacement reinstates the behavior. Testosterone treatment of female zebra finches\r\ndoes not activate song nor induce other elements of courtship behavior.
\r\n\r\nCorrelative changes of brain and behavior in zebra finches are organized by\r\nsex hormones during development. Newly hatched zebra finch chicks were subcutaneously\r\nimplanted with Silastic pellets containing either 50 \u03bcg of dihydrotestosterone\r\nor 50 \u03bcg of 17 \u03b2-estradiol. Testosterone treatment activates song in adult\r\nfemales which were implanted with estradiol when chicks, but fails to activate song\r\nin those females which had received dihydrotestosterone. The singing females\r\napproach a sexual partner with pivoting movements, straighten to an erect posture,\r\nfluff their throat feathers, and rapidly repeat their song phrase in a behavioral\r\nsequence which closely resembles that of the male.
\r\n\r\nIn zebra finches, brain nuclei of the efferent pathway for control of song\r\nshow dramatic sex differences in their volume (Nottebohm and Arnold; Science 194\r\n[1976] 211-213). 17 \u03b2-estradiol treatment of genetically female chicks organizes\r\nmale-like cytoarchitectonic differentiation of the telencephalic song nuclei RA, HVc,\r\nMAN, and X. Dihydrotestosterone induces masculinization of the brain stem song\r\nnuclei nXII and DM. Dendritic field spread, soma size, and the consequent volume of\r\nthe brain nucleus is larger in males than females at all levels of the song system. The\r\nexposure of female chicks to either androgen or estrogen supports growth of the\r\nhormone's respective target neurons in either the brain stem or telencephalic song\r\nnuclei. These neurons reach a size identical with that of the equivalent cell type in a\r\nnormal male. Anatomical comparison of normal adult male and female song systems\r\nreveals that all cell types and all identified connections are present in both sexes.\r\nThus, the specificiation of cellular identity-i.e., position, dendritic morphology and\r\nefferent synaptic projection-is expressed independently of the hormonal environment.\r\nRather than selecting pathways of anatomical differentiation, androgens and\r\nestrogens exert a similar pleiotrophic effect on their respective target neurons.\r\nAlthough in the female song system we identify all the cell types and connections of\r\nthe male, testosterone does not activate song. Thus, 17 \u03b2-estradiol may also exert a\r\nspecific inductive effect on the telencephalic song neurons which renders them\r\nphysiologically competent to respond to testosterone in the adult.
" }, { "name": "Johnson, Larry E.", "degree": "PhD", "year": "1980", "title": "Interhemispheric Visual Communication in Human Comissurotomy Subjects", "advisor": "Sperry, Roger Wolcott", "url": "https://resolver.caltech.edu/CaltechTHESIS:07162025-170515256", "creators": [ { "name": { "family": "Johnson", "given": "Larry E." }, "id": "Johnson-Larry-E", "display_name": "Johnson, Larry E." } ], "advisors": [ { "name": { "family": "Sperry", "given": "Roger Wolcott" }, "id": "Sperry-R-W", "role": "advisor", "display_name": "Sperry, Roger Wolcott" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/ccfk-jg81", "abstract": "Several problems concerning the transfer of visual information between\r\nthe cerebral hemispheres in human forebrain commissurotomy patients were\r\nexamined. These patients, 10-15 years post-surgery, were compared with normal\r\nand partially-split (splenium-intact) control subjects both on their ability to verbally\r\ncategorize or name stimuli tachistoscopically presented unilaterally into either\r\nthe left or right visual half-field (LVF or RVF), several degrees from the fixation\r\npoint, and on their ability to compare or name simultaneously presented bilateral\r\nvisual stimuli. The aim of these experiments was (1) to determine the extent to\r\nwhich stimuli presented to the left visual field could be orally described, (2) to\r\nlearn whether visual information presented separately to each \"disconnected\"\r\nhemisphere could be compared, and (3) to explain the findings in context with\r\nprevious neuroanatomical, physiological, and behavioral studies.
\r\n\r\nUsing either a box tachistoscope or a back-projection screen, a wide\r\nvariety of stimuli (brightness, colors, numbers, letters, patterns, schematic faces,\r\nand photographs of human faces) were flashed to one or both visual half-fields,\r\nand both the accuracy and the speed of several kinds of manual and verbal responses\r\nwere measured.
\r\n\r\nThree of the four split-brain patients were able to orally categorize\r\nunilaterally presented stimuli (\"yes\"-\"no\", \"odd\"-\"even\") in both visual fields.\r\nIn addition, all four patients responded as rapidly to LVF stimuli as to RVF stimuli\r\nin categorization experiments. The control subjects also accurately categorized\r\nstimuli and responded equally fast to LVF and RVF stimuli.
\r\n\r\nWhen the patients were asked to~ the unilateral stimulus, those in\r\nthe RVF were easily named but the ability to name LVF stimuli was found to vary\r\nbetween patients for different stimuli and sample sizes. However, despite these\r\ndifferences in accuracy, all of the split-brain subjects responded significantly\r\nmore slowly to LVF than to RVF stimuli, except for facial stimuli. Control subjects,\r\non the other hand, continued to respond equally fast to stimuli in either visual\r\nhalf-field.
\r\n\r\nFinally, when the patients were required to compare a variety of bilaterally\r\npresented visual stimuli as same or different, there was again a wide range of\r\nabilities. In general, it was found that those patients who were best at naming\r\nstimuli were worst at cross-comparing them, and vice versa. One subject (LB)\r\nwas able to name two bilaterally flashed stimuli, and yet was unable to compare\r\nthem as same or different beforehand, while another (NG) could cross-match two\r\nstimuli by name identity as well as physical identity, but could not name the LVF\r\nstimulus.
\r\n\r\nThese results most easily suggest the following hypotheses: (1) Some\r\ncommissurotomy patients can make oral categorization responses to unilateral\r\nLVF stimuli, perhaps using their left hemisphere by way of midbrain pathways,\r\nwhile (2) naming LVF stimuli most likely requires a different mechanism, probably\r\ninvolving right hemisphere speech. In addition, (3) some patients can cross-compare\r\nstimuli between the two visual fields. In these cases, the oral responses likely\r\ncome from left hemisphere verbal centers since these patients are also poorest\r\nat naming LVF stimuli.
" }, { "name": "Kehry, Marilyn Rose", "degree": "PhD", "year": "1980", "title": "Structure and Function of Murine Immunoglobulin M from Serum and Cell Membrane", "advisor": "Hood, Leroy E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:07232025-144534720", "creators": [ { "name": { "family": "Kehry", "given": "Marilyn Rose" }, "id": "Kehry-Marilyn-Rose", "display_name": "Kehry, Marilyn Rose" } ], "advisors": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "advisor", "display_name": "Hood, Leroy E." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/nyc2-gv40", "abstract": "Immunoglobulin M (IgM) molecules are secreted into the bloodstream by\r\nplasma cells as soluble pentamers and also exist as monomeric integral membrane\r\nreceptor proteins on the surface of B lymphocytes. Investigation of the structure\r\nof membrane and secreted \u03bc chains has provided an understanding of the basis\r\nfor the existence of IgM molecules in two very different physical environments.
\r\n\r\nThe complete amino acid sequence of a \u03bc chain secreted by the murine\r\nmyeloma MOPC 104E has been determined. When the \u03bc chains of mouse, human\r\nand dog are compared, there is a striking gradient of increasing amino acid sequence\r\nhomology from the NH2-terminus to the COOR-terminus of the \u03bc chain, reflecting\r\na functional conservation of structure. There are five sites of carbohydrate attachment\r\nin the mouse \u03bc chain constant region, one of which is present 14 residues from\r\nthe carboxyterminus. The secreted \u03bc chain (\u03bcs) contains no stretches of unchanged\r\namino acids long enough to allow it to exist as an integral membrane protein.
\r\n\r\nIgM molecules synthesized by the murine B lymphoma, WEHI 279, have\r\nbeen characterized. The cells synthesize internal precursors to secreted \u03bc chains\r\nwhich contain incompletely glycosylated complex carbohydrate moieties but in\r\nall other respects are identical to secreted \u03bc chains. WEHI 279 membrane IgM\r\nis monomeric and contains mature complex carbohydrate structures. The \u03bcm\r\nand \u03bcs chains differ in the structure of their COOR-terminal regions. The carbos\r\nhydrate moiety and methionine residue present in the COOR-terminal 19 amino\r\nacids of \u03bcs chains are absent in \u03bcm chains. In addition, four COOR-terminal amino\r\nacids which are different from the carboxyterminus of \u03bcs chains are released by\r\ncarboxypeptidase treatment of \u03bcm chains. Based on these protein and other\r\nnucleic acid data, we believe \u03bcm chains possess an uncharged and hydrophobic\r\nC-membrane terminal domain which allows monomeric IgM molecules to be\r\nintegral receptor proteins in the B cell plasma membrane.
" }, { "name": "Marton, Kenneth Lawrence", "degree": "PhD", "year": "1980", "title": "Binocular Facilitation and Inhibition in the Lateral Geniculate Nucleus of the Cat", "advisor": "Pettigrew, John D.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10292025-013330758", "creators": [ { "name": { "family": "Marton", "given": "Kenneth Lawrence" }, "id": "Marton-Kenneth-Lawrence", "display_name": "Marton, Kenneth Lawrence" } ], "advisors": [ { "name": { "family": "Pettigrew", "given": "John D." }, "id": "Pettigrew-J-D", "role": "advisor", "display_name": "Pettigrew, John D." } ], "committee": [ { "name": { "family": "Pettigrew", "given": "John D." }, "id": "Pettigrew-J-D", "role": "chair", "display_name": "Pettigrew, John D." }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "member", "display_name": "Van Essen, David C." }, { "name": { "family": "Fender", "given": "Derek H." }, "id": "Fender-D-H", "role": "member", "display_name": "Fender, Derek H." }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" } ], "option_major": [ "neurobio" ], "doi": "10.7907/w5yj-2531", "abstract": "The lateral geniculate nucleus (LGN) relays visual information from the retinas to the cortex, segregating input from each eye into separate laminae. The LGN receives an equally large input back from the visual cortex, whose cells are driven from both eyes. Therefore, binocular interactions in the LGN were studied by systematically varying visual stimuli known to fire cortical neurons. Binocular to monocular responses were compared by interleaving them using computer driven shutters in order to eliminate errors due to LGN cell response variability. Full statistical analysis was used to identify significant binocular facilitation and inhibition.
\r\n\r\nSignificantly more and stronger binocular feedback (BF) was seen with this approach than in previous studies. The vast majority of LGN cells showed both binocular facilitation and inhibition; as many as half showed BF amplitudes exceeding 50% of their typical monocular firing rate. Importantly, BF was found to be well tuned to velocity, relative retinal disparity, and sweep direction, parameters known to profoundly affect cortical firing. Multiple regions of BF were found for most cells, with the majority located near the monocular receptive fields in visual space. Regions of facilitation required zero retinal disparity twice as often as inhibition. Further, most BF reached maximum amplitudes at 6\u00b0/sec to 12\u00b0/sec. These results are a strong indication that the BF is cortical in origin.
\r\n\r\nIt is likely that this BF has a role in highlighting visual features on the plane of fixation. Because BF is very sensitive to parameters of motion, it is also conceivable that it is involved in interpreting the visual signals generated by eyes constantly in motion. This and other possible roles of BF are discussed.
" }, { "name": "Moller, Galina Dmitrieyvna", "degree": "PhD", "year": "1980", "title": "Development and Protein Synthesis in Drosophila", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02022017-130805731", "creators": [ { "name": { "family": "Moller", "given": "Galina Dmitrieyvna" }, "id": "Moller-Galina-Dmitrieyvna", "display_name": "Moller, Galina Dmitrieyvna" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/b64c-b473", "abstract": "CHAPTER 1
\r\n\r\nAt various times during metamorphosis, brain tissue of D. melanogaster\r\nhas been pulse-labeled in situ with 35S-Met. The composition of the newly synthesized\r\nstage-specific proteins and of the total bulk of Coomassie stained polypeptides\r\npresent at the same time in the tissue, or the parts of it, were analyzed on SDS-polyacrylamide\r\ngels.
\r\n\r\nThe striking similarity and constancy of the obtained patterns, and the\r\nfinding that most of the observed accumulating polypeptides are glycosalated,\r\nlead to the conclusion that the predominant change in the larval Drosophila brain\r\nis dendritic and/or axonal reorganization.
\r\n\r\nCHAPTER II
\r\n\r\nPupae of Drosophila melanogaster were heat shocked under conditions\r\nrequired to induce phenocopies in more than 90% of the flies that subsequently\r\nemerge. The effects of these treatments on protein synthesis in two tissues (thoracic\r\nepithelium and brain) were followed for several hours after the heat treatments.\r\nResults from pulse labeling and protein separations on SDS acrylamide gels showed\r\na virtually complete cessation of protein synthesis immediately after the shock\r\nfollowed by a non-coordinate resumption of the starting pattern. Similar experiments\r\nfollowing double heat shocks demonstrated a more rapid resumption of synthesis\r\nof heat shock proteins after two successive heat treatments than after a single\r\none.
\r\n\r\nCHAPTER III
\r\n\r\nWe describe variants of three heat shock proteins of Drosophila melanogaster\r\nand the use of these to map the chromosome regions which contain the coding\r\nsequences for these proteins. All three map to a region on chromosome 3L which\r\nincludes only one heat shock puff, that designated as 67B. The results imply that\r\nthe genes which code for at least three heat shock proteins are included within\r\nthe 67B region.
\r\n\r\nCHAPTER IV
\r\n\r\nMild heat treatments applied to whole animals or cell cultures of Drosophila\r\nmelanogaster prior to lethal heat shocks result in both survival and protection\r\nagainst phenocopy induction. From an examination of these heat shock effects\r\non transcriptional and translational activities in tissues and cells it appears that\r\nthe protective action of pretreatment is due to sequestering of mRNAs in a masked\r\nform as RNPs. Heat shock proteins are evidently involved in the masking process\r\neither directly or indirectly.
" }, { "name": "Murphy, Robert Francis", "degree": "PhD", "year": "1980", "title": "Chromosomal Protein-DNA Interactions", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechTHESIS:09102025-214417021", "creators": [ { "name": { "family": "Murphy", "given": "Robert Francis" }, "id": "Murphy-Robert-Francis", "orcid": "0000-0003-0358-901X", "display_name": "Murphy, Robert Francis" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "chair", "display_name": "Bonner, James Frederick" }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "member", "display_name": "Mitchell, Herschel K." }, { "name": { "family": "Lazarides", "given": "Elias" }, "id": "Lazarides-E", "role": "member", "display_name": "Lazarides, Elias" }, { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "member", "display_name": "Strauss, James H." }, { "name": { "family": "Richards", "given": "John H." }, "id": "Richards-J-H", "role": "member", "display_name": "Richards, John H." } ], "option_major": [ "bioch" ], "doi": "10.7907/3ecg-z764", "abstract": "The nature and function of some of the protein-DNA interactions\r\nin eukaryotic chromatin were investigated. The nucleosome structure\r\nof isolated template-active chromatin was determined. In vitro chemical\r\nacetylation of chromatin was shown to result in a structure similar\r\nto that of deproteinized DNA, which represents a shift towards the\r\nproperties of isolated template active chromatin, and chromatin \r\ncontaining specific transcribed genes.
\r\n\r\nThe process of chromatin replication was shown to include a\r\nshortening of the internucleosomal spacer, resulting in decreased\r\nnuclease sensitivity. Newly-replicated chromatin was separated\r\nfrom bulk chromatin in shallow metrizamide density gradients.\r\nNewly-synthesized histone and newly-acetylated protein were shown\r\nto be present predominantly in the unreplicated chromatin fraction.
\r\n\r\nThe accuracy and reproducibility of non-linear least squares\r\ndeterminations of the thermal denaturation transitions of DNA and\r\nchromatin were determined using computer programs designed for ease of\r\nuse and adaptability to mini-computer configurations. Direct fitting\r\nof melt data to a normalized error function gave results very similar\r\nto those obtained by fitting Gaussian curves to derivatized data. This\r\napproach avoids errors introduced by the derivatization method, and\r\nrequires fewer data points.
" }, { "name": "Newsome, William Thomas, III", "degree": "PhD", "year": "1980", "title": "Studies on Primate Extrastriate Visual Cortex. I. The Interhemispheric Connections of Visual Cortex in the Owl Monkey, Aotus trivirgatus, and the Bushbaby, Galago senegalensis. II. A Functional Localization of Neuronal Response Properties in Extrastriate Cortex of the Owl Monkey, Aotus trivirgatus", "advisor": "Allman, John Morgan", "url": "https://resolver.caltech.edu/CaltechETD:etd-09152004-141810", "creators": [ { "name": { "family": "Newsome", "given": "William Thomas, III" }, "id": "Newsome-William-Thomas-III", "display_name": "Newsome, William Thomas, III" } ], "advisors": [ { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "advisor", "display_name": "Allman, John Morgan" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/N9F5-SV06", "abstract": "Anatomical techniques have been used to map within visual cortex the pattern of degenerating axonal terminals produced by surgical section of the selenium of the corpus callosum in the owl monkey, Aotus trivirgatus, and the bushbaby, Galago senegalensis: To the extent to which degenerating callosal terminals correspond to physiological representations of the visual vertical meridian, the pattern of interhemispheric connections can be used as a powerful aid in locating visual area boundaries. The goals of this study have been 1) to assess the degree of correspondence between degenerating callosal terminals and anatomically identifiable vertical meridian representations, and 2) to gain information concerning the existence and organization of as yet unknown extrastriate visual areas. In both the owl monkey and the bushbaby, a discrete band of degeneration corresponds precisely to the vertical meridian representation at the V1-V2 border, and a less precise increase in the density of axonal terminals corresponds to the vertical meridian representation of extrastriate area MT. A well-defined band of degeneration on the ventral surface of the owl monkey's cerebral hemisphere corresponds to a previously unknown vertical meridian representation which is shared by two newly defined extrastriate visual areas. Over much of areas DL and DI in the owl monkey, and in the region where the center-of-gaze representations of V2, DL and MT are in close proximity in both the owl monkey and the bushbaby, the pattern of callosal terminals is complex and has little value for determining precise areal boundaries.\r\n\r\nThe response properties of 275 single neurons from four extrastriate visual areas have been quantitatively studied in chronically prepared, sedated owl monkeys. During experimental sessions, computer controlled visual stimuli were presented to the animal while the computer recorded the responses of single cortical neurons. The responses were stored on magnetic tape for later analysis. The major result of this study is the demonstration of a striking localization of direction-selective neurons to extrastriate area MT of the owl monkey. MT neurons responded maximally to stimuli tuned to a single optimal direction of motion whereas neurons of the medial third tier areas (DM, DI and M) responded maximally to oriented stimuli moving in either of two directions perpendicular to the optimal axis of orientation. Other differences were noted between neurons of MT and neurons of the medial third tier areas. A systematic difference exists in the tightness of tuning to the optimal direction of motion. Neurons in MT tended to be less well tuned to the optimal direction of motion than did neurons of the medial third tier areas. Neurons in MT also exhibited a different pattern of preferences for stimulus velocity than did neurons of the medial third tier areas. A distinct group of MT cells preferred velocities of 10-25 degrees/second, while neurons of the medial third tier areas had a broad distribution of preferred velocities ranging from 10-100 degrees/second. There was no systematic difference in tuning to preferred velocity for the two regions." }, { "name": "Armstrong, David Lee", "degree": "PhD", "year": "1979", "title": "The Kinetics of Curare Action and Restricted Diffusion within the Synaptic Cleft of Motor Nerve Terminals on Frog Skeletal Muscle", "advisor": "Lester, Henry A.", "url": "https://resolver.caltech.edu/CaltechETD:etd-08142007-073818", "creators": [ { "name": { "family": "Armstrong", "given": "David Lee" }, "id": "Armstrong-David-Lee", "display_name": "Armstrong, David Lee" } ], "advisors": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "role": "advisor", "display_name": "Lester, Henry A." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "neurobio" ], "doi": "10.7907/g7kf-2m61", "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\r\n\r\n1. The kinetics of curare inhibition were measured at the postsynaptic membrane of frog sartorius and cutaneous pectoris muscle fibers. Acetyleholine (ACh) and d-tubocurarine (dTC) were iontophoresed from twin-barrel micropipettes, and the muscle fiber's membrane potential was recorded intracellularly. By itself dTC produced no change in membrane potential, but dTC-receptor binding was assayed by observing changes in the response to a constant pulse of ACh.\r\n\r\n2. The responses to both ACh and dTC had brief latencies, reached their maxima rapidly, and were highly sensitive to the dose. Under these conditions, the kinetics of drug action are not slowed by access of the drugs to the synaptic cleft.\r\n\r\n3. After a pulse of dTC, recovery from inhibition proceeds slowly along an exponential time course with a rate constant, [...]. The recovery rate does not depend on the maximal level of inhibition and varies only slightly with temperature (Q10 = 1.25).\r\n\r\n4. After a sudden maintained increase in dTC release, inhibition develops approximately exponentially until a steady-state level of inhibition is reached. The apparent rate constant for the onset of inhibition, [...], is greater than [...]. When the steady-state inhibition reduces the ACh response to 1/n of its control value, [...] = [...].\r\n\r\n5. When the ACh sensitivity is reduced with cobra toxin, both [...] and [...] increase. Thus, the kinetics of dTC inhibition depend on the density of acetyleholine receptors in the synaptic cleft. If the density of acetylcholinesterase is reduced in the cleft by collagenase, [...] increases only twofold.\r\n\r\n6. When the nerve terminal is removed after collagenase action, and the drugs are iontophoresed directly onto the exposed postsynaptic membrane, [...] increases more than tenfold.\r\n\r\n7. Bath-applied dTC competitively inhibits the responses to brief iontophoretic ACh pulses with an apparent equilibrium dissociation constant, KD = 0.5 [...]. This suggests that dTC molecules equilibrate with the receptors on a millisecond time scale.\r\n\r\n8. On denervated frog muscle cells, extrasynaptic receptors have a lower apparent affinity for dTC. After a pulse of dTC, inhibition decays tenfold more rapidly at these extrasynaptic sites than at the nerve-muscle synapse.\r\n\r\n9. It is suggested that dTC inhibits synaptic receptors more effectively because the nerve terminal restricts diffusion within the synaptic cleft and each dTC molecule binds repeatedly to several acetylcholine receptors before escaping from the cleft. Consequently, the receptors transiently buffer the concentration of dTC in the cleft, and the macroscopic kinetics of inhibition are much slower than the molecular rates of dTC binding." }, { "name": "Blankenhorn, Elizabeth Peters", "degree": "PhD", "year": "1979", "title": "Immunogenetic Studies of the Mouse and the Rat", "advisor": "Owen, Ray David", "url": "https://resolver.caltech.edu/CaltechTHESIS:02182026-231225145", "creators": [ { "name": { "family": "Blankenhorn", "given": "Elizabeth Peters" }, "id": "Blankenhorn-Elizabeth-Peters", "orcid": "0000-0002-6331-0319", "display_name": "Blankenhorn, Elizabeth Peters" } ], "advisors": [ { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "advisor", "display_name": "Owen, Ray David" } ], "committee": [ { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "chair", "display_name": "Owen, Ray David" }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "member", "display_name": "Mitchell, Herschel K." }, { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "member", "display_name": "Lewis, Edward B." } ], "option_major": [ "immun" ], "doi": "10.7907/cymp-wa54", "abstract": "This thesis includes immunogenetic investigations of three systems in\r\nmice and rats. The first part deals with theta (Thy-1) antigens in mice. Results\r\nand conclusions are presented from three separate experiments: 1) Crosses were\r\nmade between inbred mice, and electrophoretic analysis of serum transferrin\r\nand kidney malic enzyme, along with serological detection of Thy-1, aligned the\r\nthree loci in the following order on chromosome 9: Trf --- Mod-1 --- Thy-1.\r\nThis work led to a revision of the previously-accepted map of chromosome 9 in\r\nthe mouse. 2) Sublines of inbred AKR mice, which differ in susceptibility to\r\nleukemias, were found to differ from one another by two of these same chromosome\r\n9 genetic markers (Mod-1 and Thy-1). 3) Two alleles of Thy-1 were found to\r\nbe segregating in one wild mouse colony (from the Southern California area) which\r\nhas an unusually high incidence of lymphomas. Only one allelic product was\r\ndetected in another wild mouse colony, which is lymphoma-resistant. However,\r\nthe inheritance of either allele of Thy-1 could not be associated with an individual\r\npredisposition to lymphoma.
\r\n\r\nThe second part of the thesis deals with rat lymphoid cell antigens.\r\nIn rats, the major histocompatibility complex is called H-1. A study of the membrane-associated\r\nglycoprotein products of this genetic region was conducted, employing\r\na number of biochemical techniques, including electrophoretic separation on one-\r\ndimensional and two-dimensional gels, and microsequence analysis. The study\r\nshowed that the major transplantation antigens in rats are closely similar to one\r\nanother, and homologous to those of other species. The results demonstrate that\r\nthere may be two transplantation antigens encoded by each of three H-1 haplotypes.\r\nThe alloantigens associated with the H-1 linked immune response region in rats\r\n(Ia antigens) were also studied, and evidence is presented for the existence of\r\ntwo separate Ia antigens in each rat haplotype studied.
\r\n\r\nAn immunogenetic analysis of the levels of a mouse carcino-embryonic\r\nantigen known as alpha fetoprotein (AFP) is contained in the last part of this\r\nthesis. An extremely sensitive radioimmunoassay was developed to detect levels\r\nof AFP in inbred strains and genetic crosses of mice which produce high or low\r\nadult concentrations of this fetal protein. The results from this study indicate\r\nno genetic association between the structural gene for albumin and the factor(s)\r\nregulating the level of serum AFP. There is also evidence that the high AFP level\r\nseen in some of the backcross progeny of the particular cross investigated is not\r\ndetermined by a single recessive gene.
" }, { "name": "Chomyn, Anne", "degree": "PhD", "year": "1979", "title": "Studies on Protein Synthesis after Heat Shock in Drosophila melanogaster", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05222003-103429", "creators": [ { "name": { "family": "Chomyn", "given": "Anne" }, "id": "Chomyn-Anne", "display_name": "Chomyn, Anne" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "chair", "display_name": "Mitchell, Herschel K." }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Dreyer", "given": "William J." }, "id": "Dreyer-W-J", "role": "member", "display_name": "Dreyer, William J." }, { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "member", "display_name": "Lewis, Edward B." }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "member", "display_name": "Owen, Ray David" } ], "option_major": [ "bioch" ], "doi": "10.7907/ree4-cz17", "abstract": "The synthesis of the heat shock proteins on cytoplasmic rather than mitochondrial ribosomes has been shown. The time course of the synthesis of heat shock proteins in prepupal and pupal stages of Drosophila melanogaster has been analyzed. Prepupae were heat shocked at 37.5[degrees]C for 20 minutes and pupae, either at 37.5[degrees]C for 20 minutes or at 40.2[degrees]C for 40 minutes. In prepupae, all of the heat shock proteins are synthesized immediately after the shock and continue to be synthesized for two to three hours. By three hours after the shock, the synthesis of normal proteins has resumed. In pupae, the time course is similar after the milder shock. However, the more severe shock causes a drastic reduction in total protein synthesis for at least one hour after the shock. It has been shown previously that this shock causes the production in pupae of stage specific phenocopies at high penetrance, an effect which is not observed after a shock at 37.5[degrees]C for 20 minutes. The drastic decrease in total protein synthesis and the subsequent occurrence of anomalies in the resumed program of gene expression and their possible relation to phenocopy production are discussed.\r\n\r\nThe increase in activity of phenol oxidase after a shock of 40[degrees]C for 40 minutes has been investigated to determine whether a component of the enzyme is a heat shock protein. This hypothesis was tested by measuring the extent of co-banding of [superscript 35]S-methionine labeled proteins from heat shocked and non-heat shocked cells with partially purified phenol oxidase in a sucrose gradient. The results do not support such a role for any heat shock protein; a mechanism whereby phenol oxidase activity could increase after heat shock is discussed.\r\n\r\nThe 84,000 dalton heat shock protein has been purified by ammonium sulfate fractionation, chromatography on hydroxylapatite, and one- or two-dimensional gel electrophoresis. This purified protein has been used to produce antibodies in rabbits. The antibodies have been used to show, by indirect immunoprecipitation experiments, that this heat shock protein is normally synthesized in at least three D. melanogaster tissues. Indirect immunofluorescence experiments using these antibodies indicate that the 84,000 dalton protein is also present on the chromosomes of normal and heat shocked salivary glands at the interband regions. Evidence is presented to show that this binding pattern may simply reflect the high concentration of this protein in the cell.\r\n\r\nIn a separate investigation, the effects of heat shock on the protein synthesis pattern in HeLa cells have also been analyzed." }, { "name": "Conrad, Susan Ellen", "degree": "PhD", "year": "1979", "title": "I. Sequence Organization of Drosophila melanogaster 5S rRNA and 4S RNA Genes. II. In Vitro Studies on Replication of Plasmid DNAs", "advisor": "Campbell, Judith L.; Davidson, Norman R.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03062026-161324227", "creators": [ { "name": { "family": "Conrad", "given": "Susan Ellen" }, "id": "Conrad-Susan-Ellen", "display_name": "Conrad, Susan Ellen" } ], "advisors": [ { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "orcid": "0000-0001-8291-5551", "role": "advisor", "display_name": "Campbell, Judith L." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "co-advisor", "display_name": "Davidson, Norman R." } ], "committee": [ { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "orcid": "0000-0001-8291-5551", "role": "chair", "display_name": "Campbell, Judith L." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "co-chair", "display_name": "Davidson, Norman R." }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Maniatis", "given": "Thomas P." }, "id": "Maniatis-T-P", "role": "member", "display_name": "Maniatis, Thomas P." }, { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "member", "display_name": "Strauss, James H." } ], "option_major": [ "bioch" ], "doi": "10.7907/d94z-c749", "abstract": "I. The sequence organizations of Drosophila melanogaster 4S RNA (tRNA)\r\nand 5S rRN A genes have been investigated. Segments of Drosophila DNA containing\r\nthese genes have been propagated in recombinant plasmids using Escherichia coli\r\nas a host and Co1E1 as a vector.
\r\n\r\nElectron microscope partial denaturation mapping, mapping by ferritin\r\nlabeling and restriction enzyme-gel electrophoresis analysis all indicate that the\r\nDrosophila DNA inserts of the 5S rRNA gene containing plasmids consist of tandem\r\nrepeats of 5S genes and spacer regions. The repeat length is approximately 380\r\nnucleotide pairs (ntp), corresponding to a gene of length 120 ntp and a spacer\r\nof length 260 ntp. Little heterogeneity in the lengths of the repeats has been\r\ndetected.
\r\n\r\nA tRNA gene containing clone has been characterized by electron microscopic\r\nmethods and by restriction endonuclease mapping. Four tRNA genes were\r\ndetected on a 9.34 kb fragment of DNA. Three of these genes appear to be\r\nidentical and different from the fourth. No evidence was found for extensive\r\nsequence homology in the sequences surrounding the genes. In situ hybridization\r\nwith cRNA transcribed from the plasmid showed localization at region 42A of\r\nchromosome 2R.
\r\n\r\nII. An improved system for in vitro replication of Escherichia coli plasmid\r\nDNAs has been developed and characterized. Endogenous DNA is removed from\r\nthe extracts, making replication dependent upon exogenous DNA even when plasmid\r\ncontaining cells are used as a source of extracts. Replication in this system requires\r\nE. coli DNA polymerases I and III, RNA polymerase, DNA gyrase, and the products\r\nof at least five genes required for E. coli replication (dnaB, dnaC, dnaG, dnaP, dnaZ).
\r\n\r\nThis system has been used to study the replication properties of the\r\nampicillin resistant plasmid RSF1030. We have found that, like Co1E1, RSF1030\r\nreplicates unidirectionally from a unique origin. We have also investigated the\r\nreplication of pFH118, a high copy number mutant derived from Co1E1. The\r\nmutation in pFH118 is due to the insertion of 20-30 base pairs into the coding\r\nsequence for a 100 nucleotide RNA that is transcribed during DNA replication.\r\nResults of reversion studies suggest that this RN A plays a role in determining\r\nplasmid copy number. Furthermore, the RN As transcribed from Co1E1 and RSF1030\r\nhave significant sequence homology, although the plasmids were isolated independently\r\nand have been thought to have no sequence homology.
" }, { "name": "Cooper, Michael Lee", "degree": "PhD", "year": "1979", "title": "Studies in the Visual System of the Cat. I. The Retinothalamic Pathway in Normal and Siamese Cats. II. The Vertical Horopter", "advisor": "Pettigrew, John D.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03152026-135524836", "creators": [ { "name": { "family": "Cooper", "given": "Michael Lee" }, "id": "Cooper-Michael-Lee", "display_name": "Cooper, Michael Lee" } ], "advisors": [ { "name": { "family": "Pettigrew", "given": "John D." }, "id": "Pettigrew-J-D", "role": "advisor", "display_name": "Pettigrew, John D." } ], "committee": [ { "name": { "family": "Pettigrew", "given": "John D." }, "id": "Pettigrew-J-D", "role": "chair", "display_name": "Pettigrew, John D." }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Brockes", "given": "Jeremy P." }, "id": "Brockes-Jeremy-P", "orcid": "0000-0002-3395-5159", "role": "member", "display_name": "Brockes, Jeremy P." }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "member", "display_name": "Van Essen, David C." } ], "option_major": [ "neurobio" ], "doi": "10.7907/ra6s-ca04", "abstract": "The naso-temporal division of the retinothalamic pathway was\r\nstudied in normal and Siamese cats. One lateral geniculate nucleus\r\n(LGN) of each animal was filled with horseradish peroxidase (HRP)\r\nin order to visualize the retinothalamic ganglion cells over a wide\r\narea of retina. Whole-mounted retinae of normal animals showed clear\r\nvertical decussation lines between areas projecting ipsilaterally\r\nor contralaterally. The ipsilateral decussation was sharp and passed\r\nthrough the center of the area centralis; the contralateral decussation\r\nwas somewhat less sharp, with scattered cells extending up to a few\r\ndegrees into the temporal retina. However, in contrast to the findings\r\nin Stone's ('66) tract section material, no significant numbers of\r\nHRP-filled cells were found beyond a few degrees into the contralateral\r\ntemporal retina.
\r\n\r\nIn the Siamese cat retina there was no sharp vertical decussation\r\nline between areas sending axons ipsilaterally or contralaterally;\r\nrather there was overlap in the temporal retina between cell populations\r\nprojecting to the two hemispheres. Thus there is no region of exclusive\r\ncontralateral misprojection. extending 20\u00b0 temporally from the zero\r\nazimuthal meridian; in fact, there is a smooth, gradient-like decrease\r\nin the percentage of retinothalamic cells misprojecting contralaterally\r\nas one proceeds into the temporal retina. Cell-size measurements\r\nindicated that the large (presumably Y-type) ganglion cells are more\r\naffected by the Siamese defect than the rest of the retinothalamic\r\npopulation. Use of the anterograde transport of tritiated amino acids\r\nconfirmed the existence of bilateral projection to the LGN from the\r\ncentral temporal retina of Siamese cats.
\r\n\r\nThe second part of this thesis was an attempt to determine the\r\nform of the vertical horopter in the cat and burrowing owl by electro-physiologically\r\nmapping the receptive field positions of binocular\r\ncortical neurons at various elevations along the zero azLmuthal meridian.\r\nOur recordings indicated that, in the alert, unparalyzed cat and owl,\r\nmidline binocular units in the lower visual field have crossed receptive\r\nfields compared to the fixation point, while upper field cells have\r\nuncrossed receptive fields. It follows from our data that the vertical\r\nhoropter is a straight line tilted away from the animal in both species,\r\nwith the lower field horopter closer to the animal and the upper field\r\nhoropter farther away.
" }, { "name": "Hill, Alvin Joseph", "degree": "PhD", "year": "1979", "title": "Investigations of \"Spatial\" Firing in Dorsal Hippocampus of the Rat", "advisor": "Unknown, Unknown", "url": "https://resolver.caltech.edu/CaltechTHESIS:04132026-225043368", "creators": [ { "name": { "family": "Hill", "given": "Alvin Joseph" }, "id": "Hill-Alvin-Joseph", "display_name": "Hill, Alvin Joseph" } ], "advisors": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/ycm5-cc17", "abstract": "\"Spatial\" units have been identified in micro-electrode recordings from\r\ndorsal hippocampus of rats. Such units fire at their maximum rates only when\r\nrats are in specific regions of the recording space, called the units' \"fields\", and\r\nseem to be independent of details of the animals' behavior. Spatial firing is consistent\r\nwith a recent suggestion that the hippocampus functions as a \"map\" of\r\nthe environment.
\r\n\r\nRecordings were made from dorsal hippocampus of rats as the animals\r\nperformed a spatial alternation task in an enclosed T-maze. Spatial firing was\r\nidentified in rats which had been selectively deprived of either visual, vibrissal,\r\nauditory or olfactory sensory inputs. Results are consistent with other evidence\r\nthat hippocampal spatial firing is based upon multi-modal sensory cues.
\r\n\r\nRecordings were made from dorsal hippocampus of rats as the animals\r\nlearned to shuttle and alternate in the T-maze when they had not been there before.\r\nIn 11 out of 15 cases, spatial firing occurred during the first passage of the rat\r\nthrough the unit's field. In the other 4 cases, firing occurred for the first time\r\nwithin 15 minutes of the rat's first exposure to the field. Results are discussed\r\nin relation to theories of hippocampal function.
" }, { "name": "Hubbard, Bruce D.", "degree": "PhD", "year": "1979", "title": "Biochemical Studies on the 10 nm Filaments of Avian Muscle", "advisor": "Lazarides, Elias", "url": "https://resolver.caltech.edu/CaltechTHESIS:04142026-221112421", "creators": [ { "name": { "family": "Hubbard", "given": "Bruce D." }, "id": "Hubbard-Bruce-D", "display_name": "Hubbard, Bruce D." } ], "advisors": [ { "name": { "family": "Lazarides", "given": "Elias" }, "id": "Lazarides-E", "role": "advisor", "display_name": "Lazarides, Elias" } ], "committee": [ { "name": { "family": "Lazarides", "given": "Elias" }, "id": "Lazarides-E", "role": "chair", "display_name": "Lazarides, Elias" }, { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "member", "display_name": "Revel, Jean-Paul" }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "member", "display_name": "Owen, Ray David" }, { "name": { "family": "Campbell", "given": "Judith L." }, "id": "Campbell-J-L", "orcid": "0000-0001-8291-5551", "role": "member", "display_name": "Campbell, Judith L." }, { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "member", "display_name": "Hood, Leroy E." } ], "option_major": [ "biology" ], "doi": "10.7907/bq1f-ch51", "abstract": "This thesis describes the purification and initial biochemical characterization\r\nof desmin, a major component of avian muscle 10 nm filaments.
\r\n\r\n1. Extraction of chicken gizzard with KI solubilizes most of the actomyosin\r\nand other fibrillar proteins, leaving behind a KI insoluble residue containing\r\n10 nm filaments, desmin, and a small amount of insoluble actin. Antibodies prepared\r\nagainst electrophoretically purified desmin do not cross-react with other\r\nmuscle proteins. In conjunction with indirect immunofluorescence, they demonstrate\r\nthe presence of desmin in close association with the Z-lines and plasma membrane\r\nof skeletal muscle cells. Desmin also is associated with the Z-lines, intercalated\r\ndiscs, and intercellular adhesion sites of cardiac muscle cells.
\r\n\r\n2. Both desmin and the actin that remain in the KI extracted gizzard\r\nare insoluble in high salt, but are soluble at low pH or in agents that dissociate\r\nhydrophobic bonds. Desmin may be purified by repeated cycles of solubilization\r\nby 1 M acetic acid and subsequent precipitation by neutralization to pH 4. During\r\nthis process, a constant ratio of actin to desmin is attained.
\r\n\r\n3. Gel filtration on Bio-Gel P300 in the presence of 1 M acetic acid\r\nreveals that actin is dissociated from the vast majority of desmin under these\r\nconditions. When the acetic acid is removed from actin-desmin solutions by dialysis\r\nagainst water, a gel forms that is composed of 12-14 nm diameter filaments.\r\nBoth anti-actin and anti-desmin react uniformly with these filaments to a resolution\r\nlimit of 250 nm.
\r\n\r\n4. Actin and desmin can be extracted from homogenized chicken gizzard\r\nin soluble form by 10 mM EGTA. Both proteins are retained on a DNase I affinity\r\ncolumn and also coprecipitate in indirect immunoprecipitation with either anti-actin\r\nor anti-desmin. Velocity sedimentation of the EGTA extract shows that the actin\r\nand desmin cosediment as a series of stable complexes that range in size from\r\n6 to 60 S. The 50-60 S family has an actin to desmin stoichiometry of approximately\r\n5 to 1.
\r\n\r\n5. Several proteases are investigated that selectively cleave desmin\r\n(mwt 50,000) to sequentially produce fragments of 43,500 mwt, 40,000 mwt, and\r\na 35,000 mwt limit digestion product that lacks 82% of the tyrosine of the original\r\ndesmin. The cleavage process results in the conversion of gelled desmin to a non-cohesive,\r\npowdery, precipitate. These proteases remove the desmin from isolated\r\nmyofibril bundles and cause their lateral separation into individual myofibrils.
\r\n\r\n6. Based upon these observations, it is proposed that desmin is a major\r\ncomponent of muscle 10 nm filaments and that it forms stable complexes with\r\nactin. It is further proposed that the cellular function of desmin is to mechanically\r\ninterlink individual actomyosin contractile units, both to each other and to the\r\nplasma membrane.
" }, { "name": "Loh, Elwyn Yuan", "degree": "PhD", "year": "1979", "title": "Amino Acid Sequence Studies of Immunoglobulins: Implications for the Storage, Processing, and Expression of Genetic Information", "advisor": "Hood, Leroy E.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03022026-225028437", "creators": [ { "name": { "family": "Loh", "given": "Elwyn Yuan" }, "id": "Loh-Elwyn-Yuan", "display_name": "Loh, Elwyn Yuan" } ], "advisors": [ { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "advisor", "display_name": "Hood, Leroy E." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/k7yv-gs86", "abstract": "Antibody molecules form a highly complex set of proteins. A central\r\nproblem in immunology has been how the information coding for these proteins\r\nis stored, processed, and expressed.
\r\n\r\nThe constant region sequences of two rat K light chain allotypes\r\nhave been partially sequenced. These allotypes segregate in the Mendelian\r\nfashion. In the eighty-one constant region residues compared, 10 amino acid\r\nsubstitutions and one size difference were found. This large number of substitutions\r\nraise the possibility that the structural genes for both forms may exist in all\r\nrats and the inherited marker is a regulatory gene controlling the expression\r\nof one or the other forms.
\r\n\r\nThe diversity of immunoglobulins is reflected in the diversity of myeloma\r\nproteins and much of our knowledge of antibodies comes from studies of myeloma\r\nproteins. However, the window, created by the myeloma tumors, may be a\r\nbiased one. By comparing the N-terminal amino acid sequences of myeloma\r\nlight chains from two inbred strains of mice, BALB/c and NZB, we have found\r\ndifferences which suggest that different populations of lymphocytes are being\r\ntransformed in the two strains. Thus the true diversity of immunoglobulins\r\nmay be greater than that seen in myeloma proteins.
\r\n\r\nBy sequencing a set of closely related variable regions, one can ask\r\nthe question -- what are the protein products co9ed by a single germ line gene?\r\nThe nearly complete variable regions of twenty-two K chains have been\r\nsequenced using newly developed automated sequencing technology. This data\r\nshows that at least six genes code for this set, assuming that the somatic diversity\r\ngenerating mechanisms cannot produce multiple parallel mutations. Within\r\neach subset of these sequences, coded for by at least one gene, additional\r\nvariations occur both inside and outside the hypervariable regions, although\r\npredominately inside. In addition, the sequence of the approximately twelve\r\nresidues preceeding the constant region do not correlate with the rest of the\r\nvariable region. We have termed this region the S or switch region and suggest\r\nthat it is coded for by a separate segment of DNA that is reorganized during\r\ndifferentiation much in the same way as V and C regions are rearranged.
" }, { "name": "Novitski, Charles Edward", "degree": "PhD", "year": "1979", "title": "Aspects of Regulation of Mitochondrial DNA Replication and Transcription in Mammalian Cells", "advisor": "Attardi, Giuseppe", "url": "https://resolver.caltech.edu/CaltechETD:etd-08092006-103935", "creators": [ { "name": { "family": "Novitski", "given": "Charles Edward" }, "id": "Novitski-Charles-Edward", "display_name": "Novitski, Charles Edward" } ], "advisors": [ { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "advisor", "display_name": "Attardi, Giuseppe" } ], "committee": [ { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "chair", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Maniatis", "given": "Thomas P." }, "id": "Maniatis-T-P", "role": "member", "display_name": "Maniatis, Thomas P." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "member", "display_name": "Bonner, James Frederick" }, { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "member", "display_name": "Strauss, James H." } ], "option_major": [ "biology" ], "doi": "10.7907/QCWQ-AJ90", "abstract": "The first part of the thesis deals with a study of the synthesis of RNA in isolated HeLa cell mitochondria. Isolated mitochondria were capable of supporting close to linear incorporation of the radioactive precursor [5-3H]UTP or [5-3H]ATP for at least 1 hour. Virtually all of the RNA labeled in vitro was shown to consist of mitochondrial DNA transcripts complementary to one or the other of the two strands. At least 81% of the RNA labeled in the presence of [5-3H]UTP and 72% labeled in the presence of [5-3H]ATP hybridized to mitochondrial DNA; 70% of the RNA homologous to mitochondrial DNA hybridized to the \"H\" strand and 30% to the \"L\" strand. Sucrose gradient analysis of the products labeled with either [5-3H]UTP or [5-3H]ATP showed the presence of mitochondria-specific ribosomal 16S and 12S RNAs.\r\n\r\nThe effect of cycloheximide pretreatment of the cells on the RNA synthetic capactiy of isolated organelles was investigated. Incorporation of [5-3H]UTP into RNA of mitochondria isolated from HeLa cells treated with 200 [mu]g cycloheximide/ml up to 4 hours was found to decrease very modestly. Addition of normal cytoplasm to these mitochondria resulted in a stimulatory effect on RNA synthesis independent of the length of cycloheximide treatment.\r\n\r\nThe second part of the thesis concerns an investigation of the timing of mitochondrial DNA synthesis during the cell cycle in mouse cells. In LM(TK-) Cl1D cells synchronized by selective detachment, a fairly constant rate of incorporation of [methyl-3H]thymidine or [5-3H]deoxycytidine into mitochondrial DNA was observed. Due to low levels of uptake of the radioactive precursors, however, the mitochondrial triphosphate precursor pool specific activities could not be measured and, thus, the rate of mitochondrial DNA replication could not be determined. On the other hand, in A9 cells, the rate of incorporation of [methyl-3H]thymidine into mitochondrial DNA was found to increase by at least a factor of 5 during the late-S and G2 phases relative to the G1 phase. In addition, the mitochondrial pool specific ctivity decreased by a factor of 3 during the same period, indicating a substantial increase in the rate of mitochondrial DNA synthesis in the late-S and G2 phase cells in agreement with previous evidence obtained in HeLa cells. A mathematical discussion is presented which indicates that a recent study of unsynchronized A9 cells (Bogenhagen and Clayton, Cell 11, 719, 1977), erroneously interpreted as indicating a constant rate of mitochondrial DNA replication during the cell cycle, is not inconsistent with the results presented here.\r\n\r\nIn the third part of the thesis, the results are presented of preliminary experiments with the goal of developing an approach for studying the nature of the cell cycle dependence of mitochondrial DNA synthesis. This approach is based on an analysis of mitochondrial DNA synthesis in heterokaryons formed by fusion of mouse L cells at different stages of the cell cycle. The mitochondrial DNAs of the two parental cells are distinguished by using, as one of the parental cell types in the fusion, synchronized cells which had been grown in the presence of 30 [mu]g BrdU/ml; the BrdU substituted mitochondrial DNA of these cells is separable from unsubstituted DNA by CsCl density gradient centrifugation. Cell fusion with a fast sedimenting fraction of Sendai virus was shown to result in a much higher proportion of parental cells in heterokaryons than in fusions produced by standard Sendai virus. The results of a pilot experiment carried out with Cl1D cells using the above described approach are presented." }, { "name": "Parker, Richard Carl", "degree": "PhD", "year": "1979", "title": "I. Methods for Restriction Endonuclease Studies of DNA Structure. II. Restriction Endonucleolytic Characterization of Animal Mitochondrial DNAs and Human Globin Genes", "advisor": "Vinograd, Jerome Rubin; Baldeschwieler, John D.; Maniatis, Tom", "url": "https://resolver.caltech.edu/CaltechTHESIS:04302026-160335841", "creators": [ { "name": { "family": "Parker", "given": "Richard Carl" }, "id": "Parker-Richard-Carl", "display_name": "Parker, Richard Carl" } ], "advisors": [ { "name": { "family": "Vinograd", "given": "Jerome Rubin" }, "id": "Vinograd-J", "role": "advisor", "display_name": "Vinograd, Jerome Rubin" }, { "name": { "family": "Baldeschwieler", "given": "John D." }, "id": "Baldeschwieler-J-D", "role": "advisor", "display_name": "Baldeschwieler, John D." }, { "name": { "family": "Maniatis", "given": "Tom" }, "id": "Maniatis-T", "orcid": "0000-0002-2722-8633", "role": "advisor", "display_name": "Maniatis, Tom" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/pphq-jv67", "abstract": "An initial approach to the structural organization of DNA is restriction\r\nendonuclease site mapping and gel electrophoretic analysis. This is true for genomes\r\nof the simplest or greatest complexities.
\r\n\r\nTwo techniques are presented in this thesis that facilitate this approach.\r\nThe first uses ethidium bromide to limit the action of a restriction endonuclease\r\non a closed circular DNA in order to derive a set of circularly permuted linear\r\nmolecules. These molecules, after appropriate treatment, can be used to orient\r\nthe restriction endonuclease sites and to calibrate the relationship between\r\nelectrophoretic mobility and DNA fragment size without the introduction of\r\nexternal standards.
\r\n\r\nThe second technique utilizes a low melting temperature agarose. It\r\nprovides a simple system for two-dimensional electrophoretic analysis of DNA\r\nmolecules with restriction endonuclease digestion of the DNA occurring after\r\nthe first electrophoretic separation and before the second.
\r\n\r\nThese techniques and others were used to study mitochondrial DNA from\r\nmice and rats. Some of these data explore the evolutionary divergence of the\r\nmtDNA in these animals. This information can be compared to evolutionary\r\nstudies with nuclear DNA.
\r\n\r\nAdditionally, chromosomal DNA from patients with normal hemoglobin\r\nand hemoglobin Lepore was studied. Using this type of analysis we were able\r\nto demonstrate that a change in the amino acid sequence of some of the \u03b2-related\r\nglobin chains in hemoglobin Lepore is associated with a change in DNA structure.
" }, { "name": "Presti, David Eugene", "degree": "PhD", "year": "1979", "title": "Studies of the Blue Light Receptor in Phycomyces", "advisor": "Delbruck, Max", "url": "https://resolver.caltech.edu/CaltechETD:etd-08042006-134847", "creators": [ { "name": { "family": "Presti", "given": "David Eugene" }, "id": "Presti-David-Eugene", "display_name": "Presti, David Eugene" } ], "advisors": [ { "name": { "family": "Delbruck", "given": "Max" }, "id": "Delbr\u00fcck-M", "role": "advisor", "display_name": "Delbruck, Max" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/M4MP-0R15", "abstract": "A large number of organisms possess a variety of responses to blue and long-wavelength ultraviolet light. Riboflavin and [beta]-carotene have long been considered the major candidates for the photoreceptor mediating these so-called blue-light physiological responses. The fungus Phycomyces possesses several responses to blue light, among which are induction of [beta]-carotene synthesis in the mycelium and sporangiophore growth and tropism. In this thesis, it is conclusively demonstrated that a carotenoid is not the photoreceptor for the responses of Phycomyces sporangiophores to blue light. The contention that the photoreceptor is riboflavin is further strengthened by action spectral evidence consistent with the direct excitation of the photochemically active flavin triplet state. Light-induced cytochrome optical absorbance changes are investigated in in vivo preparations of Phycomyces; an action spectrum determination shows such absorbance changes to be mediated by a flavin photoreceptor. However, no evidence linking these absorbance changes to possible steps in the sensory transduction pathway was found. Indeed, the findings that the flavin-mediated cytochrome absorbance changes occur with a low quantum yield in Phycomyces and that they also occur in cells from a human cervical carcinoma speak against their possible relevance to the physiological blue-light receptor. Electron paramagnetic resonance spectroscopy and fluorescence lifetime spectroscopy are also investigated as possible probes of the flavin photoreceptor. However, the widespread occurrence of riboflavin in cellular roles other than photoreception makes it difficult to separate out that particular flavin which functions as the physiological blue-light receptor. It represents a case of a photoreceptor which is at once ubiquitous and elusive. Finally, blue-light-induced synthesis of [beta]-carotene is investigated in the wildtype and in several sensory mutants of Phycomyces." }, { "name": "Shure, Mavis", "degree": "PhD", "year": "1979", "title": "I. Studies of Closed Circular DNA : Physical and Biological Implications of Heterogeneity in the Topological Winding Number, \u03b1. II. The Structure of Virion SV40 DNA in Situ Examined by Chemical Modification with Dimethylsulfate", "advisor": "Vinograd, Jerome Rubin", "url": "https://resolver.caltech.edu/CaltechTHESIS:03232026-172104871", "creators": [ { "name": { "family": "Shure", "given": "Mavis" }, "id": "Shure-Mavis", "display_name": "Shure, Mavis" } ], "advisors": [ { "name": { "family": "Vinograd", "given": "Jerome Rubin" }, "id": "Vinograd-J-R", "role": "advisor", "display_name": "Vinograd, Jerome Rubin" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/mn6t-xq83", "abstract": "Methods for gel electrophoresis have been developed which permit the\r\nresolution of closed circular DNA molecules differing by unit values in their\r\ntopological winding number, \u03b1. Relaxed or nonsupercoiled closed circular DNAs,\r\nnative supercoiled DNAs, as well as DNAs having intermediate numbers of super-helical\r\nturns, can be resolved into sets of bands by one or more of the different\r\nelectrophoresis conditions. In addition, a method based on theory (presented\r\nin Chapter IV) has been developed for the photographic quantitation of fluorescent\r\nsubstances. DNA stained with ethidium in agarose gels is used as an example.\r\nIn the course of developing this method, it has been demonstrated that the empirical\r\nmethods employed by other authors can give rise to large systematic errors.
\r\n\r\nThe methods for electrophoresis, used in conjunction with the method\r\nfor photographic quantitation of fluorescence, have permitted a quantitative\r\nexamination of closed circular DNA. The results of such studies (presented in\r\nChapters I, II, and III) are summarized below.
\r\n\r\nThe limit product of the action of nicking-closing (N-C) enzyme on\r\nclosed circular DNA is not a homogeneous species but rather a distribution.\r\nEach distribution has a mean degree of supercoiling of approximately zero. The\r\nindividual species within the distributions differ by \u0394\u03c4 = \u00b1 1, \u00b1 2, etc., and the\r\nrelative masses fit a Boltzmann distribution. It has also been demonstrated that\r\n\"nonsupercoiled\" closed circular duplex molecules serve as substrates for N-C\r\nenzyme and that a distribution of topological isomers is generated. Polynucleotide\r\nligase, acting on nicked circular DNA, forms under the same conditions, the\r\nsame set of closed DNAs. The latter enzyme freezes the population into sets\r\nof molecules otherwise in configurational equilibrium in solution.
\r\n\r\nBy a method of overlapping the results obtained after agarose gel electrophoresis\r\nunder two different sets of conditions, it has become possible to determine\r\nthe number of superhelical turns in a given DNA by counting the bands present\r\nafter partially relaxing the DNA with N-C enzyme. Because native supercoiled\r\nDNA is heterogeneous with respect to \u03b1, an average number of superhelical turns\r\nwas determined. Virion SV40 DNA contains 26 \u00b1 0.5 superhelical turns in 0.2 M\r\nNaCl and at 37\u00b0C, the conditions under which the enzymatic relaxations were\r\nperformed. Under this same set of conditions, virion polyoma DNA contains\r\n26 \u00b1 1 superhelical turns, which is consistent with the observations that polyoma\r\nDNA has a higher molecular weight, a lower superhelix density, but the same\r\nnumber of nucleosomes as has SV40 DNA.
\r\n\r\nThe average number of superhelical turns in SV40, 26, combined with\r\nthe value, 21, for the average number of nucleosomes per SV40 genome, yields\r\nan average of 1.25 superhelical turns per 1/21 of the SV40 genome. If the regions\r\nof internucleosomal DNA are fully relaxed, 1.25 corresponds to the average number\r\nof superhelical turns within a nucleosome.
\r\n\r\nThe superhelix densities determined by the band counting method have\r\nbeen compared with those determined by buoyant equilibrium in propidium diiodide\r\n(PDI)-CsCl gradients. A comparison of the values obtained by the two methods\r\npermits a calculation of an unwinding angle for ethidium. The mean value determined\r\nfrom experiments with SV40 DNA is 23 \u00b1 3\u00b0.
\r\n\r\nSystems for gel electrophoresis in the presence of one of the intercalative\r\nunwinding ligands, ethidium or chloroquine, have been developed which permit\r\nthe resolution of highly supercoiled closed circular DNA molecules differing\r\nby unit values of the topological winding number, \u03b1. All native closed circular\r\nDNAs examined, including the viral and intracellular forms of SY40 and polyoma\r\nDNAs, bacterial plasmid DNAs, and the double stranded closed circular DNA\r\ngenome of the marine bacteriophage, PM2, are more heterogeneous with respect\r\nto the number of superhelical turns present than are the thermal distributions\r\nobserved in the limit products of the action of nicking-closing enzyme on the\r\nrespective DNAs. In addition, the distributions within the virion and the intracellular\r\nform I DNAs of SY40 were found to be indistinguishable. This was also found\r\nto be the case for the virion and the intracellular form I DNAs of polyoma.
\r\n\r\nIn the cases of both SY 40 and polyoma, where it has been shown that\r\nthe supercoiling is a combined consequence of the binding of the four nucleosomal\r\nhistones, H2a, H2b, H3 and H4, and the action of N-C enzyme, the breadth of\r\nthe distributions within the form I DN As poses specific problems since the work\r\nof other laboratories indicates that the number of nucleosomes on the respective\r\nminichromosomes falls within a narrow distribution around 21. If it is assumed\r\nthat all nucleosomes have identical structures, and that the DNA within a nucleosome\r\nis not free to rotate, the native DN As would be anticipated to be less heterogeneous\r\nthan the thermal equilibrium mixtures present in the N-C enzyme relaxed\r\nSY40 and polyoma DNAs.
\r\n\r\nFinally (see Chapter V), the structure of virion SY40 DNA in situ has\r\nbeen examined by chemical modification with dimethylsulfate (DMS). Virions\r\nare permeable to DMS and remain physically intact while the DNA (specifically,\r\nthe bases adenine and guanine) within the virus particles becomes methylated.\r\nThis approach permits the examination of specific protein-DNA interactions\r\nin the absence of artifacts of isolation and reconstitution.
\r\n\r\nA total length of approximately 3600 nucleotides (35% of the nucleotides\r\nin SV40 DNA) was scanned using this method. A total of 15 segments of the\r\nSV40 genome (obtained by kinasing 15 different 5' termini produced by treatment\r\nwith different restriction endonucleases) was screened for decreases or increases\r\nin levels of methylation relative to naked SV40 DNA. The segments represented\r\nboth strands of the DNA and were chosen to obtain samples representative of\r\nthe entire genome, with a particular emphasis on fragments covering and surrounding\r\nthe origin(s) of DNA replication and both late and early RNA transcription.\r\nNo major or convincing differences were ever observed between purine patterns\r\nobtained after parallel treatment of corresponding samples derived from naked\r\nSV40 DNA which had been treated with DMS and from DNA within SV40 virions\r\nwhich had been treated with DMS.
" }, { "name": "Stumph, William Edward", "degree": "PhD", "year": "1979", "title": "Gene Enrichment Using Antibodies to DNA/RNA Hybrids: Mapping the Ribosomal DNA of Slime Mold and Rat", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechTHESIS:02182014-134518249", "creators": [ { "name": { "family": "Stumph", "given": "William Edward" }, "id": "Stumph-William-Edward", "display_name": "Stumph, William Edward" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "chair", "display_name": "Bonner, James Frederick" }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "member", "display_name": "Owen, Ray David" }, { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "member", "display_name": "Hood, Leroy E." }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "member", "display_name": "Mitchell, Herschel K." } ], "option_major": [ "bioch" ], "doi": "10.7907/9V3M-QH52", "abstract": "A novel method for gene enrichment has been developed and applied\r\nto mapping the rRNA genes of two eucaryotic organisms. The method makes\r\nuse of antibodies to DNA/RNA hybrids prepared by injecting rabbits with the\r\nsynthetic hybrid poly(rA)\u2022poly(dT). Antibodies which cross-react with non-hybrid\r\nnucleic acids were removed from the purified IgG fraction by adsorption on columns\r\nof DNA-Sepharose, oligo(dT)-cellulose, and poly(rA)-Sepharose. Subsequent\r\npurification of the specific DNA/RNA hybrid antibody was carried out on a column\r\nof oligo(dT)-cellulose to which poly(rA) was hybridized. Attachment of these\r\nantibodies to CNBr-activated Sepharose produced an affinity resin which specifically\r\nbinds DNA/RNA hybrids.
\r\n\r\nIn order to map the rDNA of the slime mold Dictyostelium discoideum,\r\nR-loops were formed using unsheared nuclear DNA and the 178 and 268 rRNAs\r\nof this organism. This mixture was passed through a column containing the affinity\r\nresin, and bound molecules containing R- loops were eluted by high salt. This purified\r\nrDN A was observed directly in the electron microscope. Evidence was obtained\r\nthat there is a physical end to Dictyostelium rDN A molecules approximately\r\n10 kilobase pairs (kbp) from the region which codes for the 268 rRNA. This finding\r\nis consistent with reports of other investigators that the rRNA genes exist as\r\ninverse repeats on extra-chromosomal molecules of DNA unattached to the remainder\r\nof the nuclear DNA in this organism.
\r\n\r\nThe same general procedure was used to map the rRNA genes of the rat.\r\nMolecules of DNA which contained R-loops formed with the 188 and 288 rRNAs\r\nwere enriched approximately 150- fold from total genomal rat DNA by two cycles\r\nof purification on the affinity column. Electron microscopic measurements of\r\nthese molecules enabled the construction of an R-loop map of rat rDNA. Eleven\r\nof the observed molecules contained three or four R-loops or else two R-loops\r\nseparated by a long spacer. These observations indicated that the rat rRNA genes\r\nare arranged as tandem repeats. The mean length of the repeating units was\r\n37.2 kbp with a standard deviation of 1.3 kbp. These eleven molecules may represent\r\nrepeating units of exactly the same length within the errors of the measurements,\r\nalthough a certain degree of length heterogeneity cannot be ruled out.\r\nIf significantly shorter or longer repeating units exist, they are probably much\r\nless common than the 37.2 kbp unit.
\r\n\r\nThe last section of the thesis describes the production of antibodies to\r\nnon-histone chromosomal proteins which have been exposed to the ionic detergent\r\nsodium dodecyl sulfate (SDS). The presence of low concentrations of SDS did\r\nnot seem to affect either production of antibodies or their general specificity.\r\nAlso, a technique is described for the in situ immunofluorescent detection of protein\r\nantigens in polyacrylamide gels.
" }, { "name": "Bender, Welcome", "degree": "PhD", "year": "1978", "title": "Electron Microscopic Studies of Sequence Arrangement: Poly(A) Mapping, RNA Tumor Viruses, and Slime Mold Actin Genes", "advisor": "Davidson, Norman R.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01282026-233715971", "creators": [ { "name": { "family": "Bender", "given": "Welcome" }, "id": "Bender-Welcome", "display_name": "Bender, Welcome" } ], "advisors": [ { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "advisor", "display_name": "Davidson, Norman R." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/ssje-ve39", "abstract": "No abstract." }, { "name": "Crewther, Sheila Gillard", "degree": "PhD", "year": "1978", "title": "Plasticity in the Cat Visual System", "advisor": "Sperry, Roger Wolcott", "url": "https://resolver.caltech.edu/CaltechETD:etd-11222004-094932", "creators": [ { "name": { "family": "Crewther", "given": "Sheila Gillard" }, "id": "Crewther-Sheila-Gillard", "display_name": "Crewther, Sheila Gillard" } ], "advisors": [ { "name": { "family": "Sperry", "given": "Roger Wolcott" }, "id": "Sperry-R-W", "role": "advisor", "display_name": "Sperry, Roger Wolcott" } ], "committee": [ { "name": { "family": "Sperry", "given": "Roger Wolcott" }, "id": "Sperry-R-W", "role": "chair", "display_name": "Sperry, Roger Wolcott" }, { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "member", "display_name": "Van Essen, David C." }, { "name": { "family": "Pettigrew", "given": "John D." }, "id": "Pettigrew-J-D", "role": "member", "display_name": "Pettigrew, John D." }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "member", "display_name": "Owen, Ray David" } ], "option_major": [ "biology" ], "doi": "10.7907/1YK9-0Z09", "abstract": "Behavioral and electrophysiological techniques have been used to study plasticity in the cat visual system both during and after the critical period for development of cortical binocularity. Both kittens reared with unilateral or bilateral eye rotation and adult cats given monocular rotations after the critical period showed good visuomotor adaptation to the rearranged visual information. All animals were able to learn a brightness discrimination. However, the three animals with monocular rotations of more than 120[degrees] were unable to learn oriented pattern discriminations although kittens raised with combined bilateral rotations of approximately 160[degrees] were able to learn even the most difficult orientation-discrimination. Apart from this there did not appear to be any significant correlation between rate of learning and angle of rotation.
\r\n\r\nAll animals who learned the pattern discriminations showed considerable, though incomplete interocular transfer as assessed by improvement in initial performance and by savings and errors to criterion with the second eye as compared with the first.
\r\n\r\nElectrophysiological examination of area 17, in the kittens raised with monocular or binocular rotations, indicated the involvement of factors other than disparate visual input to the two retinas and asymmetrical eye movements per se in the breakdown of cortical binocularity seen in strabismic animals. Although most of the neuronal properties of area 17 were comparable to those reported previously for animals with unilateral rotations, some evidence for physiological adaptation to monocular and binocular rotation was seen in the corticotectal cells of layer V. A large proportion of the binocular corticotectal cells seen in animals with a total rotation angle of approximately 90 degrees in both experimental groups showed significant shifts in direction selectivity such that the optimal stimulus direction was very similar in both eyes.
\r\n\r\nPlasticity of the visual cortex after the critical period was also examined electrophysiologically and it was found that the cortical effects of early monocular deprivation could be reversed when both the visual input to the brain and the extraocular afferents of the experienced eye were removed. After the combined treatments of pressure blinding and retrobulbar block of the normal eye approximately 50% of the units encountered could be shown to be influenced by the formerly deprived eye.
" }, { "name": "Edelman, Jay Barry", "degree": "PhD", "year": "1978", "title": "Statistical Mechanics of Biological Membranes: Protein Aggregation and Lipid Ordering", "advisor": "Chan, Sunney I.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02042026-222046404", "creators": [ { "name": { "family": "Edelman", "given": "Jay Barry" }, "id": "Edelman-Jay-Barry", "display_name": "Edelman, Jay Barry" } ], "advisors": [ { "name": { "family": "Chan", "given": "Sunney I." }, "id": "Chan-S-I", "orcid": "0000-0002-5348-2723", "role": "advisor", "display_name": "Chan, Sunney I." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/jgm1-cd33", "abstract": "It is known that membrane proteins are surrounded by\r\na halo of perturbed lipids. When two protein particles\r\napproach one another, their effects are superimposed.\r\nThis produces a force between them. We construct a Landau\r\nmodel of the membrane order parameter, and use it to\r\ncalculate the interaction potential. Protein aggregation\r\nis predicted to be inherently favorable, though sometimes\r\nopposed by a barrier. Because experiments can be done only\r\nin large populations of particles, the theory is generalized\r\nto that situation. This allows us to calculate the protein\r\nchemical potential, and to study how the interaction between\r\ntwo given particles is modified by proximity to others.\r\nThe protein diffusion equation predicts when the lipid-mediated\r\nforces lead to protein precipitation rather than\r\na slight clustering tendency.
\r\n\r\nNext we turn to experimental tests, based on the\r\nprotein pair correlation function. We describe its relation\r\nto the potential and develop a method for measuring it. We\r\nfind a repulsion between protein particles, extending\r\nseveral nm beyond their apparent edges, and an attraction,\r\nextending 10 to 20 nm. The prediction of when precipitation\r\noccurs is shown to be at least qualitatively accurate.
\r\n\r\nThe preceding analysis is primarily thermodynamic, and\r\nthus neglects molecular details. We next study them,\r\ndiscovering that the behavior of our order parameter coincides\r\nwith that of membrane thickness. On a molecular level,\r\nit can be controlled by either lipid conformation or tilt.\r\nAfter deriving the model from statistical mechanics, we\r\nfind the former to be implausible, suggesting that tilt\r\nmediates protein interactions.
\r\n\r\nLastly, the model is supplemented with a general\r\ndescription of lipid phase transitions. We find that it\r\ncorrectly predicts the phase behavior of protein-lipid\r\nsystems. It also reveals that the usual analysis\r\nunderestimates the amount of lipid perturbed by each protein\r\nparticle. Furthermore, we demonstrate that those lipids\r\nare usually constrained between the disordered and ordered\r\nstates, closer to the former.
" }, { "name": "Frey, Teryl Kenneth", "degree": "PhD", "year": "1978", "title": "Biochemical and Biophysical Studies on the RNA Species of Sindbis Virus", "advisor": "Strauss, James H.; Strauss, Ellen G.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03032017-150817762", "creators": [ { "name": { "family": "Frey", "given": "Teryl Kenneth" }, "id": "Frey-Teryl-Kenneth", "display_name": "Frey, Teryl Kenneth" } ], "advisors": [ { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "advisor", "display_name": "Strauss, James H." }, { "name": { "family": "Strauss", "given": "Ellen G." }, "id": "Strauss-E-G", "role": "advisor", "display_name": "Strauss, Ellen G." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/18xf-7p07", "abstract": "Sindbis virus, a togavirus, contains an infectious, single stranded RNA with a molecular weight of 4.3 x 106 (498), which in infected cells serves as a messenger RNA for the translation of the virus onstructural proteins. Also present in infected cells is an\r\nRNA with a molecular weight of 1.6 x 106 (268) which consists of the 3' terminal third of the 49S RNA and functions as a messenger RNA\r\nfor the translation of the virus structural proteins. In this thesis,\r\nthree features of these RNA species of Sindbis virus are studied:\r\nthe 3' terminal poly A tracts on the RNAs and the corresponding poly\r\nU tracts on the complementary strand of RNA; the methylation of\r\ncytidine residues in the RNA; and the circularization of the 49S RNA\r\nmolecule.
\r\n\r\nBoth 49S and 26S RNA contain 3' terminal stretches of polyadenylic\r\nacid (poly A), which are heterogeneous in size, having a mean length\r\nof 70 nucleotides and a size range of from 40 to 200 nucleotides.\r\nPoly A isolated from Sindbis virion 49S RNA grown in chicken, hamster\r\nand mosquito culture cells have similar size distributions. Although\r\nmost 49S and 26S RNA molecules contain poly A, a small fraction of\r\nintact 49S and 26S RNA molecules contain little poly A. The fact that\r\nthe fraction of 49S which lacks poly A is only 10% to 20% as infectious\r\nas the fraction which contains poly A suggests that poly A is essential\r\nfor replication of the virus. Sindbis virus double stranded RNA\r\nspecies also contain poly A with a size distribution similar to that\r\nof poly A from viral single stranded RNA. The double stranded RNA species also contain stretches of polyuridylic acid (poly U) which\r\nare on the minus strand and have a size distribution identical to\r\nSindbis virus poly A. This indicates that the poly A in Sindbis\r\nvirus RNA is synthesized by transcription of a poly U template by the\r\nvirus transcriptase.
\r\n\r\nSindbis virus intracellular 26S and 49S RNA contain internal\r\n5-methyl cytidine (m5c) residues. Sindbis virion 49S RNA contains\r\nmuch less m5c than intracellular 49S RNA. In the 26S RNA, m5c residues\r\noccur in five oligonucleotides, which are found distributed between\r\ntwo locations, one approximately 4000 nucleotides from the 3' end and\r\nthe other about 1200 nucleotides from the 31 end (out of a total length\r\nof 5000 nucleotides). The distribution of label between these two\r\nlocations suggests that each contains at least two of the methylated\r\nsequences. It thus appears that there are five specific sites for\r\nmethylation on the 26S RNA. However, only a minority of these sites\r\nare modified. Polysomal and nonpolysomal 26S RNA contain equal amounts\r\nof m5c while polysomal 49S RNA contains 60% to 80% more m5c than\r\nnonpolysomal 49S RNA, indicating that m5c may have a function in\r\ntranslation.
\r\n\r\nSindbis virus 49S RNA is capable of assuming a circular configuration\r\nthrough the hydrogen bonding of complementary nucleotide sequences\r\nlocated at the 5' and 3' ends of the 49s RNA molecule. The circular\r\nand linear forms of 49S RNA are separable on sucrose gradients\r\ncontaining 0.01 M NaCl. Sindbis virus 49S RNA extracted from virions\r\nis completely in the circular form. The melting temperature (Tm) of the circles is 39.5\u00b0C in 0.023 M NaCl and 53.5\u00b0C in 0.1 M NaCl. The 6H\r\nfor circularization is -160 kcal/mole and the \u0394S for circularization is\r\napproximately 500 eu. These parameters indicate that the length of the\r\ndouble-stranded region which is formed upon circularization of the\r\nmolecule is most likely short, on the order of 10 to 20 nucleotides.\r\nOur data indicate that extensive mismatching in this double stranded\r\nregion is unlikely. Intact linear 49S RNA molecules readily renature\r\nto form circles under appropriate conditions, the energy of activation\r\nfor this process being 42.6 kcal/mole. From the measured rate constants\r\nfor circularization, it is clear that Sindbis virus 49S RNA will\r\ncircularize readily under physiological conditions of temperature and\r\nionic strength. The virion RNA from Semliki Forest virus also forms\r\ncircles whose Tm is very similar to that of Sindbis virus RNA circles, suggesting that the sequences involved in circularization have been\r\nconserved.
" }, { "name": "Greif, Karen Faye", "degree": "PhD", "year": "1978", "title": "Memory Processing in Chickens and Goldfish", "advisor": "Sperry, Roger Wolcott", "url": "https://resolver.caltech.edu/CaltechTHESIS:02052026-164314544", "creators": [ { "name": { "family": "Greif", "given": "Karen Faye" }, "id": "Greif-Karen-Faye", "display_name": "Greif, Karen Faye" } ], "advisors": [ { "name": { "family": "Sperry", "given": "Roger Wolcott" }, "id": "Sperry-R-W", "role": "advisor", "display_name": "Sperry, Roger Wolcott" } ], "committee": [ { "name": { "family": "Sperry", "given": "Roger Wolcott" }, "id": "Sperry-R-W", "role": "chair", "display_name": "Sperry, Roger Wolcott" }, { "name": { "family": "Allman", "given": "John Morgan" }, "id": "Allman-J-M", "role": "member", "display_name": "Allman, John Morgan" }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "member", "display_name": "Owen, Ray David" }, { "name": { "family": "Van Essen", "given": "David C." }, "id": "Van-Essen-D-C", "orcid": "0000-0001-7044-4721", "role": "member", "display_name": "Van Essen, David C." } ], "option_major": [ "biology" ], "doi": "10.7907/yjt7-gq74", "abstract": "Memory processing has been investigated in day-old chickens and in\r\nadult goldfish. In chicks, bilateral vs. unilateral storage of monocularly-acquired\r\none-trial passive avoidance was examined using unilateral lesions of the avian\r\ntelencephalon. Interocular transfer tested 24 hr after training was observed\r\nafter lesions of the trained hemisphere delayed 2-18 hr after the training trial.\r\nBilateral lesions abolished task performance. It is concluded that memory for\r\none-trial passive avoidance is established bilaterally in the brain and that the\r\ntelencephalon contributes either as a storage area or as a relay in retrieval.\r\nResults of unilateral lesions delayed 2 min after training also suggested bilateral\r\nstorage, in contrast to published reports of apparent unilateral storage of the\r\nsame task after unilateral. injection of cycloheximide 2 min after training. The\r\ndisparity between results may be the result of more general disruption of brain\r\nfunction produced by protein synthesis inhibitors; cycloheximide may interfere\r\nwith transfer of sensory information or depress brain function so as to prevent\r\nthe normal establishment of bilateral traces.
\r\n\r\nAdult goldfish were used to examine the roles of the optic tectum and\r\nforebrain in memory storage of color discrimination using conditioned respiratory\r\nsuppression as the behavioral measure. Highly localized training of discrete\r\ntectal areas was followed by lesions of trained tectal regions or by bilateral\r\nforebrain lesions. Excellent intraretinal generalization to the remainder of the\r\nvisual field of the trained eye was observed. Interocular transfer across the\r\nentire visual field of the untrained eye occurred after training in the posterior\r\nvisual field. Complete tectal ablation after localized training did not produce\r\ndeficits in interocular transfer. It is concluded that binocular regions of the\r\nvisual field in goldfish do not integrate color discrimination better than do monocular\r\nregions and that memory storage may occur bilaterally in the brain.
\r\n\r\nFindings of apparent bilateral memory storage in birds and fish are\r\nrelated to studies of memory in mammalian species. It is suggested that the\r\nparallel processing methods observed may reflect selection procedures which\r\noccurred early in the evolutionary development of behavior in vertebrates.
" }, { "name": "Sheridan, Robert Edward, Jr.", "degree": "PhD", "year": "1978", "title": "Kinetics and Equilibria at Nicotinic Receptors in Electrophorus Electricus and Raia Erinacea Electroplaques", "advisor": "Lester, Henry A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04152026-170728834", "creators": [ { "name": { "family": "Sheridan", "given": "Robert Edward, Jr." }, "id": "Sheridan-Robert-Edward-Jr.", "display_name": "Sheridan, Robert Edward, Jr." } ], "advisors": [ { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "advisor", "display_name": "Lester, Henry A." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "neurobio" ], "doi": "10.7907/dvbb-4m95", "abstract": "This study utilizes relaxation kinetics and equilibrium conductance\r\nto measure the response of the nicotinic receptor to cholinergic agonists.\r\nAgonist-induced conductance, postsynaptic currents (PSC's), and voltage-jump\r\nrelaxations are studied in electroplaques of Electrophorus\r\nelectricus. Agonist-induced conductance is instantaneously ohmic at\r\nvoltages more negative than -30 mV. In bath-applied acetylcholine (ACh),\r\nthe dose vs. conductance relation is sigmoid. At 15\u00b0, the apparent\r\ndissociation constant for ACh decreases e-fold for every 87 mV of\r\nhyperpolarization, hence agonist-induced conductance increases as the\r\nelectroplaque is hyperpolarized. In other experiments, presynaptic\r\nterminals are stimulated to produce PSC's. Peak PSC changes linearly\r\nwith membrane voltages more negative than -30 mV. The estimated reversal\r\npotential for all the above agonist-induced currants is about +10 mV.\r\nAfter the peak, PSC's decay with a single exponential rate, \u221d. At 15\u00b0,\r\n\u221d equals 1.2 msec-1 at 0 mv and decreases e-fold for every 86 mV of\r\nhyperpolarization. In still another series of experiments, an\r\ninstantaneous jump in membrane voltage causes agonist-induced conductance\r\nto relax to a new equilibrium along a simple exponential time course.\r\nThe rate constant, 1/\u03c4, for such relaxations varies with the nature of\r\nthe agonist, its concentration, and the final membrane voltage. This\r\nrelaxation rate is interpreted as the sum of the closing rate of\r\nreceptors, \u221d, and a first-order, voltage-independent rate constant tor\r\nreceptor opening. As expected from this interpretation: (a) 1/\u03c4\r\napproaches \u221d at low ACh concentrations, (b) 1/\u03c4 increases linearly\r\nwith agonist concentration, and (c) 1/\u03c4 is unaffected by blockade of\r\nreceptors with \u221d-bungarotoxin. Several kinetic models of the nicotinic\r\nreceptor are tested. The one which best fits the data assumes: (a) that\r\nthe open state of the receptor forms as the receptor binds the second in\r\na series of two agonist molecules, (b) that this process constitutes the\r\nrate-limiting step in receptor activation, and (c) that dissociation of\r\neither agonist molecule closes the receptor. In ACh, the rate-limiting\r\nstep proceeds with an opening rate or 107 M-1sec-1 and a closing rate\r\nof 102 to 103 sec-1.
\r\n\r\nAgonist-induced conductance and postsynaptic currents are also\r\nstudied in electroplaques or Raia erinacea. In Raia electroplaques,\r\ndelayed rectification prevents 11easurement of conductance at voltages\r\nmore positive than -.50 mV. At voltages more negative than -50 mV, bath-applied\r\ncarbachol (3 to 9 \u00b5M) produces a steady-state conductance which\r\nis independent of membrane voltage. In ether experiments, PSC's are\r\nproduced by electrical stimulation of presynaptic nerves. Peak PSC\r\nvaries linearly with membrane voltage. At 20\u00b0, the extrapolated reversal\r\npotential for all the above agonist-induced currents is about 0 mV. PSC's\r\ndecay exponentially after the peak. The rate constant of the decay, \u221d, \r\ndoes not vary with membrane voltage and equals 0.23 msec-1 at 20\u00b0. This\r\nrate constant does increase with temperature and has a Q10 of 1.95.\r\nD-tubocurarine reduces the peak PSC but does not change the decay rate.\r\nNeostigmine reduces the decay rate but does not change the peak PSC.\r\nThese results imply that the opening and closing rates of the nicotinic\r\nreceptor are voltage insensitive in Raia electroplaques.
" }, { "name": "Stuart, Duncan Knight", "degree": "PhD", "year": "1978", "title": "The Neurosecretion of the Polypeptide Egg-Laying Hormone (ELH) from the Bag Cells, Neuronal Sites of Action of ELH, and Circadian Release of Polypeptides from the Eye of Aplysia californica", "advisor": "Strumwasser, Felix", "url": "https://resolver.caltech.edu/CaltechTHESIS:02172026-214230970", "creators": [ { "name": { "family": "Stuart", "given": "Duncan Knight" }, "id": "Stuart-Duncan-Knight", "display_name": "Stuart, Duncan Knight" } ], "advisors": [ { "name": { "family": "Strumwasser", "given": "Felix" }, "id": "Strumwasser-F", "role": "advisor", "display_name": "Strumwasser, Felix" } ], "committee": [ { "name": { "family": "Strumwasser", "given": "Felix" }, "id": "Strumwasser-F", "role": "chair", "display_name": "Strumwasser, Felix" }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "member", "display_name": "Mitchell, Herschel K." }, { "name": { "family": "Hudspeth", "given": "A. James" }, "id": "Hudspeth-A-J", "role": "member", "display_name": "Hudspeth, A. James" }, { "name": { "family": "Konishi", "given": "Masakazu" }, "id": "Konishi-M", "role": "member", "display_name": "Konishi, Masakazu" }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "member", "display_name": "Owen, Ray David" } ], "option_major": [ "neurobio" ], "doi": "10.7907/c981-k394", "abstract": "The thesis is about the neurosecretion of Aplysia peptides and demonstrates\r\nthat one of them acts directly upon the nervous system. Such neuronal\r\neffects of neurosecretory substances may prove to be a general phenomenon; this is\r\ndiscussed in the introduction.
\r\n\r\nIn chapter 1, radiolabeled peptides released from electrically active bag\r\ncell neurons in isolated bag cell clusters were compared with the polypeptide egg-laying\r\nhormone (ELH), 6,000 daltons, pl 9.0-9.3, as purified from homogenates of\r\nbag cell clusters. A labeled peptide which is selectively released from electrically\r\nactive bag cell clusters comigrates with ELH from cluster homogenates on P-6 gel\r\nfiltration columns and subsequent isoelectric focusing gels. When bag cells are\r\nactivated, a released factor(s) induces egg-laying and comigrates with ELH from\r\ncluster homogenates on P-6 columns. At least three other presumed peptides of\r\nunknown function are also released. These experiments demonstrate that ELH\r\n(6,000 m.w., pl 9.0-9.3) as purified from bag cell cluster homogenates is the major,\r\nactive form secreted from bag cells.
\r\n\r\nIn chapter 2, the effects of ELH on neuronal activity of the attached head\r\nganglia (buccal, cerebral, pleural, and pedal), on the isolated buccal ganglia, as well\r\nas on feeding in intact Aplysia were studied. Starved animals (n = 7) injected at\r\n20\u00b0C with a crude- extract containing ELH stopped eating algae at 17 \u00b1 4 min and\r\ntheir eggs first appeared at 29 \u00b1 4 min after injection. These data clearly indicate\r\nthat a suppression of feeding activity occurs before the appearance of eggs. ELH\r\napplied to the paired buccal ganglia in vitro activates a pair of neurons into a tonic\r\npacing mode (~1 spike/sec). The time for the full appearance of this activity in\r\nvitro correlates well with the time for suppression of feeding in vivo. These\r\nneurons each have an ipsilateral axon in buccal nerve 3. ELH increases the rate of\r\nfiring of a second pair of buccal neurons, each with an ipsilateral axon in the\r\ncerebrobuccal connective. ELH when applied to the attached head ganglia causes\r\nlarge bursts of neuronal activity in pedal nerves to the foot, and increased activity\r\nin the nerve to the penis. These in vitro effects were produced by ELH partially\r\npurified from bag cell cluster homogenates using ammonium sulfate precipitation,\r\nan anion exchange column, and a gel filtration column or by ELH released from\r\nactivated bag cells in isolated abdominal ganglia and then frationated by gel\r\nfiltration. The ELH effects upon the in vitro nervous system support the working ~\r\nhypothesis that ELH in vivo acts directly on the nervous system to suppress feeding\r\nactivity. ELH may also activate neural circuits in the pedal and probably cerebral\r\nganglia that produce characteristic movements of the head during egg-laying; the\r\nrelevant neurons remain to be identified.
\r\n\r\nIn chapter 3, a circadian rhythm (CR) of release of presumed peptides\r\nfrom the isolated eye of Aplysia is demonstrated. This isolated eye is known to\r\nhave a CR of compound action potentials (CAPs) as recorded from its optic nerve.\r\nSubstances labeled with radioactive amino acids and released into the perfusate\r\nwere separated on gel filtration columns and SDS polyacrylamide gels. In the CR\r\nexperiments, the perfusate of a single, labeled, dark-maintained eye was collected\r\nevery 3 h for two days while simultaneously recording the CR of CAPs. Each 3-h\r\nperfusate was applied to a P-2 gel filtration column. Excluded substances\r\n(m.w. ~ 2000) and material fractionated in the region of m.w. ~ 1000 showed a CR\r\nwhich was in phase with the CR of CAPs. Much of these labeled substances can be\r\nprecipitated with trichloroacetic acid. Their release is stimulated by a high\r\npotassium solution and inhibited by a low calcium solution that also inhibits CAP\r\nactivity. This and other previously published evidence suggests that the CAPs and\r\nthe peptide release are directly produced by electrically coupled neurosecretory\r\ncells which may also contain the CR oscillator. One or more of these peptides may\r\nbe a neurohormone and/or transmitter used for synchronizing, entraining and/or\r\ndriving the rest of the animal's CRs.
" }, { "name": "Tang, David", "degree": "PhD", "year": "1978", "title": "A DNA Nicking-Closing Enzyme from Mouse L Cells", "advisor": "Vinograd, Jerome Rubin", "url": "https://resolver.caltech.edu/CaltechTHESIS:02042026-194623147", "creators": [ { "name": { "family": "Tang", "given": "David" }, "id": "Tang-David", "display_name": "Tang, David" } ], "advisors": [ { "name": { "family": "Vinograd", "given": "Jerome Rubin" }, "id": "Vinograd-J-R", "role": "advisor", "display_name": "Vinograd, Jerome Rubin" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/797t-bh81", "abstract": "A DNA nicking-closing activity was isolated from mouse LA9 cells.\r\nThis activity converted native closed circular DNA into a limit product\r\nset of topological isomers having a mean degree of supercoiling of ~0.\r\nThese isomers differed from each other by single superhelical turns, and\r\ntheir relative masses fit a Boltzmann distribution. By re-reacting each\r\nisomer with the activity, the original distribution was regenerated.\r\nTherefore supercoiling of the DNA substrate was not required for the\r\naction of the enzyme. Polynucleotide ligase, acting on nicked circular\r\nDNA, formed under the same conditions, the same distribution of topological\r\nisomers. Therefore the nicking-closing enzyme must have generated nicked\r\nintermediates in which rotations at the nick were constantly occurring\r\nas a result of the thermal fluctuation of the DNA polymer chain. During\r\nresealing of the DNA, the enzyme froze the intermediates into a set of\r\nisomers differing by integral number of turns.
\r\n\r\nInitial purification of the mouse enzyme showed copurification of the\r\nactivity with histone Hl. It was not clear whether Hl was the enzyme or\r\na trivial contamination. In Chapter 2, I present a purification of the\r\nnicking-closing enzyme identifying it as a monopolypeptide of molecular\r\nweight 68,000. I show that the mouse enzyme is not similar to histone\r\nHl, that it is a rather acidic protein, and that its molecular weight is\r\nsimilar to that of the rat liver and the human KB cell enzymes.
" }, { "name": "West, Christopher Mark", "degree": "PhD", "year": "1978", "title": "(1) Glycoproteins and Development in the Cellular Slime Mold Dictyostelium discoideum. (2) Separation of Cells Using Isopycnic Centrifugation in Linear Density Gradients of Colloidal Silica", "advisor": "McMahon, Daniel; Dreyer, William J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-08082006-082425", "creators": [ { "name": { "family": "West", "given": "Christopher Mark" }, "id": "West-Christopher-Mark", "display_name": "West, Christopher Mark" } ], "advisors": [ { "name": { "family": "McMahon", "given": "Daniel" }, "id": "McMahon-D", "role": "advisor", "display_name": "McMahon, Daniel" }, { "name": { "family": "Dreyer", "given": "William J." }, "id": "Dreyer-W-J", "role": "advisor", "display_name": "Dreyer, William J." } ], "committee": [ { "name": { "family": "McMahon", "given": "Daniel" }, "id": "McMahon-D", "role": "chair", "display_name": "McMahon, Daniel" }, { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "member", "display_name": "Revel, Jean-Paul" }, { "name": { "family": "Delbruck", "given": "Max" }, "id": "Delbr\u00fcck-M", "role": "member", "display_name": "Delbruck, Max" }, { "name": { "family": "Dreyer", "given": "William J." }, "id": "Dreyer-W-J", "role": "member", "display_name": "Dreyer, William J." } ], "option_major": [ "biology" ], "doi": "10.7907/NM0K-3F42", "abstract": "I: Two methods were adapted for the study of glycoproteins in Dictyostelium discoideum. Both techniques relied upon prior separation of glycoproteins on one- or two-dimensional SDS-polyacrylamide gels. The first method was a modified form of crossed immunoelectrophoresis which substituted the carbohydrate-binding protein (lectin) concanavalin A (Con A) as the precipitating agent. In the second method, polyacrylamide gels were simply fixed and incubated in the presence of fluorescein-tagged lectins. After washing away free lectin, the gels were photographed and stained for protein. Identified glycoproteins were defined by their apparent molecular weights, identity and, anomeric linkage of some of their monosaccharides and in two-dimensional gels, their apparent isoelectric points. The ability of these techniques to specifically identify authentic glycoproteins was confirmed using a defined membrane system, the erythrocyte ghost, other known proteins, and hapten inhibitors. Of the two techniques, lectin diffusion was capable of higher resolution but did not give information about receptor multivalency. Both methods were more sensitive than the commonly-used periodic acid-schiff's base stain.\r\n\r\nMore than fifty different glycoproteins were detected in vegetative cells on the basis of their labeling with Con A or wheat germ agglutinin (WGA) and their sensitivity to proteolysis. These glycoproteins were distributed throughout the cell, giving each of several subfractions, including the plasma membrane, its own profile of glycoproteins. WGA receptors were apparently membrane bound and predominantly localized in the plasma membrane. A small number of glycoproteins were also detected with a galactose-binding protein, but these glycoproteins were not present in the plasma membrane or on the cell surface as determined by an independent technique. Receptors for L-fucose binding proteins were also absent from the plasma membrane of these cells. In contrast, another eukaryotic cell plasma membrane, the erythrocyte ghost, contained receptors to all lectins tested.\r\n\r\nDuring the formation of the pseudoplasmodium from vegetative cells, many glycoproteins were lost, modified, or many new ones were synthesized in all cell subfractions, including the plasma membrane. In addition, there was an asymmetry of distribution of glycoproteins in the pseudoplasmodium. There were three prestalk-cell specific glycoconjugates, which were all restricted to the plasma membrane, and two prespore-specific glycoproteins, which were present in plasma membranes as well as in a potential plasma membrane precursor. These plasma membrane differences occurred prior to overt differentiation of any stalk or spore cells. The region-specific glycoproteins were also present in the plasma membranes of cells which had not yet formed pseudoplasmodia, but were absent from vegetative cells.\r\n\r\nIn order to reinforce the temporal evidence that these region-specific glycoproteins were involved in the creation of the pseudoplasmodium, dissociated pseudoplasmodial cells were treated with the lectin which originally identified the region-specific molecules (WGA). Although dissociated cells typically reformed pseudoplasmodia, in the presence of WGA, they did not. This effect of WGA was blocked by a hapten inhibitor.\r\n\r\nII: In other work, separation of cells with different densities was attempted. When cells of Dictyostelium discoideum were centrifuged to density equilibrium in linear gradients of colloidal silica (Ludox), approximately 40 discrete bands appeared. A similar result was found for formalinized red blood cells and plastic beads. Isolated bands of cells rebanded faithfully in new gradients and band spacing depended upon gradient steepness. It was found that cell bands resulted from microscopic discontinuities in the linear gradients caused by centrifugation. When the gradients were analyzed in the analytical ultracentrifuge, absorbance scans revealed that cell bands coincided with \"bands\" of Ludox, which formed even without cells. Evidence ruling out other possible causes for cell bands is presented and procedures which avoid this condition are described." }, { "name": "Wold, Barbara J.", "degree": "PhD", "year": "1978", "title": "Studies of Structural Gene Transcripts in Sea Urchin Embryos and Adult Tissues", "advisor": "Davidson, Eric H.", "url": "https://resolver.caltech.edu/CaltechETD:etd-02272006-084615", "creators": [ { "name": { "family": "Wold", "given": "Barbara J." }, "id": "Wold-Barbara-J", "display_name": "Wold, Barbara J." } ], "advisors": [ { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "advisor", "display_name": "Davidson, Eric H." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biodev" ], "doi": "10.7907/rc6x-ar52", "abstract": "The RNA stored in the mature sea urchin oocyte includes sequences transcribed from about 6% of the single copy DNA or approximately 3.7 x 10(7) nucleotide pairs. It is estimated that there are about 1500 transcripts of each sequence per oocyte. A (3)H-labeled single copy DNA fraction highly enriched for sequences complementary to mature oocyte RNA was prepared. This DNA, referred to as oDNA, was reacted with excess polysomal mRNA from sea urchin embryos at 16-cell, blastula, and gastrula stages. Sixteen-cell embryo mRNA reacted with oDNA to a level which is 73% of that observed for the reaction of oDNA with oocyte RNA. This represents about 2.7 x 10(7) nucleotides of maternal sequence, or 12-15,000 different mRNAs of average size. Blastula and gastrula stage mRNA reacted with oDNA to about 56% and 53%, respectively, representing 2.1 x 10(7) and 1.9 x 10(7) nucleotides of oocyte RNA sequence. From the kinetics of hybridization, we calculate that the concentration of mRNAs belonging to the maternal sequence set is about 500 copies per embryo. This is typical of the \"complex class\" of embryo mRNA which contains the vast majority of diverse messenger RNA sequences but comprises only a small fraction (about 10%) of the total mRNA mass.\r\n\r\nTotal cytoplasmic RNA was extracted from 16-cell, blastula, prism and pluteus stages and reacted in excess with (3)H-oDNA. The amount of hybridization of oDNA with 16-cell cytoplasmic RNA was 100% of the value obtained when oDNA was reacted with oocyte RNA. Hybridization levels observed with cytoplasmic RNAs from later stages were progressively less, but were higher than the corresponding mRNA hybridization levels through the blastula stage.\r\n\r\nTo detect and quantitate embryo messenger RNA sequences which are not homologous with maternal RNAs, a labeled single copy DNA fraction entirely devoid of oocyte RNA complementary sequences was prepared. This selected DNA, termed null oDNA, was reacted with excess polysomal mRNA from 16-cell, blastula and gastrula embryos. Non-maternal sequences could not be detected in the mRNAs from 16-cell and gastrula stage embryos. Mesenchyme blastula mRNA, however, hybridized about 3.6 x 10(6) nucleotides of null-oDNA sequence which is sufficient to code for approximately 2,000 mRNAs of average size. These nonmaternal mRNAs are complex class sequences present on blastula polysomes at about the same concentration (500 copies per embryo) as are most maternal sequence mRNAs. Thus, a significant number of new, non-maternal structural genes are expressed in blastula stage embryos.\r\n\r\nBecause the sea urchin displays large differences between embryo and adult tissue messenger RNA sequence sets, it is possible to test the proposition that structural gene sequences are transcribed only in cells where the transcripts are translated. A (3)H-labeled single copy DNA highly enriched for sequences complementary to blastula polysomal messenger RNA was prepared. This selected DNA fraction, referred to as mDNA, represents about 2.6 x 10(7) nucleotides of embryo messenger RNA sequence. A maximum of 16% of the blastula sequences are present in coelomocyte and intestine messenger RNAs while 40% of the blastula sequences are represented in gastrula mRNA. (3)H-mDNA was hybridized with excess nuclear RNA from coelomocytes, intestine, and gastrula embryos. Within our limits of detection, all mDNA sequences were hybridized by each of the nuclear RNAs. We conclude from this result that virtually all of the blastula mRNA sequences are transcribed in three heterologous tissues in which the transcripts are not utilized as messenger RNAs. The kinetics of hybridization show that the blastula structural gene sequences are present in each nuclear RNA at about the same concentration as are the majority of the complex single copy nuclear transcripts." }, { "name": "Fischer, Ernst Peter", "degree": "PhD", "year": "1977", "title": "Serine Proteases, their Inhibitors and Chitin Synthetase in Phycomyces", "advisor": "Delbruck, Max; Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechETD:etd-08042006-131928", "creators": [ { "name": { "family": "Fischer", "given": "Ernst Peter" }, "id": "Fischer-Ernst-Peter", "display_name": "Fischer, Ernst Peter" } ], "advisors": [ { "name": { "family": "Delbruck", "given": "Max" }, "id": "Delbr\u00fcck-M", "role": "advisor", "display_name": "Delbruck, Max" }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/4T38-3H24", "abstract": "Phycomyces shows a positive growth response to blue light. Mutants are available which exhibit abnormal phototropism. They have been analysed genetically by complementation analysis. Seven complementation groups have been shown to be involved in the light information channel. They are designated mad A, -, G. Four groups are connected with the output end.\r\n\r\nThese findings encourage biochemical investigations. We make the hypothesis that light directly regulates the activity of chitin synthetase. It is investigated whether limited proteolysis represents one mode of such regulation.\r\n\r\nThree serine proteases are isolated and characterized. The molecular weights are determined to be 18.000, 22.000, and 60.000 daltons. The proteases are solubilized by detergent or salt treatment. There are two specific soluble inhibitors present, which are also isolated and characterized. Both these proteins have a MW of 10.000 daltons. Each protease forms a 1:1 complex with its inhibitor. The inhibitors are present in excess in the cells. An acid protease is able to take the inhibitor off a serine protease-inhibitor complex. This protease has been partially purified.\r\n\r\nThe proteases and inhibitors of three mutant strains have been partially purified and compared with wild type. These mutants are disturbed at the output end of the light information channel. Mutant specific changes are detected but a connection to the changed behavioral responses has not been demonstrated.\r\n\r\nChitin synthetase activity is detected in three different fractions. One form appears to be soluble; the two particulate forms can be separated by density gradient centrifugation. The high density material is probably a plasma membrane fraction.\r\n\r\nThe serine proteases are able to activate all three forms of chitin synthetase.\r\n\r\nA hypothetical cascade for chitin synthetase activation is discussed.\r\n\r\nIn an appendix the amino acid composition of cytochrome c of Phycomyces is presented." }, { "name": "Hall, David Howard", "degree": "PhD", "year": "1977", "title": "The Posterior Nervous System of the Nematode Caenorhabditis elegans", "advisor": "Russell, Richard L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:12092025-220135147", "creators": [ { "name": { "family": "Hall", "given": "David Howard" }, "id": "Hall-David-Howard", "orcid": "0000-0001-8459-9820", "display_name": "Hall, David Howard" } ], "advisors": [ { "name": { "family": "Russell", "given": "Richard L." }, "id": "Russell-R-L", "role": "advisor", "display_name": "Russell, Richard L." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/sx21-x768", "abstract": "Two young adult C. elegans have been serially sectioned\r\nand reconstructed from the tail tip forward through the\r\nanterior end of the pre-anal ganglion. Thirty-nine neurons\r\ncan be identified in the tail, twelve cells in each lumbar\r\nganglion, twelve cells in the pre-anal ganglion, and three\r\ncells in the dorso-rectal ganglion. Each cell in the tail\r\ncan be reproducibly identified on the basis of a set of\r\nmorphological features, including cell body position, fiber\r\nprojections, fiber size, and cytoplasmic appearance.\r\nEleven neurons in each lumbar ganglion are bilaterally\r\nhomologous. Many lumbar cells have sensory dendrites in\r\nthe tail. Two pairs of lumbar cells which lack sensory\r\ndendrites are prominent interneurons in the synaptic interactions\r\nof the tail. Virtually all synaptic contacts in\r\nthe tail are found in the pre-anal. ganglion. Most synapses\r\ninvolve lumbar fibers and fibers from cells whose cell\r\nbodies lie anterior to the reconstructed region. Pre-anal\r\nganglion cells themselves are relatively minor participants\r\nin these synaptic interactions .
\r\n\r\nA complete connectivity matrix has been constructed\r\nfor both animals, involving about 150 synapses in each case.\r\nCertain cells make repeated contacts with one another (up\r\nto thirteen contacts) in both animals. Other instances of\r\nnon-reproducible synapses are found, usually involving one\r\ncontact in one animal and none in the other. No self-synapses\r\nare observed, but sensory cells frequently synapse\r\nonto their bilateral homologues. Homologously paired cells\r\nmake similar sets of synaptic contacts. One class of\r\nreciprocal synapse formation is found.
\r\n\r\nEighty per cent of the contacts are dyadic, with one\r\npre-synaptic cell and two post-synaptic ones. Ten per cent\r\nof the contacts are triadic; the remaining ten per cent are\r\napparently conventional synapses with a single post-synaptic\r\nelement. Each dyadic synapse generally involves three\r\ndifferent types of neurons - none homologous to another -\r\nsuch that A \u2192 BC. Each type of pre-synaptic neuron (A)\r\ncontacts only a few preferred pairs of fibers (B,C). Most\r\ndyadic contacts are involved in multiple routes of information\r\nflow, such that A \u2192 BC and, elsewhere, B \u2192 C. The\r\nformation of dyadic synapses appears to follow strict rules\r\nwhich may reflect important factors in the development of\r\nthe nervous system.
\r\n\r\nMost synaptic interactions can be included in a simple\r\nwiring diagram by which information flows from sensory cells\r\nthrough multiple routes to converge on a pair of interneurons\r\nwhich project forward into the ventral cord.\r\nPositional information is used to identify three pairs of\r\ninterneurons which are important both in ventral cord\r\nsynaptic patterns and in the synaptic interactions of the\r\npre-anal ganglion (White et al., 1976). The nematode's\r\nbehavioral responses to sensory stimulation of the tail or\r\nthe head are analysed combining known circuitry of the\r\nventral cord, the tail, and the head.
\r\n\r\nMany neurons in C. elegans are derived after hatch\r\nfrom stereotyped sets of cell divisions (Sulston, 1976).\r\nCell body positions and detailed morphologies are used to\r\nidentify the cells of the lumbar ganglia which are derived\r\nfrom post-hatch cell divisions (in collaboration with Lois\r\nEdgar, John White, and John Sulston). Many of these cells\r\nare involved in sensory transduction in the adult tail;\r\nsome as supporting cells of the phasmids, others as ciliated\r\nsensory neurons with synaptic output in the pre-anal\r\nganglion.
\r\n\r\nSix cells in the pre-anal ganglion are derived from\r\nthe post-hatch cell divisions of the most posterior pair of\r\nprecursor cells of the ventral cord (Sulston, 1976).\r\nDaughter cells of homologous precursor cells which enter\r\nthe anterior ventral cord are known to form particular\r\nclasses of motoneurons (Sulston, 1976; White et al., 1976).\r\nOf the six pre-anal ganglion lineage cells (identified by\r\ntheir cell body positions a la Sulston, 1976), some do\r\nbecome motoneurons, possibly of the proper classes for their\r\nlineage. However, two of these cells definitely do not\r\nbecome motoneurons in the adult nematode, violating the\r\ngeneral pattern for their lineage.
\r\n\r\nSeven sensory neurons are identified in the tail.\r\nSeveral different modes of sensory transduction appear to\r\nbe utilized .
" }, { "name": "Isenberg, David Saul", "degree": "PhD", "year": "1977", "title": "Attention Demands of Processing Phonetic Information in the Perception of Dichotic Speech", "advisor": "Unknown, Unknown", "url": "https://resolver.caltech.edu/CaltechTHESIS:11192025-155757648", "creators": [ { "name": { "family": "Isenberg", "given": "David Saul" }, "id": "Isenberg-David-Saul", "display_name": "Isenberg, David Saul" } ], "advisors": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/n8v9-6765", "abstract": "We know intuitively and from dichotic shadowing studies that we\r\nmust actively listen for a message carried by speech to enter consciousness.\r\nIs such active listening necessary to process phonetic information? Theories\r\nof speech perception which have been developed to account for certain\r\nfacts of acoustic phonetics - notably the lack of invariant or segmented\r\nacoustic forms corresponding to phonemes - make implicit or explicit\r\nassumptions that cognitive processes are involved in the mental encoding\r\nof phonetic information which are thought to require attention. On the\r\nother hand, mental encoding operations which have been studied appear\r\nto proceed automatically.
\r\n\r\nIn order to explore this question, two studies employed dichotic\r\nlistening in conjunction with a secondary digit memory task to investigate\r\nclaims that phonetic distinctive features of stop consonants require capacity\r\nin short-term memory (STM) in dichotic speech perception. Experiment I\r\nfound no interference of a dichotic two-ear identification task upon STM\r\ncontingent upon number or type of feature contrast of the dichotic pair.\r\nInterference with STM was found in a dichotic discrimination task for pairs\r\nwhich contrast on place alone. In the absence of such differences for the\r\nidentification task, these results could not be interpreted to reflect demands\r\nof perceptual processing. Experiment II - designed to rule out certain\r\nartifacts - replicated the negative results of the identification task in\r\nExperiment I.
\r\n\r\nExperiment III used a probe reaction-time task to assess demands\r\nupon limited capacity during a dichotic one-ear stop consonant identification\r\ntask. No effect of number or type of feature contrast upon probe\r\nreaction-time was found for non-identical dichotic pairs. A difference\r\nin probe reaction-time between identical and non-identical pairs was attributed\r\nto the necessity of response selection in the latter case. Experiments\r\nI, II and III, taken together, demonstrate that attention is not necessary\r\nfor processing phonetic information in speech perception.
\r\n\r\nAutomatic processing of stop consonants was demonstrated in Experiments\r\nIV and V. A dichotic phoneme monitoring task was employed to\r\ndirect attention to one ear, and selective adaptation along the voicing\r\ndimension was used to measure processing contingent upon the phonetic\r\ncontents of the non-attended ear. Large effects of the non-attended channel\r\nupon selective adaptation were interpreted to reflect automatic speech-related\r\nprocessing of that channel.
\r\n\r\nTo the extent that active theories of speech perception may be construed\r\nto predict attentive processing, the present studies are taken as disconfirmation\r\nof such theories. Expansion of the search for acoustic-phonetic invariants\r\nand exploration of the interaction of higher linguistic levels with phonetic\r\nprocessing are proposed\u00b7 as two avenues of approach toward a viable passive\r\ntheory of speech perception.
\r\n\r\nAn appendix explores several different dichotic feature effects found\r\nin the present studies in terms of processing differences contingent upon\r\ntype of feature contrast.
" }, { "name": "Johnson, Carl Douglas", "degree": "PhD", "year": "1977", "title": "Multiple Molecular Forms of Cholinesterase from Elongated Animals", "advisor": "Russell, Richard L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:12152025-215621387", "creators": [ { "name": { "family": "Johnson", "given": "Carl Douglas" }, "id": "Johnson-Carl-Douglas", "display_name": "Johnson, Carl Douglas" } ], "advisors": [ { "name": { "family": "Russell", "given": "Richard L." }, "id": "Russell-R-L", "role": "advisor", "display_name": "Russell, Richard L." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/x6s3-4159", "abstract": "Cholinesterase activity in Electrophorus electricus\r\nand Caenorhabditis elegans has been fractionated, primarily\r\nby velocity sedimentation in sucrose gradients.\r\nThe electric organs of E. electricus contained five\r\nseparable forms (7,5 S, 9 S, 12 S, 14 Sand 18 S). Three,\r\nso-called native forms (9 S, 14 Sand 18 S) are insoluble\r\nin low-ionic strength solutions and they were all shifted\r\nto higher sedimentation constants by treatment with\r\nbacterial collagenase. The other two (globular) forms\r\nare soluble in low-ionic strength solutions and unaffected\r\nby collagenase. Trypsin converts the native forms to the\r\n12 S form. Neither collagenase nor trypsin affect the\r\nmolecular size of the active subunit as determined\r\nby SDS-polyacrylamide gel electrophoresis.
\r\n\r\nFour forms of cholinesterase activity were separated\r\nfrom extracts of the soil nematode Caenorhabditis elegans\r\n(5 S, 7 S, 11 S, 13 S). The smaller two forms as a pair\r\nand the larger two forms as a second pair are kinetically\r\nsimilar (Kms, substrate and inhibitor specificity).\r\nThere are significant differences between the pairs. A\r\nscreening procedure using the selective inactivation of\r\nthe 5 Sand 7 S forms by the anionic detergent sodium\r\ndeoxycholate has been applied to 89 mutants of C. elegans.\r\nOne uncoordinated mutant (BC46) apparently devoid of\r\n11 S or 13 S activity was identified. The 5 S and 7 S\r\nforms in this mutant are unaltered. The behavioral\r\ndefect is limited to the body region. Head movements\r\nand sensory responses to mechanical, chemical and osmotic\r\nstimuli seem unaltered. The mutation is X-linked.
" }, { "name": "Pearson, William Raymond", "degree": "PhD", "year": "1977", "title": "Studies on the Arrangement of Repeated Sequences in DNA", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechTHESIS:12072025-095838407", "creators": [ { "name": { "family": "Pearson", "given": "William Raymond" }, "id": "Pearson-William-Raymond", "orcid": "0000-0002-0727-3680", "display_name": "Pearson, William Raymond" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "chair", "display_name": "Bonner, James Frederick" }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." }, { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "member", "display_name": "Revel, Jean-Paul" } ], "option_major": [ "bioch" ], "doi": "10.7907/5v4r-gs82", "abstract": "Parameters of repetitive DNA sequence organization have been\r\nmeasured in the rat, Drosophila and the pea genomes.
\r\n\r\nExperiments using melting, hydroxyapatite binding and single strand\r\nnuclease digestion have been used to measure the number length and\r\narrangement of repeated DNA sequences in the rat. About 20% of rat DNA\r\nis repeated 3000 fold. Half of the sequences are 200 - 400 nucleotides\r\nlong while the remainder are longer than 1500 nucleotides. Rat repeated\r\nDNA sequences are interspersed among 2500 nucleotide long single copy\r\nsequences.
\r\n\r\nStudies on the long and short repeated DNA sequences of the rat\r\nshow that the long repeated sequences are also 3000-fold repeated.\r\nCross-hybridization of isolated long and short repeated sequences and\r\nhydroxyapatite binding interspersion experiments indicate long and short\r\nrepeated DNA may share sequences, although this may be due to\r\ncross-contamination. Self-renaturation, melting and electron microscopy\r\nof long repeated DNA fragments suggest some long fragments may be\r\ncomposed of arrays of shorter repeated sequences.
\r\n\r\nA similar sensitive search has been made in Drosophila melanogaster\r\nfor short repetitive sequences interspersed with single copy sequences.\r\nFive kinds of measurements all yield the conclusion that there are few\r\nshort repetitive sequences in this genome: 1) Comparison of long and\r\nshort fragment reassociation kinetics; 2) reassociation kinetics of\r\nlong fragments driven by an excess of short fragments; 3) measurement\r\nof the size of repeated fragments after S-1 nuclease digestion; 4)\r\nmeasurement of the hyperchromicity of repeat sequence bearing fragments\r\nof different lengths; 5) direct assay by kinetics of reassociation of\r\nthe amount of single copy sequence present on 1200 nucleotide long\r\nfragments which also contain repetitive sequences.
\r\n\r\nRenaturation of pea DNA has been used to estimate the size of the\r\npea genome and the fraction of pea DNA containing repeated DNA\r\nsequences. Pea DNA renaturation and single copy tracer hybridization\r\nindicate the size of the pea genome is 0.45 pg. More than 70% of pea\r\nDNA is repeated from 100 to 5000 times.
" }, { "name": "Ready, Donald Furner", "degree": "PhD", "year": "1977", "title": "Development of the Drosophila Retina", "advisor": "Benzer, Seymour", "url": "https://resolver.caltech.edu/CaltechTHESIS:11242025-222023123", "creators": [ { "name": { "family": "Ready", "given": "Donald Furner" }, "id": "Ready-Donald-Furner", "orcid": "0000-0003-3316-4207", "display_name": "Ready, Donald Furner" } ], "advisors": [ { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "advisor", "display_name": "Benzer, Seymour" } ], "committee": [ { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "chair", "display_name": "Benzer, Seymour" }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "member", "display_name": "Mitchell, Herschel K." }, { "name": { "family": "Hudspeth", "given": "A. James" }, "id": "Hudspeth-A-J", "role": "member", "display_name": "Hudspeth, A. James" }, { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "member", "display_name": "Revel, Jean-Paul" }, { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "member", "display_name": "Lewis, Edward B." }, { "name": { "family": "Russell", "given": "Richard L." }, "id": "Russell-R-L", "role": "member", "display_name": "Russell, Richard L." } ], "option_major": [ "neurobio" ], "doi": "10.7907/2n5e-5q95", "abstract": "Pattern formation in the Drosophila retina proceeds\r\nby the recruitment of cells, along a morphogenetic front,\r\ninto a lattice. At the advancing front, marked by a dorso-ventral\r\nfurrow in the eye imaginal disc, cells are organized\r\ninto ommatidial precursors, each containing cells destined\r\nto become photoreceptors 2, 3, 4, 5, and 8. Behind the\r\nfront, a mitotic wave produces photoreceptors 1, 6, and 7,\r\nplus the remaining cells needed to complete the ommatidia.\r\nDuring the third larval instar, the front sweeps anteriorly\r\nacross the eye disc, leaving a highly ordered pattern in\r\nits wake. Preceding the dorso-ventral furrow is a groove\r\nthat bisects the eye disc into dorsal and ventral halves\r\nand presumably plays a role in establishing the equatorial\r\nsymmetry line.
\r\n\r\nCell lineage plays little role in pattern formation in\r\nthe eye. Genetic mosaics show that the cells of each ommatidium\r\nare not derived from a single mother cell; the\r\ncells appear to be recruited at random at the morphogenetic\r\nfront. Similarly, the mirror symmetry above and below the\r\nequator is not established by a clonal mechanism; a single\r\nclone can contribute cells to ommatidia on both sides of\r\nthe equator.
" }, { "name": "Scott, Margaret Yoshiko Iwaki", "degree": "PhD", "year": "1977", "title": "Studies on Selective Neuroregeneration in Lower Vertebrates", "advisor": "Sperry, Roger Wolcott", "url": "https://resolver.caltech.edu/CaltechTHESIS:02092026-191025706", "creators": [ { "name": { "family": "Scott", "given": "Margaret Yoshiko Iwaki" }, "id": "Scott-Margaret-Yoshiko-Iwaki", "display_name": "Scott, Margaret Yoshiko Iwaki" } ], "advisors": [ { "name": { "family": "Sperry", "given": "Roger Wolcott" }, "id": "Sperry-R-W", "role": "advisor", "display_name": "Sperry, Roger Wolcott" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/xxnb-e862", "abstract": "The specificity with which terminal axons of the\r\nperipheral and central nervous systems functionally innervate\r\nforeign fields was studied in frog and goldfish.
\r\n\r\nThe effects of competitive reinnervation by left and\r\nright cutaneous nerves were carried out in frog in an\r\neffort to test for the existence of left-right biochemical\r\ndifferentiation of neural tissue. The dorsal cutaneous\r\nnerves were cut, transposed and allowed to regenerate under\r\ndifferent conditions of denervation and competition and the\r\nresultant reinnervation and reflex patterns were determined\r\nby behavioral and electrophysiological mapping techniques.\r\nThere was no indication that growth patterns and functional\r\nreconnections were influenced by the specific laterality of\r\nthe fibers.
\r\n\r\nIn goldfish, the course of functional recovery of\r\nvision during the compression of the retinotectal projection\r\nthat follows hemitectal ablations was investigated with\r\nfish maintained postsurgically in diurnal and continuous-light\r\nenvironments. Behavioral and electrophysiological\r\nmapping methods indicated that vision was restored throughout\r\nthe temporal half-field, originally blinded by the\r\ntectal lesion regardless of the postoperative lighting conditions.\r\nThe scotoma diminished in an orderly anteriorposterior\r\nfashion and color discriminability was concommitant\r\nwith visual recovery. Functional topographic reorganization\r\nof the retinotectal projection implies that locus-specific\r\naffinities between retinal and tectal neurons,\r\nwhich may play a prominent role in the direction of nerve\r\ngrowth and formation of synapses under more normal circumstances,\r\nare not permanently fixed even in mature goldfish.
\r\n\r\nThe results of the present investigations indicate\r\nthat factors such as availability of terminal sites and the\r\ntendency of fibers to seek terminal connections seem to override\r\nany qualifications imposed by the existence of lateral\r\nor locus specificity on the formation of terminal connections.\r\nThe return of color vision in goldfish, however,\r\nindicates prespecification of retinal and tectal cells for\r\ncolor, and selective reconnection of neurons according to\r\ntheir specificities which survived the compensatory developmental\r\npressures created by the retinotectal size disparity.
" }, { "name": "Cross, John William, Jr.", "degree": "PhD", "year": "1976", "title": "Protein Synthesis in Chlamydomonas reinhardi: 1. Characterization of Temperature-Sensitive Mutants in Protein Synthesis. 2. Action of the Drug, Chloral Hydrate", "advisor": "McMahon, Daniel", "url": "https://resolver.caltech.edu/CaltechTHESIS:11122009-095851274", "creators": [ { "name": { "family": "Cross", "given": "John William, Jr." }, "id": "Cross-John-William", "display_name": "Cross, John William, Jr." } ], "advisors": [ { "name": { "family": "McMahon", "given": "Daniel" }, "id": "McMahon-D", "role": "advisor", "display_name": "McMahon, Daniel" } ], "committee": [ { "name": { "family": "McMahon", "given": "Daniel" }, "id": "McMahon-D", "role": "chair", "display_name": "McMahon, Daniel" }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "member", "display_name": "Bonner, James Frederick" }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "member", "display_name": "Mitchell, Herschel K." }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "member", "display_name": "Owen, Ray David" } ], "option_major": [ "biology" ], "doi": "10.7907/M5NZ-6V45", "abstract": "I. Mutants of Chlamydomonas which are temperature-sensitive in protein synthesis were analyzed. These mutants were previously shown to be unaffected in RNA synthesis or in nucleotide pool levels at the growth-restrictive temperature (33\u00b0) although amino acid incorporation is blocked at least 40%. Here it is demonstrated that exposure of these cells to non-permissive conditions causes their polysomes to break down, resulting in the accumulation of 80S ribosomes which are not bound to mRNA. There was no defect detected in either mutant when their rates of charging of any of the 20 amino acids to tRNA were measured at the restrictive temperature in vitro, or in one of them whose degree of charging of tRNA was estimated in vivo at 33\u00b0. It is concluded that the defects in these mutants result from defective initiation of protein synthesis in vivo at 33\u00b0.\r\n\r\nII. The mechanism by which the anesthetic, chloral hydrate, inhibits protein synthesis was investigated in Chlamydomonas. This drug was previously shown to inhibit protein synthesis and cell division in Chlamydomonas at concentrations similar to those which produce anesthesia in vertebrates. The incorporation of amino acids into protein is rapidly inhibited after the drug is added to cells. At the same time, polysomes in the cells partially break down into monosomes, which are not bound to mRNA, but do contain nascent peptides capable of reacting with puromycin. Maximum polysome breakdown occurs in 15-30 min and is followed by a gradual reforation of polysomes, which is complete in 4-8 hours. Amino acid incorporation remains maximally inhibited during this period (85-90% inhibition at 10 mM chloral hydrate).\r\n\r\nThe inhibition of protein synthesis by two compounds which are metabolites of chloral hydrate in other organisms was examined. Protein synthesis is inhibited by trichloroethanol, but not by trichloroacetic acid at concentrations of 10 mM. Trichloroethanol produces the same effects on polysomes as does chloral hydrate. However, in Chlamydomonas no significant amount of chloral hydrate is metabolized to either of these two substances, and so it appears that the effects which are observed are due to the action of chloral hydrate itself. A preliminary investigation of the binding of chloral hydrate to cells shows that Chlamvdomonas accumulates the drug to a higher concentration than that found in the medium.\r\n\r\nIII. An effort was made to enable the selection of mutants of Chlamydomonas conditionally defective in assembly of chloroplast membranes.\r\n\r\nChlamydomonas cells which had formed active chloroplasts were found to be killed by photooxidation in an intense light beam, and inhibition of photosynthetic electron transport afforded significant protection from this killing. Therefore it was reasoned that Chlamydomonas mutants unable to form photosynthetically active chloroplasts would be protected. It was, however, found that photooxidation does not select for mutants with reducted contents of photosynthetic pigments.\r\n" }, { "name": "Gottesfeld, Joel M.", "degree": "PhD", "year": "1976", "title": "Chromatin Structure and Gene Expression", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechTHESIS:11142025-230228251", "creators": [ { "name": { "family": "Gottesfeld", "given": "Joel M." }, "id": "Gottesfeld-Joel-M", "orcid": "0000-0002-4643-5777", "display_name": "Gottesfeld, Joel M." } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "chair", "display_name": "Bonner, James Frederick" }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "member", "display_name": "Owen, Ray David" }, { "name": { "family": "Vinograd", "given": "Jerome" }, "id": "Vinograd-J", "role": "member", "display_name": "Vinograd, Jerome" }, { "name": { "family": "Britten", "given": "Roy" }, "id": "Britten-Roy", "role": "member", "display_name": "Britten, Roy" } ], "option_major": [ "bioch" ], "doi": "10.7907/3ayy-cg55", "abstract": "Rat-liver chromatin has been separated into nuclease-sensitive\r\nand resistant fractions after mild digestion with DNAase II. The\r\nnuclease-sensitive material is further fractionated into Mg++-soluble\r\nand insoluble chromatin fractions. The kinetics of production of\r\nthese chromatin fractions have been investigated. After a brief\r\nenzyme treatment (5 min under standard conditions), 11% of the input\r\nchromatin DNA is found in the Mg++-soluble fraction. This DNA has a\r\nweight-average single strand length of about 400 nucleotides and, as\r\ndetermined by renaturation kinetics, comprises a subset of middle\r\nrepetitive and nonrepetitive DNA sequences of the rat genome. Cross-reassociation\r\nexperiments show that a fractionation of whole genomal\r\nDNA sequences has been achieved. Moreover, the Mg++-soluble fraction\r\nof liver chromatin is enriched in nonrepeated sequences coding for\r\nliver RNA but not for brain RNA. Fractionation does not depend on\r\nsome general property of chromatin but is specific with regard to the\r\ntemplate activity of the tissue from which the chromatin was obtained.\r\nThe Mg++-soluble, template-active fraction is enriched five-fold in\r\nDNA sequences complementary to RNA.
\r\n\r\nThe Mg++-soluble fraction is enriched in nonhistone chromosomal\r\nproteins and depleted in histone protein. Histone I (fl) is absent\r\nfrom the Mg++-soluble active fraction. About half of the DNA of both\r\nMg++-soluble and Mg++-insoluble fractions is resistant to prolonged\r\ndigestion with DNAase II or staphylococcal nuclease. The nuclease-\r\nresistant structures of inactive (Mg++-insoluble) chromatin are DNAhistone\r\ncomplexes which sediment at 11-13S. Two nuclease-resistant\r\nspecies are present in active chromatin. These particles sediment\r\nat 15 and 20S, respectively, and contain DNA, RNA, histone and nonhistone\r\nproteins. Thermal denaturation studies suggest that the\u00b7\r\nDNA of active chromatin is complexed primarily with nonhistone proteins.
" }, { "name": "Harris, William Anthony", "degree": "PhD", "year": "1976", "title": "Color Vision in Drosophila", "advisor": "Benzer, Seymour", "url": "https://resolver.caltech.edu/CaltechTHESIS:05052026-230236453", "creators": [ { "name": { "family": "Harris", "given": "William Anthony" }, "id": "Harris-William-Anthony", "orcid": "0000-0002-9995-8096", "display_name": "Harris, William Anthony" } ], "advisors": [ { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "advisor", "display_name": "Benzer, Seymour" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "neurobio" ], "doi": "10.7907/cbww-8k03", "abstract": "In Chapter I an historical introduction to the study\r\nof invertebrate color vision is given, with emphasis on\r\ninsects, especially Drosophila.
\r\n\r\nIn Chapter II three mutations which eliminate specific\r\ntypes of photoreceptors in Drosophila are described. Of\r\nthe 8 photoreceptors in each facet, two mutations delete\r\nthe outer 6 (R1-6). The third eliminates R7, one of the\r\ntwo central photoreceptors. Double mutants were constructed\r\nin which only photoreceptor RB is present. The spectral\r\nsensitivities, photopigments, and behavioral properties of\r\nthese mutants were investigated. R1-6 have two sensitivity\r\npeaks, near 350 and 470 nm. These receptors contain a\r\nrhodopsin with these absorption peaks. It interconverts\r\nwith a metarhodopsin that absorbs around 570 nm. R7 is a\r\nUV-receptor, containing rhodopsin that absorbs around 370 nm\r\nand interconverts with a metarhodopsin which absorbs around\r\n470 nm. R8 is a non-adapting blue-receptor with a third\r\ntype of rhodopsin. The properties of these photopigments\r\naccount for the different sensitivities and spectral adaptation\r\nphenomena of the various photoreceptors. All the\r\nphotoreceptors have input into phototaxis. Spectral\r\nanalysis of this behavior provides evidence for integration\r\nof the input from the different receptors.
\r\n\r\nIn Chapter III experiments are described showing learning\r\nand color vision in Drosophila. Populations of Drosophila\r\nwere trained by alternately exposing them to two\r\nodorants, one coupled with electric shock. On testing, the\r\nflies avoided the shock associated odor. Pseudoconditioning,\r\nexcitatory states, odor preference, sensitization, habituation,\r\nand subjective bias have been eliminated as explanations.\r\nThe selective avoidance can be extinguished by\r\nretraining. All flies in the population have equal probability\r\nof expressing this behavior. Memory persists for 24 hr.\r\nAnother paradigm has been developed in which flies learn to\r\ndiscriminate between light sources of different color.
\r\n\r\nIn Chapter IV the results of these experiments are\r\ndiscussed, relating them to previous work and future directions.
" }, { "name": "Miller, Mark James", "degree": "PhD", "year": "1976", "title": "Investigations of the Mechanism of Cell Killing Induced by Actinomycin D", "advisor": "McMahon, Daniel", "url": "https://resolver.caltech.edu/CaltechTHESIS:09072017-112605896", "creators": [ { "name": { "family": "Miller", "given": "Mark James" }, "id": "Miller-Mark-James", "display_name": "Miller, Mark James" } ], "advisors": [ { "name": { "family": "McMahon", "given": "Daniel" }, "id": "McMahon-D", "role": "advisor", "display_name": "McMahon, Daniel" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/PSEK-BB55", "abstract": "At high concentration, actinomycin D kills the protozon\r\nChlamydomonas reinhardi with exponential kinetics. The rate of killing\r\nis dependent upon the temperature of incubation. This dependence\r\nis partially a function of the increased extent of binding of the \r\ndrug at the higher temperature (33\u00b0C), but sensitivity of the cell\r\nmust also be stimulated by the higher temperature. While actinomycin\r\nD probably kills cells in a reaction which requires binding to DNA,\r\nthere is no correlation between the lethal event and the inhibition\r\nof macromolecular synthesis or the breakdown of macromolecules.
\r\n\r\nI have developed a simple model to explain the difference in\r\nsensitivity of various species of RNA to actinomycin D inhibition.\r\nThis model predicts that frequently transcribed genes will be much\r\nmore sensitive to the drug than infrequently transcribed genes.
\r\n\r\nMutants of Chlamydomonas reinhardi have also been isolated which are both temperature sensitive in growth and resistant to kill\u00ading by actinomycin D. These mutants, unlike other actinomycin D\u00ad resistant cell lines, are neither impermeable to the drug nor do they excrete it at an accelerated rate. The mutants are partially temper\u00ad ature sensitive in their ability to synthesize RNA. In the presence of actinomycin D, however, RNA synthesis is partially protected at the nonpermissive temperature (and, in some cases, at the permissive temperature also) when compared to the inhibition of wild type cells.
\r\n\r\nExtraction and examination of RNA from these mutants reveals\r\nthat actinomycin D inhibits different species of RNA to different extents.
\r\n\r\nI propose that the mutants have an altered chromosomal constituent,\r\nwhich impedes the binding to the genome. At the nonpermissive\r\ntemperature the alteration is postulated to partially interfere with\r\ntranscription.
\r\n\r\nIn the course of these experiments it became necessary to determine\r\nthe maturation pathways of the ribosomal RNA species of Chlamydomonas \r\nreinhardi. Cytoplasmic rRNAs of C. reinhardi are cleaved from a\r\nsingle precursor of molecular weights 2.4 \u00b7 106 to a mature rRNA\r\n(0.69 \u00b7 106mol.wt) and a 1.4 \u00b7 106-mol. wt precursor of a mature 1.3 \u00b7 106-mol. wt rRNA. The kinetics of incorporation of radioactive label into the rRNAs suggest that the 0.69 \u00b7 106-mol. wt rRNA gene is located closer to the promotor than is the gene for the 1.4 \u00b7 106-mol. wt rRNA. The synthesis of cytoplasma rRNAs is extremely sensitive to camptothecin, an inhibitor of nuclear rRNA synthesis, but synthesis of\r\nchloroplast rRNA is quite resistant to the inhibitor. This has allowed\r\nus to demonstrate that chloroplast rRNAs are processed from precursors\r\nwhich resemble those of blue-green algae. A 1.14 \u00b7 106-mol. wt precursor\r\nis processed to the 1.07 \u00b7 106-mol. wt mature chloroplast rRNA, and\r\na 0.64 \u00b7 106-mol. wt precursor is cleaved to a 0.56 \u00b7 106-mol. wt species and then to the mature 0.54 \u00b7 106-mol. wt rRNA. This study\r\ndemonstrates two new ways in which the function of the chloroplast genome resembles those of prokaryotes more than those of the nucleus.
\r\n\r\n\r\n\r\n\r\n" }, { "name": "Audesirk, Gerald Joseph", "degree": "PhD", "year": "1975", "title": "Part I. Studies on the Organization of the Eye of Aplysia californica. Part II. Studies on the Interrelationship between Two Neuronal Circadian Oscillators in Aplysia californica", "advisor": "Strumwasser, Felix", "url": "https://resolver.caltech.edu/CaltechTHESIS:12052017-094450464", "creators": [ { "name": { "family": "Audesirk", "given": "Gerald Joseph" }, "id": "Audesirk-Gerald-Joseph", "display_name": "Audesirk, Gerald Joseph" } ], "advisors": [ { "name": { "family": "Strumwasser", "given": "Felix" }, "id": "Strumwasser-F", "role": "advisor", "display_name": "Strumwasser, Felix" } ], "committee": [ { "name": { "family": "Strumwasser", "given": "Felix" }, "id": "Strumwasser-F", "role": "chair", "display_name": "Strumwasser, Felix" }, { "name": { "family": "Wiersma", "given": "Cornelis A. G." }, "id": "Wiersma-C-A-G", "role": "member", "display_name": "Wiersma, Cornelis A. G." }, { "name": { "family": "Lester", "given": "Henry A." }, "id": "Lester-H-A", "orcid": "0000-0002-5470-5255", "role": "member", "display_name": "Lester, Henry A." }, { "name": { "family": "Pettigrew", "given": "John D." }, "id": "Pettigrew-J-D", "role": "member", "display_name": "Pettigrew, John D." } ], "option_major": [ "neurobio" ], "doi": "10.7907/BT9J-NK43", "abstract": "Part I
\r\n\r\n\r\nThe isolated eye of Aplysia californica produces a bursting\r\npattern of spontaneous compound action potentials (CAPs) when recordings \r\nare made from the optic nerve in darkness. The CAP frequency varies\r\nwith a circadian rhythm. The light response, also composed of CAPs,\r\nmay be separated into an initial phasic response and a late tonic \r\nresponse similar in form to the dark discharge. Solutions containing\r\nLa+++ or high Mg++ with low Ca++, which are expected to block chemical\r\nsynapses, stop the dark discharge and tonic light response but not the \r\nphasic light response. The suppression of dark discharge by high Mg++\r\nwith low Ca++ is usually temporary, lasting about 0.5 to 4 hours.\r\nSynchrony of the CAPs is not affected by either La+++ or high Mg++, low \r\nCa++. These results indicate that the dark discharge is driven through \r\nchemical synapses, but the light response is not. Replacement of \r\nchloride in the bathing medium by propionate, which uncouples electrical \r\njunctions in the crayfish septate axon, abolishes all CAPs for varying \r\nperiods of time, usually several hours. Propionate leaves the ERG\r\nintact and the optic nerve electrically excitable. A model for inter-neuronal \r\nconnections in the Aplysia eye is constructed from these data. \r\nIt is postulated that the light response is initiated in the photoreceptors, \r\nwith the receptor depolarization passing through electrical \r\nsynapses to higher order cells. Spikes are produced in these cells and \r\npass down their axons in the optic nerve. Spontaneous dark activity\r\nalso represents spiking in these higher order cells, but is initiated\r\nthrough chemical synapses by pacemaker cell(s). Synchrony of the CAPs \r\nis facilitated by electrical synapses between higher order cells. In\r\nlow Ca++ media, these higher order cells may become hyperexcitable to\r\nthe point of autoactivity.
\r\n\r\n\r\n\r\nPart II
\r\n\r\n\r\nThe circadian rhythm of spike output of the single neuron R15 \r\nin the isolated PVG of Aplysia californica can be entrained in vivo \r\nby light. The timing of the rhythm depends not only on the lighting\r\nschedule to which the animal was exposed prior to dissection, but also \r\non the time of dissection relative to that light schedule. Entrainment \r\nof the rhythm by light proceeds very slowly, if at all, in Aplysia \r\nwith their eyes removed. An indirect inhibitory neural pathway is\r\nshown to exist between the eyes and R15, but cutting nervous \r\nconnections containing this and any other neural paths from the eyes \r\nto R15 does not prevent entrainment by light in a majority of animals. \r\nIn vitro experiments show that the eyes can influence the activity \r\nof R15 even when the eyes and the PVG are not neurally connected.\t\r\nThe eyes therefore must release a water soluble factor which can affect\r\nR15, either directly or through some other neurons in the PVG. If the \r\neyes and PVGs from different animals are incubated together for\r\nseveral days and then separated, the subsequent spiking behavior of R15 \r\nis similar to that observed after in vivo entrainment to a light \r\nschedule equivalent in phase to the circadian rhythm of the eyes in \r\nvitro. It is a strong possibility that the factor released by the\r\neyes can entrain the circadian rhythm of R15.
\r\n\r\n \r\n\r\n" }, { "name": "Compton, John Lee", "degree": "PhD", "year": "1975", "title": "Specific DNA Restriction Fragments as Internal Markers in the Electron Microscope: Superimposing the \u0278X174 Genetic Map on \u0278X174/S13 DNA Heteroduplexes", "advisor": "Sinsheimer, Robert L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:09152021-165317789", "creators": [ { "name": { "family": "Compton", "given": "John Lee" }, "id": "Compton-John-Lee", "display_name": "Compton, John Lee" } ], "advisors": [ { "name": { "family": "Sinsheimer", "given": "Robert L." }, "id": "Sinsheimer-R-L", "role": "advisor", "display_name": "Sinsheimer, Robert L." } ], "committee": [ { "name": { "family": "Sinsheimer", "given": "Robert L." }, "id": "Sinsheimer-R-L", "role": "chair", "display_name": "Sinsheimer, Robert L." }, { "name": { "family": "Vinograd", "given": "Jerome" }, "id": "Vinograd-J", "role": "member", "display_name": "Vinograd, Jerome" }, { "name": { "family": "Wood", "given": "William Barry" }, "id": "Wood-W-B", "role": "member", "display_name": "Wood, William Barry" }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" } ], "option_major": [ "biology" ], "doi": "10.7907/nr2r-8c58", "abstract": "Chapter I
\r\n\r\n\u0278X174 RF II* molecules were constructed in vitro by annealing circular \u0278X174 viral DNA and linear \u0278X174 complementary strand DNA. When a specific fragment of \u0278X174 RF purified from a digestion with the restriction endonuclease Hind from Hemophilus influenzae was included in the renaturation mixture \u0278X174 RF II molecules containing a loop corresponding in size to the fragment were seen. This was confirmed for three different DNA fragments.
\r\n\r\n\u0278X174 RF II molecules were constructed in the presence of two restriction fragments. In addition to molecules containing one loop corresponding in size to one of the fragments molecules containing two loops were seen. The two loops corresponded in size to the two restriction fragments and the distances between the two loops corresponded to the distances between the two fragments on the Hind cleavage map of \u0278X174 RF.
\r\n\r\nBy including two fragments of distinguishable size that map asymmetrically on the cleavage map in the renaturation mixture, \u0278X174 RF II molecules were constructed which could be oriented on the cleavage map. Using the two loops as internal markers the \u0278X174 genetic map was superimposed on the electron micrographs of these molecules.
\r\n\r\nChapter II
\r\n\r\n\u0278X174/S13 DNA heteroduplexes were constructed by annealing purified \u0278X174 complementary strand DHA and S13 viral DNA. The heteroduplexes were mounted for electron microscopy under a series of increasingly denaturing conditions and photographed. The micrographs were measured and oriented to form a map showing the progressive denaturation of various regions of the molecule.
\r\n\r\nSuch heteroduplex molecules were totally denatured and mixed with a denatured specific fragment of \u0278X174 RF* purified from a digestion with the restriction endonuclease Hind of Hemophilus influenzae. After renaturation the molecules were mounted for electron microscopy under standard conditions. Each of three different \u0278X174 RF fragments could be seen in a different; specific region of the heteroduplex and could be oriented with respect to the others to correspond to the known \u0278X174 cleavage map.
\r\n\r\nThe \u0278X174 genetic map was thus superimposed on the heteroduplex denaturation map by using the included fragments as reference points. Under mildly denaturing conditions three regions of the heteroduplex are conserved as double-stranded: two regions of about 10% of the \u0278X174 genome each in genes A and H and a region of about 25% of the \u0278X174 genome covering gene E and most of gene F. Under increasingly denaturing conditions only two small duplex regions of 2 and 4% are conserved, one each in genes E and F.
" }, { "name": "Elliott, Ellen Jeanne", "degree": "PhD", "year": "1975", "title": "Chemical Properties and Physiological Activity of a Neuroactive Component from the Venom of Conus Californicus", "advisor": "Raftery, Michael A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03022022-182739139", "creators": [ { "name": { "family": "Elliott", "given": "Ellen Jeanne" }, "id": "Elliott-Ellen-Jeanne", "display_name": "Elliott, Ellen Jeanne" } ], "advisors": [ { "name": { "family": "Raftery", "given": "Michael A." }, "id": "Raftery-M-A", "role": "advisor", "display_name": "Raftery, Michael A." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/6kgj-m088", "abstract": "A small molecular weight component, named conusine, was purified from the venom of the marine snail Conus californicus. The solubility properties, enzyme susceptibilities, heat and pH sensitivity, spectral characteristics and reactivity with various indicator reagents of conusine were determined. Conusine was found to inhibit the excised, spontaneously beating heart of the clam Mercenaria mercenaria. This inhibition was blocked by those antagonists which were found to block acetylcholine inhibition of the heart. In addition, in the medial cells of the Aplysia pleural ganglion iontophoretically applied conusine was found to activate selectively a cholinergic receptor which mediates a slow hyperpolarization of the cell due to an increase in K+ permeability. Conusine was concluded to be a cholinergic agonist specific for this type of slow hyperpolarizing receptor. From additional pharmacological studies of various cholinergic agonists and antagonists on the clam heart, this tissue was hypothesized to contain the same type of slow hyperpolarizing cholinergic receptor as the medial cells of the Aplysia pleural ganglion.
" }, { "name": "Flory, P. John", "degree": "PhD", "year": "1975", "title": "Studies of the Replicative Intermediates and of the Structure of Animal Cell Mitochondrial DNA", "advisor": "Vinograd, Jerome Rubin", "url": "https://resolver.caltech.edu/CaltechETD:etd-08032006-133035", "creators": [ { "name": { "family": "Flory", "given": "P. John" }, "id": "Flory-P-John", "display_name": "Flory, P. John" } ], "advisors": [ { "name": { "family": "Vinograd", "given": "Jerome Rubin" }, "id": "Vinograd-J-R", "role": "advisor", "display_name": "Vinograd, Jerome Rubin" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/ZDPV-B755", "abstract": "Studies on the replication and structure of animal cell mitDNA are reported in the four sections of this thesis.\r\n\r\nPart I: Density Labeling of HeLa mitDNA with 5-Bromodeoxyuridine: Bromouracil labeling of the mitochondrial DNA in exponentially growing HeLa cells produces two hybrid mitochondrial DNA species, with density shifts of 41.9 and 54.0 mg/ml relative to unlabeled mitochondrial DNA, as well as heavy mitochondrial DNA, with a shift of 95.3 mg/mi. The two hybrid species result from the difference in thymine composition of the complementary strands of mitochondrial DNA. In addition, mitochondrial DNA with a density intermediate between the hybrid and unlabeled species was found. This quarter heavy mitochondrial DNA represents 25% (w/w) of the total DNA after eight hours of labeling, and forms two peaks with shifts of 20.6 and 27.0 mg/ml relative to unlabeled mitochondrial DNA. 70% (w/w) of the quarter heavy mitochondrial DNA is in catenated forms, while 30% (w/w) is monomeric. Degradation of the catenanes by shearing of purified quarter heavy mitochondrial DNA results in the appearance of hybrid and unlabeled mitochondrial DNA bands, demonstrating that the quarter heavy catenanes contain both hybrid and unlabeled submolecules. The implications of the structure of the quarter heavy catenanes on the mechanism of formation of catenanes are discussed.\r\n\r\nPart II: The Isolation of Complementary Strands of mitDNA and the Mapping of the rDNA and tRNA's on the L Strand Relative to the Origin of Replication: The alkali lability of mitDNA makes the preparation of intact separated strands for electron microscope mapping studies difficult. Even at pH 11.6 (the minimum pH required for strand separation of singly nicked HeLa mitDNA) the strands suffered an average of one additional nick per strand during the alkaline buoyant CsCl banding. Covalently closed mitDNA did not nick under these conditions. The rapid nicking of the BrUra-labeled strands enabled preparations of L strands containing 30 wt% single stranded circles to be obtained from covalently closed BrUra hybrid mitDNA.\r\n\r\nThe relative positions of the ribosomal RNA gene complement (rDNA) and the tRNA sites on the L strand relative to the origin of replication (7S DNA) were determined by hybrid mapping in the electron microscope using the above L strand preparation. The center of the rDNA is located almost directly opposite (0.5 G) the 7S DNA, and the L3 tRNA site is very near to one end of the 7S DNA.\r\n\r\nPart III: MitDNA Replicative Intermediates: Isolation by Benzoylated DEAE-Cellulose Chromatography and Enzymatic Analysis of Structure: Replicative intermediates of LA9 mitDNA were partially purified from clean duplex DNA by virtue of the binding of their single stranded regions to benzoylated DEAE-cellulose. The purification was incomplete because the clean duplex DNA elutes as though a portion of the molecules contain denatured regions (or regions which denature in response to the column environment). Similar results were obtained with closed and nicked PM2 viral DNA. SV40 viral DNA eluted normally.\r\n\r\nThese enriched preparations of replicative intermediates and the clean duplex DNA free of replicating forms were subjected to enzymatic analysis with T4 DNA polymerase. The results show that: (1) the nicks in the upper band clean duplex DNA are not preferentially located in either strand; (2) the gapped molecules in the upper band have a greater overall deficiency of L than H strand DNA by a ratio of 2.5 to 1 (implying that these molecules probably represent displaced H strands with incomplete complement synthesis); and (3) the 7S DNA in the lower band D-loop forms is not measurably extended by the enzyme, although label was incorporated (suggesting that a superhelix restriction on the further extension of the D-loop exists in isolated mitDNA). Attempts to covalently close the upper band clean duplex DNA with combinations of E. coli exonuclease III, polymerase I, and ligase, as well as T4 polymerase, were unsuccessful.\r\n\r\nPart IV: A Method for the Detection of Ribonucleotides in mitDNA: A method for the detection of ribonucleotides in mitDNA has been partially developed. The procedure involves alkaline hydrolysis to expose ribonucleotides at the 3' ends of mitDNA fragments, removal of terminal phosphates with alkaline phosphatase, labeling by oxidation-reduction with NaIO4-3H-NaBH4, removal of unincorporated 3H counts by extensive dialysis, spleen acid DNase II and spleen exonuclease digestion to ribonucleoside trialcohols and deoxynucleoside-3'-monophosphates, removal of the latter on DEAE-cellulose, and identification of labeled ribonucleoside trialcohols by thin layer chromatography and fluorography. The feasibility of the analysis is demonstrated, but attempts to label mitDNA were unsuccessful due to the unreliability of commercial preparations of 3H-NaBH4 of the high specific activity required for these experiments. The method is reported here in the event that high specific activity 3H-NaBH4 becomes available." }, { "name": "Jan, Yuh Nung", "degree": "PhD", "year": "1975", "title": "I. Chitin Synthetase and Sensory Transduction in Phycomyces. II. The Avoidance Response, the House Growth Response and the Rheotropic Response of Phycomyces", "advisor": "Delbruck, Max", "url": "https://resolver.caltech.edu/CaltechETD:etd-05182006-082819", "creators": [ { "name": { "family": "Jan", "given": "Yuh Nung" }, "id": "Jan-Yuh-Nung", "orcid": "0000-0003-1367-6299", "display_name": "Jan, Yuh Nung" } ], "advisors": [ { "name": { "family": "Delbruck", "given": "Max" }, "id": "Delbr\u00fcck-M", "role": "advisor", "display_name": "Delbruck, Max" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/16PP-Y948", "abstract": "Part I: Chitin Synthetase and the Sensor Transduction Processes in Phycomyces:\r\n\r\nThe response of Phycomyces sporangiophores to various stimuli shows up as changes in the elongation rate of the cell wall, a structure mainly composed of chitin fibrils. The enzyme chitin synthetase was chosen as the subject of this study for the possible role that regulation of its activity might play in the behavioral output. Properties of the enzyme: A simple assay for chitin synthetase has been developed for the flycomyces system. Enzyme prepared from Phycomyces was found to catalyze the synthesis of chitin from UDP-N-acetyl-D-glucosamine. The ion requirements, temperature dependence, buffer and pH dependence, and kinetics for this enzyme were investigated. An antibiotic Polyoxin D was found to be a competitive inhibitor of this enzyme.\r\n\r\nCellular localization: To localize the enzyme in the cell: (1) Phycomyces homogenates were fractioned through a series of differential aril isopycnic centrifugations. Each fraction was assayed for its specific activities of chitin synthetase and some membrane marker enzymes. The results suggest that chitin synthetase is a plasma membrane bound enzyme. (2) Autoradiography studies of the sporangiophone showed that this enzyme is located mainly in the growing zone of this structure.\r\n\r\nRegulations: In vivo, Phycomyces shows a positive growth response to blue light. It is demonstrated that blue light can increase the chitin synthetase activity in vitro This finding supports the idea that regulation of chitin synthetase activity plays a central role in the responses of Phycomyces sensory stimuli. Finally, the possibility of using the chitin synthetase assay as an in vitro photoresponse system in the dissection of the sensory transduction processes of Phycomces is discussed.\r\n\r\nPart II: The Avoidance Response, the House Growth Response, and the Rheotropic Response of Phycomyces:\r\n\r\nIf an object is placed about 1 mm from the growing zone of a Phycomces sporangiophore growing in air, in about 2 minutes the sporangiophore starts to bend away at a rate of about 2\u00b0/minute for as long as half an hour or more. This is called the avoidance response of Phycomyces. The purpose of this study is to find out how a sporangiophore detects a nearby object and avoids it. Electric field, electromagnetic radiation (UV, visible and IR), temperature, humidity and pressure are all excluded as the avoidance eliciting signal. The avoidance response is found to be dependent on the size and the distance of the nearby objects and independent of the compositions and surface properties of the objects.\r\n\r\nAn air current parallel to the barrier and the sporangiophore can eliminate the avoidance response. In conjunction with this key observation, the Phycomyces responses to wind (rheotropic response and wind growth response) and to enclosure (house growth response) were characterized. The key parameter in all these responses seems to be the air movement in the vicinity of the sporangiophore growing zone. All observations are compatible with the assertion that faster growth is associated with slower wind velocity. To specify in which way air movement can become a signal directly received by the sensor, we propose that the avoidance is mediated by a volatile growth effector emitted somewhere along the sporangiophore and sensed by the growing zone (the chemical self guidance hypothesis). Various possible alternative forms of the hypothesis were tested experimentally. The only remaining viable one is that the sporangiophore emits volatile growth promoting molecules continuously. The majority of the molecules are readsorbed by the growing zone before they diffuse away, and the barriers modify the distribution of the molecules by altering the ambient wind pattern. Future tests are discussed and a quantitative formulation of the model is presented in Appendices B and C." }, { "name": "Lee, Amy So-Ming Shiu", "degree": "PhD", "year": "1975", "title": "The Cleavage of \u0278X174 DNA with Restriction Endonucleases", "advisor": "Sinsheimer, Robert L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03252022-213533588", "creators": [ { "name": { "family": "Lee", "given": "Amy So-Ming Shiu" }, "id": "Lee-Amy-So-Ming-Shiu", "display_name": "Lee, Amy So-Ming Shiu" } ], "advisors": [ { "name": { "family": "Sinsheimer", "given": "Robert L." }, "id": "Sinsheimer-R-L", "role": "advisor", "display_name": "Sinsheimer, Robert L." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/3qag-c766", "abstract": "Restriction endonucleases from Hemophilus influenzae (Hin), H. parainfluenzae (Hpa) and H. aegyptius (Hae) have been prepared and purified from contaminating exonuclease activities. Using \u0278X174 RFI as substrate, Hin produces 13 specific fragments, Hpa produces 8 and Hae produces 11 fragments. These have been analyzed by polyacrylamide gel electrophoresis and their molecular size determined to be within the range of 1600 to 70 base pairs. The 5' end group of the fragments produced by the action of Hin on \u0278X RF have been identified to be adenine (50-57%), and guanine (30-40%), whereas in the case of Hpa, preliminary results show that a majority of the fragments terminate at the 5' end with cytosine.
\r\n\r\nBy analyzing partial digestion products, as well as overlapping sets of fragments produced by two different restriction enzymes, the physical order of the three sets of DNA fragments produced by hydrolysis of \u0278X RF by the Hin, Hpa and Hae restriction endonucleases have been determined. This analysis has been facilitated by the adaptation of a continuous electro-elution device to separate and recover individual DNA restriction enzyme fragments.
\r\n\r\nThe physical cleavage map has been in turn related to the \u0278X genetic map by the fact that many \u0278X174 mutations are available and that methods have been developed by which restriction enzyme fragments may be assayed to determine which genetic loci they contain.
\r\n\r\nWith the availability of the \u0278X174 cleavage map, it is possible to locate the unique, methylated cytosine of the \u0278X genome. Bacteriophage \u0278X DNA is labeled in vivo with [3H-methyl]methionine and used as template for in vitro synthesis of the complementary strand. The resultant RF DNA is then cleaved in separate experiments with the Hin and Hae enzymes. The DNA fragments are analyzed by polyacrylamide gel electrophoresis. It is concluded that the single methyl group in the viral DNA is located at a specific region of the \u0278Xl74 genome, very likely in gene H.
" }, { "name": "Rohwer, Robert George", "degree": "PhD", "year": "1975", "title": "The Maturation of Bacteriophage \u0278Xl74: the Isolation and Characterization of Subviral Particles", "advisor": "Sinsheimer, Robert L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:11052021-183302539", "creators": [ { "name": { "family": "Rohwer", "given": "Robert George" }, "id": "Rowher-Robert-George", "display_name": "Rohwer, Robert George" } ], "advisors": [ { "name": { "family": "Sinsheimer", "given": "Robert L." }, "id": "Sinsheimer-R-L", "role": "advisor", "display_name": "Sinsheimer, Robert L." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/csff-mr85", "abstract": "A search was conducted for possible intermediates in the assembly of bacteriophage \u0278Xl74. Artificial lysates of \u0278Xl74 infected cells labeled with either amino acids or nucleotides were fractionated by velocity sedimentation and the viral DNAs and infection specific proteins analysed by recentrifugations and electrophoreses. Infection specific proteins were detected by comparing the incorporation patterns of differentially labeled infected and uninfected cells. Many experiments employed 3H, 14C and 32P labels simultaneously and an exact solution to the discriminator ratios equations for three channel scintillation counting was derived to process these data.
\r\n\r\nA major proportion of the phage structural proteins that had not yet been incorporated into phage was observed in a very unstable 111S particle composed of \u0278X proteins F, G and D all three of which are also required for \u0278X SS DNA synthesis. The 111S particle does not appear to contain DNA. The D protein, present in large proportional amounts, is not found in whole virions. Depending upon the storage buffer, the 111S particle degrades into 12S (F,G protein), 9S (G protein) and 2.5S (D protein) subunits. These same particles are also observed in whole lysates. However, conditions can be found for which the 9S and 12S particles are not present suggesting that they may be 111S particle decomposition artifacts as opposed to in vivo particles. The properties of the 111S particle are those expected of a procapsid although this role in the infection has not been demonstrated. Analysis of the proteins in the 70S region of the gradient revealed that a similar D protein containing particle (distinct from the 70S lysis artifact) sediments there.
\r\n\r\nApproximately 10% of the total \u0278X replicative intermediate DNA in these gradients sedimented in the 70S - 114S regions. After deproteinization this DNA resedimented at the slower velocities expected for free replicative intermediates, indicating that it may have been associated with \u0278X proteins. This associated protein moiety is obscured by the much more extensively labeled pools of whole virus, 1118 particle and the 70S lysis artifact. It could, however, be the same as the 111S particle.
\r\n\r\nA radioimmunoassay was developed for \u0278X viral proteins and was employed to show that replicating intermediates in SS DNA synthesis (sedimenting from 20 - 30S), but not the \"resting\" RF II form DNA, are tightly complexed with a \u0278X viral antigen. A major peak (the 20S particle) of infection specific protein also sediments in this region. However any association of the DNA replicative intermediates with this protein peak is too fragile to account for the serum binding result. The concentration of DNA-binding antigen is below the resolving power of the amino acid labeling technique.
\r\n\r\nThe infection specific 20S protein peak contains \u0278X gene F protein, lesser amounts of other infection-specific components and a host protein (the major constituent); the host protein is also observed to sediment at 208 in uninfected cells. This protein has a molecular weight of about 67,000 daltons and is one of three \u0278X stimulated host proteins with molecular weights greater than that of the largest \u0278X coded protein, A'.
\r\n\r\nThe \u0278X174 literature is reviewed with an emphasis on those observations that bear upon the \u0278X174 assembly process. A model of the \u0278X maturation process incorporating the major features of our present knowledge is proposed as a working hypothesis for future study.
" }, { "name": "Rosenberg, Suzanne Thelma Ostrand", "degree": "PhD", "year": "1975", "title": "Studies of Bovine Blood Cell Surfaces", "advisor": "Owen, Ray David", "url": "https://resolver.caltech.edu/CaltechETD:etd-09222004-140527", "creators": [ { "name": { "family": "Rosenberg", "given": "Suzanne Thelma Ostrand" }, "id": "Rosenberg-Suzanne-Thelma-Ostrand", "display_name": "Rosenberg, Suzanne Thelma Ostrand" } ], "advisors": [ { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "advisor", "display_name": "Owen, Ray David" } ], "committee": [ { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "chair", "display_name": "Owen, Ray David" }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "member", "display_name": "Hood, Leroy E." }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "member", "display_name": "Mitchell, Herschel K." }, { "name": { "family": "Revel", "given": "Jean-Paul" }, "id": "Revel-J-P", "role": "member", "display_name": "Revel, Jean-Paul" } ], "option_major": [ "immun" ], "doi": "10.7907/SCPF-E841", "abstract": "The cell surface expression of genetically defined bovine red cell antigens has been studied by electron microscopic and serological techniques. Electron microscopic cell surface localization of specific antigens on bovine red cells has been achieved with the use of an indirect labeling reagent: hemocyanin glutaraldehyde coupled to rabbit-antibovineimmunoglobulin (Hcy-RABI). When the specific antigenic sites on the cell surface are combined with their corresponding antibodies, the secondary application of Hcy-RABI serves to visualize these sites for electron microscopy.\r\n\r\nSerological dosage reagents for the Z antigen are known to differentiate Z homozygotes (Z/Z) from heterozygotes (Z/-) in terms of the kinetics of complement mediated hemolysis. In the present study, cells homozygous for the Z antigen and saturated with anti-Z antibody were found to take up approximately twice as much Hcy-RABI as cells heterozygous for Z; cells negative for Z showed only background labeling values. Cells possessing the J antigen, a soluble serum substance secondarily absorbed to the red cell surface, were also examined for their quantitative uptake of hemocyanin. Many intergrades of J positive cells exist, ranging from cells which require large amounts of anti-J antibody for complement mediated lysis to cells which are lysed by minute quantities of specific antibody. The quantity of label taken up by a sampling of cells was found to be inversely related to the amount of antibody necessary to lyse those cells.\r\n\r\nSequential double labeling studies were conducted to characterize the cell surface steric configurations of antigens whose genes reside in 1) the same blood group system; 2) different blood group systems; and 3) cis versus trans conformations within a system. In no case was steric hindrance found. This result indicates that each antigenic determinant examined is spacially distant from others; it suggests that the determinants may be coded for by distinct genes, and that the antigens labeled are not a series of determinants on a common backbone macromolecule. Sequential double labeling of one set of antigens gave a value which was twice the sum of the two single label values. This phenomenon was noted only for one particular pair of antigens, and only on cells treated initially with one of the antisera. The increased uptake of the second antibody was highly specific. This observation suggests that new antigenic sites are revealed in the presence of bound antibody directed against another specificity, on cells labeled for this particular pair of antigens.\r\n\r\nConcanavalin A (Con A) binding experiments on trypsinized and nontrypsinized cells strongly indicate that the Con A receptor and the A antigen are molecular unique cell surface entities. Trypsinization of A positive cells caused increased binding of anti-A, accompanied by clustering of the A antigen sites and of the intramembranous particles seen in freeze-fracture experiments. These phenomena were accompanied by cell agglutination.\r\n\r\nUsing bovine red cell blood typing reagents in a leukocyte microcytotoxicity system, bovine leukocytes were found to have specific surface antigens. In this preliminary study there is no obvious association between leukocyte antigens and red cell antigens of any individual animal. The leukocyte and erythrocyte antigenic systems appear to be distinct from each other." }, { "name": "Birdwell, Charles Ray", "degree": "PhD", "year": "1974", "title": "Studies on the Infection of Animal Cells with Sindbis Virus: Adsorption, Cell Surface Modification, and Maturation", "advisor": "Strauss, James H.", "url": "https://resolver.caltech.edu/CaltechETD:etd-09292005-083645", "creators": [ { "name": { "family": "Birdwell", "given": "Charles Ray" }, "id": "Birdwell-Charles-Ray", "display_name": "Birdwell, Charles Ray" } ], "advisors": [ { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "advisor", "display_name": "Strauss, James H." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/W867-FB70", "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\r\n\r\nThe surface replica technique was used to study the distribution of Sindbis virus receptors on chick embryo fibroblasts. Using the position of adsorbed Sindbis virions on the cell surface to indicate the position of Sindbis virus receptors, an even distribution of Sindbis virus receptors over the cell surface was observed if the cells had been previously fixed with glutaraldehyde before adsorption of virus. The number of receptors observed varied from cell to cell, being on the order of [...] per cell. Occasionally areas of the cell surface with different concentrations of virus receptors were seen on the same cell. The virus particle-receptor complexes were found to move laterally within the plasma membrane, and the virus particles appeared clustered on the cell surface, when unfixed cells were adsorbed with Sindbis virus at 4 C.\r\n\r\nThe insertion of Sindbis virus glycoproteins into the cell surface was studied by an indirect electron microscopic labeling technique, which involved treating infected cells sequentially with rabbit anti-Sindbis IgG followed by hemocyanin-conjugated goat (anti-rabbit IgG) IgG, and examining the cells in the electron microscope by the surface replica technique; the position of hemocyanin on the cell surface was used to indicate the position of viral glycoproteins. Sindbis virus glycoproteins were detected at the surface of chick embryo fibroblasts by 2 hours after infection. When infected cells were prefixed with glutaraldehyde before labeling at 37 C, the distribution of virus glycoproteins in the cell surface was fairly even, although a slight clustering was noticed early in infection. However, when infected cells were labeled at 37 C without prefixation, the hemocyanin was clustered, suggesting that the viral glycoproteins moved laterally within the plasma membrane after treatment with the antibody.\r\n\r\nThe modification of the cell surface by Sindbis virus was also studied by examining the agglutination of infected cells by two of the plant lectins, concanavalin A and Ricinus communis agglutinin. By 3-3.5 hours after infection, Sindbis virus-infected chick embryo fibroblasts were more agglutinable by these lectins than uninfected cells. Evidence is presented that this increase in agglutination was not due to an increase in the number of lectin binding sites on the cell surface.\r\n\r\nUsing the surface replica technique, the maturation of Sindbis virus in baby hamster kidney cells and chick embryo fibroblasts was studied. Sindbis virus was found to bud through certain areas of the cell surface in patches, and through virus-specific processes - extending only from the cell periphery; budding virions were also seen on the undersides of infected cells. The virus-specific processes were also seen in cells infected with vesicular stomatitis virus, but budding through processes was not a major mode of virus release in these cells. Electron microscopic examination of thin-sections of Sindbis virus-infected cells revealed that these processes contained viral nucleocapsids in the process of budding. The maturation of Sindbis virus may be somewhat dependent on the structural integrity of cellular microtubules and microfilaments, as both colchicine and cytochalasin B were found to inhibit Sindbis virus release.\r\n\r\nA temperature-sensitive mutant of Sindbis virus, ts-103, was found to bud much differently than wild type Sindbis virus. Cells infected with ts-103 contained abnormal virus-specific processes extending from the cell edge; these mutant processes often contained large virus particles containing many viral nucleocapsids." }, { "name": "Chong, Ming Ta", "degree": "PhD", "year": "1974", "title": "Investigation of Chromatin Bound Enzymes", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechETD:etd-11152005-155825", "creators": [ { "name": { "family": "Chong", "given": "Ming Ta" }, "id": "Chong-Ming-Ta", "display_name": "Chong, Ming Ta" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/18PT-FD16", "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\r\n\r\nPart I. Rat liver chromatin contains a neutral protease with a marked preference for chromosomal proteins as substrates. The enzyme has been purified 705-fold from chromatin by salt extraction, chromatography on Bio-Rex 70, Sepharose 6B, calcium phosphate gel and QAE Sephadex. The enzyme has a molecular weight of 200,000 with two identical subunits of molecular weight 100,000. It attacks rat liver histones, NHC proteins and L-poly-lysine preferentially, is essentially inactive with rat liver cytosol proteins, and slowly degrades caseine, L-poly-arginine and protamines. The [...] for histones is 0.5 mg/ml; for NHC proteins [...] is 1 mg/ml. The enzyme is quite stable when stored at -20[degrees] for 4 months. Activity is diminished to 50% by heating to 62[degrees] for 15 min and totally destroyed at 70[degrees]. The enzyme has an optimal pH at 7.0 and a half maximal activity at pH 6.0, suggesting that a histidine residue is involved in catalysis. It is inhibited by DFP and PMSF, suggesting that a serine residue is involved. Hg++ inhibits enzyme activity, suggesting sulfhydryl group is important for enzyme activity. High salt (above 1M NaCl) inhibits enzyme activity completely but reversibly. The enzyme needs divalent ions as activators; especially potent is Mn++ (6-8 mM) which stimulates activity about 2 fold. The isolated enzyme appears to be similar to that responsible for the endogeneous degradation of histones in chromatin. The susceptibility of the five histone fractions to proteolysis is critically dependent upon whether or not the histones are complexed with DNA. In the intact nucleohistone four major histones are rather resistant to proteolytic attack, while histone I is rapidly attacked. If histones are freed from DNA all the histone molecules are attacked at about the same rate except histone I, which is relatively resistant.\r\n\r\nPart II. Rat liver chromatin also contains a nonspecific esterase which cleaves the artificial substrate--[...]-tosyl arginine methyl ester (TAME). The enzyme has been purified to 510 fold from chromatin by salt extraction, chromatography on Bio-Rex 70, Sephadex G-200, calcium phosphate gel and SE Sephadex. The molecular weight of the purified enzyme is estimated to be about 15,000. The enzyme has an optimal pH at 8.2 and half maximal activities at 6.9 and 10.5, suggesting that histidine and lysine residue might be involved in catalysis. It is inhibited by DFP and PMSF, suggesting that a serine residue is involved. At high substrate concentrations inhibition is noted. The [...] for TAME is 0.16 mM.\r\n\r\nPart III. Ferritin tagged c-RNA molecules were hybridized to rat ascites nuclear DNA in an effort to map the arrangement of these sequences in the rat genome by electron microscopy. The ferritin acts as an electron dense marker. The interferritin distance is also the inter RNA distance and c-RNA hybridizes specifically to middle repetitive DNA sequences. Thus measuring the distances between ferritin molecules attached to c-RNA reveals that middle repetitive DNA sequences (about 300 nucleotides in length) were arranged either singly or in tandem and immediately followed by a unique sequence about 500-1500 nucleotides in length." }, { "name": "Douglas, Tommy Charles", "degree": "PhD", "year": "1974", "title": "The Theta Antigen of Mice and Its Analog in Rats", "advisor": "Owen, Ray David", "url": "https://resolver.caltech.edu/CaltechETD:etd-10252005-102646", "creators": [ { "name": { "family": "Douglas", "given": "Tommy Charles" }, "id": "Douglas-Tommy-Charles", "display_name": "Douglas, Tommy Charles" } ], "advisors": [ { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "advisor", "display_name": "Owen, Ray David" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "immun" ], "doi": "10.7907/M2MS-4902", "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\r\n\r\nThe Thy-1 antigen is a normal cell surface component of mouse fibroblasts, brain cells, and thymus-derived lymphocytes (T-cells). This thesis presents the results of genetic, phylogenetic, developmental, and physiological studies of this antigen and its rat analog, [theta]-R.\r\n\r\nThe Thy-1 locus, which controls the expression of the Thy-1 antigen, was previously shown to be located on Chromosome 9 of the mouse. It has been mapped more precisely by following its segregation relative to the linked markers Trf and Mod-1. This group of genes will probably be useful in future genetic studies of this chromosome.\r\n\r\nSublines of the inbred AKR mouse strain have been typed for a variety of genetic markers. Differences were found in Thy-1 antigen, cytoplasmic malic enzyme, kidney esterase-3, principal urinary protein, expression of Murine Leukemia Virus-gs antigen and incidence of leukemias. Those sublines homozygous for [...] appeared to show an increased susceptibility to spontaneous leukemia as compared with those homozygous for [...].\r\n\r\nThymocytes from rats of 13 different inbred and 2 random-bred populations were found to express an antigen indistinguishable from Thy-l.l. At birth little or none of this antigen was present in rat brain. Soon afterward an approximately logarithmic increase in antigen expression began. This continued until about day 20 when the adult level, which exceeded that in neonatal brain by more than 100-fold, was reached.\r\n\r\nAbsorption and complement-mediated cytotoxicity tests failed to detect the Thy-l.l-like antigen ([theta]-R) on rat lymph node, spleen, or bone marrow cells. Pretreatment of immune rat peritoneal exudate cells with anti-Thy-1.1 antibodies in the presence of complement did not abolish their ability to kill tumor cells in vitro. These results suggest that the expression of the [theta]-R antigen by rat thymus-derived lymphocytes not only decreases during maturation, as is the case in the mouse, but ceases altogether.\r\n\r\nContrary to this conclusion is the finding that [...]-homozygous mice immunized against rat peripheral lymphocytes made antibodies directed against Thy-1.1. It therefore appears that small amounts of the [theta]-R antigen are in fact expressed by rat peripheral thymus-derived lymphocytes.\r\n\r\nFollowing suitable absorptions, antisera prepared in rats against thymocytes from mice homozygous for the [...] allele could be rendered specific for Thy-1.2. Antibodies directed against one or more other mouse alloantigens could also be detected in these antisera.\r\n\r\nThe relationship of these results to the current literature on Thy-1 is discussed." }, { "name": "Driskell, William Jack", "degree": "PhD", "year": "1974", "title": "The Role of Tyrosine in the Sclerotization and Tanning of the Puparium of Drosophila melanogaster", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechETD:etd-10242005-153220", "creators": [ { "name": { "family": "Driskell", "given": "William Jack" }, "id": "Driskell-William-Jack", "display_name": "Driskell, William Jack" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/Q7PS-1450", "abstract": "The cuticle of insects is sclerotized and tanned by crosslinks between proteins formed by tyrosine derivatives. The pathway from tyrosine to the catechols incorporated into the crosslinks in the puparium of Drosophila, the structure of the crosslink, and other aspects of sclerotization and tanning of the Drosophila puparium were studied.\r\n\r\nAt puparium formation there is an influx of tyrosine into the puparium, where it is incorporated as a catechol. The turnover of this catechol is very rapid. Synthesis of N-acetyldopamine, the sclerotizing catechol of Calliphora, was observed. No storage form in the larva for the tyrosine incorporated at puparium formation exists other than the pool of free tyrosine and tyrosine-0-phosphate. No differences in the larval and prepuapl tyrosine metabolisms of wild type and ebony were observed.\r\n\r\nPuparia were degraded by acid treatment and by enzymolysis and the degradation products were analyzed to determine the nature of the sclerotizing crosslink. A ketocatechol was extracted in considerable quantity from the puparium by acid. The catechol extractable as a ketocatechol is incorporated into the puparium at puparium formation. The amount of this catechol incorporated is equal to the amount of free tyrosine and tyrosine-0-phosphate lost from the hemolymph at puparium formation. The amount and type of ketocatechol as well as other tyrosine derivatives extracted by acid from puparium are the same for ebony and wild type.\r\n\r\nEnzymolysis of the puparium yielded soluble products which contain the crosslink extractable as ketocatechol. These products were fractionated and found to be heterogeneous.\r\n\r\nPossible structures and syntheses of the sclerotizing crosslink are discussed." }, { "name": "Geltosky, John Edward", "degree": "PhD", "year": "1974", "title": "A Study of Some of the Enzymes Involved in the Synthesis and Use of Tyrosine in Drosophila", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechETD:etd-10142005-092513", "creators": [ { "name": { "family": "Geltosky", "given": "John Edward" }, "id": "Geltosky-John-Edward", "display_name": "Geltosky, John Edward" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/Q5DA-4P39", "abstract": "Tyrosine is an integral component in scierotization and melanization, two developmentally controlled processes which are necessary for synthesis and pigmentation of the insect cuticle. Studies were undertaken to investigate the means by which tyrosine arises and, also, the nature of the reaction whereby tyrosine is hydroxylated to yield dopa, the subsequent step in both of the above mentioned processes in Drosophila melanogaster.\r\n\r\nIt was found that phenylalanine is rapidly converted to tyrosine in vivo and the enzyme(s) responsible for the catalysis is readily extractable. The in vitro conversion is dependent upon the presence of tetrahydropteridine (DMPH4), which is a cofactor in mammalian phenylalanine hydroxylase systems. Other common biological reductants are ineffective in this respect. The concentration of hydroxylase in the animal is developmentally regulated, the level building up during early third instar and peaking at puparium formation. Detectable quantities of enzyme are not found in pupae. The activity of the crude enzyme responds to the concentration of enzyme, substrate and DMPH4 in a linear fashion (up to the point of saturation).\r\n\r\nTyrosine may give rise to dopa via two distinct enzymatic means: tyrosinase or tyrosine hydroxylase. Drosophila are known to possess a very active tyrosinase system; however, this molecule also contains a dopa oxidase function which rapidly dehydrogenates dopa to yield dopaquinone, an irreversible step in melanization. In the absence of suitable regulation, this system does not allow for accumulation of dopa which is also necessary for sclerotization.\r\n\r\nCrude extracts are capable of supporting the conversion of tyrosine to dopa in a DMPH4 stimulated reaction. A high concentration of pteridine also serves to arrest dopa oxidase activity in vitro. DMPH4 is an essential cofactor for mammalian tyrosine hydroxylase activity. The rates of accrual and utilization of dopa (by dopa oxidase) in crude extracts indicate that there are at least two synthetic pathways by which dopa arises. The ratio of the abilities to hydroxylate tyrosine and oxidize dopa varies as a function of the age of the animal, the highest ratio observed in early third instar larvae and the lowest at puparium formation.\r\n\r\nThe activity responsible for catalyzing the hydroxylation of tyrosine behaves in a manner similar to tyrosinase under a number of conditions. Pure tyrosinase, which is derived by sucrose gradient centrifugation, catalyzes a DMPH4 stimulated hydroxylation reaction. Multiple forms of tyrosinase are derived in sucrose gradients and heterogeneity with respect to substrate utilization is observed in these species, a situation which may have significance in a consideration of tyrosinase's involvement in supplying dopa for sclerotization. This possibility is discussed.\r\n\r\nSince DMPH4 plays such a crucial role in these reactions (stimulation of hydroxylation and inhibition of dopa oxidase activity), the nature of these involvements was investigated. There are some indications that the pteridine is not functioning to stimulate the hydroxylation reaction by serving as a non-specific reductant (reductants, such as, ascorbate and DPNH are known to stimulate the hydroxylase activity of pure tyrosinase), but may be serving in a more direct fashion with the enzymatic mechanism. The nature of this interaction is discussed.\r\n\r\nBanded tyrosinase, using dopa as a substrate, catalyzes the synthesis of an unidentified compound. This reaction is stimulated by DMPH4. The nature of this material and its significance to aromatic amino acid metabolism are discussed." }, { "name": "Jan, Lily Kung-Chung Yeh", "degree": "PhD", "year": "1974", "title": "Investigations on Rhodopsin and Bacteriorhodopsin: I. Ultrastructural Localization of Rhodopsin in Vertebrate Retina. II. The Isomeric Configuration of the Bacteriorhodopsin Chromophore", "advisor": "Delbruck, Max", "url": "https://resolver.caltech.edu/CaltechETD:etd-05182006-080145", "creators": [ { "name": { "family": "Jan", "given": "Lily Kung-Chung Yeh" }, "id": "Jan-Lily-Kung-Chung-Yeh", "display_name": "Jan, Lily Kung-Chung Yeh" } ], "advisors": [ { "name": { "family": "Delbruck", "given": "Max" }, "id": "Delbr\u00fcck-M", "role": "advisor", "display_name": "Delbruck, Max" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/NTA3-9886", "abstract": "Chapter I:\r\n\r\nEarly work by Dewey and collaborators has shown the distribution of rhodopsin in the frog retina. We have repeated these experiments on cow and mouse eyes using antibodies specific to rhodopsin alone. Bovine rhodopsin in emulphogene was purified on an hydroxyapatite column. The purity of this reagent was established by spectrophotometric criteria, by SDS gel electrophoresis and by isoelectric focusing. This rhodopsin was used as an immunoadsorbent to isolate specific antibodies from the antisera of rabbits immunized with bovine rod outer segments solubilized in 2% digitonin. The antibody so prepared was shown by immunoelectrophoresis to be in the IgG class and did not cross-react with lipid extracts of bovine rod outer segments. Papain-digested univalent antibodies (Fab) coupled with peroxidase were used to label rhodopsin in formaldehyde-fixed bovine and murine retinas. In addition to the disc membranes, the plasma membrane of the outer segment, the connecting cilium and part of the rod inner segment membrane were labeled. We observed staining on both sides of the rod outer segment plasma membrane and the disc membrane. Discrepancies were observed between results of immunolabeling experiments, and observations of membrane particles seen in freeze-cleaved specimens. Our experiments indicate that the distribution of membrane particles in freeze cleaving experiments reflects the distribution of membrane proteins. Immunolabeling on the other hand can introduce several types of artifact, unless controlled with extreme care.\r\n\r\nChapter II:\r\n\r\nA method for cutting thin sections of frozen glutaraldehyde-fixed tissue developed by Tokuyasu and collaborators has made it possible to label antigens on thin sections of a tissue. Thin sections of frozen retina obtained by this method were treated first with rabbit antibodies specific for bovine rhodopsin, then with conjugates of ferritin and goat antibodies specific for rabbit antibody. Both mouse and bovine retina were labeled specifically by this method. The ferritin label on mouse retina was less heavy than that on bovine retina. Chicken retina was not labeled at all by these reagents. Ferritin label was formed on the disc membranes, on rod outer and inner segment cell membranes, on the inside of the rod inner segment, on the outer membrane of the connecting cilium and in the cytoplasmic bridge of the mouse retina.\r\n\r\nChapter III:\r\n\r\nOesterhelt and Stoeckenius in 1971 found a pigment in the cell membrane of Halobacterium halobium which they called bacteriorhodopsin because it resembled rhodopsin in many aspects. We have studied the isomeric configurations of its chromophore by thin layer chromatography of the retinal itself and of the retinal oxime derivatives. Under physiological conditions the dark-adapted bacteriorhodopsin contains 13-cis retinal, in contrast to the 11-cis retinal of rhodopsin. Upon illumination the 13-cis retinal is converted to all-trans retinal. Substantial thermal isomerization of retinal occurs if the extraction procedure is performed at room temperature. Implications of the different isomeric forms found in bacteriorhodopsin and rhodopsin are discussed." }, { "name": "Melvin, Susan Leah", "degree": "PhD", "year": "1974", "title": "Studies in Cellular Immunology", "advisor": "Owen, Ray David", "url": "https://resolver.caltech.edu/CaltechTHESIS:07302021-215238238", "creators": [ { "name": { "family": "Melvin", "given": "Susan Leah" }, "id": "Melvin-Susan-Leah", "display_name": "Melvin, Susan Leah" } ], "advisors": [ { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "advisor", "display_name": "Owen, Ray David" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "immun" ], "doi": "10.7907/krmh-a250", "abstract": "Antisera were prepared against pigeon thymus and bursa lymphocytes and against saline extracts of thymus and bursa. All unabsorbed sera were screened for differential reactivity against thymus and bursa-derived tissue by several techniques. Selected sera were absorbed with thymus and bursa tissue to demonstrate specificity. One serum with specificity against thymus extracts was identified by immunodiffusion. The thymus specificity was absent from extracts of pigeon bursa, brain, liver and breast muscle, but present in spleen extracts. This thymus specificity does not appear analogous to lymphocyte specificities identified in other species. Shared tissue specificities and a possible quantitative antigenic difference among the tissue extracts were also demonstrated by immunodiffusion and absorption analysis. In lymphocytotoxicity tests, fresh rabbit normal serum is highly toxic for pigeon thymus and bursa cells. This toxicity, in general, resembles the natural antibody present in rabbit and guinea pig sera against heterologous thymus cells. A rabbit anti-pigeon gamma globulin serum was rendered specific for bursa cells by absorption with thymus cells. Some standard antilymphocyte sera were shown to contain an antibody fraction specific for thymus cells. Some or all of these reagents may be useful for distinguishing cooperating cell populations in a variety of immune responses.
\r\n\r\nAn antigen was demonstrated on red cells from all pigeon squabs less than four days old. The antigen appears not to be secondarily adsorbed to the red cells from the fluids of the egg or the embryo. In vitro, masking of the antigen by components of adult serum does not occur under the conditions tested. Although the squab antigen behaves similarly to a known fetal red cell antigen in doves, it is probably qualitatively different from that antigen and from the known chick red cell antigen. The squab antigen is not detectable on lymphocytes from the bursa or the thymus.
\r\n\r\nVirgin female CBA/J mice were obtained after a variety of treatments and observed for primary tumors until either tumor onset or death. Included were mice which were: (1) immunosuppressed as adults by injection of anti-thymocyte serum (ATS); (2) injected with normal rabbit serum; (3) immunized with an irrelevant antigen or (4) untreated. Data were collected on tumor histology, incidence and time of onset for all groups. No tumors appeared during the period of ATS-immunosuppression or for several months following treatment. The most frequently observed subsequent tumor was the typical mammary tumor. Although the first tumors appeared in ATS-treated mice, the mean age at tumor onset was not significantly affected by ATS-immunosuppression. No unusual tumors and no lymphomas were observed. Tumor incidences among groups of mice purchased at different times were different, but unrelated to ATS-immunosuppression. The failure of ATS-immunosuppression to affect growth is consistent with the fact that cellular immunity to mammary tumors is often specifically compromised.
" }, { "name": "Meyer, Ronald Leo", "degree": "PhD", "year": "1974", "title": "Factors Affecting Regeneration of the Retinotectal Projection", "advisor": "Sperry, Roger Wolcott", "url": "https://resolver.caltech.edu/CaltechTHESIS:02092021-000143126", "creators": [ { "name": { "family": "Meyer", "given": "Ronald Leo" }, "id": "Meyer-Ronald-Leo", "display_name": "Meyer, Ronald Leo" } ], "advisors": [ { "name": { "family": "Sperry", "given": "Roger Wolcott" }, "id": "Sperry-R-W", "role": "advisor", "display_name": "Sperry, Roger Wolcott" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/qyhf-s343", "abstract": "Factors affecting the orderliness of the regenerated retinotectal projection were studied by inducing various size disparities between the retina and tectum of goldfish and tree frog. Behavioral, autoradiographic, and electrophysiological measurements showed that the plastic compression of the projection previously found and here confirmed for goldfish does not occur in half tectum frog, In fish, eye lesions and optic fiber transplants between tecta confirm Sperry's chemoaffinity hypothesis but show that this retinotectal affinity is not sufficient to prevent misgrowth. In addition, competitive and possibly chemoaffinity interactions between optic fibers were found that might explain the plasticity of the goldfish retinotectal system.
" }, { "name": "Murphy, William Ignatius, III", "degree": "PhD", "year": "1974", "title": "Studies of the Mechanism and Products of Transcription of the Nuclear Genome in Animal Cells", "advisor": "Attardi, Giuseppe", "url": "https://resolver.caltech.edu/CaltechTHESIS:06042021-220829673", "creators": [ { "name": { "family": "Murphy", "given": "William Ignatius, III" }, "id": "Murphy-William-Ignatius-III", "display_name": "Murphy, William Ignatius, III" } ], "advisors": [ { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "advisor", "display_name": "Attardi, Giuseppe" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/r002-ng85", "abstract": "Part I
\r\n\r\nChapter 1. The metabolic stability of pulse labeled or long-term labeled\r\nmRNA from cytoplasmic free polysomes has been measured in HeLa cells, using\r\nchase conditions which do not involve inhibitors of RNA synthesis, and\r\nchromatography on benzoylated-DEAE-cellulose or poly(T)-cellulose for the\r\nisolation of mRNA. For these studies, a new chase technique has been developed\r\nwhich allows the analysis of the stability of mRNA labeled during a short\r\n3H-uridine pulse. Pulse labeled and long-term labeled mRNA have been found\r\nto decay with an estimated average half-life of about 2 and 3 days, respectively,\r\nmuch longer than hitherto assumed.
\r\n\r\nChapter 2. Polyadenylated messenger RNA extracted from HeLa cells was\r\nhybridized with a mass excess of HeLa DNA. The kinetics of the hybridization\r\nreaction demonstrated that most of the mRNA is transcribed from nonrepetitive\r\nDNA. The amount of mRNA hybridized to DNA was measured both with and without\r\nprior RNase treatment. Comparison of the results indicates that within the\r\nlimits of detection, HeLa mRNA does not contain repetitive sequence elements\r\ncovalently linked to nonrepetitive sequence transcripts. However, a small\r\nfraction of the HeLa mRNA preparation is transcribed entirely from repetitive\r\nDNA sequences. This fraction represents about 6% of the total polyadenylated\r\nmRNA preparation.
\r\n\r\nChapter 3. The sedimentation properties of pulse-labeled and longterm\r\nlabeled mRNA from HeLa cell free-polysomes, selected for poly(A)\r\ncontent by two successive passages through poly(T)-cellulose columns, was\r\nanalyzed under native and denatured conditions. The sedimentation profile\r\nof the mRNA on both sodium dodecyl SO4-sucrose gradients and formaldehyde-sucrose\r\ngradients showed a broad distribution of components with estimated\r\nmolecular weights ranging from 2 x 105 to 5.5 x 106 daltons and a weight-average\r\nmolecular weight of 8.5 x 105 daltons.
\r\n\r\nPart II
\r\n\r\nThe >50 S HnRNA, isolated from either HeLa cells or irranature duck\r\nerythrocytes labeled for different times with [5-3H]uridine, was examined\r\nfor the presence of complementary transcripts capable of forming RNase-resistant\r\nduplexes. After extensive self-annealing of the HnRNA, carried\r\nout under conditions such that complementary RNA sequences present once or\r\na few times in the RNA population would have formed hybrids, no evidence was\r\nfound for the existence of symmetrical transcripts in either cell system.\r\nHowever, 2-3% and 4-5% of the purified duck and HeLa HnRNA, respectively,\r\ndid form RNase-resistant hybrids. These hybrids resulted from base-pairing\r\nof complementary regions within the HnRNA molecule, as judged from the lack\r\nof concentration dependence and from the kinetics of formation of the RNA-RNA\r\nduplexes. The weight-average length of the RNase-resistant fragments from\r\nthe duck HnRNA was found to be approximately 125 nucleotide pairs; however,\r\nshorter double-stranded segments as well as longer duplexes, up to 2000\r\nnucleotide pairs, were also observed. Annealing of the duck HnRNA in the\r\npresence of an excess of 10 S hemoglobin mRNA showed that 2% of the HnRNA\r\nformed RNase-resistant hybrids in excess of those expected from intramolecular\r\nhomology. The RNase-resistant complexes formed between the 10 S mRNA and HnRNA\r\nhad about the same size range as the intramolecular duplexes.
\r\n\r\nThe failure to detect any intermolecular hybridization in the short pulse\r\nlabeled HnRNA from either actively growing cells or highly differentiated,\r\nnon-dividing cells, strongly suggests that the mechanism for the synthesis of\r\nHnRNA in animal cells does not involve the production of high molecular\r\nweight complementary transcripts.
\r\n\r\nPart III
\r\n\r\nThis report describes the use of purified rDNA to map by electron\r\nmicroscopy the relative position of the 18 S and 28 S RNA regions within the\r\nduck rRNA precursor and their relationship to the nonconserved portions of\r\nthe precursor molecule. In the first part, the purification from duck\r\nerythrocytes of rDNA sequences suitable for use in the electron microscopic\r\nmapping of the rRNA precursor is discussed. By repeated fractionation of\r\nthe total DNA, based on the relative reassociation rates of the DNA sequences\r\nwith different degrees of repetition, a fraction of the rapidly renaturing\r\nDNA was obtained which comprised only 6% of the total DNA, but contained 71%\r\nof the rRNA cistrons. Further purification of the rDNA was achieved by\r\nsaturation hybridization with rRNA and separation of the rRNA-rDNA hybrids\r\nby banding in CsCl. In this manner, an rDNA-rRNA fraction was obtained\r\nwhich had a buoyant density of 1.805 gm/cm3, an RNA to DNA ratio of 1.01,\r\nand a base composition for the RNA present in the hybrid identical to that\r\nof an equimolar mixture of 18 sand 28 S rRNA. The final yield of rDNA\r\nisolated by this procedure is 32%. When the purified rDNA was annealed\r\nwith a mixture of 18 sand 28 S rRNA and the hybrids spread for electron\r\nmicroscopy, they appeared as two distinct populations with a number-average\r\nlength of 0.62\u00b10.13 \u03bcm and 1.37\u00b10.18 \u03bcm, respectively. Likewise, hybrids\r\nbetween the rRNA precursor, isolated from duck embryo fibroblasts, and the\r\nrDNA appeared as structures containing two duplex regions of lengths 0.60\u00b1\r\n0.11 \u03bcm and 1.38\u00b10.15 \u03bcm, separated from each other by a single-stranded\r\nregion appearing as a large bush: this represents the portion of the precursor\r\nmolecule not conserved during processing of the parent molecule.\r\nFrom these observations a model of the structure of the avian rRNA precursor\r\nis proposed.
" }, { "name": "Ram, Jeffrey Lewis", "degree": "PhD", "year": "1974", "title": "Effects of High K\u207a Media on Leucine Incorporation into Aplysia Nervous Tissue", "advisor": "Strumwasser, Felix", "url": "https://resolver.caltech.edu/CaltechTHESIS:11202021-011429389", "creators": [ { "name": { "family": "Ram", "given": "Jeffrey Lewis" }, "id": "Ram-Jeffrey-Lewis", "display_name": "Ram, Jeffrey Lewis" } ], "advisors": [ { "name": { "family": "Strumwasser", "given": "Felix" }, "id": "Strumwasser-F", "role": "advisor", "display_name": "Strumwasser, Felix" } ], "committee": [ { "name": { "family": "Strumwasser", "given": "Felix" }, "id": "Strumwasser-F", "role": "chair", "display_name": "Strumwasser, Felix" }, { "name": { "family": "Olds", "given": "James" }, "id": "Olds-J", "role": "member", "display_name": "Olds, James" }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Russell", "given": "Richard L." }, "id": "Russell-R-L", "role": "member", "display_name": "Russell, Richard L." }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "member", "display_name": "Mitchell, Herschel K." } ], "option_major": [ "bioch" ], "doi": "10.7907/qtf0-tz26", "abstract": "To study possible coupling between membrane polarization and protein synthesis, elevated external K+ levels were used to depolarize the cell membranes in isolated Aplysia californica parieto-visceral ganglia (PVG). The effect of this treatment on the incorporation of labeled leucine into proteins in the ganglion was analyzed on sodium dodecyl sulfate polyacrylamide gels. PVGs were preincubated 3 hours and then incubated 4 hours in either control medium (14C-leucine, 10 mM [ K+]) or experimental medium (3H-leucine, 10 + x mM [K+] with equimolar [Na+] reduction). These were homogenized together, separated into aqueous soluble and aqueous insoluble fractions, and run on gels.
\r\n\r\nIn the aqueous soluble fraction of the PVG High [K+] (90-110 mM [K+]} caused relative increases in incorporation in distinct peaks at 50K (K = 1000 daltons) and 40K. The larger peak, at 50K, was studied further.
\r\n\r\nThe relative increase at 50K occurred when 14c-leucine (instead of the usual 3H-leucine) was incorporated in High [K+]. The relative increase at 50K did not occur (1) when [K+] was raised to only 50 mM; (2) when [Na+] was reduced by 80 mM, and tris+ (HCl to neutralize) was substituted instead of K+; (3) in pleura-visceral connective (PVC) nerve; and (4) in the aqueous insoluble fraction of the PVG.
\r\n\r\nThe effect of High [K+] on incorporation into the giant cell (R2) of the PVG was examined by first labeling the PVG in control medium, rinsing it, and then labeling it in experimental medium. High [K+] in the experimental medium caused a significant relative increase at 50K in whole PVGs, half PVGs, and R2s dissected from the PVG following incubation. The results in R2 were marred by great variability in the control patterns.
\r\n\r\nAutoradiography of identified cells (R2 and R15) dissected from PVGs labeled with 3H-leucine in normal [K+] showed that contaminating cells {mostly glia), which always adhere to such dissected cells, generally account for less than 20% of the total incorporated formalin-fixed label. This contamination is large enough so that a glial origin of the High [K+] effect on incorporation at 50K cannot be positively excluded. However, the presence of this effect in dissected R2s and its absence in PVC nerves, which contain axons, glia, and connective tissue, but no nerve cell bodies, lend support to the notion that the effect is neuronal in origin.
\r\n\r\nHigh [K+] caused a reduction of approximately SO% in total incorporation into both aqueous soluble and aqueous insoluble proteins of the PVG. Similar decreases of 35% were seen in dissected R2s. [Na+] reduction (by 80 mM, tris+ substitution) had no significant effect on total incorporation (measured only in the aqueous soluble fraction of the PVG). High [K+] caused a reduction of approximately 85% in total incorporation into PVC nerve. Autoradiography of the nerve showed that this reduction occurred in both the connective tissue sheath and the axonal-glial region. High [K+] caused no significant change in non-volatile TCA soluble label in either the ganglion or the nerve.
\r\n\r\nOther effects of High [K+] on the PVG: (1) a small (not large enough to have caused the relative increase at 50K) decrease in the relative amount of label in the aqueous soluble, TCA insoluble fraction compared to the aqueous insoluble fraction, and (2) a relative decrease in incorporation in higher molecular weight peptides compared to lower molecular weight peptides in both aqueous soluble and aqueous insoluble fractions.
\r\n\r\nThese results suggest, but do not prove, that High [K+] caused an increase in the synthesis of a neuronal peptide of approximately 50,000 daltons molecular weight. The possibility that this peptide may be a tubulin subunit is briefly discussed.
" }, { "name": "Simmons, Daniel Tawil", "degree": "PhD", "year": "1974", "title": "The Function and Replication of Sindbis Virus-Specific RNA's in Infected Cells", "advisor": "Strauss, James H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04132021-203938881", "creators": [ { "name": { "family": "Simmons", "given": "Daniel Tawil" }, "id": "Simmons-Daniel-Tawil", "display_name": "Simmons, Daniel Tawil" } ], "advisors": [ { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "advisor", "display_name": "Strauss, James H." } ], "committee": [ { "name": { "family": "Strauss", "given": "James H." }, "id": "Strauss-J-H", "role": "chair", "display_name": "Strauss, James H." }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Russell", "given": "Richard L." }, "id": "Russell-R-L", "role": "member", "display_name": "Russell, Richard L." }, { "name": { "family": "Wood", "given": "William Barry" }, "id": "Wood-W-B", "role": "member", "display_name": "Wood, William Barry" } ], "option_major": [ "bioch" ], "doi": "10.7907/grvs-e385", "abstract": "During infection with Sindbis virus, two species of Sindbis-specific single-stranded RNA are synthesized. One of them, 49S RNA is the RNA of the vinis and has a molecular weight of 4.3 \u00b1 0.3 x 106 daltons. This molecular weight was estimated by a variety of methods, including polyacrylamide gel electrophoresis, sedimentation after reaction with fonnaldehyde and analysis of the molecular weight of its double-stranded form. The other species of single-stranded RNA, 26S RNA, was found only in infected cells and has a molecular weight of 1.6 x 106 daltons, determined by sedimentation in dimethylsulfoxide. Hybridization-competition experiments showed that 26S RNA is a specific one-third of the 49S RNA genome.
\r\n\r\nIn infected cells, 26S RNA was primarily associated with ribosomes, and was found to be the predominant species of viral messenger RNA. A small amount (10 % by weight) of the messenger RNA in the cells was Sindbis 49S RNA. No other unique and separate species of Sindbis messenger RNA could be detected in infected cells.
\r\n\r\nThe two species of Sindbis single-stranded RNA were translated in lysates of rabbit reticulocytes. Sindbis 26S RNA was translated primarily into the nucleocapsid protein and into a protein shown by others to be the precursor of the two glycoproteins of the virus. These results indicated that Sindbis 26S RNA codes solely for the structural proteins of the virus.
\r\n\r\nSindbis 49S RNA was translated in vitro into 8 or 9 polypeptides ranging in molecular weight from 60,000 to 180,000 daltons. None of these polypeptides coincided with any known Sindbis proteins.
\r\n\r\nThe replication of Sindbis-specific RNA was studied by analyzing the forms of double-stranded RNA (or replicative forms) in infected cells. When RNA from infected cells was treated with ribonuclease, three species of Sindbis-specific double-stranded RNA (RF's I, II, and III) were isolated. Their molecular weights were determined to be 8.8 x 106 daltons for RFI, 5.6 x 106 daltons for RFII, and 2.9 x 106 daltons for RFIII.
\r\n\r\nBy hybridization-competition experiments, it was shown that RFI is the double-stranded form of 49S RNA, RFIII, the double-stranded form of 26S RNA, and RFII, the double-stranded form of a species of RNA with molecular weight of 2.8 x 106 daltons and identical to two-thirds of the genome.
\r\n\r\nThe size and structure of Sindbis replicative intermediates (RI's) were studied and found to consist of a double-stranded region the size of RFI and of various lengths of single-stranded tails. Our model for the replication of Sindbis-spccific RNA predicts that Sindbis RI's exist in two classes, called RIa and RIb. RIa is the template for the synthesis of 49S RNA and is reduced to RFI when treated with ribonuclease. When Rib is treated with ribonuclease, it is reduced to RF's II and III due to a single-stranded gap in the \"plus\" strand of the RI (the virus RNA is \"plus\"-stranded). The portion of RIb corresponding to RFIII is the template for the synthesis of 26S RNA, and the portion corresponding to RFII is the template for synthesis of a species of RNA of 2.8 x 106 daltons, which has not been detected in its single-stranded form. We hypothesize that there must be two different regions on RIb where chain synthesis is initiated since 26S RNA is synthesized at a much faster rate than the product of RFII.
" }, { "name": "Tuchman, Jessica", "degree": "PhD", "year": "1974", "title": "The Developmental Role of Membrane in the Cellular Slime Mold, Dictyostelium Discoideum", "advisor": "Lodish, Harvey F.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03062018-093229873", "creators": [ { "name": { "family": "Tuchman", "given": "Jessica" }, "id": "Tuchman-Jessica", "display_name": "Tuchman, Jessica" } ], "advisors": [ { "name": { "family": "Lodish", "given": "Harvey F." }, "id": "Lodish-Harvey-F", "role": "advisor", "display_name": "Lodish, Harvey F." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/8272-S965", "abstract": "This thesis concerns the role of the cell surface membrane\r\nin cell-cell interactions. Specifically, the purpose of these\r\nexperiments was to discover whether the aggregation of the cellular\r\nslime mold Dictyostelium discoideum is accompanied by, and\r\nperhaps dependent on, specific differentiation of the cell\r\nsurface membrane, and if so, whether such components could be isolated\r\nin an active state. To this end, partially purified cell surface\r\nmembranes were prepared from both vegetative (0 hour)\r\nand developing (14 hour)cells. The membranes were characterized\r\nby sucrose gradient centrifugation, SDS polyacrylamide gel\r\nelectrophoresis, and electron microscopy. It was found that \r\nmembranes from both 0 and 14 hour cells possessed the ability\r\nto inhibit the developmentally controlled aggregation of slime\r\nmold cells when mixed with these cells and plated under normal\r\nlaboratory condition. HeLa cell membranes, even at the highest\r\nobtainable concentrations, were inert in this respect. Aggregation\r\nphase membranes were able to prevent cell aggregation at\r\nsignificantly lower concentrations than was required for vegetative\r\nmembranes, and were also markedly more resistant to heat\r\ndegradation. It appears that aggregation phase membranes block\r\naggregation by preventing the attainment of aggregation competence\r\nin the developing cells, whereas vegetative membranes appear to\r\nact through a direct competition for available aggregation antigen\r\nreceptor sites on the cell surface.
\r\n\r\nThe effect of the differentiated membranes on several develop\u00admentally \r\ncontrolled enzymes was tested. Membrane treatment leads to the \r\ninduction of some developmentally controlled enzymes and the re\u00adpression \r\nor excretion of ethers.\tIn one case, alkaline phosphatase, enzyme \r\ninduction occurs 12 hours earlier than in nonnal cells, and the enzyme \r\nreaches approximately double its normal activity. The distribution of \r\neffected and uneffected enzymes bears no resemblance to the normal \r\nsequence of enzyme induction. The only characteristic with which the \r\nmembrane effect can be linked, is the intracellular localization of \r\nthe effected enzyme. The results indicate that there are some difficulties \r\nin the generally accepted view of the slime mold developmental program, \r\nand point out the crucial role played by the formation and maintenance \r\nof cell-cell contacts during normal development.
\r\n\r\nA study of the changes in protein synthesis during slime mold \r\ndevelopment was also undertaken. Total cell protein was displayed \r\non SDS polyacrylamide gels after a 2 hour pulse label. It was found \r\nthat during the first few hours of development, a single major band accounts \r\nfor more than 20 percent of the total protein on the gel. Actin was \r\npurified from vegetative cells by a known procedure, and was found to \r\ncorrespond to the major band in several respects. The discovery of a single \r\nprotein being synthesized in such quantity, and its identification as actin, \r\nprovide a powerful tool for the isola\u00adtion of a specific messenger RNA \r\nmolecule, and for an intensive study of all the factors involved in \r\nregulating protein synthesis in a eucaryotic organism.
\r\n\r\n" }, { "name": "Zuccarelli, Anthony Joseph", "degree": "PhD", "year": "1974", "title": "Formation of Parental Replicative Forms of \u03d5-X174 : Synthesis of the First Complementary Strand", "advisor": "Sinsheimer, Robert L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:07012021-163400628", "creators": [ { "name": { "family": "Zuccarelli", "given": "Anthony Joseph" }, "id": "Zuccarelli-Anthony-Joseph", "display_name": "Zuccarelli, Anthony Joseph" } ], "advisors": [ { "name": { "family": "Sinsheimer", "given": "Robert L." }, "id": "Sinsheimer-R-L", "role": "advisor", "display_name": "Sinsheimer, Robert L." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/gwm8-pv94", "abstract": "Part I
\r\n\r\nMutants of the bacteriophage \u03d5X174 have been isolated that are less dense than wild-type \u03d5X phage particles in CsCl. When viral strands from the mutants are hybridized with wild-type complementary strands, the resulting duplex molecules have single-stranded loops characteristic of wild type-deletion heteroduplexes. The mutant phages fail to complement \u03d5X amber mutants in cistron E, but they do complement mutants in six other cistrons. Based upon contour measurements of phage DNA and duplexes, and the buoyant density of the particles, it is estimated that the mutant viruses have deleted approximately 7% of the \u03d5X genome in the region of cistron E.
\r\n\r\nPart II
\r\n\r\nThe formation of circular, double-stranded RF (replicative form) DNA in cells has been observed in the period from 15 seconds to 20 minutes after infection with the SS (single-stranded) DNA bacteriophage \u03d5Xl74. The kinetics of appearance of RF during the first few minutes lead to the conclusion that the new, complementary DNA strand is polymerized in less than 10 seconds (viz. about 600 nucleotides per second).
\r\n\r\nThe structure of RFII (a circular duplex with at least one SS break), RF made after infection with UV damaged phage, and nascent RF (extracted 1 minute after infection) were determined by sedimentation analysis and observations made with the electron microscope. They led to the following generalizations: (a) Normal RFII molecules usually have intact circular viral strands and unit-length linear complementary strands. (b) RF made on UV damaged templates also have circular viral strands, but the complementary strand is shorter than unit-length and regions of SS template are evident. (c) The new complementary strand contains many discontinuities immediately after its synthesis, but these are eventually sealed. (d) The viral strand in nascent RF also appears to be broken. These conclusions are incorporated into a proposed mechanism for the synthesis of the first complementary strand.
\r\n\r\nPart III
\r\n\r\nParental RF molecules were pulse-labeled with [3H]thymidine under conditions expected to label the parts of the new complementary strand which are synthesized last. The RF were analyzed by digestion with a restriction enzyme isolated from Haemophilus influenzae. The pattern of 3H label in the resulting fragments led to the following conclusions: (a) Synthesis of the complementary strand is ordered and begins at one or two specific initiation sites. (b) One initiation is located in or near cistron A. A second initiation near the junction of cistrons G and H may also exist. (c) Synthesis of the new strand is counter-clockwise on the genetic map, in the 5' \u2192 3' direction catalyzed by the known DNA polymerases.
" }, { "name": "Benowitz, Larry I.", "degree": "PhD", "year": "1973", "title": "Mechanisms of Information Processing in the Chick", "advisor": "Sperry, Roger Wolcott", "url": "https://resolver.caltech.edu/CaltechTHESIS:11122019-103132853", "creators": [ { "name": { "family": "Benowitz", "given": "Larry I." }, "id": "Benowitz-Larry-I", "display_name": "Benowitz, Larry I." } ], "advisors": [ { "name": { "family": "Sperry", "given": "Roger Wolcott" }, "id": "Sperry-R-W", "role": "advisor", "display_name": "Sperry, Roger Wolcott" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/FVA1-GS21", "abstract": "The kinetics of neurobiological phenomena involved in 1he formation of a memory trace were examined in the first two experiments. Transcranial subconvulsive current was administered to large groups of chicks at various intervals following an aversive training experience. The resulting retrograde amnesia data indicate that immediately upon training a metastable mnemonic process becomes activated (STM) which then remains at a constant intensity. Within a minute STM induces a more permanent form of memory (Pre-LTM) to grow at a steadily declining rate, apparently as some restricted neural substrate of memory becomes exhausted. STM may continue to function as behaviorally accessible memory for the next few hours, during which time the behaviorally latent Pre-LTM trace undergoes a subsequent transition into permanent memory. An investigation of the retrograde amnesia resulting from a sequence of two training-current sessions provides support to the existence of these mechanisms and indicates that fractional engrams summate together in a simple fashion.
\r\n\r\nTo examine the participation of different cerebral structures in information processing, chicks having various telencephalic lesions were tested in either a passive avoidance task or an appetitive discrimination. The hippocampus was found to be involved in reversal but not acquisition of the pattern discrimination, and in acquisition but not retrieval of the passive avoidance task. On the other hand, the amygdala seems to be important both for retrieval and acquisition of passive avoidance conditioning, but only for early stages of acquiring the pattern discrimination. Frontal ablations resulted in a deficit to retrieval but not to acquisition of passive avoidance conditioning, and caused some motivational changes independent of chicks' learning ability in the performance of the appetitive task. A comparison of these results with those following lesions in homologous mammalian limbic system structures suggests that the information processing of both classes is based upon cerebral mechanisms which have remained unchanged despite their divergent evolution.
\r\n\r\nMechanisms of memory processing and interhemispheric transfer were further studied in chicks having extensive unilateral ablations of the dorsal telencephalon, a region critical for visual learning. Although chicks were able to acquire a passive avoidance response equally well using either the eye ipsilateral or that contralateral to the surgery, subsequent extinction conditioning could be learned only through the ipsilateral eye. Since retinal projections cross completely at birds' optic chiasma, these results suggest that anatomically distinct systems, one bilaterally represented, the other lateralized, respectively mediate the acquisition and extinction of the aversive response. An inability or lateralized memory to transfer through the commissures is indicated by the absence of interocular transfer for monocularly learned extinction.
\r\n" }, { "name": "Frelinger, Jeffrey Allen", "degree": "PhD", "year": "1973", "title": "Transferrin Polymorphism in Pigeons", "advisor": "Owen, Ray David", "url": "https://resolver.caltech.edu/CaltechTHESIS:07062018-091237072", "creators": [ { "name": { "family": "Frelinger", "given": "Jeffrey Allen" }, "id": "Frelinger-Jeffrey-Allen", "display_name": "Frelinger, Jeffrey Allen" } ], "advisors": [ { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "advisor", "display_name": "Owen, Ray David" } ], "committee": [ { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "chair", "display_name": "Owen, Ray David" }, { "name": { "family": "Hood", "given": "Leroy E." }, "id": "Hood-L-E", "orcid": "0000-0001-7158-3678", "role": "member", "display_name": "Hood, Leroy E." }, { "name": { "family": "Hargrave", "given": "Paul" }, "id": "Hargrave-Paul", "role": "member", "display_name": "Hargrave, Paul" }, { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "member", "display_name": "Lewis, Edward B." }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "member", "display_name": "Mitchell, Herschel K." }, { "name": { "family": "Russell", "given": "Richard L." }, "id": "Russell-R-L", "role": "member", "display_name": "Russell, Richard L." } ], "option_major": [ "immun" ], "doi": "10.7907/7J4M-ZX34", "abstract": "Transferrin polymorphism, detected by differences in electrophoretic mobility among allelic forms, is widespread in vertebrates. In pigeons, two alleles have been reported. In a developmental study, I have shown that for a period following hatching the young bird's transferrin phenotype reflects the maternal rather than its own genotype, although the squab is actively synthesizing transferrin in its liver. This probably reflects transfer of maternally derived protein through the egg. As the period of maternal transfer corresponds to the period of immunoincompetence on the part of the squab, the transferrin is known to be bacterio-and fungistatic, I investigated the possibility of differences in the funistatic effects of the three transferrin phenotypes, using yeast as an assay organism. I found that transferrin derived from heterozygotes is much more effective in the inhibition of yeast growth \u00b7than that from homozygotes. Under the breeding conditions in our flock, embryonic mortality was significantly lower among the progeny of heterozygous females. This suggests that transferrin polymorphism is maintained by selection against the progeny of homozygous females. An algebraic consideration of this form of selection leads to the prediction that a population at equilibrium for allele frequencies would be in Hardy-Weinberg equilibrium for phenotype frequencies. The published data of others, in addition to my own, show that real pigeon populations are in Hardy-Weinberg equilibrium.
\r\n\r\nTransferrin was purified (> 95% pure) from all three phenotypes; all had similar amino acid compositions and molecular weights. Peptide mapping of the allele products revealed a difference in one peptide, which on analysis was consistent with a single amino acid substitution (Ser \u2192 Asp). Mixtures of homozygous type transferrins, while appearing electrophoretically identical to the heterozygous type, do not equal its behavior in yeast inhibition. This suggests the presence of \"hybrid molecules\" synthesized in heterozygotes. I found that pigeon transferrins can be dissociated to 40,000 M.W. fragments by heating in sodium dodecyl sulfate (SDS). Removal of SDS results in reassociation into 80,000 M.W. dimers. All preparations show only a single amino terminal (alanine). CNBr cleavage produces only four fragments in spite of the fact that there are eight methionines. Sequence data may indicate multiple amino acid sequences. Quantitative hydrozanalysis yields only a single carboxyl terminal (serine). These data suggest that pigeon transferrin may be a dimer.
" }, { "name": "Froehner, Stanley Charles", "degree": "PhD", "year": "1973", "title": "The Isolation, Purification and Characterization of Three RNA Polymerases from Novikoff Hepatoma Ascites Tumor", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechTHESIS:07062018-115304742", "creators": [ { "name": { "family": "Froehner", "given": "Stanley Charles" }, "id": "Froehner-Stanley-Charles", "display_name": "Froehner, Stanley Charles" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "chair", "display_name": "Bonner, James Frederick" }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Dreyer", "given": "William J." }, "id": "Dreyer-W-J", "role": "member", "display_name": "Dreyer, William J." }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "member", "display_name": "Mitchell, Herschel K." }, { "name": { "family": "Strumwasser", "given": "Felix" }, "id": "Strumwasser-F", "role": "member", "display_name": "Strumwasser, Felix" } ], "option_major": [ "bioch" ], "doi": "10.7907/M8Q4-DB66", "abstract": "DNA-dependent RNA polymerase has been isolated from nuclei of Novikoff hepatoma ascites tumor cells and resolved into three activities, designated Ia, Ib, and II, by a combination of phosphocellulose and DEAE cellulose chromatography. Ia and Ib have been further purified by sucrose density centrifugation. Both gradient profiles exhibit coincidence of the polymerase activity and protein peaks, suggesting that the two may be homogeneous enzymes. Ia migrates as a single species on non-denaturing polyacrylamide gel electrophoresis. SDS polyacrylamide gel electrophoresis indicates that Ia contains subunits of 170,000, 125,000, 69,000, 49,000, 44,000 and 37,000 molecular weights in equimolar ratios except for the\r\n69,000 and 37,000 dalton subunits which may be present in two copies per enzyme molecule. A molecular weight of\r\n600,000 for the enzyme calculated from the molecular weights of the subunits is in good agreement with that determined by exclusion chromatography. The probable molecular structure of Ib is subunits of 190,000 and 135,000 daltons, each present twice per enzyme molecule. The enzymological characterization of these three enzymes suggests that Ia and Ib are the nucleolar polymerases while II is nucleoplasmic. Ia and Ib are most active at low ionic strength with Mg++ on native DNA and are insensitive to \u03b1-amanitin. II prefers Mn++, high ionic strength, a denatured template and is inhibited by low concentrations of \u03b1-amanitin. A factor present in the material which does not bind to the DEAE cellulose column used in the purification scheme, stimulates the activity of all three of the enzymes. Ia and Ib are inactive at low enzyme concentrations in the absence of this factor. The active agent in the factor is probably a protein, since it is heat sensitive, and may be a subunit of the enzyme.
\r\n" }, { "name": "Gordon, Harold William", "degree": "PhD", "year": "1973", "title": "Verbal and Non-Verbal Cerebral Processing in Man for Audition", "advisor": "Sperry, Roger Wolcott", "url": "https://resolver.caltech.edu/CaltechTHESIS:07132018-093853708", "creators": [ { "name": { "family": "Gordon", "given": "Harold William" }, "id": "Gordon-Harold-William", "display_name": "Gordon, Harold William" } ], "advisors": [ { "name": { "family": "Sperry", "given": "Roger Wolcott" }, "id": "Sperry-R-W", "role": "advisor", "display_name": "Sperry, Roger Wolcott" } ], "committee": [ { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "member", "display_name": "Benzer, Seymour" }, { "name": { "family": "Fender", "given": "Derek H." }, "id": "Fender-D-H", "role": "member", "display_name": "Fender, Derek H." }, { "name": { "family": "Olds", "given": "James" }, "id": "Olds-J", "role": "member", "display_name": "Olds, James" }, { "name": { "family": "Van Harreveld", "given": "Anthonie" }, "id": "Van-Harreveld-A", "role": "member", "display_name": "Van Harreveld, Anthonie" }, { "name": { "family": "Sperry", "given": "Roger Wolcott" }, "id": "Sperry-R-W", "role": "member", "display_name": "Sperry, Roger Wolcott" } ], "option_major": [ "biology" ], "doi": "10.7907/MPJG-WA43", "abstract": "Hemispheric asymmetries were investigated with various auditory techniques in several groups of subjects. The first study was a dichotic listening experiment in which two separate musical chords were presented simultaneously one to each ear of right-handed males. The subjects were required to listen to the chord stimuli and then recognize them from a multiple choice of four chords heard immediately following the dichotic presentation. More chords were recognized from the left ear than from the right implying right cerebral dominance for this task. In a similar test, dichotic presentation of melodies showed no difference between the ears. It was hypothesized that the subjects in this case were identifying the tune segments on the basis of rhythmic rather than pitch cues. It was suggested that the right hemisphere is superior to the left in processing stimuli that are \"non-temporal.\"
\r\n\r\nMusical expression was investigated in patients who had transiently lost the function of one hemisphere following intracarotid amytal injection. It was observed that after right hemisphere depression, singing was devoid of pitch at a time when speech was only minimally disturbed.\r\nConversely, singing was much less affected than speech after left hemisphere depression. This differential effect of amytal depression is supportive of the idea that the right hemisphere is used for pitch control in singing whereas the left hemisphere is used expressly for speech.
\r\n\r\nSinging was also studied in two young patients with surgical hemispherectomies for non-infantile causes. One patient who had a right hemisphere removal with no evidence of aphasia, sang most songs poorly. He also failed pitch discrimination tests wherein he could not distinguish two tones that were separated by an interval of less than one musical step. Another patient with a left hemispherectomy produced the opposite results. She had great difficulties in expressive speech yet could sing with excellent pitch control and intonation. These cases support the previous conclusion that the right hemisphere is necessary for correct pitch production in singing.
\r\n\r\nDichotic listening studies on patients with complete surgical division of the corpus callosum indicated that the right hemisphere also had some capacity to understand and manually express verbs and verbal commands. This was evidenced in instances where only the command presented to the left ear was manually performed at a time when another command presented simultaneously to the right ear was the only one that was verbally reported. The indication is that the right hemisphere understood and performed the required action when the left hemisphere was apparently unaware. However, it was also shown that for most dichotic verbal tests the left hemisphere still has dominant control over the right.
\r\n\r\nDichotic listening studies also indicated that the left hemisphere could separately monitor stimuli in the ipsilateral along with stimuli in the contralateral pathway. This was contradictory to previous conclusions that the contralateral pathway suppresses the ipsilateral in dichotic competition. Response time studies carried out in these callosum-sectioned patients investigated organization of the two cortical\r\nsystems that separately analyzed stimuli from the two ascending paths.
\r\n\r\nIt was found that response times for repeating words to the right ear were faster than for words in the left ear. Control tests showed the cause of this difference was not in delay of transmission in ascending routes, nor in differences of perception in the two systems.\r\nIt was deduced .that the cause was an asymmetrical process of memory retrieval for translation into motor impulses to the speech apparatus.
\r\n" }, { "name": "Holmes, David Salway", "degree": "PhD", "year": "1973", "title": "Studies on Nuclear RNA", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechTHESIS:07192018-114123511", "creators": [ { "name": { "family": "Holmes", "given": "David Salway" }, "id": "Holmes-David-Salway", "display_name": "Holmes, David Salway" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "chair", "display_name": "Bonner, James Frederick" }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "member", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Russell", "given": "Richard L." }, "id": "Russell-R-L", "role": "member", "display_name": "Russell, Richard L." }, { "name": { "family": "Davidson", "given": "Eric H." }, "id": "Davidson-E-H", "role": "member", "display_name": "Davidson, Eric H." } ], "option_major": [ "bioch" ], "doi": "10.7907/XDYW-S154", "abstract": "The isolation of giant nuclear RNA (HnRNA) from rat ascites cells \r\nis described. By the criteria of sedimentation through sucrose,\r\nformaldehyde and dimethyl sulfoxide, it is estimated that the\r\nmajority of the radioactivity of giant HnRNA after a 30 minute pulse\r\nof 3H-uridine is associated with molecules in the range 5-10 x 106\r\ndaltons. In the electron microscope, under denaturing conditions,\r\n84% (mass %) of giant HnRNA has a contour length of 4-9\u00b5 corresponding\r\nto a molecular weight of about 5-10 x 106 daltons.
\r\n\r\nGiant HnRNA has a \"DNA-like\" base composition (G+C = 46-54%) and\r\nhas considerable secondary structure (ca. 60% helix conformation) as\r\njudged by its melting profile and reactivity with formaldehyde.
\r\n\r\nRat nuclear DNA is characterized by its reassociation profile\r\n((Na+) = 0.18 at 62\u00b0, Tm - 23\u00b0) as judged by chromatography on\r\nhydroxyapatite. Single-copy DNA (Cot 1/2 observed = 1.5 x 103)\r\ncomprises 65% of the genome and 19% of the genome consists of sequences\r\nrepeated an average 1,800 times (middle repetitive DNA, Cot 1/2\r\nobserved = 1.0). 9% of the genome (highly repetitive DNA)\r\nreassociates faster than is measured in these experiments (Cot 1/2\r\nobserved < 2 x 10-2).
\r\n\r\nMiddle repetitive and single-copy DNA are isolated and \r\ncharacterized with respect to their reassociation kinetics and melting\r\nprofiles. They reassociate with kinetics similar to the kinetics\r\ndescribing these components when they are present in total\r\nDNA. The reassociated single-copy DNA has a high thermal stability\r\nindicative of fidelity of base pairing; the reassociated middle\r\nrepetitive DNA has a lower thermal stability which is probably\r\nattributable, in part, to base-pair mismatch.
\r\n\r\nRat giant nuclear RNA (HnRNA, 5-10 x 106 daltons) is hybridized\r\nto isolated single copy or middle repetitive DNA ((Na+) = 0.18 at 62\u00b0)\r\nHnRNA hydbridizes to about 4.5% of the single-copy and 9.4% of the\r\nmiddle repetitive DNA. The Tms of single-copy and middle repetitive\r\nhybrids are 1-2\u00b0 lower than those of the reassociated single-copy\r\nand middle repetitive DNA respectively. The DNA isolated from the\r\nsingle-copy or middle repetitive hybrids reassociates with kinetics\r\nsimilar to the input single-copy or middle repetitive DNA respectively.\r\nHnRNA is hybridized to total genomic DNA present in excess. 37% of the\r\nHnRNA hybridizes with kinetics (Cot 1/2 = 2.0 x 103) similar to\r\nsingle-copy DNa and 12% hybridizes with kinetics (Cot 1/2 = 5.6), a\r\nlittle more slowly than the major reassociating component of middle\r\nrepetitive DNA.
\r\n\r\nA chromatin-associated RNA (cRNA) prepared from rat ascites cells\r\nhybridizes to about 16% of isolated middle repetitive and 1% of\r\nisolated single copy rat DNA. In a hybridization reaction to total\r\nDNA, present in excess, at least 50% of the cRNA hybridizes at an\r\naverage rate similar to the major component of the middle repetitive\r\nDNA. These experiments indicate that the majority of cRNA consists\r\nof repetitive transcripts. Under conditions which assay essentially\r\nonly repetitive transcripts cRNA hybridizes to about 4.7% and giant\r\nnuclear RNA (HnRNA) hybridizes to about 4.6% of total nuclear rat\r\nDNA immobilized on filters. The Tm of cRNA hybrids (73.5\u00b0) and HnRNA\r\nhybrids (75.5\u00b0) are considerably lower than the Tm of native rat DNA\r\n(85.5\u00b0). This lowering of Tm is probably attributable, at least in\r\npart, to base-pair mismatch. Under the same conditions of hybridization\r\nthere is some hybridization competition for complementary DNA sites\r\nbetween cRNA and HnRNA, presumably between repetitive transcripts.\r\nDue to probable base-pair mismatch it is possible to infer only that\r\nthere is a similarity between HnRNA and cRNA transcripts and not\r\nnecessarily an identity.
" }, { "name": "Jesaitis, Algirdas Joseph", "degree": "PhD", "year": "1973", "title": "Linear Dichroism and Orientation of the Phycomyces Photopigment: I. Response and Absorption Studies. II. Fluorescence Studies", "advisor": "Delbruck, Max", "url": "https://resolver.caltech.edu/CaltechETD:etd-08092006-084855", "creators": [ { "name": { "family": "Jesaitis", "given": "Algirdas Joseph" }, "id": "Jesaitis-Algirdas-Joseph", "display_name": "Jesaitis, Algirdas Joseph" } ], "advisors": [ { "name": { "family": "Delbruck", "given": "Max" }, "id": "Delbr\u00fcck-M", "role": "advisor", "display_name": "Delbruck, Max" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/C2AX-J941", "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\r\n\r\nThe response of five strains of the fungus Phycomyces to linearly polarized light was studied. The sporangiophores of the strains, C2, C5, C9, C158, and NRRL 1555 (wildtype) differed primarily in optical attenuation. Their abilities to distinguish between longitudinally and transversely polarized blue light were found to be approximately the same.\r\n\r\nThe angle (with respect to the transverse axis) at which the [...] vector of the polarized light must be oriented to give a maximum response during perpendicular incidence into the cell was measured. It was found to be 42\u00b0 \u00b1 3\u00b0 for 280 nm light in the wild type strain. In the C158 strain this angle was 7\u00b0 \u00b1 3\u00b0 at 456 nm and 7 \u00b1 8\u00b0 at 486 nm.\r\n\r\nThe in vivo attenuation of polarized light as a function of the angle between the [...] vector and the cell axis was measured. The maximum transmission differences resulting from anisotropic attenuation were 4 \u00b1 2% at 320 nm, 3 \u00b1 2% at 456 nm, and 2 \u00b1 1% at 486 nm.\r\n\r\nThese results indicate that the polarized light effect in Phycomyces cannot arise from reflections at the cell surface, nor from attenuations due to internal screening or scattering and, therefore, must be due to the dichroism and orientation of the visual pigment.\r\n\r\nAn attempt was made to detect the presence of the photopigment in cell wall and plasmalemma fractions using fluorescence kinetics and polarization. Excitation of these preparations with high intensity 488 nm laser light and monitoring fluorescence intensity with a microphotometer, a fluorescence decay and a periodic dependence of fluorescence intensity on the [...] vector angle of exciting light was found. The relevance of this fluorescence to the in vivo photosystem is questionable because of the high excitation intensity necessary to produce any detectable fluorescence." }, { "name": "Linseman, Mary Ann Monica", "degree": "PhD", "year": "1973", "title": "Unit Activity in the Hypothalamus and Striatum of the Rat During Learning", "advisor": "Olds, James", "url": "https://resolver.caltech.edu/CaltechTHESIS:02062018-101121529", "creators": [ { "name": { "family": "Linseman", "given": "Mary Ann Monica" }, "id": "Linseman-Mary-Ann-Monica", "display_name": "Linseman, Mary Ann Monica" } ], "advisors": [ { "name": { "family": "Olds", "given": "James" }, "id": "Olds-J", "role": "advisor", "display_name": "Olds, James" } ], "committee": [ { "name": { "family": "Olds", "given": "James" }, "id": "Olds-J", "role": "chair", "display_name": "Olds, James" }, { "name": { "family": "Van Harreveld", "given": "Anthonie" }, "id": "Van-Harreveld-A", "role": "member", "display_name": "Van Harreveld, Anthonie" }, { "name": { "family": "Strumwasser", "given": "Felix" }, "id": "Strumwasser-F", "role": "member", "display_name": "Strumwasser, Felix" }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "member", "display_name": "Owen, Ray David" } ], "option_major": [ "biology" ], "doi": "10.7907/9BAS-1713", "abstract": "Unit activity was recorded from the hypothalamus and striatum\r\nof 80 freely moving rats during an appetitive classical conditioning \r\nsituation. Responses to auditory stimuli were observed from 118\r\nunits before and during a conditioning procedure in which presentation\r\nof food occurred one second after the onset of an auditory stimulus.\r\nA large proportion of units (111) showed changed responses to the CS \r\nduring conditioning. Only 8 of these, however, showed new conditioned \r\nresponses of the very shortest latency measured, 20 msec. after CS \r\nonset. These were interpreted as likely sites of rerouting of the stimulus \r\ninformation within the brain as a result of learning. They \r\nwere located largely near the intersection of hypothalamic and striatal \r\nstructures. A transient increase in rate of background firing over \r\ntrials was recorded following the onset of conditioning among hypo\u00adthalamic \r\nunits, suggesting they may temporarily represent a dynamic trace of the \r\nnew learning. No significant differences were found between areas \r\nstudied in order of appearance over trials of the conditioned responses. \r\nHowever, as a group, the conditioned responses studied here, appeared \r\nsignificantly earlier than a group of cortical neurons studied under \r\nsimilar conditions. There was greater generalization of response \r\nto the CS- by units of the basal ganglia than other areas, suggesting \r\nthey may be of importance in inhibition of response to the CS-.\r\n\r\n" }, { "name": "Segal, Menahem", "degree": "PhD", "year": "1973", "title": "The Hippocampus as a Learning Machine", "advisor": "Olds, James", "url": "https://resolver.caltech.edu/CaltechTHESIS:09172018-103619092", "creators": [ { "name": { "family": "Segal", "given": "Menahem" }, "id": "Segal-Menahem", "display_name": "Segal, Menahem" } ], "advisors": [ { "name": { "family": "Olds", "given": "James" }, "id": "Olds-J", "role": "advisor", "display_name": "Olds, James" } ], "committee": [ { "name": { "family": "Olds", "given": "James" }, "id": "Olds-J", "role": "chair", "display_name": "Olds, James" }, { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "member", "display_name": "Bonner, James Frederick" }, { "name": { "family": "Fender", "given": "Derek H." }, "id": "Fender-D-H", "role": "member", "display_name": "Fender, Derek H." }, { "name": { "family": "Sperry", "given": "Roger Wolcott" }, "id": "Sperry-R-W", "role": "member", "display_name": "Sperry, Roger Wolcott" }, { "name": { "family": "Strumwasser", "given": "Felix" }, "id": "Strumwasser-F", "role": "member", "display_name": "Strumwasser, Felix" } ], "option_major": [ "biology" ], "doi": "10.7907/P1SD-Z263", "abstract": "A series of experiments were conducted with the purposes of\r\ndescribing a functional pathway in the rat hippocampus, characterizing some conditions necessary for activating it, and identifying critical steps in this pathway. In all experiments a classical conditioning paradigm was used and the responses of units in the hippocampus and related forebrain structures to the conditioned stimulus were measured. In the first experiment a few differences between dentate, CA-3, and CA-1, the main fields of the hippocampus, were found. Units in the dentate were first to acquire a conditioned response, CA-3 followed and CA-1 was last. This order fits with the anatomical pathway. However, dentate responses were phasic, that is, did not outlast the CS-US interval, and were not specific to the conditioned stimulus. The responses of CA-3 and CA-1 units, on the other hand, were sustained and specific. The second experiment was devoted to the analysis of conditioned response latencies, in the hippocampus\r\nas well as in septum, subiculum, cingulate, entorhinal, and related structures, all known to be input stages to the hippocampus. In this experiment unconditioned short response latencies were found in the medial septum, one of the afferents of the hippocampus. These were not changed in the process of learning. The shortest conditioned response latencies were found in area CA-3 of the hippocampus. Units in area CA-1 followed, but units in dentate did not precede those of CA-3. Units in entorhinal cortex, the other main afferent to the hippocampus did not seem to precede hippocampal units either. The special relations between the hippocampus and the dentate were demonstrated in another part of this experiment, where dentate units lost their conditioned responses, in the process of extinction, before those of CA-3 and CA-1. It was postulated that septal input triggers CA-3 responses and these\r\nwould be maintained in the presence of reinforcing dentate and\r\nentorhinal inputs.
\r\n\r\nThe relations between the dentate and the hippocampus were\r\nfurther studied in two experiments in which aversive electric shock served as an unconditioned stimulus. In experiment 3 food and shock served as unconditioned stimuli on alternate days. In\r\nexperiment 4 food and shock were presented in the same sessions as unconditioned stimuli to two different CS's. Dentate units had an excitatory conditioned response to a food signal and an inhibitory conditioned response to a shock signal in both experiments. Hippocampal units had excitatory responses to both signals. Acquisition of a conditioned response was not demonstrated within the hippocampus when the conditioned stimulus preceded shock and was slow when food or shock were applied following two different signals in the same session. However, when first trained that a signal precedes food, the conditioned response would be maintained in the hippocampus even if shock is now the US. The dentate is probably involved in the initiation of a conditioned response in the hippocampus but not in the maintenance of it.
\r\n\r\nA sensory-sensory paradigm (experiment 5) has demonstrated\r\nthe presence of unconditioned unhabituated sensory responses in two of the afferents to the hippocampus, that is, the medial septum and the cingulate cortex. It failed to show signs of conditioning in the hippocampus proper. It was proposed that in the absence of an appetitive reward and the activity of the entorhinal-dentate pathway, conditioned responses in hippocampus cannot be established.
\r\n\r\nConditioned entorhinal responses (experiment 6) had long\r\nlatency but also long time constant. Their evoked activity was\r\nmaintained for periods as long as one minute. It was found that\r\nhippocampal responses were larger, if the conditioned stimulus was applied within one minute from the previous trial. Hence, a\r\ncorrelation between hippocampal responses and entorhinal firing\r\nrate was demonstrated. On the basis of these experiments it was\r\nproposed that septal input enters the hippocampus at the CA-3\r\narea, is able to selectively activate these cells only in the\r\npresence of facilitation produced by entorhinal and dentate activity. The facilitatory entorhinal activity is triggered mainly by positive reward.
\r\n" }, { "name": "Beckendorf, Steven Kent", "degree": "PhD", "year": "1972", "title": "Studies of Bacteriophage T4 Tail Fibers and Tail Fiber Genes", "advisor": "Wood, William B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03072016-145346915", "creators": [ { "name": { "family": "Beckendorf", "given": "Steven Kent" }, "id": "Beckendorf-Steven-Kent", "display_name": "Beckendorf, Steven Kent" } ], "advisors": [ { "name": { "family": "Wood", "given": "William B." }, "id": "Wood-William-B", "role": "advisor", "display_name": "Wood, William B." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/HGWS-XX32", "abstract": "The distal half of the bacteriophage T4 tail fiber interacts with the surface of the bacterium during adsorption. The largest polypeptide in this half fiber is the product of gene 37 (P37). During assembly of the tail fiber, P37 interacts with the product of gene 38 (P38). These two gene products are incompatible with the corresponding gene products from the related phage T2. T2 P37 does not interact with T4 P38 and T2 P38 does not interact with T4 P37. Crosses between T2 and T4 phages mutant in genes 37 and 38 have shown that the carboxyl end of P37 interacts with P38 and with the bacterial surface. In the corresponding region of gene 37 and in gene 38 there is no recombination between T2 and T4. In the rest of gene 37 there are two small regions with relatively high recombination and a region of low recombination.
\r\n\r\nWhen T2/T4 heteroduplex DNA molecules are examined in the electron microscope four nonhomologous loops appear in the region of genes 37 and 38. Heteroduplexes between hybrid phages which have part of gene 37 from T4 and part from T2 have roughly located gene 37 mutations in the heteroduplex pattern. For a more precise location of the , mutations a physical map of gene 37 was constructed by determining the molecular weights of amber polypeptide fragments on polyacrylamide gels in the presence of sodium dodecyl sulfate. When the physical and heteroduplex maps are aligned, the regions of low recombination correspond to regions of nonhomology between T2 and T4. Regions with relatively high recombination are homologous.
\r\n\r\nThe molecular weight of T2 P37 is about 13,000 greater than that of T4 P37. Analysis of hybrid phage has shown that this molecular weight difference is all at the carboxyl end of P37.
\r\n\r\nAn antiserum has been prepared which is specific for the distal half fiber of T4. Tests of the ability of gene 37 hybrids to block this antiserum show that there are at least 4 subclasses of antigen specified by different parts of P37.
\r\n\r\nObservations in the electron microscope of the tailfiber - anti- body complexes formed by the gene 37 hybrids and the specific anti- serum have shown that P37 is oriented linearly in the distal half fiber with its N-terminus near the joint between the two half fibers and its C-terminus near the tip of the fiber. These observations lead to a simple model for the structure of the distal half fiber.
\r\n\r\nThe high recombination in T4 gene 34 was also investigated. A comparison of genetic and physical maps of gene 34 showed that there is a gradient of increasing recombination near one end of the gene.
" }, { "name": "Benbow, Robert Michael", "degree": "PhD", "year": "1972", "title": "On the Genetic Recombination of Bacteriophage \u03a6X174 DNA Molecules", "advisor": "Sinsheimer, Robert L.", "url": "https://resolver.caltech.edu/CaltechETD:etd-08082006-132253", "creators": [ { "name": { "family": "Benbow", "given": "Robert Michael" }, "id": "Benbow-Robert-Michael", "display_name": "Benbow, Robert Michael" } ], "advisors": [ { "name": { "family": "Sinsheimer", "given": "Robert L." }, "id": "Sinsheimer-R-L", "role": "advisor", "display_name": "Sinsheimer, Robert L." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/M0E3-WG80", "abstract": "Genetic recombination between two [phi]X174 parental replicative form DNA molecules primarily occurs by the following sequence of events: (i) a single strand scission in one HF DNA molecule; (ii) displacement synthesis; (iii) unimolecular branch migration; (iv) attack; (v) formation of a hydrogen bonded joint molecule; (vi) bimolecular branch migration and further DNA (repair?) synthesis; (vii and viii) covalent bond formation and single strand scission (order not certain); (ix) restoration of recombinant DNA molecules to the parental HF configuration. The net result is an asymmetric non-reciprocal recombination event yielding one parent and one recombinant. The average net DNA synthesis is less than 600 nucleotides; breakage and reunion occurs; over 50% of all genetic exchanges involve regions less than 4.00 nucleotides in length. Single strand insertion is (probably) the most common recombination event. Recombinant formation is complete at or shortly after the initiation of progeny HF DNA synthesis; the completed recombinant DNA molecule and surviving parental molecule then resume normal [phi]X174 DNA replication.\r\n\r\nA (minor) secondary recombination mechanism exists in which recombinant formation occurs between [phi]X174 progeny HF DNA molecules." }, { "name": "Bergman, Kostia", "degree": "PhD", "year": "1972", "title": "Sensory Responses of Phycomyces: I. Blue-Light Control of Sporangiophore Initiation. II. Classification of mad Mutants", "advisor": "Delbruck, Max", "url": "https://resolver.caltech.edu/CaltechETD:etd-08092006-110158", "creators": [ { "name": { "family": "Bergman", "given": "Kostia" }, "id": "Bergman-Kostia", "display_name": "Bergman, Kostia" } ], "advisors": [ { "name": { "family": "Delbruck", "given": "Max" }, "id": "Delbr\u00fcck-M", "role": "advisor", "display_name": "Delbruck, Max" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/K2PW-6W48", "abstract": "Part I. Blue-Light Control of Sporangiophore Initiation:\r\n\r\nMany fungi produce spores or spore-bearing structures under the control of blue light. Phycomyces sporangiophores are produced continuously along racing tube cultures grown in constant darkness or constant light. However, if a dark-grown culture is exposed to light for a short time on one day a narrow, dense band of sporangiophores is observed the next day at that point of the tube occupied by the mycelial tips during the light pulse. A periodic program with \"short days\", (e.g. 4-hour light/20-hour dark) leads to periodic bands of sporangiophores spaced at intervals corresponding to one period-length (in this case 24 hours) of mycelial growth. Sporangiophore initiation is inhibited by a light to dark transition and is stimulated by a dark to light transition. A partial action spectrum of the initiation response, covering the critical 480 nm to 540 nm region, strongly suggests that the same photo-receptor-pigment is involved as in the phototropic response and light growth response of sporangiophores. Mutants with altered light control of sporangiophore initiation have been found among those selected for altered phototropism. This joint elimination of these two responses to blue light by a single mutation is evidence for a common early transduction system. The extensive literature on the effects of light on fungal sporulation is reviewed.\r\n\r\nPart II. Classification of mad Mutants:\r\n\r\nMutants of Phycomyces with altered responses of their sporangiophores to light are called mad. Simple quantitative tests have been devised and used to separate the mad mutants into phenotypic classes based on three sensory responses: phototropism of sporangiophores, avoidance of solid objects by sporangiophores (or auto-chemotropism), and light control of sporangiophore initiation. Class 1 mutants have altered phototropism but normal avoidance. Some of these mutants show phototropism only at intensities 10(6) times higher than the wild type threshold, but none are completely non-phototropic. Class 1 is divided into two subclasses. Class 1-1 mutants are also deficient in light control of sporangiophore initiation. Class 1-2 mutants show normal light control of sporangiophore initiation. Class 2 mutants have altered phototropism and altered avoidance but normal light control of sporangiophore initiation. A simple model of the organization of the sensory systems of Phycomyces is proposed." }, { "name": "Elgin, Sarah Carlisle Roberts", "degree": "PhD", "year": "1972", "title": "Investigations of Nonhistone Chromosomal Proteins", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechTHESIS:04182016-080802139", "creators": [ { "name": { "family": "Elgin", "given": "Sarah Carlisle Roberts" }, "id": "Elgin-Sarah-Carlisle-Roberts", "display_name": "Elgin, Sarah Carlisle Roberts" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/6VGH-RZ17", "abstract": "The major nonhistone chromosomal proteins (NHC proteins) are a group of 14-20 acidic proteins associated with DNA in eukaryotic chromatin. In comparisons by SDS gel electrophoresis (molecular weight sieving) one observes a high degree of homology among the NHC protein fractions of different tissues from a given species. Tissue-specific protein bands are also observed. The appearance of a new\r\nNHC protein, A, in the NHC proteins of rat liver stimulated to divide by partial hepatectomy and of rat ascites cells suggests that this protein may play a role in preparing the cell for division. The NHC proteins of the same tissue from different species are also very similar. Quantitative but not qualitative changes in the NHC proteins of rat uterus are observed on stimulation (in vivo) with estrogen. These observations suggest that the major NHC proteins play a general role in chromatin structure and the regulation of genome expression; several may be enzymes of nucleic acid and histone metabolism and/or structural proteins analogous to histones. One such enzyme, a protease which readily and preferentially degrades histones, can be extracted from chromatin with 0.7 N NaCl.
\r\n\r\nAlthough the NHC proteins readily aggregate, they can be separated from histone and fractionated by ion exchange chromatography on Sephadex SE C-25 resin in 10 M urea-25% formic acid (pH 2.5). Following further purification, four fractions of NHC protein are obtained; two of these are single purified proteins, and the other two contain 4-6 and 4-7 different proteins. These NHC proteins show a ratio of acidic to basic amino acids from 2.7 to 1.2 and isoelectric points from apparently less than 3.7 to 8.0. These isolated fractions appear more soluble and easier to work with than any whole NHC protein preparation.
\r\n\r\n" }, { "name": "Firtel, Richard Alan", "degree": "PhD", "year": "1972", "title": "Part I. Regulation of Development in the Cellular Slime Mold Dictyostelium discoideum. Part II. Polysomes and RNA SyntheSiS During Early Development of the Surf Clam Spisula solidissima", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechTHESIS:04112016-144248288", "creators": [ { "name": { "family": "Firtel", "given": "Richard Alan" }, "id": "Firtel-Richard-Alan", "display_name": "Firtel, Richard Alan" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/G15P-H973", "abstract": "Part I. The cellular slime mold Dictyostelium discoideum is a simple eukaryote which undergoes a multi-cellular developmental process. Single cell myxamoebae divide vegetatively in the presence of a food source. When the food is depleted or removed, the cells aggregate, forming a migrating pseudoplasmodium which differentiates into a fruiting body containing stalk and spore cells. I have shown that during the developmental cycle glycogen phosphorylase, aminopeptidase, and alanine transaminase are developmentally regulated, that is their specific activities increased at a specific time in the developmental cycle. Phosphorylase activity is undetectable in developing cells until mid-aggregation whereupon it increases and reaches a maximum at mid-culmination. Thereafter the enzyme disappears. Actinomycin D and cycloheximide studies as well as studies with morphologically aberrant and temporally deranged mutants indicate that prior RNA and concomitant protein synthesis are necessary for the rise and decrease in activity and support the view that the appearance of the enzyme is regulated at the transcriptional level. Aminopeptidase and alanine transaminase increase 3 fold starting at starvation and reach maximum activity at 18 and 5 hours respectively.
\r\n\r\nThe cellular DNA s of D. discoideum were characterized by CsC1 buoyant density gradient centrifugation and by renaturation kinetics. Whole cell DNA exhibits three bands in CsCl: \u03c1 = 1.676 g/cc (nuclear main band), 1.687 (nuclear satellite), and 1.682 (mitochondrial). Reassociation kinetics at a criterion of Tm -23\u00b0C indicates that the nuclear reiterated sequences make up 30% of the genome (Cot1/2 (pure) 0.28) and the single-copy DNA 70% (Cot1/2(pure) 70). The complexity of the nuclear genome is 30 x 109 daltons and that of the mitochondrial DNA is 35-40 x 106 daltons (Cot1/2 0.15). rRNA cistrons constitute 2.2% of nuclear DNA and have a \u03c1 = 1.682.
\r\n\r\nRNA extracted from 4 stages during developmental cycle of Dictyostelium was hybridized with purified single-copy nuclear DNA. The hybrids had properties indicative of single-copy DNA-RNA hybrids. These studies indicate that there are, during development, qualitative and quantitative changes in the portion of the single-copy of the genome transcribed. Overall, 56% of the genome is represented by transcripts between the amoeba and mid-culmination stages. Some 19% are sequences which are represented at all stages while 37% of the genome consists of stage specific sequences.
\r\n\r\nPart II. RNA and protein synthesis and polysome formation were studied during early development of the surf clam Spisula solidissima embryos. The oocyte has a small number of polysomes and a low but measurable rate of protein synthesis (leucine-3H incorporation). After fertilization, there is a continual increase in the percentage of ribosomes sedimenting in the polysome region. Newly synthesized RNA (uridine-5-3H incorporation) was found in polysomes as early as the 2-cell stage. During cleavage, the newly formed RNA is associated mainly with the light polysomes.
\r\n\r\nRNA extracted from polysomes labeled at the 4-cell stage is polydisperse, nonribosomal, and non-4 S. Actinomycin D causes a reduction of about 30% of the polysomes formed between fertilization and the 16-cell stage.
\r\n\r\nIn the early cleavage stages the light polysomes are mostly affected by actinomycin.
\r\n\r\n" }, { "name": "Foster, Kenneth William", "degree": "PhD", "year": "1972", "title": "The Photoresponse of Phycomyces: Analysis Using Manual Techniques and an Automated Machine Which Precisely Tracks and Measures Growth During Programmed Stimuli", "advisor": "Delbruck, Max", "url": "https://resolver.caltech.edu/CaltechETD:etd-08072006-150918", "creators": [ { "name": { "family": "Foster", "given": "Kenneth William" }, "id": "Foster-Kenneth-William", "display_name": "Foster, Kenneth William" } ], "advisors": [ { "name": { "family": "Delbruck", "given": "Max" }, "id": "Delbr\u00fcck-M", "role": "advisor", "display_name": "Delbruck, Max" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/SR6R-P445", "abstract": "The sporangiophores of the fungus Phycomyces are sensitive to light, to gravity, to mechanical stretch, and possibly to a gas. In order to understand the processes by which these stimuli evoke response from the organism, the relationships between these input stimuli, and the output responses of the fungus have beenn studied. The light stimulus was chosen because of its ease of quantification.\r\n\r\nTo study these responses better than in the past, automated equipment has been developed to improve the precision of observation, the control of the environment, and the collection of data. The heart of this equipment is a tracking system which stabilizes in space the sporangium, which serves as a convenient spherical marker just above the sensing and responding growing zone. Stabilization of this zone makes uniform stimulation relatively easy. Further the automatic system makes possible 6 or more hours of measurement on a single sporangiophore without requiring much operator intervention. After suppression of noise by means of a low pass filter of bandwidth 0.07 Hz, the growth velocity of typically 50 microns/min was measured to a precision of better than 5%. This compares favorably with manual techniques which optimally have a precision of about 20% for the average growth velocity over a 30 sec period.\r\n\r\nUse of this machine as well as of conventional experimental techniques on the newly available mutants of Phycomyces has provided a clearer understanding of the stimulus-response system. The effect of light scattering and of absorption of light inside the sporangiore on the response to light has been investigated. These studies have resulted in appreciation of the quantitative effect of the lens focusing of the cell and in an improved action spectrum.\r\n\r\nEvidence is presented that a major factor causing phototropism is the cell's natural twist (spiral growth) of about 6\u00b0/min in the middle of the growing zone. Each region of the cell locally adapts to the local light intensity. However with the twist of the cell, at one edge of the focused band which is produced by the lens focusing of the cell there will be a continuous entry of dark adapted area into the higher intensity band, thus giving rise to transient positive growth responses in a succession of surface elements. The response to this stimulus is expressed slowly in time and as it is expressed, it is carried around at the twist rate of the cell. This model has successfully explained both our experiments and a number from the literature.\r\n\r\nUsing the machine, latency and shape of response have been characterized as a function of temperature, stimulus size, level of adaptation and incident light wavelength. Latency is dependent on the logarithm of the absolute adaptation level and there is no wavelength dependence of the stimulus response curve. For larger stimuli the shape of the response changes markedly, with the rising of an early peak in the growth velocity and inhibition of a later second peak.\r\n\r\nStudy of mutants defective in the chemical pathway from light stimulation to response show a great variety of mutant types implying a relatively large number of biochemical steps from stimulus to response." }, { "name": "Hiatt, David Ellis", "degree": "PhD", "year": "1972", "title": "Investigations of Operant Conditioning of Single Unit Activity in the Rat Brain", "advisor": "Olds, James", "url": "https://resolver.caltech.edu/CaltechETD:etd-08302007-114218", "creators": [ { "name": { "family": "Hiatt", "given": "David Ellis" }, "id": "Hiatt-David-Ellis", "display_name": "Hiatt, David Ellis" } ], "advisors": [ { "name": { "family": "Olds", "given": "James" }, "id": "Olds-J", "role": "advisor", "display_name": "Olds, James" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/FQEA-6371", "abstract": "The aim of these studies was to show that the capacity for operant responses is distributed differentially in the brain and that such capacity is maintained in the absence of feedback from movement in specific parts of the brain. The experimental subjects were rats chronically implanted with microelectrodes for single unit recording from several different brain structures. There were three experimental paradigms. In Experiments I and II positive reinforcement was applied following bursts of activity of an arbitrarily selected unit during periods indicated by a discriminative stimulus. All such units in cerebellum and brain stem displayed significant conditioned rate increases while only about half those in hippocampus, midbrain and superior colliculus did so indicating that operant conditioning is more a property of \"motor\" units. Experiment II was a direct continuation of Experiment I with some of the rats which had conditioned units. The contribution of the bodily movement which seemed inevitably correlated with the conditioned unit response was determined by inducing skeletal muscle paralysis with Flaxedil. Conditioned responses were maintained under paralysis in all 5 rats with an experimental unit in the brain stem but in only one of the 6 rats with an experimental unit in the cerebellum, and none in the other 7 rats with experimental units divided among hippocampus, midbrain and superior colliculus. This indicated that the conditioned responses of most of the units were fed-back from movement which the conditioned activity of the brain stem units probably preceded. A control experiment with non-contingent reinforcement showed that these conditioned responses were probably not entirely due to operant conditioning. This ambiguity was absent in Experiment III which showed clearly operant activity. The rate of units, predominantly in the cerebellum, was increased or decreased depending upon the contingency of reinforcement. However, Experiment III used active animals and tested no units in the brain stem. A final experiment demonstrated clearly operant activity of a brain stem unit under paralysis. Reinforcement was made contingent upon rapid alternation between activity and inactivity of the unit. After acquisition, this behavior was brought under the control of a discriminative stimulus, and then maintained under paralysis, which eliminated the alternation of stereotyped movements that had been correlated with the unit activity.\r\n\r\n(Photographic materials on pages 18, 22, 66, 68, 144, 146, and 148 are essential and will not reproduce clearly on Xerox copies. Photographic copies should be ordered.)" }, { "name": "Konopka, Ronald Jerome", "degree": "PhD", "year": "1972", "title": "Circadian Clock Mutants of Drosophila melanogaster", "advisor": "Benzer, Seymour", "url": "https://resolver.caltech.edu/CaltechTHESIS:04012016-120503899", "creators": [ { "name": { "family": "Konopka", "given": "Ronald Jerome" }, "id": "Konopka-Ronald-Jerome", "display_name": "Konopka, Ronald Jerome" } ], "advisors": [ { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "advisor", "display_name": "Benzer, Seymour" } ], "committee": [ { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "chair", "display_name": "Benzer, Seymour" }, { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "member", "display_name": "Lewis, Edward B." }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "member", "display_name": "Mitchell, Herschel K." }, { "name": { "family": "Strumwasser", "given": "Felix" }, "id": "Strumwasser-F", "role": "member", "display_name": "Strumwasser, Felix" }, { "name": { "family": "Vinograd", "given": "Jerome" }, "id": "Vinograd-J", "role": "member", "display_name": "Vinograd, Jerome" } ], "option_major": [ "bioch" ], "doi": "10.7907/R04B-3425", "abstract": "Three mutants of Drosophila melanogaster have been isolated in which the free-running period of the circadian eclosion rhythm and the adult locomotor activity rhythm is affected. One mutant is arrhythmic, another has a short period of 19 hours, and the third has a long period of 28 hours. The mutants retain their phenotypes over the temperature range 18\u00b0 to 25\u00b0 C. All three mutants map near the tip of the X chromosome (distal to the centromere). By deficiency mapping, the short-period mutation has been localized to the 3B1-2 region. Complementation tests show that all three mutations affect the same functional gene.
\r\n\r\nAnalysis of activity rhythms of individual mosaic flies indicates that the site of action of the short-period mutation is probably located in the head of the fly. A few activity patterns of split-head and mixed-head mosaics appear to possess both mutant and heterozygous components, suggesting that the fly head may contain\r\ntwo complete clocks capable of maintaining their periodicities independently.
\r\n\r\nThe short-period mutation affects both the duration of the light-insensitive part of the oscillation and the degree to which the clock can be reset during the light-sensitive part of the oscillation.
\r\n\r\nBoth the short-period and long-period mutant eclosion rhythms can be entrained to a period of 24 hours by a 12:12 light-dark cycle having a light intensity at least two orders of magnitude greater than that required to entrain the normal rhythm. The arrhythmic mutant does not entrain under these conditions. In the presence of a temperature cycle, however, the arrhythmic mutant does entrain, but its rhythm damps out when the temperature cycle is removed.
\r\n\r\nEvidence is presented that Pittendrigh's two-oscillator model for the clock in D. pseudoobscura applies to D. melanogaster as well. The three clock mutations primarily affect the light- sensitive driving oscillator. The arrhythmic mutation appears to have eliminated the driving oscillator while leaving the temperature-sensitive driven oscillator relatively intact.
\r\n" }, { "name": "Kornblith, Carol Lee", "degree": "PhD", "year": "1972", "title": "Conditioned Responses in the Reticular Formation", "advisor": "Olds, James", "url": "https://resolver.caltech.edu/CaltechTHESIS:05022016-093643296", "creators": [ { "name": { "family": "Kornblith", "given": "Carol Lee" }, "id": "Kornblith-Carol-Lee", "display_name": "Kornblith, Carol Lee" } ], "advisors": [ { "name": { "family": "Olds", "given": "James" }, "id": "Olds-J", "role": "advisor", "display_name": "Olds, James" } ], "committee": [ { "name": { "family": "Olds", "given": "James" }, "id": "Olds-J", "role": "chair", "display_name": "Olds, James" }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "member", "display_name": "Owen, Ray David" }, { "name": { "family": "Strumwasser", "given": "Felix" }, "id": "Strumwasser-F", "role": "member", "display_name": "Strumwasser, Felix" }, { "name": { "family": "Van Harreveld", "given": "Anthonie" }, "id": "Van-Harreveld-A", "role": "member", "display_name": "Van Harreveld, Anthonie" }, { "name": { "family": "Wiersma", "given": "Cornelis A. G." }, "id": "Wiersma-C-A-G", "role": "member", "display_name": "Wiersma, Cornelis A. G." } ], "option_major": [ "biology" ], "doi": "10.7907/J6N6-EV96", "abstract": "Unit activity was recorded from the midbrain and pons of 40 freely moving rats in an appetitive classical conditioning situation. Responses to auditory stimuli were observed from 100 units before and during a conditioning procedure in which presentation of food occurred 1 sec after the onset of the auditory stimulus. Conditioned unit responses (i.e., spike rate accelerations or decelerations) were considered to be positive when 1) no similar responses appeared prior to conditioning, and 2) latencies were equal to or less than those of sensory responses derived from the inferior colliculus. Such short latency conditioned unit responses were recorded from 11 probes located in the mid-lateral pert of the ventral region of the brain stem. This region was differentiated from paramedian, far lateral and dorsal parts of the brain stem reticular formation. Conditioned unit responses of considerably longer latencies were recorded from 76 probe located in these other regions. Among the longer latency responses interesting differences appeared in experiments conducted after the first conditioning series was completed. With additional training, units in the \"reticular activating system\" of midbrain and pons tended to yield stabilized responses in the early portion of the\r\nCS-US interval closely related in time to the orientation responses evoked by the CS. In contrast, the responses of units in the limbic midbrain tended to stabilize in the later part of the CS-US interval closely related in time to preparatory responses tied to the US. During extinction when the auditory stimulus was no longer followed by presentation of food, many of the responses were reduced to their pre-conditioning levels. However, there was a tendency for units which had displayed short latency responses on the first conditioning day to be more resistant to extinction than units which had displayed longer latency conditioned responses. The data were interpreted as indicating a local correlate of learning in the reticular formation of midbrain end pons and a separation of the midbrain system into at least two areas: 1) the classical \"reticular activating system\" related to orienting reactions, and 2) the limbic midbrain areas related to drives and rewards. Because the ventral and mid-lateral area with very short latency conditioned responses was not clearly tied to either of these; it was considered as possibly representing a third division." }, { "name": "Kow, Lee-Ming", "degree": "PhD", "year": "1972", "title": "Study of Ion Movements in Isolated Chicken Retinas During Spreading Depression", "advisor": "Van Harreveld, Anthonie", "url": "https://resolver.caltech.edu/CaltechTHESIS:03212016-155122153", "creators": [ { "name": { "family": "Kow", "given": "Lee-Ming" }, "id": "Kow-Lee-Ming", "display_name": "Kow, Lee-Ming" } ], "advisors": [ { "name": { "family": "Van Harreveld", "given": "Anthonie" }, "id": "Van-Harreveld-A", "role": "advisor", "display_name": "Van Harreveld, Anthonie" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "neurobio" ], "doi": "10.7907/T0TR-Z517", "abstract": "Spreading depression (SD) is a phenomenon observed in several sections of vertebrate central nervous system. It can occur spontaneously or be evoked by a variety of stimuli, and consists of a wave of depression of the normal electrical activity of the nervous tissue which spreads slowly in all directions in the tissue. This wave of depression is accompanied by several concomitants including ion movements. All the concomitants of SD can be explained by an increase in the sodium permeability of the plasma membranes of cellular elements involved in this phenomenon.
\r\n\r\nIn the chicken retina, SD is accompanied by a transparency change which can be detected with the naked eye. The isolated retina is a thin (0.1 mm) membrane in which the extracellular fluid quickly and completely equilibrates with the incubation solutions. This preparation was therefore used to study the ion movements during SD by measuring and comparing the ion contents and the extracellular space (ECS) of retinas incubated in various solutions of which some inhibited SD, whereas others allowed this phenomenon to occur.
\r\n\r\nThe present study has shown that during SD there is a shift of extracellular sodium into the intracellular compartment of the retina, a release of intracellular K and a decrease in the magnitude of ECS. These results are in agreement with previous postulates about SD, although the in vitro experimental condition makes the ion movements appear larger and the loss of ECS smaller than observed in the intact cortical tissue. The movements of Na and K, in opposite directions, are reversible. The development and magnitudes of SD is very little affected by deprivation of the oxygen supply.
\r\n\r\nIt was established that the inward sodium shift is not a consequence of an arrest of the Na-pump. It can be prevented, together with SD by the membrane stabilizers, magnesium and procaine. Spreading depression and the ion movements are incompletely inhibited by tetrodotoxin, which blocks the sodium influx into nerve fibers during the action potential. The replacement of Na in the bathing solution by Li does not prevent SD, which is accompanied by Li accumulation in the intracellular compartment. From these experiments and others it was concluded that the mechanism underlying SD and the ion shifts is an increase in the sodium permeability of cell membranes.
\r\n" }, { "name": "Lu, Cary", "degree": "PhD", "year": "1972", "title": "The Interaction of Color and Luminance in Stereoscopic Vision", "advisor": "Fender, Derek H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05022016-163223190", "creators": [ { "name": { "family": "Lu", "given": "Cary" }, "id": "Lu-Cary", "display_name": "Lu, Cary" } ], "advisors": [ { "name": { "family": "Fender", "given": "Derek H." }, "id": "Fender-D-H", "role": "advisor", "display_name": "Fender, Derek H." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/MSFY-AH42", "abstract": "Experiments are described using the random dot stereo patterns devised by Julesz, but substituting various colors and luminances for the usual black and white random squares. The ability to perceive the patterns in depth depends on a luminance difference between the colors used. If two colors are the same luminance, then depth is not perceived although each of the individual squares which make up the patterns is easily seen due to the color difference. This is true for any combination of different colors. If different colors are used for corresponding random squares between the left and right eye patterns, stereopsis is possible for all combinations of binocular rivalry in color, provided the luminance difference is large enough. Rivalry in luminance always precludes stereopsis, regardless of the colors involved.
\r\n" }, { "name": "Meltzer, Paul Stuart", "degree": "PhD", "year": "1972", "title": "Studies on the A Components of Drosophila Phenol Oxidase", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06102016-143545592", "creators": [ { "name": { "family": "Meltzer", "given": "Paul Stuart" }, "id": "Meltzer-Paul-Stuart", "display_name": "Meltzer, Paul Stuart" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/RYW9-R750", "abstract": "In insects, the enzyme phenol oxidase is involved in the hardening and darkening of the cuticle. Drosophila phenol oxidase occurs in a latent form. Phenol oxidase activity is produced as a consequence of the interaction of several proteins. Three of these proteins are designated as the A components (A1, A2, A3). A quantitative assay for the A components has been developed. The A1 component has been prepared in stable and highly purified form by gel filtration, preparative electrofocusing, and preparative electrophoresis. No major contaminants could be detected by analytical polyacrylamide gel electrophoresis, analytical electrofocusing, SDS gel electrophoresis, and analytical ultracentrifugation. The A1 component has a molecular weight of approximately 77,000 daltons and an isoelectric point of 5.1. The isoelectric point of the A2 component is 6.0, and its molecular weight is similar to that of the A1 component. The A1 component has no detectable phenol oxidase activity and cannot be converted to active phenol oxidase by the S (salivary gland) component. The A1 component contains approximately 0.15% copper and does not contain large amounts of fatty acids or phosphate. The amino acid composition of A1 is reported. Microheterogeneity was observed in preparations of purified A1. The relationship of the A components to active phenol oxidase and the mechanism of the activation reaction are discussed.
\r\n\r\n" }, { "name": "Robberson, Donald Lewis", "degree": "PhD", "year": "1972", "title": "I. A Study by Electron Microscopy of the RNA and RNA-DNA Hybrids of Hela Mitochondria. II. Replication of Closed Circular DNA in Mouse L Cells", "advisor": "Davidson, Norman R.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06212016-081819448", "creators": [ { "name": { "family": "Robberson", "given": "Donald Lewis" }, "id": "Robberson-Donald-Lewis", "display_name": "Robberson, Donald Lewis" } ], "advisors": [ { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "advisor", "display_name": "Davidson, Norman R." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/JKBR-M955", "abstract": "The thesis consists of two parts. In Part I, electron microscope analysis of HeLa mitochondrial RNA-DNA hybrids isolated from CsSO4 gradients demonstrates that more than 85% of the heavy strand of HeLa mitochondrial DNA is transcribed in vivo.
\r\n\r\nA modified basic protein film method of spreading RNA in a strongly denaturing solvent for examination in the electron microscope has been developed and applied to determine the size of the HeLa mitochondrial specific ribosomal RNA (rRNA) components. Length measurements on purified 12S and 16S mitochondrial rRNA, on mixtures of the two, and on mixtures of 12S with 18S cytoplasmic rRNA have given molecular lengths of 0.27 \u03bc, 0.42 \u03bc, and 0. 55 \u03bc for the 12S, 16S, and 18S rRNAs, respectively. If these molecular lengths are proportional to molecular weight, and if the molecular weight of 18S cytoplasmic rRNA is taken as 0.71 x 106, as determined by sedimentation equilibrium, the molecular weights of the 12S and 16S components are 0.35 x 106 and 0.54 x 106, respectively. These molecular weight values are in good agreement with the relative values predicted from sedimentation velocity measurements, but not with the relative values based on gel electrophoresis.
\r\n\r\nElectron microscopy of hybrids between the heavy strand of HeLa mitochondrial DNA and HeLa mitochondrial rRNA demonstrates that the genes for 16S and 12S HeLa mitochondrial rRNAs are situated adjacent, or very close, to each other on mitochondrial DNA. The length of the DNA segment separating the two genes is estimated to correspond to less than 500 nucleotide pairs.
\r\n\r\nA coupling procedure has been developed for the efficient covalent attachment of periodate oxidized RNA to an insoluble matrix of hydrazide-derived sepharose, which is subsequently available for nucleic acid hybridization.
\r\n\r\nIn Part II of the thesis, the properties of a new structure of mitochondrial DNA are discussed. In two strains of mouse L cells, it was found that approximately one half of the closed mitochondrial DNA molecules contain a short three-stranded DNA region in which a short single strand of progeny DNA containing about 350 nucleotides is inserted into the closed circular duplex with the displacement of a corresponding stretch of the still circular parental strand. The three-stranded region is called a D-loop because of its formal shape and because it appears to\r\nhave been formed by displacement replication. Double-length mitochondrial DNA molecules often contain two D-loops which are always at diametrically opposed positions on the 20 megadalton circle. This latter result is taken to indicate that one of the forks in each D-loop marks a unique site for the initiation of replication.
\r\n\r\n" }, { "name": "Seybold, William Davidson", "degree": "PhD", "year": "1972", "title": "Part I. Studies on the Activation of Drosophila Phenol Oxidase. Part II. Studies On Serum Insulin in Normal Subjects and Diabetics", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:03212016-102830515", "creators": [ { "name": { "family": "Seybold", "given": "William Davidson" }, "id": "Seybold-William-Davidson", "display_name": "Seybold, William Davidson" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/WXV5-S223", "abstract": "Part I
\r\n\r\nPhenol oxidase is the enzyme responsible for hardening and pigmentation of the insect cuticle. In Drosophila, phenol oxidase is a latent enzyme. Enzyme activity is produced by the interaction of a number of protein components. A minimal activation scheme consisting of six protein components, designated Pre S, S activator, S, P. P' and \u02451 is described. Quantitative assays have been developed for the S activator, S, P and P' proteins and these components have been partially purified. Experiments describing the interactions of the six components have been conducted and a model for the activation of phenol oxidase in a minimal system is proposed. Possible mechanisms of the reactions between the constituents of the activating system and potential regulatory mechanisms involved in phenol oxidase production and function are discussed.
\r\n\r\nPart II
\r\n\r\nA method has been developed for the partial purification of insulin from human serum. A procedure for the determination of the electrophoretic mobility of serum insulin on polyacrylamide gels is described. An electrophoretic analysis of insulin isolated from a normal subject is reported and in addition to a major band, the existence of a number of minor bands of immunoreactive insulin is described. A comparison of the electrophoretic patterns of insulin isolated from normal and diabetic subjects was carried out and indications that differences between them\r\nmay occur are reported.
\r\n" }, { "name": "Smith, Charles Allen", "degree": "PhD", "year": "1972", "title": "Closed Circular DNA in Animal Cells: I. Complex Mitochondrial DNA in Normal and Malignant Tissue and the in vivo Effects of Drugs on the Superhelix Density of Mitochondrial DNA. II. Small Polydisperse Circular DNA of HeLa Cells. III. Sequence Heterogeneity in Simian Virus 40 Deoxyribonucleic Acid", "advisor": "Unknown, Unknown", "url": "https://resolver.caltech.edu/CaltechTHESIS:06212016-092618206", "creators": [ { "name": { "family": "Smith", "given": "Charles Allen" }, "id": "Smith-Charles-Allen", "display_name": "Smith, Charles Allen" } ], "advisors": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/RHBK-9856", "abstract": "Part I of the thesis is concerned with mitochondrial DNA (mtDNA) of animal cells. Complex mtDNA includes catenated oligomers two or more of the basic 5-\u03bc circles linked as in a chain, and circular dimers, 10-\u03bc circles composed of two monomer genomes in tandem. The mtDNA of human placenta and several organs of rabbits, guinea pigs, and mice was found to contain 6 to 9% catenated dimers and 0.5 to 2% higher oligomers. Circular dimers were absent or below 0.2%; In contrast, the mtDNA from 12 of 15 human tumors contained the circular dimer in frequencies from 0.2 to 9%, in addition to catenated molecules in the above frequency ranges. Two lines of mouse L cells were found to contain circular dimers in frequencies of 5 and almost 100%. These frequencies were not changed when cells were treated with cycloheximide or maintained at high cell density.
\r\n\r\nEthidium bromide was shown to inhibit mtDNA synthesis in HeLa and SV3T3 cells, but not to affect base composition or complexity of the pre-existing mtDNA. With increasing treatment time or dosage, the pre-existing mtDNA undergoes a gradual change in the superhelix density from about -0.025 superhelical turns per ten base pairs to maxima of -0.088 in HeLa and -0.114 in SV3T3 cells. The nicking-closing\r\ncycle demonstrated by these results operates at least every 30 minutes and was shown not to be an artifact of mtDNA isolation. The change is reversible and can be demonstrated with other known intercalators. Similar changes in superhelix density were found in the mtDNA of livers, spleens, and kidneys of mice treated with ethidium bromide. It is postulated that the effect on superhelix density is the result of in vivo intercalation of the drugs into mtDNA.
\r\n\r\nPart ll is a study of the small polydisperse closed circular DNA in HeLa cells. This DNA was found to range in size from 0.05 to greater than 2 \u03bc, with an average length of 0.32 \u03bc and a weight average molecular weight of 1.0 x 106. This latter value was corroborated in sedimentation studies in neutral and alkaline solvents. The buoyant density, 1.692 g/ml, indicates a G-f-C content of 38 mole %. The separated strands do not have detectably different buoyant densities at pH 12.5. The superhelix density of the DNA (-0.039 to -0.045) is significantly greater than that of HeLa mtDNA. Renaturation kinetics studies have shown that the circles are not composed of varying numbers of a sequence the size of the smallest molecules in the population. The DNA may be prepared from whole cell extracts or cytoplasmic fractions; it is not associated with purified mitochondria or nuclei. There are a minimum of 50 circles per growing cell; treatment with cycloheximide results in a 20- to 30-fold increase. Labeling experiments showed that the cycloheximide-induced small circles are not newly replicated, but are formed from pre-existing DNA.
\r\n\r\nPart III is a study of sequence heterogeneity in closed circular SV40 viral DNA. Denatured singly nicked DNA was reannealed and the heteroduplexes formed were examined for regions of nonhomology by formamide-protein film electron microscopy. Substituted and deleted sequences longer than about 50 nucleotides are detected by this method. DNA from viruses passaged twice at multiplicities of infection much less than one p.f.u./cell contained 2% deletions and no detectable\r\nsubstitutions. In contrast, DNA from viruses grown by several passages of undiluted lysates or by infecting cells with stock virus at 5 p.f.u./cell contained 13 and 11% deleted molecules and 12 and 7% substituted molecules, respectively. The substitutions appear to have arisen as the result of integration of SV40 into chromosomal DNA, followed by excision of molecules containing stretches of chromosomal DNA. On the average, the substituted sequence is somewhat shorter\r\nthan the native SV40 sequence it replaces.
\r\n\r\n" }, { "name": "Tibbetts, Clark Joseph Bullock", "degree": "PhD", "year": "1972", "title": "In Vitro Studies of the Synthesis of Mitochondrial Deoxyribonucleic Acid", "advisor": "Vinograd, Jerome Rubin", "url": "https://resolver.caltech.edu/CaltechTHESIS:09062016-133706695", "creators": [ { "name": { "family": "Tibbetts", "given": "Clark Joseph Bullock" }, "id": "Tibbetts-Clark-Joseph-Bullock", "display_name": "Tibbetts, Clark Joseph Bullock" } ], "advisors": [ { "name": { "family": "Vinograd", "given": "Jerome Rubin" }, "id": "Vinograd-J-R", "role": "advisor", "display_name": "Vinograd, Jerome Rubin" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/1XDN-S884", "abstract": "A DNA polymerase has been partially purified from the mitochondria of HeLa cells. Properties, including low levels of nuclease activity and a preference for native duplex DNA templates, were favorable for the study of in vitro DNA synthesis using circular duplex DNA templates. Initiation of DNA synthesis occurs predominately at single-strand scissions with covalent addition of nucleotides to the priming template strand. Centrifugation and electron microscopy have established that the template DNA strand ahead of the growing point is displaced rather than degraded. Hairpin structures are not formed in the course of DNA synthesis on duplex DNA templates. Studies with HeLa cell mitochondrial DNA template have indicated base composition-complement fidelity of the product of DNA synthesis as well as a preferential synthesis of DNA corresponding to the denser complement (in CsCl solution) of the template. This asymmetric synthesis of mitochondrial DNA appears to arise from a bias in the number of single-strand scissions sustained by the complementary strands.
\r\n\r\nDNA synthesis in isolated HeLa cell mitochondria has also been investigated. Product DNA appears in closed and nicked circular mitochondrial DNA. Preferential synthesis of DNA corresponding to the less dense complement (in CsCI solution) was observed.
\r\n\r\nThe mode of action of the partially purified mitochondrial DNA polymerase and the asymmetric synthesis of mitochonarial DNA, observed in vitro and in situ, are discussed with regard to the results of recent studies of the replication of mitochondrial DNA in vivo.
\r\n\r\n" }, { "name": "Attardi, Barbara Joan Furman", "degree": "PhD", "year": "1971", "title": "Part I. Characterization of the RNA from the Mitochondrial Fraction of HeLa Cells. Part II. Properties of Membrane-Bound Ribosomes in HeLa Cells", "advisor": "Owen, Ray David; Attardi, Giuseppe", "url": "https://resolver.caltech.edu/CaltechTHESIS:11212017-100113540", "creators": [ { "name": { "family": "Attardi", "given": "Barbara Joan Furman" }, "id": "Attardi-Barbara-Joan-Furman", "display_name": "Attardi, Barbara Joan Furman" } ], "advisors": [ { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "advisor", "display_name": "Owen, Ray David" }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "co-advisor", "display_name": "Attardi, Giuseppe" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/QMSK-Y603", "abstract": "Part I of this thesis is concerned with the characterization\r\nof RNA components from the mitochondrial fraction of HeLa cells. \r\nMitochondria prepared by differential centrifugation followed by \r\nbuoyant density fractionation in sucrose gradient are contaminated \r\nby elements of the rough endoplasmic reticulum.\tIn order to identify \r\nRNA components of mitochondrial origin in this fraction the\r\nfollowing criteria were used: association of newly synthesized RNA \r\nwith mitochondria (recognized by cytochrome oxidase as say), insensitivity \r\nin situ to ribonuclease digestion, linear kinetics of labeling \r\nafter [3H]-uridine pulses, sensitivity of their synthesis to\r\nethidium bromide, and, especially, capacity to hybridize with purified \r\nclosed-circular mitochondrial DNA.
\r\n\r\nIt has been found that the RNA synthesized on a mit-DNA template \r\nconsists of both rapidly labeled heterogeneous RNA components, \r\nvarying in sedimentation constant from 4 to 50 S or more, and discrete\r\nRNA species, migrating at 16 S, 12 S, and 4 S in sucrose gradient. Analysis \r\nof the sedimentation behavior of the 16 S and 12 S species under denaturing \r\nconditions has indicated that they are represented by continuous \r\npolynucleotide chains. From their migration in polyacrylamide gels\r\nrelative to ribosomal RNA markers, the respective molecular weights \r\nof 16 S and 12 S have been estimated to be 0.7 x 106 and 0.4 x 106\r\ndaltons. The 16 S, 12 S, and 4 S RNA's appear to be methylated.\tBoth \r\nthe discrete and heterogeneous mit-DNA coded RNA components have\r\na base composition clearly different from that of ribosomal RNA and\r\ncytoplasmic messenger RNA and complementary, as concerns the\r\nA and U content, to that of the heavy strand of mit-DNA.
\r\n\r\nThe results of an investigation concerning the properties \r\nof the ribosomes associated with the endoplasmic reticulum \r\nin HeLa cells are reported in Part II. From the distribution of\r\nribosomal RNA among the subcellular fractions, it has been estimated \r\nthat 10 -15% of the total ribosomes in HeLa cells are membrane-bound;\r\n65-70% of these can be recovered in the form of polysomes after\r\nmembrane lysis with sodium deoxycholate. The 18 S and 28 S RNA \r\ncomponents of the membrane-bound ribosomes have similar kinetics \r\nof labeling and identical sedimentation properties and nucleotide \r\ncomposition to the homologous components of free ribosomes, these \r\nresults pointing to a common nuclear origin. The ribosomal RNA\r\nof membrane-bound ribosomes is made acid-soluble to about the \r\nsame extent as the ribosomal RNA of free polysomes under the conditions \r\nof ribonuclease digestion in situ enployed here. Treatment \r\nwith EDTA releases about 85-90% of the small ribosomal subunits \r\nand 70% of the large ribosomal subunits, a situation which is similar \r\nto that which has been observed for rat liver microsomes.\r\nThese results suggest that the great majority of the ribosomes in\r\nthe mitochondrial fraction of HeLa cells are extramitochondrial, i.e. \r\nbound to the endoplasmic reticulum, but the existence of a small number \r\nof intramitochondrial ribosomes is not excluded.
\r\n\r\n\r\n\r\n\r\n" }, { "name": "Burke, Patricia Virginia", "degree": "PhD", "year": "1971", "title": "Freezing Phycomyces Sporangiophores in Superfluid Helium for Ultrastructure Studies", "advisor": "Delbruck, Max", "url": "https://resolver.caltech.edu/CaltechETD:etd-08082006-142417", "creators": [ { "name": { "family": "Burke", "given": "Patricia Virginia" }, "id": "Burke-Patricia-Virginia", "display_name": "Burke, Patricia Virginia" } ], "advisors": [ { "name": { "family": "Delbruck", "given": "Max" }, "id": "Delbr\u00fcck-M", "role": "advisor", "display_name": "Delbruck, Max" } ], "committee": [ { "name": { "family": "Delbruck", "given": "Max" }, "id": "Delbr\u00fcck-M", "role": "chair", "display_name": "Delbruck, Max" }, { "name": { "family": "Goodstein", "given": "David L." }, "id": "Goodstein-D-L", "role": "member", "display_name": "Goodstein, David L." }, { "name": { "family": "Liepmann", "given": "Hans Wolfgang" }, "id": "Liepmann-H-W", "role": "member", "display_name": "Liepmann, Hans Wolfgang" }, { "name": { "family": "Vinograd", "given": "Jerome" }, "id": "Vinograd-J", "role": "member", "display_name": "Vinograd, Jerome" }, { "name": { "family": "Van Harreveld", "given": "Anthonie" }, "id": "Van-Harreveld-A", "role": "member", "display_name": "Van Harreveld, Anthonie" } ], "option_major": [ "biology" ], "doi": "10.7907/PY8S-BH09", "abstract": "Knowledge of the ultrastructure of the growing zone of Phycomyces sporangiophores is a desirable adjunct to studies of their sensory physiology. This includes discovery of the type of organelles present and their spatial arrangement. Chemical fixation preserves some of the cell's organelles satisfactorily, but considerable disruption and dislocation occur. Physical fixation by freezing should preserve the spatial distribution of organelles within the cell if the freezing is rapid enough to prevent ice crystal formation. Ice crystal inhibitors do not penetrate the cell satisfactorily, so that crystal size is determined only by the freezing rate.
\r\n\r\nLiquid He II exhibits a quantum mechanical mechanism of heat transfer which is much more efficient than the normal, classical heat conduction in fluid. The heat transfer rate is qualitatively described by the Landau equations for liquid He II with additional terms (Gorter-Mellink mutual friction) to account for frictional forces within the liquid. For the case of a cylindrical heater, the maximum steady state heat transfer rate before film boiling occurs depends on the depth of the heater below the liquid surface (hydrostatic head), the pressure of the gas above the liquid, and the liquid temperature. For a gas pressure of 20 Torr or more in excess of the equilibrium vapor pressure, the appearance of the film boiling changes from a uniform gas film to a fine haze, presumably of tiny gas bubbles. If He4 gas is used to pressurize the liquid, the temperature of the liquid He bath rises to about 2 K and a layer of liquid He I forms at the gas-liquid interface. This layer of the He I grows at the expense of the bulk He II as heat is supplied by the warm gas. The phase boundary between the two liquid phases moves down through the liquid at about 5 cm/min. The conditions for optimal heat transfer occur immediately after the pressure increase. These conditions are a pressure excess of 20 Torr or more and a bath temperature of 1.9 - 2 K.
\r\n\r\nSporangiophores are suspended by an iron filing from an electromagnet in a special chamber (room temperature) above a helium cryostat. As soon as the pressure excess in the cryostat exceeds 20-60 Torr they are released and fall freely into the superfluid helium through a tube of heated gas. The sporangiophores are collected in a plastic beaker at the bottom of the cryostat and transferred to a liquid nitrogen storage dewar. Frozen sporangiophores are prepared for electron microscopy using freeze-substitution and freeze-fracture techniques.
\r\n\r\nThe thin sections and the freeze-fracture replicas show extensive ice crystal damage to stage IV sporangiophores which have a large central vacuole. Ice crystal damage is considerably less in stage I sporangiophores which have a much smaller vacuole. Presumably, the vacuole is responsible for the poor preservation of the ultra-structure.
\r\n" }, { "name": "McConnell, David John", "degree": "PhD", "year": "1971", "title": "Investigations on (i) Chromosomal Ribonucleic Acid of Ascites Tumour, (ii) RNA Polymerase of E. Coli", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechTHESIS:05252018-095226235", "creators": [ { "name": { "family": "McConnell", "given": "David John" }, "id": "McConnell-David-John", "display_name": "McConnell, David John" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/D6DK-F035", "abstract": "PART I: When chromatin isolated from rat ascites cells is \r\ndissociated in the presence of high salt and the chromosomal \r\nproteins separated from the DNA by buoyant density centrifugation, \r\na portion of the RNA contained in the chromatin remains associated \r\nwith the chromosomal proteins. This RNA (chromosomal RNA) is \r\ncharacterized by its small size, s20,w = 3.3S, \r\nits high content of dihydroribothymidine and its ability to form \r\nhybrid with about 4% of the nuclear DNA. It appears\r\nto have no sequences in common with ascites transfer, ribosomal, \r\nor messenger RNA.
\r\n\r\nA class of RNA (cytoplasmic 3S RNA) with similar proper\u00adties \r\nbut associated with the cytoplasmic proteins has also been isolated. \r\nThis RNA hybridizes to about 2% of the nuclear DNA and contains \r\nvery few, if any, sequences not also contained in chromosomal RNA.\t\r\nThis fraction of RNA is however unable to compete with about 50% of \r\nthe sequence present in chromosomal RNA indicating that a large portion \r\nof chromoso\u00admal RNA is confined to the chromatin. A further class of \r\nRNA associated with the nuclear sap proteins appears to be identical \r\nto the RNA associated with cytoplasmic proteins.
\r\n\r\nA further class of RNA (nuclear 3S RNA) with hybridiza\u00adtion \r\nproperties similar to the cytoplasmic 3S RNA has been isolated from \r\nthe nuclear sap. It hybridises to a lower extent than chromosomal \r\nRNA and is strongly competed by cyto\u00adplasmic 3S RNA.
\r\n\r\nPART II. RNA polymerase, more than 95% pure, has been prepared \r\nfrom E. coli D-10 and B. It is free from ribonu\u00adclease and phosphatase.\t\r\nIt carries out poly A synthesis on E. coli or ascites tumour DNA but \r\nnot on T7 DNA. Phosphate incorporation (TCA precipitable) from the \u03b3 phosphate of ATP \r\nin the absence of primer may be caused by a slight contami\u00adnation with \r\npolyphosphate kinase. This can be controlled. Initiation of synthesis on T7, \r\nE. coli and ascites tumour DNA\tdiffer in the response to the \u03c3 sub-unit.\t\r\nRNA synthe\u00adsized on T7 DNA can initiate with A or G. A salt- or rifam\u00ad-\r\npicin-stable complex between T7 DNA can be formed when a single nucleoside \r\ntriphosphate (commercial reagent) is present, but requires a small amount of \r\npropagation. After purification of the nucleotides the efficiency of \r\ncomplex foration is greatly reduced.
\r\n\r\n\r\n\r\n" }, { "name": "Nebes, Robert David", "degree": "PhD", "year": "1971", "title": "Investigations on Lateralization of Function in the Disconnected Hemispheres of Man", "advisor": "Sperry, Roger Wolcott", "url": "https://resolver.caltech.edu/CaltechTHESIS:06082018-123956199", "creators": [ { "name": { "family": "Nebes", "given": "Robert David" }, "id": "Nebes-Robert-David", "display_name": "Nebes, Robert David" } ], "advisors": [ { "name": { "family": "Sperry", "given": "Roger Wolcott" }, "id": "Sperry-R-W", "role": "advisor", "display_name": "Sperry, Roger Wolcott" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/AA8W-SC34", "abstract": "The effect of long standing cerebral damage upon the pattern of functional lateralization revealed by division of the forebrain commissures was investigated in a young conunissurotomy patient with birth injury to the somato-sensory region of his left hemisphere. Results from a battery of sensory - motor tasks showed that, unlike previous\r\nconunissurotomy cases, the major hemisphere of this subject had access to somesthetic information from the ipsilateral as well as the contralateral hand, thus allowing him to name objects out of sight in his left hand, and to use this hand to tactually find items, the pictures or names of which had been visually presented to only the left hemisphere. The most plausible explanation for these exceptional cross integrative abilities would be the presence of a left sided ipsilateral somesthetic projection, which, in compensation for the subject's early brain damage, has strengthened into a functional system. Additional evidence for compensatory reorganization in this boy was found in his minor hemisphere, which exhibited an enhanced capacity for expressive language, being capable of transcribing printed words into script6 and, upon occasion, of writing the name of an object.
\r\n\r\nFurther research into the lateralization of higher intellectual functions in man involved a study of the psychological processes responsible for the superiority of the right side of the brain on certain perceptual activities.\r\nThe minor hemisphere, in the several commissurotomy patients tested, was found to excel the major on tasks involving visualization, from incomplete or disjointed sensory data, of the total stimulus configuration: this was revealed by its supremacy on such problems as: judging from a tactual or visual inspection of an arc, the size of the circle from which it had come, or mentally reconstructing the contour of a geometric shape seen in a fragmented state, or perceiving the pattern inherent in a visual display due to the differential spacing of its components. Extension of this testing to normal persons established that competency in the handling part-whole relationships is, in some manner, correlated with handedness, as left handed individuals performed much worse than right handed ones.
\r\n" }, { "name": "Sedat, John W.", "degree": "PhD", "year": "1971", "title": "On Bacterial and \u03a6X-174 Messenger-RNA", "advisor": "Sinsheimer, Robert L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04172018-094635838", "creators": [ { "name": { "family": "Sedat", "given": "John W." }, "id": "Sedat-John-W", "display_name": "Sedat, John W." } ], "advisors": [ { "name": { "family": "Sinsheimer", "given": "Robert L." }, "id": "Sinsheimer-R-L", "role": "advisor", "display_name": "Sinsheimer, Robert L." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/5Z4D-EC83", "abstract": "General methods for the chromatography of nucleic \r\nacids on benzoylated (naphthoylated) DEAE cellulose are \r\ndescribed. This procedure results in well-resolved peaks \r\nwith good recovery and seems to separate nucleic acids on\r\nthe basis of their secondary structure almost independently \r\nof their molecular weight. These methods have proved to be \r\nuseful in the analysis of the replicative intermediates of \r\nMS-2 RNA, \u00d8X RFDNA, and \u00d8X single stranded DNA, for example.
\r\n\r\nSpecific methods for the purification of E. coli.\r\npulse-labeled RNA based on the chromatography on benzoylated \r\nDEAE cellulose at pH 7.5 and pH 3.5 were developed. Greater \r\nthan 60% of the pulse label with less than 4% of the mass \r\nlabel is recovered, and the RNA size distribution in a \r\ndenaturing solvent (99% dimethyl sulfoxide) after chroma\u00adtography \r\nshows that the mass label closely parallels the \r\npulse label as a function of size; there appears to be no \r\nselection or degradation. Pulse-chase experiments indicate \r\nthat a large fraction of both the pulse and mass labels are \r\nchased out, suggesting reasonable purity of the mRNA. \r\nThese data imply that there is a difference, structural or \r\nchemical, between mRNA and other known RNAs that allows\r\nall E. Coli. mRNA to act as a group regardless of size\r\nduring the purification.
\r\n\r\n\r\nThe in vivo \u00d8X mRNA has been studied using the above \r\nprocedure for purification and analysis. The results show \r\nthat the \u00d8X mRNA size distribution at early and late times \r\nafter infection is very broad ranging from a distinct maximum \r\nsize of 1.7 megadaltons (one genome length of poly-\r\ncistronic mRNA), to a peak in the distribution at 0.2-0.3\r\nmegadaltons, and with significant mRNA as small as 104\r\ndaltons. The only difference observed between the early and \r\nlate times after infection appears to be in the amount of \r\nRNA present: approximately 100 molecules of mRNA per cell \r\nare present at 4 min. after infection compared to 1000 \r\nmolecules per cell at 25 min. after infection. Little if \r\nany difference could be found in the size distribution of \r\nmRNA made by the strongly polar OP6 \u00d8X mutant or upon \r\ninfection of the \u00d8X replication-restrict. A variety of experimental approaches showed \r\nan absence of significant methylation and 5'triphosphate\r\ntermini in the mRNA.
\r\n\r\n\r\nAttempts were made to ask which RF, parental or \r\nprogeny, was the template for the transcription of the \u00d8X \r\nmRNA. One type of experiment indicated that there was a\r\nsmall amount of pulse labeled RNase-, phenol-, and detergent- \r\nresistant RNA specifically attached to the density labeled \r\nparental RF. However, other types of direct experiments \r\n(such as analysis of non-RNase treated RF in CsCl density \r\nequilibrium gradients) demonstrated that neither type of \r\nRF was shifted to higher densities due to attached nascent \r\nmRNA; nor could significant pulse labeled RNA be detected \r\nin the RF region of the CsCl density gradients. Thus,\r\nno unambiguous answer can be given to the template question.
\r\n\r\n" }, { "name": "Thomasson, William Alvis", "degree": "PhD", "year": "1971", "title": "Hormonal Control of Protein Granule Accumulation in Fat Bodies of Drosophila melanogaster Larvae", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06132018-114151376", "creators": [ { "name": { "family": "Thomasson", "given": "William Alvis" }, "id": "Thomasson-William-Alvis", "display_name": "Thomasson, William Alvis" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/VGXE-Y658", "abstract": "It has been previously shown that large granules of protein accumulate in the larval fat body of holometabolous insects shortly before pupation. In the butterfly Calpodes ethlius there is evidence that the protein in these granules is sequestered from the hemolymph and that their accumulation is controlled by the molting hormone, ecdysone. Evidence as to hormonal control of granule accumulation in Drosophila\r\nmelanosaster is conflicting.
\r\n\r\nIn this thesis it is shown that if 3H-labelled proteins and 14C labelled amino acids are injected into D. melanogaster larvae at the time of granule formation, the proteins are incorporated into the granules but the free amino acids are not. The conclusion that all proteins in the granules are preformed hemolymph proteins is reinforced by the observation that injection of cycloheximide shortly before first appearance of the granules did not in any way interfere with their formation.
\r\n\r\nIt is shown that injection of ecdysterone into early third instar larvae will induce precocious formation of granules. When fat bodies are incubated in vitro in media containing serum, addition of ecdysterone consistently causes granule formation. However, granules sometimes form in the absence of added hormone. Addition of 50 mg/ml or more protein consistently causes the formation of small granules, whether or not hormone is also present.
\r\n\r\nWhen fat bodies are incubated in media without serum, granules consistently appear in aerated media without addition of hormone. However, addition of hormone causes the formation of larger granules which appear earlier.
\r\n\r\nIt is therefore concluded that in D. melanogaster ecdysone is sufficient but not necessary for granule formation.
\r\n\r\nSince the granules appear seventeen hours before pupariation, while ecdysone is secreted only four hours before pupariation, it is suggested that ecdysone is not involved in control of the natural appearance of the granules in D. melanogaster. The hypothesis is presented that the granules form spontaneously as soon as the juvenile hormone titer falls below a certain level.
\r\n\r\nIn the course of this work it was incidentally noticed that injection of ecdysterone into early third instar larvae caused a syndrome which resembled a partial and abortive prepupal molt. No puparium was formed and the larvae died within a few days.
\r\n" }, { "name": "T\u00f6k\u00e9s, Zolt\u00e1n Andr\u00e1", "degree": "PhD", "year": "1971", "title": "Cell Surface Changes in Development: The I Blood Group Antigen in Humans", "advisor": "Dreyer, William J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08222017-093339483", "creators": [ { "name": { "family": "T\u00f6k\u00e9s", "given": "Zolt\u00e1n Andr\u00e1" }, "id": "T\u00f6k\u00e9s-Zolt\u00e1n-Andr\u00e1", "display_name": "T\u00f6k\u00e9s, Zolt\u00e1n Andr\u00e1" } ], "advisors": [ { "name": { "family": "Dreyer", "given": "William J." }, "id": "Dreyer-W-J", "role": "advisor", "display_name": "Dreyer, William J." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/RGTW-W924", "abstract": "The I blood group system in humans was investigated in order to study cell surface changes in development.
\r\n\r\n\r\nOne advantage in studying the I-antigen in man is the availability of well defined, easily purifiable cold agglu\u00adtinin molecules with anti-I specificity. Sera from patients with chronic cold agglutinin, post pneumonia cold agglutinin and post viral influenza cold agglutinin disease have been studied. The IgM agglutinins were isolated and their restric\u00adted heterogeneity established. These well characterized protein molecules were used in experiments to study the I antigen's development and in experiments designed to reveal information about the molecular basis of I antigen specifi\u00adcity.
\r\n\r\nThe development of I antigen and postnatal changes in hemoglobin were examined in human infants, to see whether the developmental changes in these two attributes are correlated individual cells in such a way as to suggest that they have common biochemical control mechanisms. The results demon\u00adstrated that the expression of I antigen on the erythrocyte surface is independent from the control mechanism of the biosynthesis of the beta chain of hemoglobin. These observa\u00adtions are explained by suggesting two separate modulatory regulations, one for the enzymes involved in the expression of I-specific molecules on the cell surface and another for the regulation of hemoglobin subunit synthesis.
\r\n\r\nThe rates of A-antigen and I antigen site development were compared using I125 labeled anti-A IgG molecules in order to test for possible common control or close association. Erythrocytes from postnatal infants were fractionated with respect to their I-agglutinability. Experimental results indicate that cells with well expressed I-specificity have more A-antigen sites; less sites were found on erythrocytes\r\nwith weak expression of I antigen. These results suggest that enzymes responsible for the expression of these two antigenic specificities are under a common control mechanism or that they are closely associated.
\r\n\r\nErythrocyte stromata from human adults and from umbilical cord blood samples were fractionated. Fingerprints of the two membrane preparations indicated that the major protein components are identical. Molecular fractionation of the stomata resulted in fractions with I antigen activity. All fractions with I-activity contained ABO blood activity, but some preparations with ABO activity failed to inhibit agglu\u00adtinins with I-specificity. Hydrophilic fractions containing glycopeptides with blood group activity were isolated.\tThe\r\nI-activity was pronase sensitive. Quantitation of molecular fractions with I blood group activity from cord and adult erythrocytes suggests that I-negative phenotype could be explained by either the two dimensional distribution of molecules on the cell surface or by the covering up of the\r\nI-specific molecules in the membrane.
\r\n\r\nThe significance of these findings are discussed with respect to embryonic cell surface specificity and with respect to the recognition of molecular patterns by IgM molecules with multiple binding sites.
" }, { "name": "Ward, Samuel", "degree": "PhD", "year": "1971", "title": "Structure and Assembly of Bacteriophage T4 Tail Fibers", "advisor": "Wood, William B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06232017-114341075", "creators": [ { "name": { "family": "Ward", "given": "Samuel" }, "id": "Ward-Samuel", "display_name": "Ward, Samuel" } ], "advisors": [ { "name": { "family": "Wood", "given": "William B." }, "id": "Wood-William-B", "role": "advisor", "display_name": "Wood, William B." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/VW5T-Z307", "abstract": "The structure and assembly of bacteriophage T4 tail\r\nfibers was examined as a model system for studying assembly\r\nof multiprotein structures. The function of six genes (34-\r\n38 and 57) is necessary for assembly of tail fibers. The\r\nrole of each of these genes in assembly was examined by isolating\r\nand characterizing whole fibers and four precursors\r\nwhich accumulate in lysates of mutant phage-infected bacteria.\r\nEach isolated structure showed a single major electrophoretic\r\ncomponent on polyacrylamide gels indicating near homogeneity.\r\nElectron microscopic examination of these isolated structures\r\nrevealed that the whole fiber consists of two halves; one,\r\nrequiring genes 57 and 34 for its assembly, is a rod 690 x\r\n27 \u00c5 with a knob on one end and an antigen A; the other,\r\nrequiring genes 35, 36, 37 and 57 for its assembly, is a rod\r\n690 x 26 \u00c5 containing antigens B and C (designated BC'). The\r\nantigen B is introduced into this rod by the action of gene\r\n36 which increases the length of a half fiber precursor, C,\r\nthe product of genes 37, 38 and 57, from 560 to 690 \u00c5. The\r\nresulting BC precursor is converted to BC' under the control\r\nof gene 35, a step necessary to allow interaction with the\r\nA half fiber, but one making no morphological or serological\r\nchange in the fiber.
\r\n\r\n\r\nThe subunit composition of the isolated fibers and\r\ntheir precursors was examined by dissociation at l00\u00b0c and\r\ngel electrophoresis in the presence of the anionic detergent\r\nsodium dodecyl sulfate (SDS). A was found to contain a major\r\npolypeptide of molecular weight 150,000. C, BC and BC' were\r\neach found to contain a major polypeptide of molecular weight\r\n123,000. Minor components were also present but they were\r\nnot reproducible. Because the major polypeptides were so\r\nlarge they could be resolved on SDS gels of crude lysates\r\nof mutant infected cells. These gels showed that amber\r\nmutations in gene 34 eliminated the 150,000 polypeptide and\r\namber mutations in gene 37 eliminated the 123,000 polypeptide.\r\nThis indicates that these polypeptides are the products of\r\ngenes 34 and 37 (P34 and P37). The other tail fiber genes\r\ndid not affect the synthesis of P34 and P37 except that\r\namber mutations in gene 36 reduced the amount of P37 by\r\n40%, suggesting that these genes are co-transcribed. Molecular\r\nweight estimates of the fiber precursors show that there are\r\ntwo copies of P34 in the A half fiber and two copies of P37\r\nin each of the other half fibers.
\r\n\r\n\r\nMutations in genes 57 and 38 affected the apparent\r\nsolubility of P34 and P37 and allow these polypeptides to be\r\ndissociated in SDS at 37\u00b0C. This is consistent with P57\r\ncontrolling the dimerization of P34 leading to the A half\r\nfiber and P38 and P57 controlling the dimerization of P37\r\nleading to the C half fiber.
\r\n\r\n\r\nA new apparatus for destaining acrylamide gels electrophoretically\r\nand a new method of fractionation and scintillation\r\ncounting of radioisotope-labeled acrylamide gels\r\nare also described.
\r\n" }, { "name": "Winicur, Sandra", "degree": "PhD", "year": "1971", "title": "I. Studies on the Motility and Biochemistry of Cilia. II. Chitinase Activity During Drosophila Development", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10102017-114354504", "creators": [ { "name": { "family": "Winicur", "given": "Sandra" }, "id": "Winicur-Sandra", "display_name": "Winicur, Sandra" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/30GS-ZK08", "abstract": "Part I
\r\n\r\nCilia isolated from Tetrahymena after inducing ciliary shedding \r\nby addition of calcium chloride to cells suspended in an ethanol\r\nsolution have been shown capable of motility in the presence of ATP. \r\nThe potential for motility was restored by treating the isolated \r\ncilia with glycerol, ethylene glycol or digitonin-sucrose solutions. \r\nUp to 80% of these cilia were motile upon addition of ATP or ADP. \r\nThese cilia showed an optimum reactivation temperature of 16\u00b0 C.
\r\n\r\n\r\nThe cilia are most motile in concentrated solution. Addition \r\nof xylose and dextrose, both of which are present in high \r\nconcen\u00adtration in native cilia, cause a sharp increas in \r\npercent motility.
\r\n\r\nThe ATPase activity was also studied under different conditions \r\nof motility.
\r\n\r\n\r\nRegeneration of cilia by Tetrahymena deciliated by pH 5 treat\u00adment \r\nwas observed. This regeneration took about 7 hours.
\r\n\r\n\r\nPart II
\r\n\r\n\r\nBefore both larval molts in Drosophila melanogaster, the chitin\r\nin the cuticle is digested to a significant degree by the molting fluid. \r\nA spurt of chitinase activity appears just before each molt, drops \r\nsharply after the first molt and begins to rise again just about the\r\ntime that chitin degradation becomes visible in electron micrographs. \r\nThe level of enzyme activity per mg of soluble protein or per mg of \r\nwet weight reached just before the second molt is about twice that \r\nbefore the first, and this declines gradually after the molt until \r\npuparium formation.
\r\n\r\n\r\nThe activity measured per individual larva however starts a slow \r\nrise after hatching with a slight peak at the second molt and continues \r\nto rise to a point mid way between the second molt and puparium for\u00admation.\t\r\nThis indicates that some of the measured chitinase activity\r\nmay be due to the activity of another enzyme, either a different chitinase, \r\na lysozyme, or possibly a chitin synthetase, which would be most \r\nactive during the third instar.
\r\n\r\n\r\nData exist\tsupporting the presence of more than one enzyme with \r\nchitinase activity. Two fractions can be separated by ammonium sulfate \r\nprecipitation and three peaks can be separated on a DEAE-Sephadex column.
\r\n\r\n\r\nThe enzyme activity is stable, with no loosely bound cofactor and \r\na single isoelectric point about 3.8. Chitinase activity was measured \r\nby a viscometric assay on a substrate of chitosan, a partially \r\ndeacety\u00adlated solubilized product of chitin.
\r\n\r\n" }, { "name": "Clayton, David Alvin", "degree": "PhD", "year": "1970", "title": "I. Occurrence and Structure of Complex Mitochondrial DNA in Human Leukemic Leukocytes and Normal Mammalian Tissues. II. Use of Alkali Metal Salts of Trichloroacetic Acid as Buoyant Denaturing Solvents for DNA", "advisor": "Vinograd, Jerome Rubin", "url": "https://resolver.caltech.edu/CaltechETD:etd-08092006-083749", "creators": [ { "name": { "family": "Clayton", "given": "David Alvin" }, "id": "Clayton-David-Alvin", "display_name": "Clayton, David Alvin" } ], "advisors": [ { "name": { "family": "Vinograd", "given": "Jerome Rubin" }, "id": "Vinograd-J-R", "role": "advisor", "display_name": "Vinograd, Jerome Rubin" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/AS6W-K850", "abstract": "This thesis is divided into two parts. Part I is concerned with the occurrence and structure of complex forms of mitochondrial DNA isolated from human leukemic leukocytes and normal mammalian tissues. Complex mitochondrial DNA (M DNA) occurs in two forms. The covalently closed double-sized circle (10 [mu]) is called the circular dimer. Oligomers composed of interlocked 5 [mu] monomer submolecules are called catenanes. The catenane form is ubiquitous in nature. It has been observed in M DNA preparations from every mammalian source examined to date. These sources include various organs from rabbits, guinea pigs, and mice. The frequency of catenated dimers in normal and leukemic tissues varies from 5 to 11 percent, and the frequency of catenated higher oligomers from 0.1 to 8.0 percent.
\r\n\r\nIn contrast, the circular dimer has not been observed in normal tissues. M DNA's from the peripheral blood of 14 patients with myelogenous leukemia contained a circular dimer form. No such structure could be found in M DNA's from three patients with nonmalignant proliferations of myeloid cells. The frequency of the circular dimer form is reduced upon treatment with antileukemic drugs. This result suggests that a significant relation exists between the formation and presence of the circular dimer M DNA form and myelogenous leukemia in man. Additional data presented in this section demonstrate that a sixteen-hour labeling of leukocyte M DNA results in a uniform labeling pattern of the circular dimer and monomer form.
\r\n\r\nDNA hybrids between the circular dimer and monomer were analyzed by electron microscopy and analytical centrifugation. The results indicate that the circular dimer and monomer are at least 90 percent homologous and that heterologous regions, insertions, or deletions exceeding 50 to 100 nucleotides in length do not occur. The electron microscope studies also show that monomer genomes in the dimer are connected in a head-to-tail structure, rather than in a head-to-head structure. In addition, the results show that leukemic leukocyte M DNA is substantially homogeneous in base sequence.
\r\n\r\nPart II contains a preliminary study of a denaturing buoyant system for DNA. It is shown that rubidium trichloroacetate is a potentially useful buoyant solvent for DNA. It is likely that most DNA's can be banded at neutral pH and room temperature in both the native and denatured states without introducing single-strand scissions. It is also concluded that Li, Na, and K trichloroacetate are potentially useful denaturing solvents for sedimentation velocity studies of DNA.
" }, { "name": "Craig, Sydney Pollock, III", "degree": "PhD", "year": "1970", "title": "Mitochondria and the Development of Sea Urchin Embryos", "advisor": "Tyler, Albert; Vinograd, Jerome Rubin; Sinsheimer, Robert L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08182015-114145046", "creators": [ { "name": { "family": "Craig", "given": "Sydney Pollock, III" }, "id": "Craig-Sydney-Pollock-III", "display_name": "Craig, Sydney Pollock, III" } ], "advisors": [ { "name": { "family": "Tyler", "given": "Albert" }, "id": "Tyler-A", "role": "advisor", "display_name": "Tyler, Albert" }, { "name": { "family": "Vinograd", "given": "Jerome Rubin" }, "id": "Vinograd-J-R", "role": "advisor", "display_name": "Vinograd, Jerome Rubin" }, { "name": { "family": "Sinsheimer", "given": "Robert L." }, "id": "Sinsheimer-R-L", "role": "advisor", "display_name": "Sinsheimer, Robert L." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biodev" ], "doi": "10.7907/AS0V-9678", "abstract": "After artificial activation or fertilization of non-nucleate fragments or eggs of the sea urchin, the mitochondria actively synthesize RNA. The RNA made in non-nucleate fragments is shown to be mostly single stranded and to be associated primarily with the low speed pellet of centrifuged cellular homogenates.
\r\n\r\nProtein synthesis is observed in non-nucleate fragments in the presence or absence of the mitochondrial RNA synthesis: it is found to be qualitatively similar but quantitatively less in the absence of the RNA synthesis. The continued syntheses of proteins in the non-nucleate fragments in the absence of mitochondrial RNA synthesis provides additional evidence for the presence of a stable messenger RNA component in the unfertilized sea urchin egg.
\r\n\r\nSince the uptake or actinomycin D was found to be inhibited by the presence of a fertilization membrane, ethidium bromide, at 10 \u03bcgs/ml, is used as an effective inhibitor of RNA synthesis in non-nucleate fragments and in early cleavage stage embryos. However, this same concentration of ethidium bromide is found to be only partially effective in blocking RNA synthesis at the mesenchyme blastula stage of development.
\r\n\r\nLow concentrations of ethidium bromide (2 and 5 \u03bcgs/ml) are found not to be lethal but to be capable of producing moderate developmental defects. In the presence of concentrations of ethidium bromide adequate to inhibit all the mitochondrial RNA synthesis (10 \u03bcgs/ml of ethidium bromide), from fertilization on, the embryos do not cleave beyond the 4-8 cell stages. When similar concentrations of ethidium bromide are added at an early mesenchyme blastula stage, the embryos do not gastrulate but continue to swim for more than 24 additional hours (adequate for control embryos to develop to a late prism stage). These results lead to the conclusion that mitochondrial RNA synthesis may be very essential for normal development to occur.
\r\n\r\nDNA is synthesized in the non-nucleate fragments of sea urchin eggs. None of the newly synthesized DNA is found in the closed circular form. When phenol extracted directly from the fragments, the DNA is found to sediment at approximately 38 and 27s in sucrose gradients but neither of these size classes could be found associated with the isolated mitochondria. The template for the synthesis of DNA in non-nucleate fragments remains unknown.
\r\n" }, { "name": "Goodell, Ernest William", "degree": "PhD", "year": "1970", "title": "Processes Affecting the Growth of Phycomyces Sporangiophores", "advisor": "Delbruck, Max", "url": "https://resolver.caltech.edu/CaltechETD:etd-08082006-104559", "creators": [ { "name": { "family": "Goodell", "given": "Ernest William" }, "id": "Goodell-Ernest-William", "display_name": "Goodell, Ernest William" } ], "advisors": [ { "name": { "family": "Delbruck", "given": "Max" }, "id": "Delbr\u00fcck-M", "role": "advisor", "display_name": "Delbruck, Max" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/DQ76-Z557", "abstract": "The stage IV sporangiophore (spph) of Phycomyces is a very large single cell. It is 0.1 mm in diameter and grows to a length of 10 cm. The sporangiophore bears asexual spores at its tip in a spherical sporangium. The sporangiophore elongates via a 2 mm long growing zone just beneath the sporangium. Most of this thesis deals with the interaction of the growing zone with the rest of the sporangiophore and with the sporangium.\r\n\r\nThe top of the sporangiophore appears to have the most active metabolism. The top 6 mm of the sporangiophore consumes 1/2 of the oxygen. The oxygen consumption of the whole spph and of this 6 mm long section does not change as the spph elongates from 25 mm to 50 mm. The oxygen consumption of the sporangium is very much less than that of the sporangiophore.\r\n\r\nThe whole sporangiophore can grow independently from the mycelium. Similiarly, the top 3 or 4 mm can elongate independently from the rest of the sporangiophore. It can grow for about 10 hours without the lower part of the sporangiophore. For the first 4 hours its rate of growth is nearly normal.\r\n\r\nThe elongation of the sporangiophore is much more dependent upon the sporangium. If the sporangium is removed, the growth of the sporangiophore decreases to 1/10 of the normal rate within 2 hours. Six to sixteen hours after sporangium removal, the sporangiophore produces a branch sporangiophore from the former growing zone (figure 1). The spores in the sporangium appear to synthesize some compound (or compounds) which stimulates the sporangiophore's elongation and inhibits its branching." }, { "name": "Griffith, Jack Denney", "degree": "PhD", "year": "1970", "title": "High Resolution Electron Microscope Studies of Chromosomal Fibers", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechTHESIS:08042015-105247195", "creators": [ { "name": { "family": "Griffith", "given": "Jack Denney" }, "id": "Griffith-Jack-Denney", "display_name": "Griffith, Jack Denney" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/RTXG-PQ59", "abstract": "Techniques are described for mounting and visualizing biological macromolecules for high resolution electron microscopy. Standard techniques are included in a discussion of new methods designed to provide the highest structural resolution. Methods are also discussed for handling samples on the grid, for making accurate size measurements at the 20 \u00c5 level, and for photographically enhancing image contrast.
\r\n\r\nThe application of these techniques to the study of the binding of DNA polymerase to DNA is described. It is shown that the electron micrographs of this material are in agreement with the model proposed by Dr. Arthur Kornberg. A model is described which locates several active sites on the enzyme.
\r\n\r\nThe chromosomal material of the protozoan tetrahymena has been isolated and characterized by biochemical techniques and by electron microscopy. This material is shown to be typical of chromatin of higher creatures.
\r\n\r\nComparison with other chromatins discloses that the genome of tetrahymena is highly template active and has a relatively simple genetic construction.
\r\n\r\nHigh resolution electron microscope procedures developed in this work have been combined with standard biochemical techniques to give a comprehensive picture of the structure of interphase chromosome fibers. The distribution of the chromosomal proteins along its DNA is discussed.
\r\n" }, { "name": "Hatlen, Loren Endicott", "degree": "PhD", "year": "1970", "title": "Studies on 4S and 5S RNA of HeLa Cells", "advisor": "Attardi, Giuseppe", "url": "https://resolver.caltech.edu/CaltechTHESIS:08032015-141200820", "creators": [ { "name": { "family": "Hatlen", "given": "Loren Endicott" }, "id": "Hatlen-Loren-Endicott", "display_name": "Hatlen, Loren Endicott" } ], "advisors": [ { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "advisor", "display_name": "Attardi, Giuseppe" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/XBGN-YT10", "abstract": "SECTION I\r\nSection I is concerned with a partial sequence analysis conducted on 5S RNA from HeLa cells. Analysis of the oligonucleotide pattern after pancreatic ribonuclease digestion of a highly-purified preparation of 5S RNA gave results which were in general agreement with those published for KB cells, both with respect to the identity and the frequency of the partial sequences. However, the presence of a trinucleotide not found in the KB 5S pattern, together with the reproducibly much lower than expected molar yield of the larger oligonucleotides strongly suggested the occurrence of alternate sequences at various sites in the 5S molecules of human cells. The presence of ppGp and pppGp at the 5'-terminus of HeLa 5S RNA was clearly demonstrated. The implications of this finding with regard to the origin of 5S RNA are discussed.
\r\n\r\nSECTION II
\r\nIn Section II the proportion of the HeLa cell genome complementary to tRNA was investigated by using RNA- DNA hybridization. The value for saturation of the HeLa DNA by tRNA was found to be 1.1 x 10-5, which corresponds to about 4900 sites for tRNA per HeLa cell in an exponentially growing culture. Analysis of the nucleotide composition of the hybridized tRNA revealed significant differences from the nucleotide composition of the input tRNA, with the purine to pyrimidine ratio indicating, however, that these differences were not produced by excessive RNase attack of the hybrid. The size of the hybridized tRNA was only moderately smaller than that of the input RNA; the average S value in formaldehyde was 2.7 (corresponding to a length of about 65 nucleotides), suggesting that a relatively small portion near the ends of the hybridized 4S chains had been removed by RNase.
\r\n\r\nSECTION III
\r\nThe proportion of the HeLa cell genome complementary to 5S RNA was investigated by using RNA-DNA hybridization. The value for saturation of the HeLa DNA by 5S RNA was found to be 2.3 x 10-5, which corresponds to about 7,000 sites for 5S RNA per HeLa cell in an exponentially growing culture. Analysis of the nucleotide composition of the hybridized 5S RNA revealed no significant difference from the nucleotide composition of the input RNA. At the RNA to DNA input ratio of 1:1000, the average S value in formaldehyde of the hybridized 5S RNA corresponded to a polynucleotide chain about two-thirds the size of the input RNA.
\r\n" }, { "name": "Josslin, Richard Douglas Kerr", "degree": "PhD", "year": "1970", "title": "The Lysis Mechanism of Phage T4", "advisor": "Delbruck, Max; Sinsheimer, Robert L.", "url": "https://resolver.caltech.edu/CaltechETD:etd-08082006-094136", "creators": [ { "name": { "family": "Josslin", "given": "Richard Douglas Kerr" }, "id": "Josslin-Richard- Douglas-Kerr", "display_name": "Josslin, Richard Douglas Kerr" } ], "advisors": [ { "name": { "family": "Delbruck", "given": "Max" }, "id": "Delbr\u00fcck-M", "role": "advisor", "display_name": "Delbruck, Max" }, { "name": { "family": "Sinsheimer", "given": "Robert L." }, "id": "Sinsheimer-R-L", "role": "advisor", "display_name": "Sinsheimer, Robert L." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/2KYN-NA14", "abstract": "Under normal conditions, phage T4-infected bacteria lyse at a characteristic time after infection, each infected cell releasing a few hundred progeny phage. To investigate the mechanism of this lysis, a selection technique was used to isolate lysis-defective mutants of phage T4. A new class of lysis-defective mutants was found which define a new T4 gene called t. During infection, t-defective mutants synthesize both phage and phage lysozyme, yet fail to either cease metabolism or lyse at the usual time. Thus, T4-induced lysis entails a sequence of at least two events: function of t gene product, resulting in the cessation of host metabolism and allowing e gene product, phage lysozyme, to degrade the host cell wall and release progeny phage.\r\n\r\nAlthough the mechanism of T4 t gene product remains to be determined, some alternatives were ruled out. Neither a metabolic poison nor exogenous lysozyme will phenotypically revert the t gene defect. These results show that the normal function of gene t involves something besides cessation of host metabolism and disruption of the host cell wall. To test whether t gene product acts by degrading host membrane phospholipid, hydrolysis of host phospholipid was measured during T4 infection. It was found that lysis, although normally accompanied by phospholipid hydrolysis, can occur in the absence of this reaction. Thus the phospholipid hydrolysis observed is a by-product of the t gene event rather that a requirement for lysis. The role of gene t in the lysis mechanism of phage T4 is discussed in light of these results." }, { "name": "Levy, Jerre", "degree": "PhD", "year": "1970", "title": "Information Processing and Higher Psychological Functions in the Disconnected Hemispheres of Human Commissurotomy Patients", "advisor": "Sperry, Roger Wolcott", "url": "https://resolver.caltech.edu/CaltechTHESIS:08072015-095846342", "creators": [ { "name": { "family": "Levy", "given": "Jerre" }, "id": "Levy-Jerre", "display_name": "Levy, Jerre" } ], "advisors": [ { "name": { "family": "Sperry", "given": "Roger Wolcott" }, "id": "Sperry-R-W", "role": "advisor", "display_name": "Sperry, Roger Wolcott" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/N7VN-TP54", "abstract": "Several patients of P. J. Vogel who had undergone cerebral commissurotomy\r\nfor the control of intractable epilepsy were tested on\r\na variety of tasks to measure aspects of cerebral organization\r\nconcerned with lateralization in hemispheric function. From tests\r\ninvolving identification of shapes it was inferred that in the absence\r\nof the neocortical commissures, the left hemisphere still has access\r\nto certain types of information from the ipsilateral field. The major\r\nhemisphere can still make crude differentiations between various\r\nleft-field stimuli, but is unable to specify exact stimulus properties.\r\nMost of the time the major hemisphere, having access to some ipsilateral \r\nstimuli, dominated the minor hemisphere in control of the body.
\r\n\r\nCompetition for control of the body between the hemispheres is\r\nseen most clearly in tests of minor hemisphere language competency,\r\nin which it was determined that though the minor hemisphere does possess\r\nsome minimal ability to express language, the major hemisphere\r\nprevented its expression much of the time. The right hemisphere was\r\nsuperior to the left in tests of perceptual visualization, and the\r\ntwo hemispheres appeared to use different strategies in attempting to\r\nsolve the problems, namely, analysis for the left hemisphere and\r\nsynthesis for the right hemisphere.
\r\n\r\nAnalysis of the patients' verbal and performance I.Q.'s, as well\r\nas observations made throughout testing, suggest that the corpus\r\ncallosum plays a critical role in activities that involve functions\r\nin which the minor hemisphere normally excels, that the motor expression\r\nof these functions may normally come through the major hemisphere\r\nby way of the corpus callosum.
\r\n\r\nLateral specialization is thought to be an evolutionary adaptation\r\nwhich overcame problems of a functional antagonism between the\r\nabilities normally associated with the two hemispheres. The tests of\r\nperception suggested that this function lateralized into the mute\r\nhemisphere because of an active counteraction by language. This\r\nlatter idea was confirmed by the finding that left-handers, in whom\r\nthere is likely to be bilateral language centers, are greatly\r\ndeficient on tests of perception.
" }, { "name": "Maxwell, Joyce Bennett", "degree": "PhD", "year": "1970", "title": "Part I. Synthesis of L-Arnino Acid Oxidase by a Serine- or Glycine-Requiring Strain of Neurospora. Part II. Studies Concerning Multiple Electrophoretic Forms of Tyrosinase in Neurospora", "advisor": "Horowitz, Norman Harold", "url": "https://resolver.caltech.edu/CaltechTHESIS:08132015-083412760", "creators": [ { "name": { "family": "Maxwell", "given": "Joyce Bennett" }, "id": "Maxwell-Joyce-Bennett", "display_name": "Maxwell, Joyce Bennett" } ], "advisors": [ { "name": { "family": "Horowitz", "given": "Norman Harold" }, "id": "Horowitz-N-H", "role": "advisor", "display_name": "Horowitz, Norman Harold" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/GHCK-W396", "abstract": "Part I: Synthesis of L-Amino Acid Oxidase by a Serine- or Glycine-Requiring\r\nStrain of Neurospora
\r\n\r\nWild-type cultures of Neurospora crassa growing on minimal\r\nmedium contain low levels of L-amino acid oxidase, tyrosinase, and\r\nnicotinarnide adenine dinucleotide glycohydrase (NADase). The enzymes\r\nare derepressed by starvation and by a number of other conditions which\r\nare inhibitory to growth. L-amino acid oxidase is, in addition, induced\r\nby growth on amino acids. A mutant which produces large quantities of\r\nboth L-amino acid oxidase and NADase when growing on minimal medium was\r\ninvestigated. Constitutive synthesis of L-amino acid oxidase was shown\r\nto be inherited as a single gene, called P110, which is separable from\r\nconstitutive synthesis of NADase. P110 maps near the centromere on\r\nlinkage group IV.
\r\n\r\nL-amino acid oxidase produced constitutively by P110 was partially\r\npurified and compared to partially purified L-amino acid oxidase\r\nproduced by derepressed wild-type cultures. The enzymes are identical\r\nwith respect to thermostability and molecular weight as judged by gel\r\nfiltration.
\r\n\r\nThe mutant P110 was shown to be an incompletely blocked auxotroph\r\nwhich requires serine or glycine. None of the enzymes involved\r\nin the synthesis of serine from 3-phosphoglyceric acid or glyceric acid\r\nwas found to be deficient in the mutant, however. An investigation of\r\nthe free intracellular amino acid pools of P110 indicated that the\r\nmutant is deficient in serine, glycine, and alanine, and accumulates\r\nthreonine and homoserine.
\r\n\r\nThe relationship between the amino acid requirement of P110 and\r\nits synthesis of L-amino acid oxidase is discussed.
\r\n\r\nPart II: Studies Concerning Multiple Electrophoretic Forms of Tyrosinase\r\nin Neurospora
\r\n\r\nSupernumerary bands shown by some crude tyrosinase preparations\r\nin paper electrophoresis were investigated. Genetic analysis indicated\r\nthat the location of the extra bands is determined by the particular T\r\nallele present. The presence of supernumerary bands varies with the\r\nmethod used to derepress tyrosinase production, and with the duration\r\nof derepression. The extra bands are unstable and may convert to the\r\nmajor electrophoretic band, suggesting that they result from modification\r\nof a single protein. Attempts to isolate the supernumerary bands\r\nby continuous flow paper electrophoresis or density gradient zonal\r\nelectrophoresis were unsuccessful.
" }, { "name": "Newbold, John Edward", "degree": "PhD", "year": "1970", "title": "Part I. The Abortive Infection of Bacteriophage \u00d8X174 at Low Temperatures. Part II. The Early Stages in the Process of Infection by Bacteriophage \u00d8X174", "advisor": "Sinsheimer, Robert L.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08122015-154248368", "creators": [ { "name": { "family": "Newbold", "given": "John Edward" }, "id": "Newbold-John-Edward", "display_name": "Newbold, John Edward" } ], "advisors": [ { "name": { "family": "Sinsheimer", "given": "Robert L." }, "id": "Sinsheimer-R-L", "role": "advisor", "display_name": "Sinsheimer, Robert L." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/XR69-BE33", "abstract": "Part I\r\nThe infection of E. coli by \u03a6X174 at 15\u00b0C is abortive; the cells are killed by the infection but neither mature phage nor SS (single-stranded) DNA are synthesized. Parental RF (replicative form) is formed and subsequently replicated at 15\u00b0C. The RF made at 15\u00b0C shows normal infectivity and full competence to act as precursor to progeny SS DNA after an increase in temperature to 37\u00b0C. The investigations suggest that all of the proteins required for SS DNA synthesis and phage maturation are present in the abortive infection at 15\u00b0C.
\r\nThree possible causes are suggested for the abortive infection at 15\u00b0C: (a) A virus-coded protein whose role is essential to the infection is made at 15\u00b0C and assumes its native conformation, but its rate of activity is too low at this temperature to sustain the infection process. (b) Virus maturation may involve the formation of a DNA-protein complex and conformational changes which have an energy threshold infrequently reached at 15\u00b0C. (c) A host-coded protein present in uninfected cells, and whose activity is essential to the infection at all temperatures, but not to the host at 15\u00b0C, is inactive at 15\u00b0C. An hypothesis of this type is offered which proposes that the temperature-limiting factor in SS DNA synthesis in vivo may reflect a temperature-dependent property of the host DNA polymerase.
\r\n\r\nPart II
\r\n\r\nThree distinct stages are demonstrated in the process whereby \u03a6X174 invades its host:\r\n(1) Attachment: The phage attach to the cell in a manner that does not irreversibly alter the phage particle and which exhibits \"single-hit\" kinetics. The total charge on the phage particle is demonstrated to be important in determining the rate at which stable attachment is effected. The proteins specified by \u03a6X cistrons II, III and VII play roles, which may be indirect, in the attachment reaction.\r\n(2) Eclipse: 'The attached phage undergo a conformational change.\r\nSome of the altered phage particles spontaneously detach from the cell (in\r\na non-infective form) while the remainder are more tightly bound to the\r\ncell. The altered phage particles detached (spontaneously or chemically)\r\nfrom such complexes have at least 40% of their DNA extruded from the\r\nphage coat. It is proposed that this particle is, or derives from, a direct\r\nintermediate in the penetration of the viral DNA.
\r\n\r\nThe kinetics for the eclipse of attached phage particles are first-order with respect to phage concentration and biphasic; about 85% of the phage eclipse at one rate (k = 0.86 min-1) and the remainder do so at a distinctly lesser rate (k = 0.21 min-1).
\r\n\r\nThe eclipse event is very temperature-dependent and has the relatively high Arrhenius activation energy of 36.6 kcal/mole, indicating the cooperative nature of the process. The temperature threshold for eclipse is 17 to 18\u00b0C.
\r\n\r\nAt present no specific \u03a6X cistron is identified as affecting the eclipse process.\r\n(3) DNA penetration: A fraction of the attached, eclipsed phage particles corresponding in number to the plaque-forming units complete DNA penetration. The penetrated DNA is found in the cell as RF, and the empty phage protein coat remains firmly attached to the exterior of the cell. This step is inhibited by prior irradiation of the phage with relatively high doses of UV light and is insensitive to the presence of KCN and NaN3. Temporally excluded superinfecting phages do not achieve DNA penetration.
\r\n\r\nBoth eclipsed phage particles and empty phage protein coats may be dissociated from infected cells; some of their properties are described.
\r\n" }, { "name": "Parkinson, John Stansfield", "degree": "PhD", "year": "1970", "title": "Organization and Function of the Phage Lambda Chromosome", "advisor": "Edgar, Robert S.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08112015-161712404", "creators": [ { "name": { "family": "Parkinson", "given": "John Stansfield" }, "id": "Parkinson-John-Stansfield", "display_name": "Parkinson, John Stansfield" } ], "advisors": [ { "name": { "family": "Edgar", "given": "Robert S." }, "id": "Edgar-R-S", "role": "advisor", "display_name": "Edgar, Robert S." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/V4T1-KJ37", "abstract": "The process of prophage integration by phage \u03bb and the\r\nfunction and structure of the chromosomal elements required for \u03bb\r\nintegration have been studied with the use of \u03bb deletion mutants.\r\nSince att\u03c6, the substrate of the integration enzymes, is not essential\r\nfor \u03bb growth, and since att\u03c6 resides in a portion of the \u03bb chromosome\r\nwhich is not necessary for vegetative growth, viable \u03bb deletion\r\nmutants were isolated and examined to dissect the structure of att\u03c6.
\r\n\r\nDeletion mutants were selected from wild type populations by\r\ntreating the phage under conditions where phage are inactivated at a\r\nrate dependent on the DNA content of the particles. A number of\r\ndeletion mutants were obtained in this way, and many of these mutants\r\nproved to have defects in integration. These defects were defined by\r\nanalyzing the properties of Int-promoted recombination in these att\r\nmutants.
\r\n\r\nThe types of mutants found and their properties indicated that\r\natt\u03c6 has three components: a cross-over point which is bordered on\r\neither side by recognition elements whose sequence is specifically\r\nrequired for normal integration. The interactions of the recognition\r\nelements in Int-promoted recombination between att mutants was\r\nexamined and proved to be quite complex. In general, however, it\r\nappears that the \u03bb integration system can function with a diverse\r\narray of mutant att sites.
\r\n\r\nThe structure of att\u03c6 was examined by comparing the genetic\r\nproperties of various att mutants with their location in the \u03bb chromosome.\r\nTo map these mutants, the techniques of heteroduplex DNA\r\nformation and electron microscopy were employed. It was found that\r\nintegration cross-overs occur at only one point in att\u03c6 and that the\r\nrecognition sequences that direct the integration enzymes to their\r\nsite of action are quite small, less than 2000 nucleotides each.\r\nFurthermore, no base pair homology was detected between att\u03c6\r\nand its bacterial analog, attB. This result clearly demonstrates\r\nthat \u03bb integration can occur between chromosomes which have little,\r\nif any, homology. In this respect, \u03bb integration is unique as a\r\nsystem of recombination since most forms of generalized recombination\r\nrequire extensive base pair homology.
\r\n\r\nAn additional study on the genetic and physical distances in the\r\nleft arm of the \u03bb genome was described. Here, a large number of\r\nconditional lethal nonsense mutants were isolated and mapped, and\r\na genetic map of the entire left arm, comprising a total of 18 genes,\r\nwas constructed. Four of these genes were discovered in this study.\r\nA series of \u03bbdg transducing phages was mapped by heteroduplex\r\nelectron microscopy and the relationship between physical and\r\ngenetic distances in the left arm was determined. The results\r\nindicate that recombination frequency in the left arm is an accurate\r\nreflection of physical distances, and moreover, there do not appear\r\nto be any undiscovered genes in this segment of the genome.
\r\n" }, { "name": "Smart, John Edward", "degree": "PhD", "year": "1970", "title": "Studies on the Role of Histones in the Structure and Function of Chromatin", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechTHESIS:09032015-162652124", "creators": [ { "name": { "family": "Smart", "given": "John Edward" }, "id": "Smart-John-Edward", "display_name": "Smart, John Edward" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/DQN1-VN19", "abstract": "Studies on the dissociation of histones from chromatin by increasing concentrations of sodium deoxycholate (DOC) have shown that histrone II is removed at lowest concentrations of DOC, while slightly higher concentrations remove histones III and IV. Still higher concentrations remove histone I.
\r\n\r\nThe complete separation of chromatin and 14C-DOC by sucrose sedimentation indicated that the binding of DOC to chromatin is readily and completely reversible.
\r\n\r\nThe dissociation of histones from chromatin by increasing concentrations of related cholanic acids and some of their conjugated derivatives were studied. The results suggested that the driving force for the interaction between the cholanic acid anion and histones is the lowering of the activity coefficient of the cholanic acid anion which occurs when it is partially removed from solution by interaction with hydrophobic regions of the positively charged histones.
\r\n\r\nThe role of histones in the structure of chromatin has been studied by comparing the effects of selective removal of histones from chromatin by increasing concentrations of DOC with those caused by NaCl (removes histone I at lowest concentrations, while higher concentrations remove histones II, III, and IV). Properties studied included thermal denaturation, sedimentation velocity, flow dichroism, relaxation times of molecules oriented in a flow field, and the irreversible disruption of a 130 S, cross-linked component of sheared chromatin. The data indicated that none of the structural or chemical parameters with which these properties are correlated show a dependence on the presence of one particular histone fraction.
\r\n\r\nThe template activity (ability to prime a 0.2 M KC1 DNA-dependent RNA synthesis system catalyzed by E. coli RNA polymerase) increases from that of native chromatin (approximately 25 per cent of that pure DNA) to that of pure DNA in a fashion which shows a nearly linear relationship to the amount of histone coverage of the template. The precipitability of partially dehistonized chromatin samples in 0.15 M NaCl shows a large dependence on the presence of histone I.
\r\n" }, { "name": "Toevs, Lois Schloemer", "degree": "PhD", "year": "1970", "title": "Identification and Characterization of the Egg-Laying Hormone from the Neurosecretory Bag Cells of Aplysia", "advisor": "Strumwasser, Felix", "url": "https://resolver.caltech.edu/CaltechTHESIS:08282015-154034894", "creators": [ { "name": { "family": "Toevs", "given": "Lois Schloemer" }, "id": "Toevs-Lois-Schloemer", "display_name": "Toevs, Lois Schloemer" } ], "advisors": [ { "name": { "family": "Strumwasser", "given": "Felix" }, "id": "Strumwasser-F", "role": "advisor", "display_name": "Strumwasser, Felix" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "neurobio" ], "doi": "10.7907/70RQ-P642", "abstract": "This investigation has resulted in the chemical identification\r\nand isolation of the egg-laying hormone from Aplysia californica,\r\nAplysia vaccaria, and Aplysia dactylomela. The hormone, which was\r\noriginally identified as the Bag Cell-Specific protein (BCS protein)\r\non polyacrylamide gels, is a polypeptide of molecular weight \u2248 6000,\r\nwhich is localized in the neurosecretory bag cells of the parietovisceral\r\nganglion and the surrounding connective tissue sheath which\r\ncontains the bag cell axons. All three species produce a hormone of\r\nsimilar molecular weight, but varying electrophoretic mobility as determined\r\non polyacrylamide gels. As tested, the hormone is completely\r\ncross-reactive among the three species.
\r\n\r\nAlthough the bag cells of sexually immature animals contain the\r\nactive hormone, sexual maturation of the animal results in a 10-fold\r\nincrease in the BCS protein content of these neurons.
\r\n\r\nA seasonal variation in the BCS protein content was also observed,\r\nwith 150 times more hormone contained in the bag cells of\r\nAplysia californica in August than in January. This correlates well\r\nwith the variation in the animals' ability to lay eggs throughout the\r\nyear (Strumwasser et al., 1969). There are some indications that the\r\nreceptivity of the animal to the available hormone also fluctuates\r\nduring the year, being lower in winter than in swmner. The seasonal\r\nrhythm of the other species, Aplysia vaccaria and Aplysia dactylomela,\r\nhas not been investigated.
\r\n\r\nA polyacrylamide gel electrophoresis analysis of water-soluble\r\nproteins in Aplysia californica revealed several other nerve-specific\r\nproteins. One of these is also located in the bag cell somas and stains\r\nturquoise with Amido Schwarz. The function of this protein has not been\r\ninvestigated.
" }, { "name": "Flatgaard, Jeffrey Edward", "degree": "PhD", "year": "1969", "title": "The Role of the Gene 9 Product in the Assembly and Triggering of Bacteriophage T4", "advisor": "Edgar, Robert S.", "url": "https://resolver.caltech.edu/CaltechETD:etd-08082006-090749", "creators": [ { "name": { "family": "Flatgaard", "given": "Jeffrey Edward" }, "id": "Flatgaard-Jeffrey-Edward", "display_name": "Flatgaard, Jeffrey Edward" } ], "advisors": [ { "name": { "family": "Edgar", "given": "Robert S." }, "id": "Edgar-R-S", "role": "advisor", "display_name": "Edgar, Robert S." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/5F2T-5124", "abstract": "Gene 9 of phage T4 specifies a protein needed in the assembly of the virus particle, whose site of action is on the baseplate. Assembly without gene 9 product results in the production of inactive particles which lack tail fibers. These defective particles can be converted to complete active phage in vitro by the sequential action of the gene 9 product and the addition of tail fibers. Gene 9 defective particles are unstable and spontaneously convert to an aberrant \"triggered\" form that has a contracted sheath, but retains the DNA in the head. Action of the gene 9 product on the particle stabilizes it from converting to the triggered form, provides for DNA release when the sheath contracts during infection, and provides sites on the baseplate for the attachment of tail fibers. For the particle to be subsequently active the gene 9 product must act at least three times on the gene 9 defective particle; there is some experimental evidence to suggest that the gene 9 product may act on the particle stoichiometrically rather than catalytically.\r\n\r\nA model is proposed for the action of the gene 9 product on the particle in which six gene 9 products are incorporated into the baseplate. These components join the tail plug to the baseplate vertices such that the change which occurs in baseplate configuration during infection will eliminate the tail plug by pulling it apart. Removal of the tail plug opens the end of the core to allow DNA release. The attachment site for the tail fiber on the baseplate is proposed to be at the junction of the gene 9 product and the baseplate." }, { "name": "Hutchison, Clyde Allen, III", "degree": "PhD", "year": "1969", "title": "Bacteriophage \u03a6X174: Viral Genes and Functions", "advisor": "Sinsheimer, Robert L.", "url": "https://resolver.caltech.edu/CaltechETD:etd-08092006-162535", "creators": [ { "name": { "family": "Hutchison", "given": "Clyde Allen, III" }, "id": "Hutchison-Clyde-Allen-III", "display_name": "Hutchison, Clyde Allen, III" } ], "advisors": [ { "name": { "family": "Sinsheimer", "given": "Robert L." }, "id": "Sinsheimer-R-L", "role": "advisor", "display_name": "Sinsheimer, Robert L." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/82XF-B518", "abstract": "Mutants. Conditional lethal mutants of bacteriophage [phi]X174 ([phi]X) have been selected. These include nonsense mutants of the am (amber nonsense triplet - UAG)and op (opal nonsense triplet - UGA) types. Mis-sense mutants of the ts (temperature-sensitive) and ss (solvent-sensitive) types have also been isolated. The ss mutants are abnormally sensitive, during plaque formation, to the presence of solvents such as dimethylsulfoxide, ethylene glycol, or urea in the growth medium. Host range mutants are also described. Double and triple mutant strains have been constructed from specific single mutants.
\r\n\r\nCistrons. Complementation studies (principally with am and ts mutants) have defined seven complementation groups or cistrons. It is believed that these represent the genes for seven essential [phi]X-coded proteins. At least six of these cistrons are homologous, by complementation, to cistrons of the related phage S13.
\r\n\r\nFunctions. Studies of abortive infection by conditional lethal mutants under restrictive conditions have been performed in order to elucidate the functions of the mutant cistrons. Properties of phage particles produced by mutants have been examined in order to identify cistrons which code for components of the phage coat. The cistron functions deduced from these studies are: cistron I - lysis of the host. Mutants in this cistron produce progeny at a normal rate, and for an extended time, but are not able to lyse the host cell. cistron II - spike component. This cistron codes for a protein which is a component of the spikes which project from the 12 vertices of the isometric [phi]X capsid. cistron III - spike component. This cistron codes for a second protein component of the phage spike and contains the serum-blocking antigen of the phage particle. cistron IV - phage coat. This cistron codes for a protein of the phage coat which is probably, by elimination, the main structural component of the capsid. cistron V - ?. The function of this cistron product is not known. cistron VI - RF replication. The product of this cistron is necessary for replication of the double-stranded form of [phi]X-DNA (RF). cistron VII - spike component. The product of this cistron is believed to be a component of the phage spike which determines the host range of the particle.
\r\n\r\nThese physiological studies suggest that assembly of phage, or defective phage particles is necessary for the synthesis of viral single-stranded DNA. RF replication (which requires cistron VI function and cistron V function are also necessary for single-strand synthesis.
\r\n\r\nRecombination experiments (involving both two and three factor crosses) indicate that the order of the cistrons is II-III-VII-V-I-IV-VI, possibly joined end to end to form a circular map. It is of interest that cistrons which code for the spike components (II, III, and VII) appear to be contiguous on the map. A polar am mutant in cistron IV is deficient also in cistron VI function. This is interpreted to mean that reading proceeds from left to right on the map as shown (5' to 3' direction of the viral DNA and [phi]X mRNA).
" }, { "name": "Logan, James Barrie", "degree": "PhD", "year": "1969", "title": "Biochemistry and Genetics of Canavanine Resistance in Neurospora", "advisor": "Horowitz, Norman Harold", "url": "https://resolver.caltech.edu/CaltechTHESIS:06022017-085722120", "creators": [ { "name": { "family": "Logan", "given": "James Barrie" }, "id": "Logan-James-Barrie", "display_name": "Logan, James Barrie" } ], "advisors": [ { "name": { "family": "Horowitz", "given": "Norman Harold" }, "id": "Horowitz-N-H", "role": "advisor", "display_name": "Horowitz, Norman Harold" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/2j3p-es09", "abstract": "The pattern of inheritance of resistance to growth inhibition\r\nby canavanine in Neurospora crassa is shown to result from interactions\r\nbetween a major gene and several modifiers. The major gene controls\r\nthe production of a constitutive enzyme that destroys canavanine. The\r\nmodifiers affect the rate of uptake of the analog from the medium.\r\nStrains which lack the enzyme activity are canavanine sensitive; strains\r\nwhich possess it are resistant, but the level of resistance is dependent\r\non the rate of uptake.
\r\n\r\n\r\nThe canavanine degrading enzyme was partially purified and its\r\nproperties studied. The detoxification reaction was shown to be a\r\ncleavage of canavanine yielding hydroxyguanidine.
" }, { "name": "Lyon, Alexander Newell", "degree": "PhD", "year": "1969", "title": "Studies on the Isolation of Messenger RNA", "advisor": "Borsook, Henry", "url": "https://resolver.caltech.edu/CaltechTHESIS:06072017-100016945", "creators": [ { "name": { "family": "Lyon", "given": "Alexander Newell" }, "id": "Lyon-Alexander-Newell", "display_name": "Lyon, Alexander Newell" } ], "advisors": [ { "name": { "family": "Borsook", "given": "Henry" }, "id": "Borsook-H", "role": "advisor", "display_name": "Borsook, Henry" } ], "committee": [ { "name": { "family": "Horowitz", "given": "Norman Harold" }, "id": "Horowitz-N-H", "role": "chair", "display_name": "Horowitz, Norman Harold" } ], "option_major": [ "bioch" ], "doi": "10.7907/HPJ4-J340", "abstract": "The rapidly-labeled RNA fraction of E. coli has been\r\npurified approximately 15-fold on benzoylated DEAE cellulose\r\ncolumns (BC). It is metabolically unstable (as shown\r\nby a pulse/chase experiment) and is considered to represent\r\nmRNA. The yield of pulse-labeled RNA is about 70% and\r\ncomprises 4-5% of the RNA of the cell.
\r\n\r\n\r\nThe true size distribution of this RNA, determined by\r\nsedimentation in a denaturing solvent (99% DMSO), does not\r\nchange during purification. This result indicates that\r\nneither degradation nor selection for molecules of a particular\r\nsize has occurred. Upon sedimentation of the final\r\npreparation in DMSO, the distribution of pulse label is\r\nthe same as that of RNA mass, indicating nearly complete\r\nseparation from longer-lived RNA components.
\r\n\r\n\r\nThe isolation of globin-specific mRNA from rabbit\r\nreticulocytes has been attempted, both by a modification\r\nof BC chromatography and by the previously published\r\nsucrose gradient method of Marbaix, Burny, and Chantrenne,\r\nbut the results (in both cases) were inconclusive. The\r\nfinal preparation sedimented heterogeneously, with an\r\nestimated mean sedimentation coefficient (s20,w) of 8.4 S.\r\nTests of the possible identity of this material as mRNA by\r\nbiological assay in a cell-free protein synthesizing\r\nsystem have not been attempted.
" }, { "name": "Nesson, Michael Harvey", "degree": "PhD", "year": "1969", "title": "Studies on Radula Tooth Mineralization in the Polyplacophora", "advisor": "Lowenstam, Heinz A.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05182012-113748142", "creators": [ { "name": { "family": "Nesson", "given": "Michael Harvey" }, "id": "Nesson-Michael-Harvey", "display_name": "Nesson, Michael Harvey" } ], "advisors": [ { "name": { "family": "Lowenstam", "given": "Heinz A." }, "id": "Lowenstam-H-A", "role": "advisor", "display_name": "Lowenstam, Heinz A." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/CQNC-FZ32", "abstract": "This report is concerned with several aspects of the process of iron mineralization of the radula teeth of chitons (Polyplacophora.
\r\n\r\nIn Part I, the cusp cells of the radula sac, which are responsible for the deposition of iron into the major lateral teeth, are studied with the electron microscope. In Mopalia muscosa and Lepidochitona (Cyanoplax) hartwegi, it is found that these cells extend from a dorsal blood sinus to the surface of the teeth. The basal ends of the cusp cells, near the dorsal sinus, contain numerous rhopheocytotic vesicles filled with ferritin. Near the apical ends of the cells, there is a high concentration of iron-containing membrane-bound granules. Some granules contain ferritin, others are filled with ferruginous particles of electron-dense material, and others contain ferritin cores with an outer layer of ferruginous particles. In each cusp cell, the granule region is separated from the tooth surface by a bundle of microvilli that arises from a layer of mitochondria-rich cytoplasm and terminates on the tooth surface. No electron-dense material is found in the microvilli. Double-membrane structures are observed in regions where iron-containing granules occur near the microvilli. A model of the pathway of iron through the cusp cells is deduced from the observations on their ultrastructure.
\r\n\r\nIn Part II, it is shown that at least 90% of the iron contained in the blood of Mopalia muscosa (40 to 110 \u03bcg Fe per ml) occurs in the form of the protein-iron complex, ferritin. The blood ferritin is purified and compared with ferritin isolated from the superior epithelial cells and with vertebrate ferritins.
\r\n\r\nIn Part III, it is determined, by Fe59-labeling experiments, that the radula replacement rate of Mopalia muscosa is approximately 0.6 rows per day. The amount of iron contained in the major lateral teeth of Mopalia muscosa radulas is measured.
\r\n" }, { "name": "Shearn, Allen David", "degree": "PhD", "year": "1969", "title": "A Study of the Possible Role of Transfer RNA in the Regulation of Enzyme Synthesis in Neurospora", "advisor": "Horowitz, Norman Harold", "url": "https://resolver.caltech.edu/CaltechTHESIS:03282017-143303527", "creators": [ { "name": { "family": "Shearn", "given": "Allen David" }, "id": "Shearn-Allen-David", "display_name": "Shearn, Allen David" } ], "advisors": [ { "name": { "family": "Horowitz", "given": "Norman Harold" }, "id": "Horowitz-N-H", "role": "advisor", "display_name": "Horowitz, Norman Harold" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/9TVD-N609", "abstract": "In several organisms, developmental transitions are accompanied\r\nby transfer RNA (tRNA) alterations. These alterations are usually\r\nobserved in the chromatographic profile of amino acyl-tRNA specific\r\nfor one or more amino acids and are of interest because of their possible\r\nsignificance in the regulation, at the translation level, of\r\nspecific protein synthesis and cell differentiation. I have investigated\r\nwhether such alterations accompany the biochemical differentiation of\r\nvegetative cultures of Neurspora crassa which occurs in response to\r\n\"hard-times,\" e.g., starvation or inhibition by amino acid analogs or\r\ncycloheximide. The synthesis of tyrosinase is a well-known characteristic\r\nof this developmental transition.
\r\n\r\n\r\nAfter determining the conditions required for the complete\r\ncharging of all 20 amino acids to Neurospora tRNA, I compared the\r\nchromatographic profile on methylated albumin-Kieselguhr columns of\r\namino acyl-tRNA's from vegetative cultures to those of cultures which\r\nwere derepressed for tyrosinase with ethionine, a methionine analog.\r\nNo qualitative tRNA alterations were observed; the same number of\r\ncomponents for each amino acid were found in cultures of both developmental\r\nstates and they had the same chromatographic mobilities.\r\nHowever, quantitative changes of acceptor activity were observed for\r\nseveral amino acids. The time course of the pattern of quantitative\r\nalteration suggests that the observed changes result from partial ribonuclease\r\ndigestion of the tRNA complement. I believe this ribonuclease\r\nis synthesized in response to the deprived environment and its function\r\nis to hydrolyze the RNA which is present in excess, in order that the\r\ncatabolic products may be used as building blocks for the synthesis\r\nof other kinds of molecules.
" }, { "name": "Shih, Thomas Yu-tzong", "degree": "PhD", "year": "1969", "title": "I. Chromosomal RNA of Calf Thymus Chromatin. II. The Template Properties of DNA-Polypeptide Complexes. III. Studies on DNA Complexes with Purified Histone Fractions", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechTHESIS:03312017-130624894", "creators": [ { "name": { "family": "Shih", "given": "Thomas Yu-tzong" }, "id": "Shih-Thomas-Yu-tzong", "display_name": "Shih, Thomas Yu-tzong" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/HQQY-2J93", "abstract": "Part I. Chromosomal RNA of Calf Thymus Chromatin
\r\n\r\nCalf thymus chromatin is shown to contain an associated chromosomal\r\nRNA as do the chromatins of other species. The chromosomal RNA\r\nof calf thymus chromatin is present in an amount of 1% of that of DNA.\r\nThe purified material eluted from DEAE-Sephadex column at a NaCl\r\nconcentration of 0.56 M as do the chromosomal RNA's of other organisms.\r\nIts average chain length by end-group assay is approximately 40 nucleotides\r\nand it contains approximately 3-4 dihydrouridylic acid residues\r\nper chain. Calf thumus chromosomal RNA is associated with chromosomal\r\nprotein in a form not dissociable by high salt concentration.
\r\n\r\nPart II. The Template Properties of DNA-Polypeptide Complexes
\r\n\r\nDNA complexes with poly-L-lysine, poly-L-arginine and protamine\r\nwere prepared by a salt gradient dialysis. The complexes possess\r\nthe stoichiometry of one lysine or arginine residue per nucleotide\r\nresidue as determined from the biphasic melting profiles. The template\r\nactivity of a complex in support of RNA synthesis in the presence of\r\nexcess RNA polymerase and required substrates is proportional to its\r\nfractional content of free DNA segments. The complexed DNA region is\r\nquantitatively blocked and does not act as template. Kinetic analysis\r\nof the template behavior reveals two different modes of inhibition by\r\nthe polypeptides. If the template is in a finely dispersed state, it\r\nis available to the enzyme as shown by the fact that the equal template\r\nconcentrations of complex and of pure DNA are required for half\r\nsaturation of a given amount of enzyme (K). Inhibition of RNA synthesis\r\nis, we propose, due to interference with local untwisting of DNA.\r\nIf the template is in a highly aggregated state, K is drastically increased\r\nand it is unavailable to the enzyme. The several species of\r\nhistone molecules normally complexed with DNA in the eucaryotic organisms\r\ndiffer among themselves in content of lysine and arginine. The\r\npresent studies show that the arginine residues are as effective as\r\nthe lysine residues in abolishing DNA template activity.
\r\n\r\nPart III. Studies on DNA Complexes with Purified Histone Fractions
\r\n\r\nWell-defined DNA complexes with calf thymus histones Ia, Ib,\r\nIIb and IV have been prepared by a salt gradient dialysis in the\r\npresence of 5 M urea. The complexes with subequivalent histone/DNA\r\nratio exhibit biphasic melting profiles. Tm,1 is the melting of free\r\nDNA segments, and Tm,2 that of the histone-complexed regions. Tm,2\r\nis characteristic for each DNA-basic protein complex. Tm,2(\u00b0C) of,\r\nthe complexes in 2.5 x 10-4 M sodium EDTA, pH 8.0 is as follows: DNA,\r\n47.2; chromatin, 74.3; DNA-histone Ia, 75.4; DNA-histone Ib, 76.3;\r\nDNA-histone IIb, 81.5; DNA-histone IV, 83.7; DNA-protamine, 92.5; DNA-polyarginine,\r\n98.0; and DNA-polylysine, 99.5. The stoichiometric\r\nratio (histone lysine plus arginine to nucleotide) of the equivalent\r\ncomplexes as detennined from the biphasic melting profiles is DNA-histone\r\nIa and Ib, 0.8; DNA-histone IIb, 1.2 and DNA-histone IV, 1.5.\r\nThe general shape of UV spectrum of DNA is not changed by complexing\r\nwith various histone species. DNA-histone IV complex is inactive in\r\npriming RNA synthesis in E.coli RNA polymerase system. Possible\r\nstructures of the DNA-binding parts of histone molecules have been\r\ndiscussed and illustrated with CPK molecular models in the case of\r\nhistone IV (pp. 157, 158).
" }, { "name": "Ahmed, Nawal Abd El-Hay", "degree": "PhD", "year": "1968", "title": "The Iodide Space in Rabbit Brain", "advisor": "Van Harreveld, Anthonie", "url": "https://resolver.caltech.edu/CaltechTHESIS:08302016-104724672", "creators": [ { "name": { "family": "Ahmed", "given": "Nawal Abd El-Hay" }, "id": "Ahmed-Nawal-Abd-El-Hay", "display_name": "Ahmed, Nawal Abd El-Hay" } ], "advisors": [ { "name": { "family": "Van Harreveld", "given": "Anthonie" }, "id": "Van-Harreveld-A", "role": "advisor", "display_name": "Van Harreveld, Anthonie" } ], "committee": [ { "name": { "family": "Van Harreveld", "given": "Anthonie" }, "id": "Van-Harreveld-A", "role": "chair", "display_name": "Van Harreveld, Anthonie" }, { "name": { "family": "Benzer", "given": "Seymour" }, "id": "Benzer-S", "role": "member", "display_name": "Benzer, Seymour" }, { "name": { "family": "Dreyer", "given": "William J." }, "id": "Dreyer-W-J", "role": "member", "display_name": "Dreyer, William J." }, { "name": { "family": "Roberts", "given": "John D." }, "id": "Roberts-J-D", "role": "member", "display_name": "Roberts, John D." }, { "name": { "family": "Strumwasser", "given": "Felix" }, "id": "Strumwasser-F", "role": "member", "display_name": "Strumwasser, Felix" } ], "option_major": [ "neurobio" ], "doi": "10.7907/E49K-K156", "abstract": "In the present investigation labeled iodide was used to investigate the interrelationship between brain, blood and cerebrospinal fluid, to examine active transport across the blood-brain- and the blood-cerebrospinal fluid barriers, and to estimate the extracellular space of the brain.
\r\n\r\nThe iodide space in the brain and the iodide concentration in cerebrospinal fluid after intravenous administration of radioactive iodide are determined by the following mechanisms. Iodide passes into the cerebrospinal fluid but active transport in the choroid plexus moves most of the iodide back again into the plasma, keeping the concentration at a very law value. An extracellular fluid is formed at the blood-brain barrier possibly in a similar way. The iodide concentration of this fluid is unknown but is probably higher than that in the cerebrospinal fluid. Diffusion of iodide across the brain-cerebrospinal fluid barrier transports this ion from the brain into the cerebrospinal fluid which is constantly renewed \"sink action\".
\r\n\r\nThe iodide space was found to be 2.4% four to five hours after the intravenous administration of 131I, the iodide content of the cerebrospinal fluid was 1.2% of that of the TCA serum filtrate. The iodide space increased to 10.6% in preparations in which in addition to 131I unlabeled iodide (to a serum concentration of 25 to 50 mM) was administered to saturate the active transport processes in the choroid plexuses and blood-brain barrier. The iodide activity of the cerebrospinal fluid in these experiments increased to 29.3% of that in the TCA serum filtrate. In experiments in which the inhibitor of iodide transport, perchlorate (8 mM), was injected intravenously with the 131I-, the iodide space was 8.2% and the iodide concentration in the cerebrospinal fluid 26.4%. These experiments demonstrate the effect of saturation and inhibition of active transport on the iodide space. They show furthermore that the depression of the active transport did not raise the iodide concentration in the cerebrospinal fluid to the plasma concentration. The relatively low (1/3 of that in the serum TCA filtrate) iodide concentration in the cerebrospinal fluid under these circumstances was ascribed to a differential permeability of the blood-cerebrospinal fluid barrier for iodide and chloride.
\r\n\r\nThe sink action can be eliminated by perfusion of the ventricles with an artificial cerebrospinal fluid containing iodide. Ventriculocisternal perfusion with 131I- alone resulted in an iodide space of 7.2% after 4.5 hours. An iodide space of 10.2% was determined by a combined intravenous administration and ventricular perfusion with an artificial cerebrospinal fluid containing the same concentration of 131I as present in the plasma. When in similar experiments perchlorate was administered intravenously, the iodide space rose to 16.8%. The iodide space determined by simultaneous intravenous injection and ventricular perfusion with both labeled and unlabeled iodide, in a concentration sufficient to saturate the active transport, was 20.8%. In the latter instances the sink action is eliminated and also active transport is inhibited or saturated. It was postulated that under these conditions the iodide concentration in plasma and brain extracellular fluid are approximately the same. The use of the iodide space as a measure of the brain extracellular space was discussed.
" }, { "name": "Dahmus, Michael Edward", "degree": "PhD", "year": "1968", "title": "Chapter I. Studies on Chromosomal RNA. Chapter II. Effect of Hydrocortisone on the Template Activity of Liver Chromatin", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechETD:etd-10032002-102109", "creators": [ { "name": { "family": "Dahmus", "given": "Michael Edward" }, "id": "Dahmus-Michael-Edward", "display_name": "Dahmus, Michael Edward" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/APSP-5851", "abstract": "Chapter I.
\r\n\r\nChapter I of this thesis is concerned with the isolation and characterization of ascites chromosomal RNA. The isolation of rat ascites chromosomal RNA as well as some of its physical and chemical properties are described in Section 1. Chromosomal RNA is characterized by its small size, lack of amino acid acceptor activity, and, relative to transfer RNA, low content of methylated bases. It has a base composition similar to that of ascites ribosomal RNA and is not labeled when the cells are exposed to a short pulse of \u00b3\u00b2P. An RNA (3S cytoplasmic RNA), with properties similar to those of chromosomal RNA but contained in the cytoplasm, has also been isolated.
\r\n\r\nSection 2 is concerned with the hybridization properties of ascites chromosomal RNA to denatured ascites nuclear DNA. Chromosomal RNA hybridizes to about 4% of ascites nuclear DNA and has no sites in common with ascites messenger RNA. 3S cytoplasmic RNA hybridizes to about 20 of ascites nuclear DNA and contains no sequences not also contained in chromosomal RNA. The 3S RNA contained in the nuclear sap is homologous to 3S cytoplasmic RNA. Therefore, it appears that a fraction of chromosomal RNA is confined to the chromatin while the remainder is homologous to an RNA contained in both the cytoplasm and nuclear sap.
\r\n\r\nChapter II.
\r\n\r\nThis chapter is concerned with the effect of hydrocortisone on the template activity of liver chromatin. Hydrocortisone administered to an adrenalectomized rat causes a two- to threefold increase in the rate of RNA synthesis in the liver. Chromatin isolated from the liver of hydrocortisone-treated rats possesses a 30 % greater template activity for DNA-dependent RNA synthesis than does chromatin isolated from control rats. The difference in template activity is abolished by removal of the proteins associated with the DNA. Hydrocortisone, administered to isolated purified chromatin, does not alter its template activity.
" }, { "name": "David, Charles Newbold", "degree": "PhD", "year": "1968", "title": "Ferritin in the Fungus Phycomyces", "advisor": "Delbruck, Max", "url": "https://resolver.caltech.edu/CaltechETD:etd-10032002-103720", "creators": [ { "name": { "family": "David", "given": "Charles Newbold" }, "id": "David-Charles-Newbold", "display_name": "David, Charles Newbold" } ], "advisors": [ { "name": { "family": "Delbruck", "given": "Max" }, "id": "Delbr\u00fcck-M", "role": "advisor", "display_name": "Delbruck, Max" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/NNQ9-TM81", "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\r\n\r\nThe iron storage protein ferritin has been purified from mycelium, sporangiophores, and spores of the fungus Phycomyces blakesleeanus. Its morphology in the electron microscope and the electron diffraction pattern of its iron core are almost indistinguishable from those of horse ferritin. Its protein/iron ratio is 4.7. It has a sedimentation coefficient of 55S and a buoyant density in CsCl density gradients of 1.82 g/[cubic]cm.\r\n\r\nIn the cytoplasm of Phycomyces, ferritin is located on the surface of lipid droplets (0.5-2.0[micron] diameter) where it forms crystalline monolayers which are conspicuous in electron micrographs of sporangiophore thin sections. The ferritin-lipid complex can be isolated from sporangiophore lysates. Treatment of the complex with detergent separates ferritin from the lipid.\r\n\r\nThe level of ferritin iron in Phycomyces is regulated by the iron level in the growth medium. A 20-40 fold increase in ferritin has been achieved by supplementing the medium with iron.\r\n\r\nFerritin and iron are concentrated in spores. In lysates of sporangiophores grown on low-iron growth medium about 80% of the iron is recoverable in the spores. At least one half of this spore iron can be extracted and is shown to be ferritin. The remaining iron is unextractable. There is no extractable low molecular weight iron in ungerminated spores.\r\n\r\nWhen ferritin is present in \"limiting\" amounts in spores, it disappears rapidly upon germination and simultaneously a low molecular weight form of iron becomes extractable. When present in large amounts in spores, ferritin does not disappear following germination although low molecular weight iron does become extractable. These results suggest that ferritin in Phycomyces spores functions as a source of iron in the early stages of germination.\r\n\r\nBecause the initial intracellular ferritin concentration and the external iron concentration of the germination medium can be varied, Phycomyces spores are potentially a very useful system in which to study the mechanism and control of ferritin iron metabolism." }, { "name": "Eakin, Richard Timothy", "degree": "PhD", "year": "1968", "title": "Mitochondrial Oxidase Systems in Neurospora", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:11042016-151827606", "creators": [ { "name": { "family": "Eakin", "given": "Richard Timothy" }, "id": "Eakin-Richard-Timothy", "display_name": "Eakin, Richard Timothy" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/YPY7-V492", "abstract": "Mitochondrial oxidase systems of Neurospora crassa were investigated\r\nwith respect to their mechanisms of electron transport and their\r\nrelationships to respiratory metabolism.
\r\n\r\nA mitochondrial oxidase system able to utilize dihydroorotic acid\r\nas a primary substrate was found and characterized. Another substrate\r\nfor this system was isolated from yeast extract and identified as 5-N-methylformamido-L-dihydroorotic acid. This system was found to occur\r\nin both wildtype and poky, a respiratory mutant. No linkage to the\r\ncytochrome chain or to oxidative phosphorylation could be detected.
\r\n\r\nA succinate oxidase system and NADH oxidase system independent of\r\ncytochromes b,a, and a3 were found to occur in both wildtype and in\r\nthe poky mutant. The system was partially characterized using the\r\npoky mutant, which is deficient in cytochromes b,a and a3.\r\nIt was found that this oxidase system was a part of respiratory metabolism and\r\nwas linked to oxidative phosphorylation.
\r\n\r\nMechanisms of electron transport in these oxidase systems are discussed\r\nand models presented.
\r\n\r\nPossible biological origins and the biological significance of\r\n5 N-methylformamidodihydroorotic acid are also discussed.
\r\n\r\nThe development of poky and wildtype mitochondria during the growth\r\ncycle were studied and compared. Differences in morphology were detected\r\nusing electron microscopy and differences in cytochrome content were\r\ndetected by absorption spectroscopy. The relationships between the oxidase\r\nsystems and mitochondrial development are discussed.
\r\n\r\n" }, { "name": "Fambrough, Douglas McIntosh, Jr.", "degree": "PhD", "year": "1968", "title": "Studies on Plant and Animal Histones", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechTHESIS:04262018-155935430", "creators": [ { "name": { "family": "Fambrough", "given": "Douglas McIntosh, Jr." }, "id": "Fambrough-Douglas-McIntosh-Jr", "display_name": "Fambrough, Douglas McIntosh, Jr." } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/C95J-PM40", "abstract": "The histones of the pea plant, Pisum sativum L., and of calf thymus have been fractionated and further characterized in order to determine the extent of heterogeneity and the main chemical features of these basic nuclear proteins. Histones were fractionated by chromatography on Amberlite CG-50 and by preparative disc electrophoresis. The resulting highly purified histone fractions were further characterized by analytical disc electrophoresis, amino acid analysis, N-terminal and C-terminal analyses, and the preparation of tryptic peptide maps. Calf thymus histones Ia, Ib, IIb1, III and IV (f1, f1, f2a2, f3, and f2a1 in the nomenclature of Johns, Phillips and Butler) and pea bud histones Ib, IIb, III and IV were obtained as electrophoretically pure components and each appears to be a single molecular species on the basis of N-terminal and C-terminal analysis and the number of tryptic peptides. The total number of major histones in calf thymus appears to be six, in pea bud, \r\neight. The apparent heterogeneity of calf thymus histones demonstrated by disc electrophoresis is largely due to the formation of histone III complexes by disulfide bridges between histone III monomers. While calf thymus histone III contains two cysteines per molecule pea bud histone III contains but one and thus can form only dimers.
\r\n\r\nFor each calf thymus histone there appears to be an homologous pea bud histone. It is proposed that the homologous pea and calf histones are related by evolution and perform identical biological functions. This hypothesis is based upon remarkable similarities in chromatographic and electrophoretic behavior, amino acid compositions, terminal amino acids, and in some cases even peptide maps of corresponding pea and calf histones. Peptide maps of the arginine-rich histone III contain 29 soluble peptides of which 26 are common to calf and pea; maps of histone IV contain 32 peptides of which 27 are common to calf and pea.
\r\n\r\nBy chromatography and electrophoresis the histones of various pea tissues are qualitatively identical to those of pea bud. There are, however, quantitative differences and these have been accurately measured by a method of quantitative analytical disc electrophoresis. Young pea cotyledons contain only about a third as much lysine-rich histone as do mature cotyledons. Exploratory experiments on the synthesis of histone in pea cotyledons as a function of development and in relation to other macromolecular parameters are described in an appendix.
\r\n\r\nThe dissociation of histones from pea bud nucleohistone by NaCl was studied, employing quantitative disc electrophoresis. Histone I (lysine-rich) is selectively dissociated by 0.5 M NaCl and the remaining histones are non-selectively dissociated primarily over the range 0.5 - 1.5 M NaCl. These data are compared with data for the dissociation of calf thymus histones from nucleohistone by NaCl and the general similarities are noted.
\r\n\r\n\r\n" }, { "name": "Gilbert, David Scott", "degree": "PhD", "year": "1968", "title": "Visual Acuity and Eye Movements", "advisor": "Fender, Derek H.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04242018-112335463", "creators": [ { "name": { "family": "Gilbert", "given": "David Scott" }, "id": "Gilbert-David-Scott", "display_name": "Gilbert, David Scott" } ], "advisors": [ { "name": { "family": "Fender", "given": "Derek H." }, "id": "Fender-D-H", "role": "advisor", "display_name": "Fender, Derek H." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "neurobio" ], "doi": "10.7907/EYE4-WX79", "abstract": "Several longstanding theories and some recently published\r\nexperimental evidence support the hypothesis that eye movements\r\nserve to improve acuity. By measuring eye movements during a\r\nsimple acuity task, and during a control non-acuity task, we have\r\nshown that certain patterns of eye movement are characteristic of \r\nacuity tasks. Similarly, specific patterns of eye movement are\r\ngenerated during spatial localization tasks. These observations \r\nprovide circumstantial evidence for the existence of mechanisms \r\nby which eye movements mediate acuity and spatial localization \r\ninformation.
\r\n\r\nThrough a comparison of acuity for stabilized retinal images \r\nwith acuity for normal retinal images we have found that eye movements \r\nimprove acuity very slightly at most, and that even this small \r\nimprovement may be adequately accounted for by the residual fade \u00ad \r\nout effects commonly observed during prolonged viewing of stabilized \r\nimages. Measurement of distance and angle estimation ability in\r\nboth normal and stabilized vision reveals much the same result. \r\nStabilization diminishes the accuracy of these estimates only slightly, \r\nas might be expected from the persistent fade effects observed during \r\nthe stabilized trials. Residual retinal image movement in the \r\nstabilized trials was less than approximately 3 min arc. If such \r\nacuity improving mechanisms exist, they either operate on very\r\nsmall retinal image movements (less than 3 min arc), or they\r\nimprove acuity only slightly (e.g., by less than 0.1 log unit in sine \r\nwave grating contrast sensitivity). Thus eye movements serve to \r\nsustain all sensory visual inflow by countering the slow process of \r\nfading of a stabilized image. They do not, however, play a vital role \r\nin the much more rapid processes which determine visual acuity as \r\nwell as distance and angle estimation ability.
\r\n\r\n" }, { "name": "Goldstein, Stuart Frederick", "degree": "PhD", "year": "1968", "title": "Local Activation and Inactivation Experiments of Flagella", "advisor": "Brokaw, Charles J.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05082018-134636609", "creators": [ { "name": { "family": "Goldstein", "given": "Stuart Frederick" }, "id": "Goldstein-Stuart-Frederick", "display_name": "Goldstein, Stuart Frederick" } ], "advisors": [ { "name": { "family": "Brokaw", "given": "Charles J." }, "id": "Brokaw-C-J", "role": "advisor", "display_name": "Brokaw, Charles J." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/EGRE-9774", "abstract": "The mechanisms of flagellar movement were investigated by studying the ability of various regions of a flagellum to initiate bends, and to propagate bends independently of activities in other regions. Two experimental approaches were used: the establishment of an artificial gradient of ATP along a flagellum, and the inactivation of a small region of a flagellum by localized irradiation. Flagella of the spermatozoa of sea urchins and a few other marine invertebrates were used in this study. Glycerinated flagella were activated by ATP gradients established by means of diffusion from the tips of micropipettes. These gradients could be made broad enough to produce a gradual decrease in ATP concentration along the entire flagellum, or narrow enough to supply ATP to only part of the flagellum. Localized supply of ATP to regions of the flagell11m other than the basal end produced no beating. Beating properties along the flagellum appeared quite sensitive to ATP concentration at the basal end, but rather insensitive to ATP concentration at other points, and centering a gradient about points other than the basal end did not cause beating to start at those points.
\r\n\r\nSmall regions of flagella were irradiated at preselected phases of beating by means of a pulsed ruby laser microbeam. Multiple-exposure dark field photographs of the spermatozoa were taken immediately before and after irradiation. The region of a flagellum between the head and the irradiated point continued beating for at least a few beats if that region was at least a quarter of the length of the tail, and stopped immediately if it was shorter. Bends which were already established beyond the irradiated point continued to the tip, but showed a variety of changes in their properties. No new bends were formed in this region. Irradiation within a bent region caused that region to straighten immediately.
\r\n\r\nThese experiments indicate that the basal end of the flagellum is necessary for bend initiation, and largely responsible for the determination of wave properties. Although a portion of a flagellum can independently propagate established bends, the bend properties at any point are influenced by activities along the rest of the flagellum.
\r\n\r\nThe relevance of these observations to current models of flagellar beating is discussed.
\r\n" }, { "name": "Harding, Roy Woodrow, Jr.", "degree": "PhD", "year": "1968", "title": "Carotenoid Biosynthesis in Neurospora Crassa", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04202018-161806669", "creators": [ { "name": { "family": "Harding", "given": "Roy Woodrow, Jr." }, "id": "Harding-Roy-Woodrow", "display_name": "Harding, Roy Woodrow, Jr." } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/FY91-G279", "abstract": "As a means of inducing a reproducible amount of carotenoid synthesis in large quantities of mycelium, a new illumination-incubation technique was developed and is described. In addition, procedures are presented for obtaining phytoene and each of the carotenoid pigments spectroscopically and radiochemically pure when 2-C14_ mevalonate is used as a labelled precursor. The criteria of purity which were employed are discussed.
\r\n\r\nUsing these and other basic procedures, data have been obtained which were all consistent with one particular pathway of carotenoid synthesis. The pathway involves conversion of phytoene to the carotenoid pigments by a series of sequential reactions which include dehydrogenation, cyclization, and oxidative cleavage of a C=C bond.
\r\n\r\nThe proposed pathway is supported by labelling data, time course measurements of each carotenoid in mycelium illuminated and incubated under various physiological conditions, studies with color mutants, and the chemical structures of the compounds involved. In addition, the carotenoid beta-zeacarotene, which had not been previously reported in Neurospora, was shown to be present in mycelium under certain conditions, and this fact added additional support to part of the proposed pathway.
\r\n\r\nSome information was also obtained about the mechanism of induction of carotenoid synthesis in Neurospora by light. It was confirmed that under well aerated conditions the initial light reaction occurs very rapidly (30 seconds or less). Using cycloheximide, an inhibitor of protein synthesis, evidence was obtained which strongly suggests that one effect of light is to induce the de novo synthesis of an enzyme (or enzymes) which is required for the synthesis of carotenoids and is absent in dark-grown mycelium. Possible mechanisms for such an induction by light are discussed.
" }, { "name": "Hood, Leroy Edward", "degree": "PhD", "year": "1968", "title": "Immunoglobulins: Structure, Genetics and Evolution", "advisor": "Dreyer, William J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-09162004-110314", "creators": [ { "name": { "family": "Hood", "given": "Leroy Edward" }, "id": "Hood-Leroy-Edward", "orcid": "0000-0001-7158-3678", "display_name": "Hood, Leroy Edward" } ], "advisors": [ { "name": { "family": "Dreyer", "given": "William J." }, "id": "Dreyer-W-J", "role": "advisor", "display_name": "Dreyer, William J." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/3ZM8-A807", "abstract": "The immune system is capable of generating an immense number of different antibody molecules. The nature of the genetic machinery responsible for this diversity has been studied by selective amino acid sequence analysis of homogeneous immunoglobulin light chains (derived from myeloma tumors). The evolution of the immune system has also been examined through chemical studies of normal pooled light chains derived from various mammalian and avian species. These studies place constraints on proposed genetic mechanisms for antibody diversity. The theories, the structural constraints, and the evolutionary implications of these observations are discussed.\r\n" }, { "name": "Huberman, Joel Anthony", "degree": "PhD", "year": "1968", "title": "Studies on the Structure and Function of Mammalian Chromosomes", "advisor": "Attardi, Giuseppe", "url": "https://resolver.caltech.edu/CaltechTHESIS:04232018-145543593", "creators": [ { "name": { "family": "Huberman", "given": "Joel Anthony" }, "id": "Huberman-Joel-Anthony", "display_name": "Huberman, Joel Anthony" } ], "advisors": [ { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "advisor", "display_name": "Attardi, Giuseppe" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/1XDD-6803", "abstract": "At levels of organization between the Watson-Crick model of DNA \r\non the one hand, and the microscopically visible mitotic or meiotic \r\nchromosome on the other, very little is known about the structure or \r\nfunction of chromosomes in eukaryotic organisms. The studies reported \r\nin this thesis were an attempt to learn more about the arrangement\r\nof certain DNA sequences in mammalian chromosomes, about the size of \r\nthe DNA molecules in such chromosomes, and about the replication of \r\nthese DNA molecules.
\r\n\r\n\r\nPart I contains the results of experiments designed to determine\r\nthe distribution of the hundreds of genes (DNA sequences) for ribosomal \r\nRNA among the chromosomes of HeLa cells. In the course of these experi\u00adments, \r\nmethods were developed for isolating metaphase chromosomes on\r\na large scale from HeLa cells and for fractionating them on the basis \r\nof sedimentation velocity. Hybridization experiments between ribo\u00adsomal \r\nRNA and DNA from the various fractions of isolated chromosomes \r\nshowed that the genes for ribosomal RNA are confined entirely to small \r\nHeLa cell chromosomes.
\r\n\r\n\r\nIn Part II are reported the results of autoradiographic experi\u00adments \r\nintended to help determine the size and manner of replication of \r\nthe DNA in mammalian chromosomes. All the experiments described in \r\nPart II are the result of collaboration with Dr. Arthur D. Riggs. We\r\nused a modification (by Dr. Riggs) of the technique for autoradiography \r\nof individual DNA molecules which had been developed by Cairns (J. Mol.\r\nBiol. 6, 208 (1963)). Our application of this technique to the DNA\r\nof Chinese hamster cells demonstrated the presence in Chinese hamster \r\ncell chromosomes of DNA fibers up to 1,800 \u00b5 long. Subsequent pulse\u00ad \r\nlabeling studies showed that such long fibers are divided into many \r\nshorter replication units, and that DNA replication probably starts\r\nin the interior of each unit and then proceeds outward in both direc\u00adtions, \r\nat fork-like growing points, to the ends of the unit.
\r\n" }, { "name": "Huskey, Robert John", "degree": "PhD", "year": "1968", "title": "Isolation and Characterization of Deletion Mutants of Bacteriophage Lambda", "advisor": "Edgar, Robert S.", "url": "https://resolver.caltech.edu/CaltechETD:etd-08102006-130739", "creators": [ { "name": { "family": "Huskey", "given": "Robert John" }, "id": "Huskey-Robert-John", "display_name": "Huskey, Robert John" } ], "advisors": [ { "name": { "family": "Edgar", "given": "Robert S." }, "id": "Edgar-R-S", "role": "advisor", "display_name": "Edgar, Robert S." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/436X-JK20", "abstract": "A method for the isolation of deletion mutants of lambda is reported utilizing thermal inactivation as a selective enrichment technique. Thermal inactivation of lambda is shown to depend upon the amount of DNA in the phage, the temperature of inactivation and the ionic make-up of the medium. A phenotypically heat resistant, less dense class of phage is reported which, upon growth, returns to the density and heat characteristics of wild type phage.\r\n\r\nThe several hundred deletion mutants isolated have many properties in common with the lambda b2 mutant. All mutants are of lambda immunity and host restriction type. In these mutants, the amount of DNA deleted, calculated from their density, varies from 10 to 30%. All of the mutants like b2 form abortive lysogens. Preliminary experiments indicate that the mutants retain the recombination function of lambda. Some of the deletion mutants show reduced recombination for the region deleted, the middle segment of the genome.\r\n\r\nHowever, a number of the deletion mutants exhibit abnormally high recombination for the middle segment of the genome and in one of these mutants, lambda b130, a physical discontinuity in the DNA of the phage occurs at or near the locus which manifests high recombination. This discontinuity resembles in structure the cohesive ends of lambda DNA.\r\n\r\nIn an attempt to explain the properties and bizarre structure of the chromosome of b130, a model for lambda integration and excision is proposed. As a result of this model, the high recombination of certain lambda deletion mutants is thought to be due to the inclusion of a small region of host DNA into the middle segment of the DNA of these mutants. It is proposed that this included region resembles closely the cohesive ends of lambda leading to the production of a new set of cohesive ends and localized high recombination. The model introduces several new notions concerning the integration of lambda into the chromosome which are experimentally testable." }, { "name": "Kiger, John Andrew", "degree": "PhD", "year": "1968", "title": "Part I. The Transcription of Simple and Complex DNAs by the RNA Polymerase of Escherichia coli. Part II. The Structure and Replication of Intracellular Bacteriophage Lambda DNA", "advisor": "Sinsheimer, Robert L.", "url": "https://resolver.caltech.edu/CaltechETD:etd-09242002-102646", "creators": [ { "name": { "family": "Kiger", "given": "John Andrew" }, "id": "Kiger-John-Andrew", "display_name": "Kiger, John Andrew" } ], "advisors": [ { "name": { "family": "Sinsheimer", "given": "Robert L." }, "id": "Sinsheimer-R-L", "role": "advisor", "display_name": "Sinsheimer, Robert L." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/1HYJ-RC42", "abstract": "Part I of this thesis is a study of the in vitro transcription of DNA by Escherichia coli RNA polymerase. Experiments on the transcription of phage lambda DNA form Chapter 1. The transcription of lambda DNA is partially asymmetric. When symmetric transcription occurs, RNA so transcribed is found in a double-stranded form which is resistant to digestion by ribonuclease. The temperature of synthesis and the presence of Mn\u207a\u207a in the synthetic mixture affect the frequency of symmetric transcription. RNA-DNA hybridization experiments indicate that no more than 50% of the lambda genome (25% of the DNA) is transcribed in vitro.
\r\n\r\nExperiments on the transcription of some eucaryote DNAs are presented in Chapter 2. Only a small fraction (15-20%) of the RNA transcribed from these DNAs is observed to form hybrid with complementary DNA under a variety of conditions. That RNA which does form hybrid is transcribed asymmetrically from a limited portion (about 10%) of the DNA. Evidence is presented for heterogeneity in the rate at which various RNA and DNA sequences form hybrid. This heterogeneity is believed to be of the same nature as the heterogeneity revealed by renaturation studies of eucaryote DNA.
\r\n\r\nPart II of this thesis is a study of the structure and replication of intracellular phage lambda DNA. Studies on purified non-replicating intracellular lambda DNA are presented in Chapter 1. Three species of lambda DNA are present following infection of immune bacteria by lambda phage: a closed-circular molecule, component I; an open-circular molecule containing one or more single-strand breaks, component II; and linear phage DNA, component III. The physical and infective properties of these molecules are studied. Component I in the native or denatured state is found to be almost equally infective to spheroplasts. Component II, however, is infective in the native state and shows a large increase in infectivity upon denaturation. This is due to the liberation of single-stranded rings which are more efficient in infecting spheroplasts than are native DNA molecules. Component III decreases greatly in infectivity following denaturation. Evidence is presented from sedimentation studies of components I, II, and III which suggests that the pitch of the Watson-Crick helix is variable in solution.
\r\n\r\nExperiments on the fractionation of replicating intracellular lambda DNA by chromatography on benzoylated-naphthoylated DEAE cellulose (BNC) are presented in Chapter 2. Intracellular lambda DNA is labeled following induction and mitomycin C treatment (to suppress host DNA synthesis) of lysogens and the purified DNA is adsorbed to BNC. Components II and III along with native phage DNA are eluted from BNC by a gradient of 0.3-1.0M\u0332 NaCl. A subsequent gradient of 0-2% caffeine elutes a heterogeneous species of intracellular DNA. This species is rapidly labeled by short pulses of \u00b3H-thymidine and is virtually non-infective in the native state to spheroplasts. Denaturation of this species renders it very infective to spheroplasts suggesting that it contains single-strand rings. Analysis of the single-strand composition of this species by alkaline sedimentation reveals material sedimenting up to 1.5 times the rate of single-strand phage DNA. A model for DNA replication, involving initiation of one daughter strand by covalent addition to the 3'-OH of the identical parent strand, is presented based on the single strand composition of the DNA eluted from BNC by caffeine.
\r\n\r\nSupporting data from pulse and pulse-chase experiments are presented in Chapter 3. Approximately half of the label incorporated in very short pulses into material sedimenting at neutral pH as intracellular lambda DNA sediments in alkali faster than phage DNA single strands.
\r\n\r\nVery short pulses have revealed the presence of a small, rapidly labeled component in induced cells which appears to be DNA and sediments at about 10S at neutral and alkaline pH. The nature of this component is obscure at present.
" }, { "name": "King, Jonathan Alan", "degree": "PhD", "year": "1968", "title": "Steps in the Assembly of Bacteriophage T4", "advisor": "Edgar, Robert S.", "url": "https://resolver.caltech.edu/CaltechETD:etd-09242002-105334", "creators": [ { "name": { "family": "King", "given": "Jonathan Alan" }, "id": "King-Jonathan-Alan", "display_name": "King, Jonathan Alan" } ], "advisors": [ { "name": { "family": "Edgar", "given": "Robert S." }, "id": "Edgar-R-S", "role": "advisor", "display_name": "Edgar, Robert S." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/X9HX-V431", "abstract": "The formation of bacteriophage T4 has been studied by examining the phage components accumulating in cells infected - under restrictive conditions - with mutants blocked at different stages of the assembly process. Structural intermediates in the pathway for tail and tail fiber assembly have been partially characterized by serology, electron microscopy, sedimentation, and in vitro complementation behavior.\r\n\r\nTail assembly: After the baseplate (80S) is completed, the core forms on the baseplate. The functions of genes 19, 48, and 54 are required for this conversion. The gene 18 product, a major sheath structural subunit, polymerizes on the core-baseplate (80S) and then the 3 and 15 gene products fix the sheath subunits in the polymerized form, yielding a sheathed tail (130S).\r\n\r\nTail fiber assembly: The tail fiber (10S) is made up of 8S and 9S components. The 8S component is the product of genes 38, 37, 36 and 35. The 9S component is the product of gene 34. Two precursors to the 8S component, both also 8S, are identified. A pathway for the steps in tail fiber assembly is proposed.\r\n\r\nThe tail fibers attach to the tail only after the head has joined with the tail. Particles which have not been acted upon by the gene 11 or 12 product absorb to bacteria but do not kill them.\r\n\r\nElectron microscopic observations on the state of phage heads in mutant lysates are also presented. Mutations in three genes result in the accumulation of head membranes empty of DNA.\r\n\r\nAll the evidence supports the view that phage heads, tails, and tail fibers are formed independently of each other." }, { "name": "Pall, Martin Lawrence", "degree": "PhD", "year": "1968", "title": "Part I. Tyrosinase Induction by Antimetabolites in Neurospora. Part II. Amino Acid Transport in Neurospora", "advisor": "Horowitz, Norman Harold", "url": "https://resolver.caltech.edu/CaltechTHESIS:04202018-102319427", "creators": [ { "name": { "family": "Pall", "given": "Martin Lawrence" }, "id": "Pall-Martin-Lawrence", "display_name": "Pall, Martin Lawrence" } ], "advisors": [ { "name": { "family": "Horowitz", "given": "Norman Harold" }, "id": "Horowitz-N-H", "role": "advisor", "display_name": "Horowitz, Norman Harold" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/3025-N696", "abstract": "Part I.
\r\n\r\nA technique for inducing very high levels of tyrosinase activity \r\nwith various antimetabol ites is described. A modification of this tech\u00adnique \r\nwas used for studying tyrosinase induction in Neurospora over\r\nperiods of a few hours. An amount of the antimetabolite inducer \r\nsufficient to induce the enzyme is rapidly taken up into the mycelium. \r\nFollowing uptake, however, there is a lag period of about two hours \r\nbefore tyrosi nase is synthesized. During the lag period, some active, \r\nenergy requiring process prepares the mycelium for tyrosinase synthesis. \r\nRapid synthesis of tyrosinase then ensues. High concentrations of \r\ncycloheximide, an inhibitor of protein synthesis, inhibit the develop\u00adment \r\nof any further enzyme activity when added to inducing cultures, \r\nindicating that the synthesis of tyrosinase is de novo.
\r\n\r\nLow concentrations of cycloheximide which had been previously \r\nshown to induce tyrosinase, partially inhibit general protein synthesis.\r\nEthionine and parafluorophenyl alanine appear to induce the enzyme \r\nby being incorporated into proteins in place of methionine and phenylala\u00adnine, \r\nthus lowering the functional activity of newly synthesized\r\nproteins. The partial inhibition of the synthesis of functional \r\nproteins, then, is sufficient, in some way, to induce tyrosinase.
\r\n\r\nPart II.
\r\n\r\nKinetic studies have revealed the existence of two transport sys\u00adtems \r\nfor amino acids in Neurospora. Transport system I corresponds to \r\na system previously studied by Wiley and Matchett (24). Its activity \r\nis specifically missing in mtr mutant cultures previously described \r\nby Lester (26) and Stadler (25). It is capable of transporting most\r\nneutral L-amino acids. Amino acid transport system II has not been described \r\npreviously. It has an affinity for a wide variety of amino acids. It \r\ntransports amino acids with hydrophobic and hydrophilic side\r\nchains, both basic and neutral amino acids, and D- as well as L-amino \r\nacids. Transport system II has an affinity for both \u03b2- and \u03b1-amino \r\nacids.
\r\n\r\nTransport system I has high activity in young, rapidly growing cultures. \r\nTransport system II has little or no activity in young cultures. In older, \r\ncarbon-starved cultures, however, it is more active than transport system I. \r\nThis, together with the high affinities it shows\r\nfor many amino acids, suggests that amino acid transport \r\nsystem II serves a scavenger function, removing from the \r\nmedium traces of exogen\u00adous amino acids.
\r\n\r\n\r\n\r\n" }, { "name": "Radloff, Roger James", "degree": "PhD", "year": "1968", "title": "Structure and Properties of Closed Circular DNA", "advisor": "Vinograd, Jerome Rubin", "url": "https://resolver.caltech.edu/CaltechTHESIS:05082018-082914989", "creators": [ { "name": { "family": "Radloff", "given": "Roger James" }, "id": "Radloff-Roger-James", "display_name": "Radloff, Roger James" } ], "advisors": [ { "name": { "family": "Vinograd", "given": "Jerome Rubin" }, "id": "Vinograd-J-R", "role": "advisor", "display_name": "Vinograd, Jerome Rubin" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/rr5y-yc64", "abstract": "This thesis is concerned with the structure and properties of closed circular DNA. Part I of the thesis reports on experiments performed in this laboratory by the author and others to determine the structure of polyoma DNA component II. The following results were obtained: (1) Polyoma II is a ring-shaped duplex DNA molecule. (2) It is generated by introducing one single strand scission in polyoma I by the action of pancreatic DNAase or chemical reducing agents. (3) The sedimentation coefficient of polyoma II is insensitive to several single strand scissions. (4) Polyoma II, when not excessively attacked by pancreatic DNAase or chemical reducing agents, is infective. A publication is enclosed.
\r\n\r\nThe renaturation products of polynucleotide single strands obtained from nicked polyoma DNA were examined in Part II of the thesis. It was found that complementary, linear, single-stranded DNA can renature extensively. Preparations of single-stranded DNA containing more than 50% circular single strands renature to form two bands of DNA in CsCl buoyant density experiments. The buoyant density of one band is approximately that of native polyoma DNA and the buoyant density of the other is characteristic of denatured DNA. No evidence is found for any renaturation of complementary, circular, single-stranded DNA
\r\n\r\nPart III of the thesis presents evidence indicating that nonnucleotide linkers are absent in both polyoma and \u00f8X174 DNA. This evidence was obtained by examining the exonuclease I digestion products of denatured1 pancreatic DNAase treated polyoma and \u00f8X174 DNA in alkaline CsCl velocity gradients.
\r\n\r\nPart IV of the thesis contains a publication describing a dye-buoyant density method for the isolation and detection of closed circular DNA. The method is based on the reduced binding of the intercalating dye, ethidium bromide, by closed circular DNA. In an application of the method, it was found that HeLa cells contain, in addition to closed circular mitochondrial DNA of mean length 4.81 microns, a heterogeneous group of smaller DNA molecules which vary in size from 0.2 to 3.5 microns, and a paucidisperse group of multiples of the mitochondrial length. In addition, methodological results and data not presented in the publication are presented and discussed.
\r\n" }, { "name": "Sadgopal, Anil", "degree": "PhD", "year": "1968", "title": "Part I. Synchronization of the Cell Division Cycle of HeLa Cells in Suspension Cultures. Part II. Studies on Chromosomal Proteins of HeLa Cells During the Cell Division Cycle", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechTHESIS:01042016-083704592", "creators": [ { "name": { "family": "Sadgopal", "given": "Anil" }, "id": "Sadgopal-Anil", "display_name": "Sadgopal, Anil" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/5GTY-NC55", "abstract": "Part I
\r\n\r\nThese studies investigate the potential of single and double treatments with either 5-fluorodeoxyuridine of excess thymidine to induce cell division synchrony in suspension cultures of HeLa cells. The patterns of nucleic acid synthesis and cell proliferation have been analyzed in cultures thus synchronized. Several changes in cell population during long incubation with 5-fluorodeoxyuridine or excess thymidine are also described. These results are subjected to detailed evaluation in terms of the degree and quality of synchrony finally achieved.
\r\n\r\nPart II
\r\n\r\nHistones and non-histone proteins associated with interphase and metaphase chromosomes of HeLa cells have been qualitatively and quantitatively analyzed. Histones were fractionated by chromatography on Amberlite CG-50 and further characterized by analytical disc electrophoresis and amino acid analysis of each chromatographic fraction. It is concluded that histones of HeLa cells are comprised of only a small number of major components and that these components are homologous to those of other higher organisms. Of all the histones, arginine-rich histone III alone contains cysteine and can polymerize through formation of intermolecular disulfide bridges between histone III monomers.
\r\n\r\nA detailed comparison by chromatography and disc electrophoresis established that interphase and metaphase histones are made up of similar components. However, certain quantitative differences in proportions of different histones of interphase and metaphase cells are reported. Indirect evidence indicates that a certain proportion of metaphase histone III is polymerized through intermolecular disulfide links, whereas interphase histone III occurs mainly in the monomeric form.
\r\n\r\nMetaphase chromosomes are associated with an additional acid-soluble protein fraction which is absent from interphase chromosomes. All of these additional acid-soluble proteins of metaphase chromosomes are shown to be non-histones and it is concluded that the histone/DNA ratio is identical in interphase and metaphase chromosomes. The bulk of acid-soluble non-histone proteins of metaphase chromosomes were found to be polymerized through disulfide bridges; corresponding interphase non-histone proteins displayed no evidence of similar polymerization.
\r\n\r\nThe factors responsible for the condensed configuration and metabolic inactivity of metaphase chromosomes are discussed in light of these findings.
\r\n\r\nThe relationship between histone and DNA synthesis in nondividing differentiated chicken erythrocyte cells and in rapidly dividing undifferentiated HeLa cells is also investigated. Of all the histones, only arginine-rich histones are synthesized in mature erythrocytes. Histone synthesis in HeLa cells was studied in both unsynchronized and synchronized cultures. In HeLa cells, only part of the synthesis of all histone fractions is dependent on concurrent DNA synthesis, whereas all histones are synthesized in varying degrees even in the absence of DNA synthesis.
\r\n" }, { "name": "Scott, William Addison", "degree": "PhD", "year": "1968", "title": "Cytochrome c Synthesis in Neurospora crassa", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechETD:etd-08042006-160643", "creators": [ { "name": { "family": "Scott", "given": "William Addison" }, "id": "Scott-William-Addison", "display_name": "Scott, William Addison" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/JZZG-RZ19", "abstract": "Part A: Cytochrome c synthesis was studied in the respiration deficient poky mutant of Neurospora crassa. Two cytochromes c, CI and CII, were detected. The two proteins were shown to differ by a secondary modification at residue 72. A precursor-product relationship was established; CII (lysine) is converted to CI (Lys-X). Lys-X was found to be a lysine derivative although the exact chemical nature of this residue is unknown.\r\n\r\nThe kinetics of CI and CII synthesis in poky were compared to those of wild type and the respiration deficient mutants: mi-3, cyt-l, and po-f. In comparison to wild type, poky accumulates CII during the early stages of growth and has a delayed synthesis of CI. These events in the other mutants are normal even though they accumulate cytochrome c as does poky.\r\n\r\nOnly CI was recovered from isolated poky mitochondria even though the whole cell contained both cytochromes c. In young poky over half of the cytochrome c is unbound. The conversion of CII to CI is thought to reflect a binding of cytochrome c to mitochondria since this event parallels the change of the poky phenotype to a more normal state.\r\n\r\nPart B: When wild type Neurospora is grown in high concentrations of chloramphenicol (4 mg./ml.), it exhibits a phenotype similar to that of the maternally inherited poky mutation. Both have no cytochromes a and b but have an excess of cytochrome c. Spontaneously occurring strains partially resistant to the drug were isolated. The resistance is controlled by more than one nuclear gene. The implications of these results are discussed in terms of the primary effect of the poky mutation and the differential inhibition of mitochondrial protein synthesis by chloramphenicol in cells of higher organisms." }, { "name": "Williams, Larry Gale", "degree": "PhD", "year": "1968", "title": "Thymidine Metabolism in Neurospora crassa", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04192018-163310702", "creators": [ { "name": { "family": "Williams", "given": "Larry Gale" }, "id": "Williams-Larry-Gale", "display_name": "Williams, Larry Gale" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/ZQ64-CE98", "abstract": "The metabolism of thymidine by Neurospora crassa was investigated \r\nafter radioactive labeling experiments showed that thymidine is not \r\nspecifically incorporated into the DNA of Neurospora; a finding in\r\ncontrast to the results of labeled thymidine experiments with many \r\nother organisms. Labeling experiments in which 2-C14 deoxyuridine, \r\n2-C14 thymidine and 2-C14 thymine were administered to Neurospora\r\nrevealed that in each case ninety per cent of the nucleic acid label \r\nwas in the RNA fraction. When thymidine methyl-H3 was administered \r\nno label was found in either the RNA or DNA. This and other evidence\r\nwas taken as proof that Neurospora lacks phosphorylating enzymes for \r\ndeoxyribose pyrimidine nucleosides but does possess enzymatic reactions \r\nby which these same compounds can be converted to RNA precursors.
\r\n\r\nThree mutants were isolated in which different synthetic steps \r\nare blocked in the pathway that converts the pyrimidine ring of \r\nthymidine to an RNA precursor. Evidence from genetic studies, nutri\u00adtional \r\ntests and accumulation studies with the three mutant strains \r\nshows the pathway to proceed as follows: thymidine \u2192 thymine \u2192 5-hydroxymethyluracil \u2192 \r\n5-formyluracil \u2192 uracil \u2192 uridylic acid. A mutant strain in which the \r\nthymidine to thymine conversion is pre\u00advented is also unable to utilize \r\ndeoxyuridine and deoxycytidine as pyrimidine sources and suggests a \r\ndefective deoxyribose pyrimidine nucleosidase enzyme. A second mutation \r\nblocks the pathway at the 5-hydroxymethyluracil to 5-formyluracil step \r\nand causes the accumulation of thymine in the growth medium. The third \r\nmutation prevents the utilization of uracil and the compounds \r\npreceding it in the pathway.
\r\n\r\nThree mutants were isolated in which the pyrimidine transport \r\nsystem was affected. One of these mutants could utilize the pyrimidine \r\nnucleosides (cytidine, uridine, deoxyuridine and thymidine) but could \r\nuse none of the free bases (thymine, 5-hydroxymethy luracil, 5-formyl\u00ad-uracil \r\nand uracil). A second mutant could utilize the free bases but not the \r\nnucleosides. These results can be most simply interpreted in \r\nterms of the first mutation blocking a transport system specific \r\nfor pyrimidine bases, and the second mutation blocking a system specific \r\nfor the transport of pyrimidine nucleosides. A third mutation pre\u00advented \r\nthe utilization of both pyrimidine bases and nucleosides in a \r\nmedium containing ammonium salts but permitted their use in a medium \r\ncontaining nitrate salts.
\r\n\r\nA strain was isolated carrying a mutation which influences the \r\nregulation of the thymidine to RNA precursor pathway. This mutation \r\nallows the steps of the pathway that convert thymidine to uracil to\r\nfunction in germinating conidia. A second mutation was found which \r\nsuppresses the action of the first and restores the normal condition, \r\nthe absence of the thymidine to uracil conversion in germinating \r\nconidia.
\r\n\r\n" }, { "name": "Kabat, David", "degree": "PhD", "year": "1967", "title": "Part I. Protein Synthesis During Chicken Erythrocyte Differentiation. Part II. Studies of Ordered DNA Protein Fibers", "advisor": "Attardi, Giuseppe", "url": "https://resolver.caltech.edu/CaltechETD:etd-09202002-093411", "creators": [ { "name": { "family": "Kabat", "given": "David" }, "id": "Kabat-David", "display_name": "Kabat, David" } ], "advisors": [ { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "advisor", "display_name": "Attardi, Giuseppe" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/EPST-RQ80", "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\r\n\r\nPart I: Protein Synthesis During Chicken Erthrocytes Differentiation. \r\n\r\nIt was the major purpose of this research to study changes of protein synthesis during chicken erythrocyte differentiation.\r\n\r\nIn Chapter 1, erythrocytes from the blood of normal and anemic birds were fractionated by buoyant density centrifugation in bovine serum albumin gradients. It is shown that this procedure fractionates the erythroid cells according to their physiological maturity. Reduction of RNA synthesis, RNA content, and protein synthesis are shown to accompany cell maturation. Inhibition of RNA synthesis with actinomycin D does not affect hemoglobin synthesis in erythroid cells from anemic birds. The two hemoglobins, present within single chicken erythrocytes, appear to be synthesized in constant ratio throughout eiythropoiesis, suggesting that the factors involved (at the genetic and translation levels) in the regulation of these syntheses operate in a cordinate manner.\r\n\r\nIn Chapter 2, it is shown by peptide mapping that the two chicken hemoglobins contain many common amino acid sequences; their genes presumably arose by duplication of common ancestral genes. The site of hemglobin synthesis is the cytoplasmic polyribosome, as shown in part by the similar appearance of autoradiographic peptide maps of [[superscript 14]C] leucine-labeled purified hemoglobin and of [[superscript 14]C] leucine-labeled nascent polypeptides associated with the ribosomes. Labeled growing polypeptide chains, isolated from ribosomes following pulses with [[superscript 3]H] leucine, are shown to be heterogeneous in size, ranging from very small peptides up to complete globin chains. The kinetics of labeling the different sized polypeptides is in agreement with the well-established model of protein synthesis in which amino acids are sequentially polymerized from one end of the growing chains. At 37? in vitro, it requires roughly one minute to synthesize a complete globin chain (approximately 150 amino acids). It is also demonstrated that both histone and non-histone chromosomal proteins are synthesized in non-dividing avian erythrocytes. The histones synthesized are, selectively, the \"arginine-rich\" histones.\r\n\r\nIn Chapter 3, a study is made of the nuclear hemoglobin of chicken erythrocytes. One percent of the total cell hemoglobin remains associated with nuclei isolated in low ionic strength solutions, but this bound hemoglobin fraction can be extracted with 0.14 hi NaCl. A ubiquitous and widely-studied class of nuclear protein molecules from plant and animal cells (\"nuclear soluble proteins\") has similar nuclei-binding properties. It is demonstrated that the bound hemoglobin molecules consist exclusively of one of the two hemoglobin types, that they are complexed to the erythrocyte chromosomes, and that they co-sediment with the chromatin in sucrose gradients.\r\n\r\nPart II: Studies of Ordered DNA Protein Fibers. \r\n\r\nMixtures of-chicken hemoglobin and DNA or horse heart cytochrome c and DNA form highly-ordered and tightly-packed fibrous complexes only in the presence of divalent metal ions. Polarization and electron microscopy are used to demonstrate that the DNA molecules are strongly oriented parallel to the fiber axes. Polarized visible absorption spectra are used to determine the hemoprotein orientation in the fibers; spectra of several derivatives of horse hemoglobin crystals and also horse heart ferricytochrome c crystals are analyzed as controls. A model for the role of divalent metal ions in the tightly-packed nucleoprotein fibers is deduced from these experiments. The well-known strong stabilizing effects of divalent metal ions on tightly-packed ribosome and chromosome structures are also discussed.\r\n" }, { "name": "Kelly, Regis Baker", "degree": "PhD", "year": "1967", "title": "The Replication of Bacteriophage MS2", "advisor": "Sinsheimer, Robert L.", "url": "https://resolver.caltech.edu/CaltechETD:etd-09232002-160128", "creators": [ { "name": { "family": "Kelly", "given": "Regis Baker" }, "id": "Kelly-Regis-Baker", "display_name": "Kelly, Regis Baker" } ], "advisors": [ { "name": { "family": "Sinsheimer", "given": "Robert L." }, "id": "Sinsheimer-R-L", "role": "advisor", "display_name": "Sinsheimer, Robert L." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/ZBYQ-NQ66", "abstract": "The genetic information of the small bacteriophage MS2 is stored in a single-stranded RNA molecule. This thesis is an analysis of the mechanism whereby the MS2 genetic material is replicated.\r\n\r\nIn Part I, the presence of phage-specific double-stranded RNA in MS2-infected cells is demonstrated. The kinetics of conversion of the input parental strand to a duplex form, and of the formation of progeny duplexes, is also described.\r\n\r\nIn Part II, the replication of phage-specific RNA duplexes is studied by means of density, labeling and CsC1 equilibrium density sedimentation. Neither progeny nor parental duplexes are conserved, which is strong evidence that the duplex plays some role in replication.\r\n\r\nThe above experiments were performed on material isolated after ribonuclease digestion of the nucleic acids from infected cells. In Part III, two other procedures are described which permit identification of phage-specific replicative intermediates (RI) without use of ribonuclease: the host cell RNA synthesis can be preferentially blocked by use of the antibiotic, Actinomycin D; or RI can be separated from the other infected cell components by a centrifugal method based on studies of RNA denaturation with the organic solvent DMSO. These procedures lead to the conclusion that the true RI is a heterogeneous, approximately 15S component which is only partially double-stranded.\r\n\r\nIn Part IV, use is made of this partial double-stranded nature to purify and fractionate the RI on columns of benzoylated, naphthoylated DEAE cellulose, which seem to fractionate nucleic acids on the basis of secondary structure. From measurements of the ribonuclease-resistance and the buoyant densities of purified RI fractions it was concluded that the RI consists of a duplex of constant length to which is attached one single-stranded \"tail\" of length less than the viral RNA. The RI is only infective in the spheroplast assay when denatured.\r\n\r\nThis structure for the RI is interpreted to mean that the nascent strand is still attached after purification of the RI. It was therefore possible to determine the mode of replication by determining whether the \"tail\" consisted of the nascent strand (conservative replication) or the displaced portion of the viral strand (semi-conservative replication). Appropriate labeling experiments indicated that the tail could arise from either origin, equally often.\r\n\r\nIt was concluded that double-stranded RNA is involved in phage RNA replication and that replication is both conservative and semi-conservative, equally often" }, { "name": "Piatigorsky, Joram Paul", "degree": "PhD", "year": "1967", "title": "Studies on Nucleoside and Amino Acid Uptake and on RNA and Protein Synthesis by Growing Oocytes, Unfertilized and Fertilized Sea\r\nUrchin Eggs", "advisor": "Tyler, Albert; Owen, Ray David", "url": "https://resolver.caltech.edu/CaltechETD:etd-10032002-110729", "creators": [ { "name": { "family": "Piatigorsky", "given": "Joram Paul" }, "id": "Piatigorsky-Joram-Paul", "display_name": "Piatigorsky, Joram Paul" } ], "advisors": [ { "name": { "family": "Tyler", "given": "Albert" }, "id": "Tyler-A", "role": "advisor", "display_name": "Tyler, Albert" }, { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "advisor", "display_name": "Owen, Ray David" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biodev" ], "doi": "10.7907/ZWTJ-CW39", "abstract": "The principal theme of these investigations concerns the inhibited state of mature unfertilized sea urchin eggs with respect to uridine uptake and protein synthesis. \r\n \r\nPart I demonstrates that unfertilized-eggs are relatively impermeable to uridine. Fertilized eggs, however, develop during the first hour an energy-dependent, uptake mechanism for uridine accumulation. Labeled uridine assimilated by fertilized eggs is recovered as phosphorylated nucleosides, primarily triphosphates. Experiments support the idea that uridine penetration into sea urchin eggs depends upon the phosphorylation of the 5' carbon atom at the cell surface. Tests with puromycin show that protein synthesis is unnecessary for the generation of uridine uptake after fertilization. The evidence favors the view that uridine kinase is sequestered within the unfertilized egg and thus incapable of activity at the cell surface until after fertilization.\r\n \r\nParts II and III use biochemical and autoradiographic methods to show that growing oocytes of sea urchins, in contrast to many other organisms, undergo considerable RNA and protein synthesis. Protein synthesis in isolated oocytes occurs throughout the germinal vesicle and cytoplasm and takes place on polyribosomes. RNA synthesis is localized in the nucleolus. Mature eggs, however, synthesize only little protein even in mixed suspensions with oocytes. Long-term maintenance of spawned female sea urchins, after but one injection of labeled uridine, produces ripe unfertilized eggs possessing highly radioactive RNA. The distribution of label in the extracted RNAs is 70-80% ribosomal, 10-20% heterogeneous, and 5-10% soluble.\r\n \r\nPart IV is an electron microscopic and biochemical examination of RNA-labeled mature unfertilized and fertilized eggs. The findings are correlated with the difference in protein synthesizing activity before and after fertilization. The results show that unfertilized eggs synthesize protein upon RNase-sensitive polyribosomes. The large increase in protein synthesis after fertilization occurs in association with the assembly of additional polyribosomes. Homogenates of unfertilized eggs also possess synthetically inactive, RNase-resistant, ribosomal aggregates. Evidence suggests that trypsin followed by RNase disperses the aggregates. Homogenates of fertilized eggs, however, contain very few RNase-resistant ribosomal aggregates. By forty minutes after fertilization, about 70% of the new protein synthesis can be attributed to the new polyribosomes. The weight of the evidence indicates that the remaining 30% of the stimulation of protein synthesis is due to the activation of \"masked\" polyribosomes.\r\n \r\nAppendix 1 shows that, for unfertilized and fertilized eggs, competition for uptake of amino acids occurs primarily among those belonging to the same charge group. Appendix 2 demonstrates that one amino acid can displace another of the same category from intact eggs both before and after fertilization. By combination of these facts, then, it is possible to achieve greater labeling of egg-proteins than has been previously realized." }, { "name": "Russell, Richard Lawson", "degree": "PhD", "year": "1967", "title": "Speciation Among the T-Even Bacteriophages", "advisor": "Edgar, Robert S.", "url": "https://resolver.caltech.edu/CaltechETD:etd-10012002-154154", "creators": [ { "name": { "family": "Russell", "given": "Richard Lawson" }, "id": "Russell-Richard-Lawson", "display_name": "Russell, Richard Lawson" } ], "advisors": [ { "name": { "family": "Edgar", "given": "Robert S." }, "id": "Edgar-R-S", "role": "advisor", "display_name": "Edgar, Robert S." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/SHD0-FM17", "abstract": "A system of amber mutants has been developed for each of the T-even phages T2, T6, and RB69. In T2 these mutants identify 53 genes, of which 46 are homologous with T4 genes. The order of genes in the T2 map is virtually identical to that of T4, and recombination in T2 strongly resembles that in T4.\r\n\r\nIn T6 the mutants identify 42 genes, of which 34 are homologous to T4 genes and 3 are homologous to T2 genes not yet identified in T4. The few T6 gene orders which have been determined are the same as in T2 and T4, and T6 recombination resembles that of T2 and T4.\r\n\r\nRB69 is judged to be a member of the T-even phage species by its serological properties, its particle morphology, and various physical parameters of the RB69 particle and its contained DNA. RB69 mutants identify 37 genes, and their phenotypes are the same as those of T4 mutants. Recombination in RB69 strongly resembles that in T2 and T4, and the distribution of mutant phenotypes around the RB69 map is very much like that of T4.\r\n\r\nT2, T4, and T6 differ most noticeably in the tail fiber region. Genes 37 and 38 are a unique example of a pair of coadapted genes; T2-T6 combinations of the products of these genes are compatible, whereas T2-T4 and T4-T6 combinations are not. The host range differences between the phages are determined by gene 38. Genes 34 and 35 are genetically much smaller in T2 and T6 than in T4, but there is considerable homology between T2 gene 34 and T4 gene 34. The degree of homology between T2 and T4 decreases from gene 34 to gene 38.\r\n\r\nIn T2-T4 mixed infections T4 excludes T2 by acting against localized exclusion sensitivity determinants near early genes in the T2 genome. This action prevents the T2 genome from participating in the formation of the replicating structure, and it prevents the determinants from appearing among the progeny, but it does not prevent T2 genes from functioning. Markers from the T2 genome appear among the progeny by recombination away from these determinants. The specificity of the T4 action is not controlled by any known T4 gene, nor does it depend on differences between T2 and T4 in DNA glucosylation.\r\n\r\nThe T4-T6 mixed infection is characterized by a depressor effect, and T4 excludes T6 strongly. T6 excludes T2 weakly, and RB69 excludes T2, T4, and T6 very strongly. T4 excludes thirty other newly-isolated T-even phages fairly strongly. A general model has been developed to account for all of these cases of exclusion, and it also accounts for cases of partial exclusion in the T5 and T3-T7 phage species.\r\n\r\nFrom the relationships among the T-even phages and their interactions in mixed infections, an attempt is made to reconstruct the evolution of exclusion as a bacteriophage isolating mechanism. Analogies between exclusion and host-controlled modification as a bacterial isolating mechanism are discussed." }, { "name": "Strauss, James Henry", "degree": "PhD", "year": "1967", "title": "Studies on the RNA of Bacteriophage MS2", "advisor": "Delbruck, Max", "url": "https://resolver.caltech.edu/CaltechETD:etd-10102002-121043", "creators": [ { "name": { "family": "Strauss", "given": "James Henry" }, "id": "Strauss-James-Henry", "display_name": "Strauss, James Henry" } ], "advisors": [ { "name": { "family": "Delbruck", "given": "Max" }, "id": "Delbr\u00fcck-M", "role": "advisor", "display_name": "Delbruck, Max" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biochem" ], "doi": "10.7907/1WZB-AY91", "abstract": "The first part of this dissertation contains a publication which discusses the purification and properties of bacteriophage MS2 and its RNA, including the RNA content of the virus, and light scattering particle weights and sedimentation properties of the virus and its RNA. A short discussion of this publication in light of later results is included.
\r\n\r\nAn infectivity assay for MS2-RNA is characterized in Part II. The results of varying several parameters of the assay are presented, and the effects on the assay of competing RNA's and external ribonuclease are discussed.
\r\n\r\nPart III presents the initial kinetics of degradation of MS2-RNA by ribonuclease, heat, and alkali, as followed by the exponential decline in RNA infectivity. The turnover number of pancreatic ribonuclease is independent of RNA concentration between 10\u00b9\u00b9 and 10\u00b9\u2075 RNA molecules per ml. Inactivation at high pH is almost proportional to the hydroxyl ion concentration between pH 11 and 12.3. The activation energy for thermal inactivation is 22 kcal./mole.
\r\n\r\nPart IV contains studies on the denaturation of MS2-RNA, of a double-stranded form of RNA, and of rG:rC by thermal means and with organic solvents. Evidence is presented that in dimethylsulfoxide RNA is completely denatured at room temperature.
\r\n\r\nPart V describes the centrifugal properties of MS2-RNA. The RNA is shown to possess no covalent configurational restraints by the presence of but a single component when sedimented under denaturing conditions, by the correspondence between the decline in infectivity produced by ribonuclease and the production of RNA fragments, and the coincident sedimentation of the RNA infectivity with the bulk RNA under a variety of conditions, including sedimentation through dimethylsulfoxide. The RNA chain does hydrogen-bond to itself, however, giving rise to multiple, homogeneous, infective components under certain conditions.
" }, { "name": "Tuan, Dorothy Yung-Hsun", "degree": "PhD", "year": "1967", "title": "Part I. Interaction of DNA and Histone in Native Nucleohistone. Part II. Dormancy Associated with the Repression of Genetic Activity", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechETD:etd-10042002-162144", "creators": [ { "name": { "family": "Tuan", "given": "Dorothy Yung-Hsun" }, "id": "Tuan-Dorothy-Yung-Hsun", "display_name": "Tuan, Dorothy Yung-Hsun" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/X7X3-QH34", "abstract": "Part I. Interaction of DNA and histone in nucleohistone \r\n\r\nChapter 1. SELECTIVE DISSOCIATION OF HISTONE FROM NUCLEOHISTONE\r\n\r\nWith increasing concentration of NaCl solution, an increasing amount of histone is dissociated from dissolved nucleohistone. The dissociated histone fractions are identified by gel electrophoresis. The lysine rich histone fraction I is dissociated from nucleohistone in the range 0.3-0.5 F NaCl; slightly lysine rich histone II in the range 0.8-1.6 F NaCl; arginine rich histone III+IV in the range 0.9-1.6 F NaCl. The results suggest that both electrostatic and non-electrostatic interactions contribute to the strength of binding between DNA and histones.\r\n\r\nChapter 2. OPTICAL ROTATORY DISPERSION STUDIES ON HISTONES\r\n\r\nThe optical rotatory dispersion spectra of histones free and in reconstituted nucleohistone (in which histone is complexed to DNA) are recorded. By the criterion of optical rotatory dispersion at wavelengths below 220 mu, histone I is the least helical of the histones, histone II the most helical. The helicity of DNA-bound histones in reconstituted nucleohistone is greater than that of free histones, but the order, histone II most helical and histone I least, is still preserved.\r\n\r\nChapter 3. OPTICAL ROTATORY DISPERSION STUDIES ON THE DNA OF NATIVE NUCLEOHISTONE AND OF PARTIALLY DEHISTONIZED NUCLEOHISTONES\r\n\r\nThe conformation of DNA in native nucleohistone is altered by the DNA-histone interaction. The dissociation of histone I does not produce significant conformational change in DNA of nucleohistone but removal of histone II and of histone III+IV bring about changes which cause the conformation of DNA in nucleohistone to resume virtually that characteristic of free DNA. The possibility of DNA supercoiling in nucleohistone is discussed.\r\n\r\nPart II. Dormancy associated with repression of genetic activity\r\n\r\nChapter 1. THE DORMANCY OF POTATO BUDS\r\n\r\nChromatin of the buds of dormant potato tubers is almost totally incapable of the support of DNA-dependent RNA synthesis in the presence of added exogenous RNA polymerase. The chromatin of non-dormant buds of potato tubers (in which dormancy has been broken by treatment with ethylene chlorohydrin) is highly effective in the support of DNA-dependent RNA synthesis by added exogenous RNA polymerase. It is therefore concluded that the genetic material of the buds of dormant potato tubers is largely in a repressed state, and that the breaking of dormancy is accompanied by derepression of the genetic material.\r\n\r\nChapter 2. THE DORMANCY OF ONION BULBS\r\n\r\nThe chromosomal material of non-growing and non-dividing onion buds possesses template activity in support of in vitro DNA-dependent RNA synthesis. If we define dormancy not only by the absence of visible growth and mitotic division, but also by the lack of ability to direct in vitro DNA-dependent RNA synthesis, potato buds are then dormant but onion buds are not. And the block to onion bud growth must lie somewhere else than in the repression of genetic material.\r\n\r\nChapter 3. ISOLATION OF GLADIOLUS CHROMATIN\r\n\r\nGladiolus corms contain very little isolatable chromatin material and the isolated chromatin is highly contaminated by the presence of starchy material." }, { "name": "Young, Elton Theodore, II", "degree": "PhD", "year": "1967", "title": "Structure and Synthesis of Bacteriophage Lambda DNA", "advisor": "Sinsheimer, Robert L.", "url": "https://resolver.caltech.edu/CaltechETD:etd-08082006-151750", "creators": [ { "name": { "family": "Young", "given": "Elton Theodore, II" }, "id": "Young-Elton-Theodore", "display_name": "Young, Elton Theodore, II" } ], "advisors": [ { "name": { "family": "Sinsheimer", "given": "Robert L." }, "id": "Sinsheimer-R-L", "role": "advisor", "display_name": "Sinsheimer, Robert L." } ], "committee": [ { "name": { "family": "Sinsheimer", "given": "Robert L." }, "id": "Sinsheimer-R-L", "role": "chair", "display_name": "Sinsheimer, Robert L." }, { "name": { "family": "Weigle", "given": "Jean J." }, "id": "Weigle-J-J", "role": "member", "display_name": "Weigle, Jean J." }, { "name": { "family": "Vinograd", "given": "Jerome" }, "id": "Vinograd-J", "role": "member", "display_name": "Vinograd, Jerome" }, { "name": { "family": "Delbruck", "given": "Max" }, "id": "Delbr\u00fcck-M", "role": "member", "display_name": "Delbruck, Max" } ], "option_major": [ "biology" ], "doi": "10.7907/MHCY-S381", "abstract": "The research in this thesis represents an attempt to study the replication of bacteriophage lambda in terms of the structure of the vegetative DNA. A spheroplast assay for lambda DNA is described in Part I and several parameters affecting the efficiency of the assay are investigated. The biological activity of intracellular lambda DNA is then studied using this assay. In contrast to the results obtained with the transformation or helper assay, no eclipse of infectivity of vegetative lambda DNA occurs in the spheroplast assay. The DNA species responsible for most of the infectivity in the spheroplast assay of DNA extracted from infected, immune bacteria, is the fast-sedimenting \"super-coiled\" form of lambda DNA.\r\n\r\nIn Part II the structure and replication of the fast-sedimenting species of vegetative lambda DNA is examined physically and biologically. Its physical properties are those of a twisted, circular molecule each of whose two strands is closed upon itself. Using a mitomycin C technique to inhibit host DNA synthesis, the accumulation and semi-conservative replication of closed-circular lambda DNA specifically early in the infection is demonstrated with radio-isotopes. Biological analysis using the spheroplast assay verifies this conclusion. Chloramphenicol inhibits the synthesis of viral DNA at a lower-concentration than that needed to inhibit the synthesis of closed-circular DNA. Chloramphenicol does not prevent the conversion from viral to closed-circular DNA.\r\n\r\nThe data presented in Part III suggest that open-circular lambda DNA may act as a precursor to, both closed-circular and viral DNA. The closed-circular species is not a major precursor to viral DNA. Sedimentation analysis also reveals the existence of as yet unidentified intermediates in the replication of viral DNA.\r\n\r\nPart IV describes several methods for purifying intracellular lambda DNA and examines the biological activity of the purified DNA in the spheroplast assay. The circular DNA is infective before and after denaturation, whereas denaturation inactivates viral DNA. The increased infectivity of open-circular DNA after denaturation (relative to the native state) appears to be due to the appearance of biologicallyactive single-stranded rings of lambda DNA. The closed-circular DNA usually has the same infectivity before and after denaturation." }, { "name": "Benjamin, Thomas Livingston", "degree": "PhD", "year": "1966", "title": "Radiobiological and Biochemical Investigations of Polyoma Virus-Cell Interactions", "advisor": "Dulbecco, Renato; Attardi, Giuseppe; Vinograd, Jerome Rubin", "url": "https://resolver.caltech.edu/CaltechTHESIS:09182015-084245868", "creators": [ { "name": { "family": "Benjamin", "given": "Thomas Livingston" }, "id": "Benjamin-Thomas-Livingston", "display_name": "Benjamin, Thomas Livingston" } ], "advisors": [ { "name": { "family": "Dulbecco", "given": "Renato" }, "id": "Dulbecco-R", "role": "advisor", "display_name": "Dulbecco, Renato" }, { "name": { "family": "Attardi", "given": "Giuseppe" }, "id": "Attardi-G", "role": "advisor", "display_name": "Attardi, Giuseppe" }, { "name": { "family": "Vinograd", "given": "Jerome Rubin" }, "id": "Vinograd-J-R", "role": "advisor", "display_name": "Vinograd, Jerome Rubin" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/BBKM-MN37", "abstract": "The cytolytic interaction of Polyoma virus with mouse embryo cells\r\nhas been studied by radiobiological methods known to distinguish temperate \r\nfrom virulent bacteriophage. No evidence for \"temperate\" properties\r\nof Polyoma was found. During the course of these studies, it was\r\nobserved that the curve of inactivation of Polyoma virus by ultraviolet\r\nlight had two components - a more sensitive one at low doses, and a\r\nless sensitive one at higher doses. Virus which survives a low dose\r\nhas an eclipse period similar to that of unirradiated virus, while\r\nvirus surviving higher doses shows a significantly longer eclipse period.\r\nIf Puromycin is present during the early part of the eclipse period,\r\nthe survival curve becomes a single exponential with the sensitivity\r\nof the less sensitive component. These results suggest a repair mechanism\r\nin mouse cells which operates more effectively if virus development\r\nis delayed.
\r\n\r\nA comparison of the rates of inactivation of the cytolytic and\r\ntransforming abilities of Polyoma by ultraviolet light, X-rays, nitrous\r\nacid treatment, or the decay of incorporated P32, showed that the transforming \r\nability has a target size roughly 60% of that of the plaque-forming\r\nability. It is thus concluded that only a fraction of the viral\r\ngenes are necessary for causing transformation.
\r\n\r\nThe appearance of virus-specific RNA in productively infected\r\nmouse kidney cells has been followed by means of hybridization between\r\npulse-labelled RNA from the infected cells and the purified virus DNA.\r\nThe results show a sharp increase in the amount of virus-specific RNA\r\naround the time of virus DNA synthesis. The presence of a small amount\r\nof virus-specific RNA in virus-free transformed cells has also been\r\nshown. This result offers strong evidence for the persistence of at\r\nleast part of the viral genome in transformed cells.
" }, { "name": "Boyd, James Brown", "degree": "PhD", "year": "1966", "title": "Part I. Turnover of the Hemolymph Proteins of Drosophila melanogaster. Part II. A New Method for the Detection of Deoxyribonucleases and its\r\nApplication to Studies of Drosophila melanogaster", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechETD:etd-09102002-133403", "creators": [ { "name": { "family": "Boyd", "given": "James Brown" }, "id": "Boyd-James-Brown", "display_name": "Boyd, James Brown" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/4ZS0-CV69", "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\r\n\r\nPart I\r\n\r\nA method has been developed for measuring the radioactivity of proteins labeled with[.....] following their separation by disc electrophoresis. The radioactivity is measured directly in acrylamide gel with scintillation techniques after the water in the gel has been replaced with a toluene-based scintillator solution. Seventy-four gel slices can be prepared with a minimum of handling in 9 to 18 hours depending upon the size of the slices.\r\n\r\nThis technique has been used in combination with densitometry measurements of separately stained gels to study the turnover of the hemolymph proteins of [Drosophila melanogaster]. By injecting labeled homologous proteins into unlabeled animals, active turnover of most of the hemolymph proteins has been demonstrated in both larvae and pupae. One particular group of proteins begins to turn over rapidly after puparium formation with a half life of 13 hours. Another group of proteins has been shown to be relatively inert in pupae. A postulated mechanism for protein turnover in metamorphosing insects is discussed.\r\n\r\nPart II\r\n\r\nA method is described for detecting deoxyribonucleases which have been separated in acrylamide gel containing trapped DNA. After electrophoresis the enzymes are incubated at their final resting positions with the substrate found at those positions. The remaining DNA is stained and recorded with a densitometer. The assay is sensitive to less than 0. 25 nanograms of crystallized deoxyribonuclease I and is quantitatively reproducible.\r\n\r\nAn investigation of the deoxyribonucleases of [Drosophila melanogaster] with this technique has revealed a large number of peaks of enzymatic activity which undergo marked changes during the course of development. By varying the substrate and the magnesium concentration used for incubation, it has been shown that this organism probably produces at least eight different enzymes which can degrade DNA. A preliminary determination of the properties of some of these enzymes has been made." }, { "name": "Denny, Paul Claire", "degree": "PhD", "year": "1966", "title": "I. Studies on Alkaline Phosphatase Activity in Developing Sea Urchin Embryos. II. Studies on the Activation of Protein Biosynthesis in Sea Urchin Eggs at Fertilization", "advisor": "Tyler, Albert", "url": "https://resolver.caltech.edu/CaltechTHESIS:09152015-083120183", "creators": [ { "name": { "family": "Denny", "given": "Paul Claire" }, "id": "Denny-Paul-Claire", "display_name": "Denny, Paul Claire" } ], "advisors": [ { "name": { "family": "Tyler", "given": "Albert" }, "id": "Tyler-A", "role": "advisor", "display_name": "Tyler, Albert" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biodev" ], "doi": "10.7907/J3KZ-P058", "abstract": "I. Alkaline phosphatase activity in the developing sea urchin Lytechinus pictus has been investigated with respect to intensity at various stages, ionic requirements and intracellular localization. The activity per embryo remains the same in the unfertilized egg, fertilized egg and cleavage stages. At a time just prior to gastrulation (about 10 hours after fertilization) the activity per embryo begins to rise and increases after 300 times over the activity in the cleavage stages during the next 60 hours.
\r\n\r\nThe optimum ionic strength for enzymatic activity shows a wide peak at 0.6 to 1.0. Calcium and magnesium show an additional optimum at a concentration in the range of 0.02 to 0.07 molar. EDTA at concentrations of 0.0001 molar and higher shows a definite inhibition of activity.
\r\n\r\nThe intracellular localization of alkaline phosphatase in homogenates of 72-hour embryos has been studied employing the differential centrifugation method. The major portion of the total activity in these homogenates was found in mitochondrial and microsomal fractions with less than 5% in the nuclear fraction and less than 2% in the final supernatant. The activity could be released from all fractions by treatment with sodium deoxycholate.
\r\n\r\nII. The activation of protein biosynthesis at fertilization in eggs of the sea urchins Lytechinus pictus and Strongylocentrotus purpuratus has been studied in both intact eggs and cell-free homogenates. It is shown that homogenates from both unfertilized and fertilized eggs are dependent on potassium and magnesium ions for optimum amino acid incorporation activity and in the case of the latter the concentration range is quite narrow. Though the optimum magnesium concentrations appear to differ slightly in homogenates of unfertilized and fertilized eggs, in no case was it observed that unfertilized egg homogenates were stimulated to incorporate at a level comparable to that of the fertilized eggs.
\r\n\r\nAn activation of amino acid incorporation into protein has also been shown to occur in parthenogenetically activated non-nucleate sea urchin egg fragments or homogenates thereof. This activation resembles that in the fertilized whole egg or fragment both in amount and pattern of activation. Furthermore, it is shown that polyribosomes form in these non-nucleate fragments upon artificial activation. These findings are discussed along with possible mechanisms for activation of the system at fertilization.
\r\n" }, { "name": "Egbert, Larre Nyman", "degree": "PhD", "year": "1966", "title": "Isolation and Characteristics of a Bacteriophage for Bacillus stearothermophilus", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01282016-143942913", "creators": [ { "name": { "family": "Egbert", "given": "Larre Nyman" }, "id": "Egbert-Larre-Nyman", "display_name": "Egbert, Larre Nyman" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/YHC4-5P08", "abstract": "A bacteriophage (T\u00d83) which infects the thermophilic bacterium\r\nBacillus stearothermophilus ATCC 8005 was isolated and characterized.\r\nInfection of the bacterium by the bacteriophage was carried out at 60\u00b0C,\r\nthe optimum growth temperature of the host. At 60\u00b0C the phage has a\r\nlatent period of 18 minutes and a burst size of about 200. The phage\r\nis comparatively thermostable in broth. The half life of the phage is\r\n400 minutes at 60\u00b0C, 120 minutes at 65\u00b0C, 40 minutes at 70\u00b0C and 12\r\nminutes at 75\u00b0C. The activation energy for the heat inactivation of\r\nT\u00d83 is 56,000 cal. The buoyant density of T\u00d83 in a cesium chloride\r\ndensity gradient is 1.526.
\r\n\r\nElectron micrographs of T\u00d83 indicate that the phage has a regular\r\nhexagonal shaped head 57 m\u03bc long. The morphology of the head is\r\ncompatible with icosahedral symmetry. Each edge of the head is 29 m\u03bc\r\nlong, and there are 6 or 7 subunits along each edge. The tail of T\u00d83\r\nis 125 m\u03bc long and 10 m\u03bc wide. There are about 30 cross striations that\r\nare spaced at 3.9 m\u03bc intervals along the tail.
\r\n\r\nThe DNA of phage T\u00d83 has a melting temperature of 88.5\u00b0C. Heat\r\ndenatured T\u00d83 DNA can be extensively annealed in a high ionic strength\r\nenvironment. The buoyant density of T\u00d83 DNA in a cesium chloride\r\ndensity gradient is 1.695. T\u00d83 DNA contains: 42.7% guanine plus cytosine,\r\nas determined from the melting temperature; 43% guanine plus\r\ncytosine, as determined from the buoyant density; and 40.2% guanine\r\nplus cytosine, as determined by chromatographic separation and spectrophotometric\r\nestimation of the bases. The molecular weight of T\u00d83\r\nDNA is 16.7 X 106 as determined from the band width of the T\u00d83 DNA\r\nconcentration distribution in a cesium chloride density gradient. Electron\r\nmicroscopy of T\u00d83 DNA revealed a single linear molecule that is\r\n11.7 \u03bc long. This corresponds to a molecular weight of 22.5 X 106.
\r\n\r\nHeat denatured T\u00d83 DNA forms two bands in a cesium chloride density\r\ngradient, one at a density of 1.707 and the other at a density of 1.715.\r\nAfter the separated bands are mixed and annealed in the centrifuge cell,\r\nthe renatured T\u00d83 DNA forms a single band at a density of 1.699. These\r\nresults indicate that the two complementary strands of T\u00d83 DNA have\r\ndifferent buoyant densities in cesium chloride, presumably because they\r\nhave different base compositions.
\r\n\r\nThe characteristics of T\u00d83 are compared with those of other phages.\r\nA hypothesis is presented for a relationship between the base composition\r\nof one strand of T\u00d83 DNA and the amino acid composition of the proteins\r\nof T\u00d83.
" }, { "name": "Fried, Michael", "degree": "PhD", "year": "1966", "title": "Studies of Temperature Sensitive Mutants of Polyoma Virus", "advisor": "Dulbecco, Renato", "url": "https://resolver.caltech.edu/CaltechTHESIS:09292015-103236585", "creators": [ { "name": { "family": "Fried", "given": "Michael" }, "id": "Fried-Michael", "display_name": "Fried, Michael" } ], "advisors": [ { "name": { "family": "Dulbecco", "given": "Renato" }, "id": "Dulbecco-R", "role": "advisor", "display_name": "Dulbecco, Renato" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/MP3Y-5Q07", "abstract": "Polyoma virus can undergo two different types of interactions with susceptible cells; one type of interaction leads to the production of new infectious virus and eventual cell death while the other leads to a neoplastically transformed cell which is able to continue to divide under conditions that inhibit the multiplication of uninfected normal cells. In order to study the viral genes involved in both of these virus-cell interactions the isolation of temperature sensitive mutants of polyoma virus was undertaken.
\r\nTwo strains (TS-a, TS-b) which were temperature sensitive in their plaque forming ability at 38.5\u02daC, but not at 31.5\u02daC, were isolated from a mutagenized stock of the polyoma wild type virus (PY). TS-a was studied in further detail.
\r\nTS-a grown at 31.5\u02daC was found to be indistinguishable from PY in a number of physical characteristics including the heat sensitivity of the completed viral components. TS-a was inhibited in its ability to produce infectious virus in mouse cells when incubated at 38.5\u02daC; this inhibition could be overcome by infection with high multiplicities.
\r\nThe nature of the intracellular temperature sensitive step of TS-a was analysed to some degree. It was found that this step occurs after uncoating of the infecting virus particles and about the time of new viral DNA synthesis. New infectious viral DNA does not appear to be made at the nonpermissive temperature; in contrast noninfectious capsids are made at 38.5\u02daC, but in amounts smaller than a full yield, such as made by TS-a at 31.5\u02daC or by PY at both the high and low temperature.
\r\nTS-a has also been found to be temperature sensitive in its transforming ability in vitro. Cells transformed at 31.5\u02daC by TS-a retain their transformed characteristics upon cultivation at 38.5\u02daC. Thus the temperature sensitive function seems to be important for the initiation of transformation, but not essential for the maintenance of the transformed state. TS-a also appears to be temperature sensitive in the production of tumors in newborn hamsters.
\r\n" }, { "name": "Glowacki, Ellen Rose", "degree": "PhD", "year": "1966", "title": "Studies on Rabbit Reticulocyte Ribosomes and Polyribosomes and their Relation to Hemoglobin Synthesis", "advisor": "Borsook, Henry", "url": "https://resolver.caltech.edu/CaltechTHESIS:02122016-082048849", "creators": [ { "name": { "family": "Glowacki", "given": "Ellen Rose" }, "id": "Glowacki-Ellen-Rose", "display_name": "Glowacki, Ellen Rose" } ], "advisors": [ { "name": { "family": "Borsook", "given": "Henry" }, "id": "Borsook-H", "role": "advisor", "display_name": "Borsook, Henry" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/H4BA-4M16", "abstract": "This dissertation is divided into three parts.
\r\n\r\nThe first section is concerned with protein synthesis in cellfree\r\nsystems from reticulocytes. The sub-cellular reticulocyte fractions,\r\nreagents, etc. have been examined for the presence of traces of ribonuclease,\r\nusing. an assay based upon the loss of infectivity of RNA\r\nfran bacteriophage MS2. This assay is sensitive to 5 x 10-7 \u03b3 RNase/ml.\r\nIn addition, the loss of synthetic capacity of an 80S ribosome on dissociation\r\nhas been studied, and can be attributed to loss of messenger\r\nRNA when the monomer is separated into subunits. The presence of\r\nribonuclease has been shown to be a major cause of polyribosome disintegration\r\nduring cell-free protein synthesis.
\r\n\r\nThe second section concerns the changes in ribosomes and polyribosomes\r\nwhich occur during the maturation of a reticulocyte into an\r\nerythrocyte. With increasing age, the cells lose a large proportion of\r\nthe ribonucleoprotein, but the percentage of ribosomes present as polyribosomes\r\nis only slightly altered. The loss of hemoglobin synthesis\r\non maturation is probably due to both the loss of total ribosomes\r\nand to the lessened specific activity of the polyribosomes.
\r\n\r\nThe third section contains analytical ultracentrifugation data\r\non 80S ribosomes, polyribosomes, and ribosomal RNA from reticulocytes.\r\nThe 60s and 40s subunits, obtained by dissociation of the 80s particle\r\nwith inorganic pyrophosphate, were also studied. The RNA from reticulocyte\r\nribosomes has been examined under a variety of denaturing conditions,\r\nincluding dimethyl sulfoxide treatment, formaldehyde reaction and thermal\r\ndenaturation. From these studies we can conclude that the 28S and\r\n16S RNA's are single polynucleotide chains and are not made up of\r\nsmaller RNA subunits hydrogen-bonded together.
" }, { "name": "Krane, Stanley Garson", "degree": "PhD", "year": "1966", "title": "Studies on the Biosynthesis of Studies of \u00d8X174 Coat Protein", "advisor": "Sinsheimer, Robert L.", "url": "https://resolver.caltech.edu/CaltechETD:etd-09272002-131251", "creators": [ { "name": { "family": "Krane", "given": "Stanley Garson" }, "id": "Krane-Stanley-Garson", "display_name": "Krane, Stanley Garson" } ], "advisors": [ { "name": { "family": "Sinsheimer", "given": "Robert L." }, "id": "Sinsheimer-R-L", "role": "advisor", "display_name": "Sinsheimer, Robert L." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/1MXF-6632", "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\r\n\r\nA serum blocking power (SBP) assay for [phi]X174 coat protein, which depends on the ability of whole phage or certain phage components to combine with antiphage serum, was developed. This assay registers complete [phi]X, both viable and nonviable, and 70S particles, but not chemically prepared 5S subunits.\r\n\r\nStudies on the growth of wild type [phi]X, at 37[degrees], showed that SBP synthesis begins at about 5 min, under conditions such that the eclipse ends at 6 to 8 min. Although the curves for SBP and intracellular phage growth have the same shape, the titer of SBP phage equivalents exceeds that of infectious progeny particles at all times. The excess SBP is found in the form of 70S particles, complete but noninfectious phage, and subunits that sediment at about 15S.\r\n\r\nChloramphenicol, 5-fluorouracil deoxyriboside, and phleomycin when present at the time of infection each produce essentially complete inhibition of SBP synthesis. These results suggest that at least one complete RF molecule (the double stranded form of [phi]X DNA) must be made in order for SBP synthesis to occur.\r\n\r\nSeveral temperature sensitive [...] mutants were studied for their ability to produce SBP at 40[degrees]C (nonpermissive conditions for production of infectious phage). Most of the twenty-six mutants examined did make SBP at 40[degrees]C; however, mutant [...] 79 definitely did not.\r\n\r\nStudies were made on the physical state of the SBP synthesized, at 40[degrees]C, by the mutants [...] Y, 9, and [...] 4 by the technique of sucrose gradient velocity centrifugation. The results showed that [...] Y, and [...] 9 make their SBP principally in the form of subunits that sediment at approximately 15S. The mutant [...] 4 makes its SBP principally in the form of particles that sediment at 71S. The [...] 4 71S particles may contain both DNA and protein." }, { "name": "Scheibe, Joseph Emil", "degree": "PhD", "year": "1966", "title": "Studies on Photoblastic Germination in Lettuce Seed", "advisor": "Lang, Anton", "url": "https://resolver.caltech.edu/CaltechTHESIS:10192015-101751719", "creators": [ { "name": { "family": "Scheibe", "given": "Joseph Emil" }, "id": "Scheibe-Joseph-Emil", "display_name": "Scheibe, Joseph Emil" } ], "advisors": [ { "name": { "family": "Lang", "given": "Anton" }, "id": "Lang-Anton", "role": "advisor", "display_name": "Lang, Anton" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/Q0PY-3Q87", "abstract": "It was shown, with the aid of osmotic inhibition of germination, that the action of the far-red-absorbing form of phytochrome (Pf) in promoting germination can be completed even if the seed is held under conditions where germination is not possible. An effect of the continuing action of Pf beyond the point of complete germination promotion was demonstrated by enhancement of germination rate after removal of the osmotically active solute.
\r\nPrevious reports that the rate of growth in water of seeds freed from the expansion-restricting endosperm is independent of the state of phytochrome were confirmed. However, a marked, phytochrome-mediated enhancement of the growth potential of such seeds was demonstrated through restricting water uptake by incubation in an osmoticum.
\r\nAn experimental system, utilizing the appearance of a geotropic curvature in the radicle of the excised axial portion of the seed, was developed for more detailed studies of the phytochrome-enhanced growth potential. It was possible to demonstrate the light effect in water as well as in osmotica; this apparently is not possible with de-endospermed entire seeds. As in intact seeds, the effect of the continuing action of Pf is to enhance the rate of the response. Secretion of a chemical inhibitor of growth by the endosperm as a possible mechanism of induction of light sensitivity has been ruled out.
\r\nThe phytochrome-dependent rate of appearance of geotropic curvature in osmotica is paralleled in time by a similar dependence of the rate of early extension growth of the embryonic axis. Only the first small increment of growth is a differentially responsive to red (R) and far-red (F); the rate of later increase in length is independent of the light regime.
\r\nIt was shown that the high concentrations of gibberellic acid required for germination promotion in the intact seed are due at least in part to a diffusion barrier in the endosperm, and that the occasional reports in the literature of the ineffectiveness of kinetin are probably due to the same phenomenon. It was shown that gibberellin, like red light, enhances the growth potential of the axis, but kinetin does not. The difference in rates of response obtained after R-irradiation or gibberellin treatment, together with other results reported in the literature, strongly suggests that gibberellic acid and red light promote germination by different means. The idea that kinetin promotes germination by yet another mechanism, probably operating in the cotyledons, was supported through two different experimental approaches.
\r\nThe phenomenon of temperature-dependent dark germination was examined in detail, using a wide range of both temperatures and incubation times. With the aid of the half-seed system, it was demonstrated that the promotive effect of low temperature on germination could not be due to a low optimum temperature for early growth of the radicle, since the rate of that process increased with increasing temperature, up to the highest temperature used.
\r\nIt was shown that phytochrome does not function at high temperatures. This fact is of considerable importance in interpreting the phenomenon of thermodormancy, since in the literature only a small part of the effect of high temperature has been ascribed to an effect on phytochrome, and at that, only to an acceleration of dark reversion of Pf to the red-absorbing form of phytochrome (Pr). Partial denaturation of phytochrome may also make some contribution.
\r\nIt was shown that the germination-promoting effect of low temperature depends on the presence of Pf, and concluded that low temperatures act by delaying or preventing transformation of Pf. Support for the assumption that Pf, not Pr, is the active form of phytochrome in lettuce seeds was drawn from the same evidence.
\r\nAttempts to stimulate germination by repeated irradiation with F over relatively prolonged incubation times resulted in failure, as have similar attempts reported in the literature. However, an enhancement of growth potential in the half-seed system by the maintenance of a small amount of Pf over long periods at ordinary temperatures by repeated irradiation with F was demonstrated.
\r\nIt was observed that cold storage of the dry seed prevents or delays loss of dark dormancy during post-harvest storage. No change in the response of the half-seed in osmoticum to R and F was observed in seeds that has lost dark dormancy; that is, no internal change took place to measurably increase the growth potential of the embryonic axis. This suggests that the endosperm is the seat of changes responsible for after-ripening of photoblastic lettuce seed.
\r\n" }, { "name": "Widholm, Jack Milton", "degree": "PhD", "year": "1966", "title": "Physical and Biological Studies of Crab Deoxyribonucleic Acid", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechETD:etd-09232002-114928", "creators": [ { "name": { "family": "Widholm", "given": "Jack Milton" }, "id": "Widholm-Jack-Milton", "display_name": "Widholm, Jack Milton" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/1276-DQ97", "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document. \r\n\r\nThe native dAT components of C. antennarius and of C. borealis DNA have been separated in pure form by a new mercury binding technique.\r\n\r\nTwo further DNA species, not heretofore described, are apparently also present in C. antennarius DNA. They have melting points and buoyant densities intermediate between those of dAT and of the major DNA component.\r\n\r\nA series of tests using optical density melting electrophoresis, buoyant density, electron microscopic, band sedimentation, exonuclease I susceptibility and actinomycin D inhibition techniques are employed to show that melted and renatured C. antennarius dAT remains partially denatured. After strand separation and recombination approximately 7% of the base pairs do not reform. These unpaired bases are predominantly guanine and cytosine (G and C) since exonuclease I hydrolysis assays and RNA synthesis inhibition by actinomycin D give the results which would be expected on this basis. The C. antennarius dAT contains 3.5% G and C bases. C. borealis dAT with 2.5% GC behaves similarly to C. antennarius dAT, except that the denatured structure is less evident, apparently because of the lower GC content.\r\n\r\nWhen the aforementioned tests are applied to synthetic dAT, which contains no G or C bases, the results indicate that this alternating dAT copolymer is fully hydrogen bonded both before and after melting.\r\n\r\ndAT is present to the extent of 10-11% in another Cancer species, C. anthonyi.\r\n\r\ndAT is found in all C. antennarius tissues and is localized in the cell nucleus.\r\n\r\nStudies with the E. coli RNA polymerase system show that dAT is copied at a rate 2.6 times as fast as is the main DNA component. dAT also has a higher affinity for the enzyme.\r\n\r\nActinomycin D inhibits the transcription of native primers more than it does that of melted ones. Incorporation of GC into RNA is preferentially inhibited.\r\n\r\nProtamine, bistones Ib, IIb, III and IV, listed in order of decreasing effectiveness, inhibit the RNA synthesis supported by DNA. These basic proteins inhibit incorporation of A and U preferentially indicating preferential binding of the inhibitors to AT rich regions of the template.\r\n\r\nThe RNA synthesized from the dAT template by E. coli RNA polymerase is double-stranded and melts at 67[degrees]C. This rAU has an S value of 5-6 after ribonuclease T1 treatment. This result is due to the low G content of the dAT, 1.8%.\r\n\r\nActinomycin D inhibition studies with crab hepatopancreas tissue indicate that rAU is not synthesized at an appreciable rate in vivo if at all.\r\n\r\nThe RNA from this tissue also contains no rAU as shown by melting and by T1 treatment. The rAU if present would melt sharply and be detectable and would show up as 5-6 S material in sucrose density gradient centrifugation of the hepatopancreas RNA after T1 treatment.\r\n\r\nChromatin is less active as template for RNA synthesis than is whole crab DNA and the chromatin dAT component is particularly repressed." }, { "name": "Yarus, Michael Jeffrey", "degree": "PhD", "year": "1966", "title": "Bacteriophage \u03a6\u03a7-174: Its Sensitivity to Ultraviolet Light and Growth in Starved and Irradiated Cells", "advisor": "Sinsheimer, Robert L.", "url": "https://resolver.caltech.edu/CaltechETD:etd-09232002-152158", "creators": [ { "name": { "family": "Yarus", "given": "Michael Jeffrey" }, "id": "Yarus-Michael-Jeffrey", "display_name": "Yarus, Michael Jeffrey" } ], "advisors": [ { "name": { "family": "Sinsheimer", "given": "Robert L." }, "id": "Sinsheimer-R-L", "role": "advisor", "display_name": "Sinsheimer, Robert L." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/KGXF-FF97", "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document. \r\n\r\nPart I\r\nUltraviolet action spectra for inactivation of [phi chi]-174 virus, its infective single-stranded DNA (SS), and an infective, intracellular and presumably double-stranded DNA (RF) have been determined. The biological activity of the irradiated DNA was measured using bacterial protoplasts.\r\n\r\nThe inactivation cross-section of the RF is nearly an order of magnitude less at all wavelengths than that of either the free single-stranded DNA or the intact virus, which have very similar cross-sections. Besides the difference in magnitude, the action spectrum of RF, when compared to that of SS [phi chi] DNA shows several differences in band shape.\r\n\r\nThe similarity of the free SS and virus sensitivities to radiation in the range 240-302 [megamicrons] suggests that energy of these wavelengths which is effective in inactivation of the virus is that absorbed by the nucleic acid. Below 240 [megamicrons] the DNA is less sensitive than the virus; UV inactivation as a consequence of energy absorbed by the virus protein is a likely explanation of the higher viral sensitivity.\r\n\r\nThe quantum yield for inactivation of the single-stranded DNA is a function of wavelength in the range of wavelengths used, 225-302 [megamicrons]. This may be understood as a result of the variable fraction of absorbed energy localized in pyrimidines. This dependence of quantum yield on wavelength is altered in the case of the whole virus, presumably because of the important role of the protein at low wavelengths. The quantum yield of SS [phi chi] DNA increases slightly with salt concentration, reflecting the existence of some process which is enhanced on contraction of the polymer and the resulting stronger interactions between bases.\r\n\r\nPart II\r\n\r\nThe ability of [phi chi] bacteriophage-infected cells to release progeny after UV irradiation has been examined using both a host possessing host cell reactivation (E. coli C(subscript)N) and one lacking it (E. coli C(subscript s). The UV sensitivity of both free [phi chi] DNA extracted from infected cells and DNA irradiated in situ in the infected cell, as judged by their infectivity to bacterial protoplasts, is sufficient to account for the intrinsic sensitivity of the host-phage complex.\r\n\r\nPart III\r\n\r\nThe burst of a starved bacterium infected with several (phi chi]-174 bacteriophage is usually found to contain descendants of only one of the parents; less often, two phage may multiply. Unstarved cells, in contrast, can support the growth of at least four phage. The unproductive phage seem to convert their parental single-stranded DNA into intracellular, double-stranded RF. These results are interpreted to mean that some factor required by [phi chi] for the production of progeny is limited in starved cells.\r\n\r\nPart IV\r\n\r\nEvidence is presented that starved, UV-irradiated E. coli C which has lost its capacity to support [phi chi] bacteriophage reproduction, has also become unsuitable for the synthesis of infective RF, though the incoming single stranded viral DNA is able to undergo the transition to an RF whose behavior on neutral density gradient analysis is the same as normal RF. Very alkaline conditions appear to activate the inactive RF, releasing infective parental single strands as well as making infective those RF whose strands do not separate." }, { "name": "Denhardt, David Tilton", "degree": "PhD", "year": "1965", "title": "A Biophysical Study of Bacteriophage \u00d8X-174 Replication", "advisor": "Sinsheimer, Robert L.", "url": "https://resolver.caltech.edu/CaltechETD:etd-08082006-140135", "creators": [ { "name": { "family": "Denhardt", "given": "David Tilton" }, "id": "Denhardt-David-Tilton", "display_name": "Denhardt, David Tilton" } ], "advisors": [ { "name": { "family": "Sinsheimer", "given": "Robert L." }, "id": "Sinsheimer-R-L", "role": "advisor", "display_name": "Sinsheimer, Robert L." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/PV89-V879", "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\r\n\r\nTechniques for synchronizing, by cyanide and starvation, the development of [phi]X-174 in all of the infected cells of a culture of E. coli C were developed. The infection of starved cells in buffer, followed by the addition of broth, was found most satisfactory. One-step growth curves of [phi]X and [...] (a plaque morphology mutant of [phi]X that has a longer mean latent period and will not lyse cells in concentrated cultures) were determined. The rate of phage maturation is constant. A probit plot of the percentage of phage released vs. the logarithm of the time after infection is linear.\r\n\r\nThe replication of [phi]X DNA was investigated using E. coli CR (a thymine requiring strain), 5-bromodeoxyuridine, the technique of equilibrium CsCl density gradient centrifugation, and the protoplast assay technique for [phi]X DNA. The DNA combining with the infecting single strand to form the first replicative form (RF) is synthesized after infection from low molecular weigh precursors from the medium. The rate of increase in the amount of infective RF in the culture during the eclipse period is constant, and the RF molecule containing the parental single strand replicates semiconservatively. The conserved subunit is a single strand of DNA. The material constituting the progeny single strands is derived directly from the medium, not from RF.\r\n\r\n[phi]X carrying 2-4 P(32) atoms per particle were used to infect cells in cold medium. The phage and early complexes were killed with an efficiency of 1; mature complexes were inactivated with an efficiency of about 0.2, even though unlabeled and infective RF had been synthesized in them. At about the same time as progeny single strands were synthesized the complexes became completely refractory to suicide. Chloramphenicol did not prevent the P32 suicide efficiency of the complexes from decreasing to about 0.2; complexes formed in chloramphenicol from cells which had not been starved became fully resistant to inactivation, whereas complexes formed from starved cells did not.\r\n\r\nThe ultraviolet radiation sensitivity of [phi]X complexes formed with E. coli C, E. coli Cs (a non host cell reactivating strain), and E. coli CR were determined at various wavelengths, at various stages of development, and under various conditions. Free [phi]X is not host cell reactivable; the target that is observed in the Luria-Latarjet curves of the complex is host cell reactivable. The most uv sensitive structure in the complex is the single strand of DNA synthesized after infection and associated with the infecting single strand. Budr present in this newly synthesized strand will sensitize the complex to uv. Experiments entailing a switch between Budr and thymidine media indicated that the uv sensitive target in the complex could be identified with the single strand of DNA associated with the infecting parental strand. The DNA constituting this strand \"turns over\" each time the parental RF replicates.\r\n\r\nThe uv sensitivity of the bacterial capacity to support [phi]X growth was also studied. Although the colony forming ability of Cs is more sensitive to uv than C, the capacity is not. There was evidence that a single nucleic acid like structure and a large number of protein like structures had to be inactivated in order to destroy the capacity. Starvation of CR for thymine, or growth in Budr, increased the uv sensitivity of the capacity; as the cells in a thymine starved culture underwent thymineless death, they also lost their capacity to support [phi]X growth.\r\n\r\nAction spectra for the complexes and for capacity are presented. The uv sensitivities of MS-2, Bu-[phi]X, and [...] (in the presence and absence of cysteamine) are described." }, { "name": "Filner, Philip", "degree": "PhD", "year": "1965", "title": "Studies on Exponential Cultures of Plant Cells.", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechETD:etd-04082003-112136", "creators": [ { "name": { "family": "Filner", "given": "Philip" }, "id": "Filner-Philip", "display_name": "Filner, Philip" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/HRVF-V763", "abstract": "The properties of tobacco cells growing exponentially in a chemically defined liquid medium were investigated for the purpose of characterizing the exponential plant cell culture and to explore its potential as a developmental model system.\r\n\r\nThe cells multiply exponentially with a generation time of 2 days, and exponential multiplication persists for 4.5 generations. The replication of DNA was studied by means of N[superscript 15] density labeling. All the DNA in the cells replicates in about one generation, and replication proceeds by a semiconservative mechanism. The average cell properties of the culture are not constant during the exponential phase, indicating that the population is not in a steady state condition. The number of cells per group, the water content per cell, the soluble protein content, and the rate of RNA synthesis vary. Enzyme levels of the soluble protein fraction do not vary in parallel with the protein content, but rather they vary in their own characteristic ways.\r\n\r\nThe changes in cell properties are related to changes in the chemical environment which the cells themselves bring about. The cells deplete the medium of phosphorus after three generations, and they deplete it of potassium and nitrogen by the end of the exponential phase. Nitrogen is the growth limiting nutrient, and nitrogen may be supplied either as nitrate or as a complete amino acid mixture. The nitrate reductase (NR) level in the culture falls as the nitrate supply is depleted, so that the enzyme must be induced before the cells can grow when subcultured into a medium in which nitrate is the sole nitrogen source. There is a lag in the onset of mitosis which correlates with the time necessary for NR induction. The initial and terminal events of the exponential phase have been tentatively identified as the induction of NR and the depletion of nitrate, respectively.\r\n\r\nThe regulation of NR involves both substrate induction and end-product repression. NR is induced by nitrate and repressed by a complete amino acid mixture in proportion to the ability of the mixture to meet the nitrogen requirement of the cells. If amino acids are available at a sub-optimal level, and nitrate is also present, sufficient NR is induced and nitrate reduced to bring the nitrogen supply up to the optimal level.\r\n\r\nThe amino acids may be classed as repressors or derepressors, according to their action in the regulation of NR. The derepressors are arginine, lysine, cysteine and isoleucine. A single repressor inhibits the growth of the cells on a nitrate medium as a consequence of the condition of nitrogen starvation which results from NR repression. In the presence of a repressor and an appropriate derepressor, NR is induced and the cells grow. Since the growth of the cells can be controlled through the NR regulatory mechanism, it has been suggested that this mechanism may be used by the whole organism to regulate the growth of its parts." }, { "name": "Gazzaniga, Michael Saunders", "degree": "PhD", "year": "1965", "title": "Some Effects of Cerebral Commissurotomy on Monkey and Man", "advisor": "Sperry, Roger Wolcott", "url": "https://resolver.caltech.edu/CaltechETD:etd-02062003-152539", "creators": [ { "name": { "family": "Gazzaniga", "given": "Michael Saunders" }, "id": "Gazzaniga-Michael-Saunders", "display_name": "Gazzaniga, Michael Saunders" } ], "advisors": [ { "name": { "family": "Sperry", "given": "Roger Wolcott" }, "id": "Sperry-R-W", "role": "advisor", "display_name": "Sperry, Roger Wolcott" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/BWV2-WE04", "abstract": "The following studies were aimed at further clarification of certain of the functional effects produced by brain bisection in primates. They involved also the application and implications of brain bisection for problems of normal cerebral organization. In particular, extensive functional testing was carried out on two human patients in whom brain bisection had been performed for the treatment of severe epileptic seizures. In addition, these human studies were combined with experiments on certain related functions in monkeys.\r\n\r\nIn the human experiments a variety of tests were administered which involved interhemispheric integration of sensory-sensory and sensory-motor information. The psychological capacities of each separated hemisphere were also examined. Results on somesthesis indicated predominately contralateral projection. Ipsilateral responses for some stimuli were apparent in one case suggesting direct afferent pathways may participate in a doubling of somesthetic representation of some body areas. Bilateral cortical projection for all types of somesthesis was indicated in results from the face and head. Visual testing revealed a complete gnostic separation of the left and right visual fields along with a complete lack of subcallosal interhemispheric interaction for any sort of visual stimuli. Motor control of both arms and both legs from one disconnected hemisphere varied but in general was possible in most testing situations. There was no interhemispheric transfer of learning and memory.\r\n\r\nDifferences in intrinsic performance capacities of each separated hemisphere with regard to visual-constructional tasks and to speech and language functions were also observed. The left hemisphere appeared capable of verbal communication by both written and spoken means while the right, although unable to express itself in language of any form, could respond to certain verbal cues. Also, only the right hemisphere was capable of performing visual-constructional tasks.\r\n\r\nRelated studies on monkeys concerning the neural mechanisms responsible for memory and visual-motor coordination, have revealed that pre-operatively learned visual discriminations are retained by only one hemisphere following brain bisection. In addition, control of the ipsilateral arm in split-brain monkeys was found to be impaired in visual learning situations but not in the habitual movements of everyday activity such as reaching for food." }, { "name": "Millette, Robert Loomis", "degree": "PhD", "year": "1965", "title": "Hemoglobin Synthesis in the Maturing Rabbit Reticulocyte", "advisor": "Borsook, Henry", "url": "https://resolver.caltech.edu/CaltechETD:etd-05162003-090149", "creators": [ { "name": { "family": "Millette", "given": "Robert Loomis" }, "id": "Millette-Robert-Loomis", "display_name": "Millette, Robert Loomis" } ], "advisors": [ { "name": { "family": "Borsook", "given": "Henry" }, "id": "Borsook-H", "role": "advisor", "display_name": "Borsook, Henry" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/G8SP-AN14", "abstract": "A method is described for fractionating rabbit reticulocytes by buoyant density centrifugation in an albumin (BSA) gradient. The progressively lighter cells contained more ribosomes and were more active in protein synthesis than the denser cells.\r\n\r\nCytological, physical, and biochemical properties of the fractions showed that the procedure separates the cells according to their degree of physiological maturity. Fractions were examined for their protein synthesizing activity and ribosomal distribution by sucrose gradient analysis. With increasing cell maturation the percent of ribonucleoprotein present as polysomes declined slightly, the pentamer persisted as the major ribosomal aggregate, and the specific activity of the polysomes decreased. The loss of hemoglobin synthesis during reticulocyte maturation is closely correlated with the loss of total ribosomal material and with an increasing percentage of inactive polysomes.\r\n\r\nA fraction of the most immature reticulocytes was labeled with H[superscript 3]-leucine and transfused into a normal rabbit for in vivo maturation. BSA-gradient analysis of blood samples taken at various time intervals confirmed that the position of the cells in a BSA gradient is a function of their age.\r\n\r\nA reticulocyte cell-free amino acid incorporating system is described which is capable, after an initial rapid incorporation, of a linear rate of protein synthesis for at least two hours. This system was used to further investigate the differences between reticulocyte fractions from a BSA gradient. Crossed incubations of ribosomes and supernatants showed that not only the ribosomes become less active but that supernatant factors limit amino acid incorporation in the more mature cells. The nature of the supernatant effect is discussed.\r\n\r\nPreliminary cell-free studies using polysomes and 80S ribosomes, isolated from a sucrose gradient, showed that 80S ribosomes are inactive by themselves. However, in the presence of polysomes they participate in amino acid incorporation. The results indicate that the rate of protein synthesis is a function of the concentrations of both \"monosomes\" and polysomes." }, { "name": "Akinrimisi, E. Olabisi", "degree": "PhD", "year": "1964", "title": "Part I. Properties of Helical Polycytidylic Acid. Part II. Interactions of Purine with Proteins and Amino Acids. Part III. Binding of Basic Proteins to DNA", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechETD:etd-09032002-103837", "creators": [ { "name": { "family": "Akinrimisi", "given": "E. Olabisi" }, "id": "Akinrimisi-E-Olabisi", "display_name": "Akinrimisi, E. Olabisi" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "chair", "display_name": "Bonner, James Frederick" }, { "name": { "family": "Davidson", "given": "Norman R." }, "id": "Davidson-N-R", "role": "member", "display_name": "Davidson, Norman R." }, { "name": { "family": "Horowitz", "given": "Norman" }, "id": "Horowitz-N", "role": "member", "display_name": "Horowitz, Norman" }, { "name": { "family": "Ts'o", "given": "Paul O. P." }, "id": "Ts'o-Paul-O-P", "role": "member", "display_name": "Ts'o, Paul O. P." }, { "name": { "family": "Vinograd", "given": "Jerome" }, "id": "Vinograd-J", "role": "member", "display_name": "Vinograd, Jerome" } ], "option_major": [ "biology" ], "doi": "10.7907/EFG9-2C60", "abstract": "This thesis is divided into three sections. The first part deals with the secondary structure of poly C in acid solution, as revealed by several of its physical-chemical properties in solution. The interpretation of the data was based on previous knowledge of the general properties of polynucleotides in solution and on the properties of poly C monomer.
\r\n\r\nThe second part of the thesis deals with the interaction of purine with the proteins. Conformation changes in the proteins are easily measurable in terms of changes in optical rotation even at the visible wavelength regions. Full use was made of this fact to study the nature of interaction of purine with the proteins. The most significant aspect of the studies is not the theoretical speculation on the mechanism of the interaction, but rather the possible practical applications of the findings. This is briefly considered in the discussion. The mechanism of urea. interaction with the proteins cannot be regarded as solved in spite of the voluminous literature on the subject. Similarly, the mechanism of purine interaction with proteins cannot be regarded as solved. The speculations presented at length on the mechanism of the interactions of purine or urea with the proteins, therefore, merely represent a simplified deduction from available data -- a deduction intended to stimulate more experiments and perhaps a modified interpretation of the nature of the interactions.
\r\n\r\nThe third part of the thesis presents initial findings on a very complex problem -- the relationship of polyvalent polymer-polymer interactions to some of their other physical-chemical and biochemical properties. The conclusions drawn from the data are not new or peculiar to current thinking about the nature of interaction of the histones or protamines to DNA. However, certain satisfactions are derived from the fact that it has been possible to carry out reliable physical-chemical measurements on the nucleohistone and nucleoprotamine systems by means of simple standard techniques. Such data are rare in the nucleohistone or nucleoprotamine literature. No doubt, the confirmation of more complex findings on these very important systems will often require these simple physical-chemical data.
\r\n" }, { "name": "Hamilton, Charles Robert", "degree": "PhD", "year": "1964", "title": "Studies on Adaptation to Deflection of the Visual Field in Split-Brain Monkeys and Man", "advisor": "Sperry, Roger Wolcott", "url": "https://resolver.caltech.edu/CaltechETD:etd-09172002-135454", "creators": [ { "name": { "family": "Hamilton", "given": "Charles Robert" }, "id": "Hamilton-Charles-Robert", "display_name": "Hamilton, Charles Robert" } ], "advisors": [ { "name": { "family": "Sperry", "given": "Roger Wolcott" }, "id": "Sperry-R-W", "role": "advisor", "display_name": "Sperry, Roger Wolcott" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/Q7G8-MN23", "abstract": "Experiments were performed with split-brain monkeys and human subjects to investigate several questions concerning the ability of higher mammals to compensate for errors in reaching produced by optical rearrangements of the visual field. The aim of these experiments was to determine 1) if the adaptive process or effect could be localized to or associated with particular structures or regions of the brain, 2) if the adaptive changes are made in visual, motor, or proprioceptive elements of coordination, and 3) how the process and effect of adaptation compare with other types of learning and memory.\r\n\r\nSubjects were required to practice localizing movements with specified combinations of eyes and limbs while looking through prisms that deflected their visual field. They then were tested for transfer of the acquired adaptation to unpracticed members of the body. Normal monkeys and monkeys with differing degrees of midline section of the cerebral and midbrain commissures and of other structures showed normal adaptive ability with all eye-hand combinations and possessed no deficits when tested for interocular transfer. Normal reaching also appeared to be unaffected by split-brain surgery. Monkeys and human subjects did not transfer the acquired compensations from the practiced to the unpracticed arm in several experiments, although partial transfer to unpracticed members was found with human subjects in certain situations. The adaptive effects did not require vision for a return toward normal coordination.\r\n\r\nIt was concluded that the adaptive process is not organized completely at cortical levels and that subcortical centers are involved in reaching with both normal and adapted coordination. The effect of adaptation seems best described as recalibrations in position sense of particular limbs of the body. Under some conditions these alterations seem to be restricted to the level of the particular joints that were practiced during adaptation, while under other conditions generalization to unpracticed members was observed. The critical factor for the occurrence of generalization appears to be the kind or amount of movement rather than differences in visual stimulation. Adaptation differs from other forms of sensory and motor learning by the presence of interocular transfer in split-brain monkeys and by the complete absence of intermanual transfer in monkeys and man under some conditions." }, { "name": "Barrett, Dennis", "degree": "PhD", "year": "1963", "title": "The Parallel Accumulation and Distribution of Two Purine-Oxidizing Enzymes During Frog Development", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02272012-160448464", "creators": [ { "name": { "family": "Barrett", "given": "Dennis" }, "id": "Barrett-Dennis", "display_name": "Barrett, Dennis" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/0TRM-B219", "abstract": "The patterns of two specialized and functionally related\r\nenzymes were investigated in the developing frog (Rana temporaria\r\nand Rana catesbeiana). Xanthine dehydrogenase (E.C. 1.2.1.) and\r\nuricase (E. C. 1. 7 . 3 . 2.) were found to remain apparently constant\r\nduring the first ten days of normal development, both rising rapidly\r\nthereafter, beginning at the same larval stage. Comparison with\r\nenzymes of similar developmental pattern suggests this phase-specific\r\nincrease is in preparation for the onset of digestive activity.
\r\n\r\nThe same parallelism between uricase and xanthine dehydrogenase\r\nwas observed when determinations were extended to the organ\r\nlevel. Each enzyme was found in quantity in liver and kidney, and in\r\ntrace amounts, all of about the same magnitude, in several other\r\norgans tested.
\r\n\r\nBy detecting radioactive tracer amounts of product, very\r\nlow levels of xanthine dehydrogenase were measured in embryonic\r\nstages, probably corresponding to the order of small numbers of\r\nmolecules per cell.
\r\n\r\nEvidence was gathered to suggest that embryonic uricase\r\naccumulation is neither induced by its substrate nor involved with\r\nthe subcellular particles in which the enzyme is found.
\r\n" }, { "name": "Cole, Thomas Alan", "degree": "PhD", "year": "1963", "title": "Peptidyl Acylating Agents in the Pupae of Drosophila melanogaster and their Possible Relationship to Protein Synthesis", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02232012-162634131", "creators": [ { "name": { "family": "Cole", "given": "Thomas Alan" }, "id": "Cole-Thomas-Alan", "display_name": "Cole, Thomas Alan" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/3V4P-DY85", "abstract": "The effects of hydroxylamine and tyrosine on homogenates\r\nof early Drosophila pupae have been studied by several criteria.\r\nBoth low speed and high speed supernatants of the\r\nhomogenized pupae give positive tests for hydroxamic acids\r\nafter incubation with hydroxylamine. A comparison of the\r\nhydroxamic acid forming abilities of the supernatants shows\r\nthat the low speed supernatant is the more active and that\r\ntyrosine increases hydroxamate formation in the low speed\r\nbut not in the high speed supernatants.
\r\n\r\nBoth hydroxylamine and tyrosine have definite effects\r\non alkaline phosphatase activity in the low speed supernatant. \r\nTryosine causes an increase in the activity upon incubation\r\nat 0\u00b0C and 25\u00b0C. Hydroxylamine decreases the activity regardless\r\nof the temperature or the presence of tyrosine.
\r\n\r\nBoth tyrosine and hydroxylamine affect the distribution\r\nof ninhydrin-positive compounds of dialyzates of low speed\r\nsupernatants. Hydroxylamine effectively reduces the amount\r\nof the acidic components, but its effect is slightly modified\r\nby tyrosine. The presence of tyrosine alone increases the\r\namount of acidic materials as compared to the untreated sample.\r\nIn general, the ninhydrin-positive compounds are more sensitive\r\nto the presence of hydroxylamine than of tyrosine.
\r\n\r\nSome preliminary work on the nature of the compounds in\r\nquestion shows that they contain more than one amino acid.\r\nThe data on the nature of these compounds is consistent with\r\nthe proposition that peptidyl hydroxamates are the Fe ^(+++)-\r\npositive materials.
\r\n\r\nNaturally-occurring peptides and the current understanding\r\nof protein synthesis are reviewed. The possibility that\r\npeptides per se are incorporated into the proteins in Drosophila\r\npupae is discussed and mechanisms for this possibility\r\nare explored.
\r\n" }, { "name": "Morr\u00e9, D. James F.", "degree": "PhD", "year": "1963", "title": "Biochemistry of Cell Extension", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechTHESIS:11022012-144309062", "creators": [ { "name": { "family": "Morr\u00e9", "given": "D. James F." }, "id": "Morr\u00e9-D-James-F", "display_name": "Morr\u00e9, D. James F." } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/G6WY-AS68", "abstract": "By subjecting various plant tissues to conditions which influence\r\ngrowth, such as treatment with the growth hormone, 3-indoleacetic acid\r\n(IAA), changes in certain cytoplasmic constituents were observed which\r\nparallel simultaneous changes in the mechanical properties of the cell\r\nwall.
\r\n\r\nTreatment of maize roots for one hour in the presence of concentrations\r\nof IAA that normally promote shoot growth and, therefore, inhibit\r\nroot growth followed by measurement of the deformability of the\r\nrapidly elongating region under artificially imposed load, demonstrated\r\na concentration-dependent, IAA-induced plasticity component, not observable\r\nin untreated roots. The response is transient; half-maximal plasticity\r\nis induced by ca. 5 x 10^(-7) M IAA, one hour after treatment (a\r\nconcentration comparable to that of Avena sections). Increased deformability\r\nis paralleled by increased growth of root sections, also transient,\r\nwith maximum IAA-induced increase in growth rate coinciding with the\r\nmaximum for increased wall plasticization. The initial response of maize\r\nroots to IAA, therefore, resembles that of Avena coleoptiles both qualitatively\r\nand quantitatively. The end result, however, is to effectively\r\nshorten the period over which the root can elongate.
\r\n\r\nAssociated with increased plasticity and growth rate of maize roots\r\nis rapid formation (or maintenance) of a protein-bound carbohydrate\r\nfraction. Disruption of the complex by such agents as extremes of pH or\r\norganic solvents results in increased turbidity of aqueous solutions and\r\nacquisition of solutility properties characteristic of lipides. Results\r\nof preliminary characterization studies also suggest that material is of\r\nlipoprotein origin.
\r\n\r\nEvidence for a similar fraction, increased in amount by treatment of\r\nthe tissue with IAA, has been extended to include Avena coleoptiles, pea\r\nepicotyls and pea embryo axes. In addition to lipide-soluble components,\r\nthese fractions contain approximately equimolar quantities of carbohydrate\r\n(including hexose) and of esterified phosphate. The material, therefore,\r\nhas been designated as a protein-bound glycolipide (PGL).
\r\n\r\nA cytoplasmic origin of PGL is suggested. Also associated with the\r\n2- to 4-fold increase in amount of protein-bound PGL is a decrease in the\r\nheat coagulability of cytoplasmic proteins. Similarily, a portion of an\r\napparent IAA-induced increase in acid phosphatase activity may be\r\nattributable to increased stabilization as well. An electron microscopic\r\nsurvey of IAA effects on the fine structure of subcellular organelles\r\nrevealed no major structural changes, however, the number of vesicles\r\nassociated with the central Golgi structure of both maize roots and Avena\r\nsections may be increased by IAA treatment.
\r\n\r\nA study of cell wall pectic constituents has revealed that although\r\nversene-soluble pectin does represent a solubility class distinct from\r\nhot water-soluble pectin, the conditions for extraction do not correspond\r\nto those of the classical residual pectin fraction. The existence of\r\npectin in the cold buffer-soluble, 70% ethanol-insoluble fraction of\r\nAvena coleoptiles (cold water-soluble pectin) seems doubtful, however.
\r\n\r\n" }, { "name": "Studier, Frederick William", "degree": "PhD", "year": "1963", "title": "Studies on the DNA of Bacteriophage T7", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechTHESIS:11092012-141743346", "creators": [ { "name": { "family": "Studier", "given": "Frederick William" }, "id": "Studier-Frederick-William", "display_name": "Studier, Frederick William" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/GV9J-HV27", "abstract": "The molecular weight of T7 DNA was determined to be 15-17 x 10^6 by\r\nthe method of sedimentation equilibrium in a CsCl density gradient. Possible\r\nsources of error in the optical reproduction of the concentration\r\ndistribution in the cell were considered and evaluated. Comparison of\r\nthe above molecular weight with the weight of the total DNA content of\r\nT7 phage determined by Davison and Freifelder (22) suggests: 1) that the\r\nentire DNA content of T7 bacteriophage can be released into solution as\r\na single molecule and 2) that the density gradient method for determining\r\nmolecular weights of homogeneous DNA samples is accurate within 10-20%.
\r\n\r\nThe denaturation and renaturation of T7 DNA was studied using\r\nformamide as the denaturing agent. The complete cycle of denaturation\r\nand renaturation can thus be studied at room temperature without degradation\r\nof the primary structure of DNA.
\r\n\r\nKinetics of denaturation were found to be essentially independent of\r\nconcentration but were not first order, the rate falling off with time.\r\nIrreversible denaturation does not commence until the DNA has reached\r\n80-90% of its full hyperchromicity. Samples of DNA isolated from denaturing\r\nconditions appear to be mixtures of fully denatured molecules and\r\nmolecules which exhibit native characteristics to a high degree. Stirring\r\nsolutions of DNA under denaturing conditions markedly accelerates the rate\r\nof denaturation. It seems possible that the molecular configuration of\r\nDNA molecules in intermediate states of denaturation may be a factor in\r\nstability to denaturation.
\r\n\r\nOptimally renatured T7 DNA has 85-90% of the native hyperchromic\r\neffect and bands at a slightly denser position than native DNA in a CsCl\r\ndensity gradient. Preliminary studies indicate second order kinetics for\r\nthe renaturation reaction.
\r\n\r\n\r\n\r\n" }, { "name": "Weisberg, Robert Avrum", "degree": "PhD", "year": "1963", "title": "Studies on Mouse Embryo Cultures Infected with Polyoma Virus", "advisor": "Dulbecco, Renato", "url": "https://resolver.caltech.edu/CaltechTHESIS:10082012-162250806", "creators": [ { "name": { "family": "Weisberg", "given": "Robert Avrum" }, "id": "Weisberg-Robert-Avrum", "display_name": "Weisberg, Robert Avrum" } ], "advisors": [ { "name": { "family": "Dulbecco", "given": "Renato" }, "id": "Dulbecco-R", "role": "advisor", "display_name": "Dulbecco, Renato" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/D4P8-0E51", "abstract": "After exposure of mouse embryo cultures to high concentrations\r\nof Py, a variable fraction of the cell population is converted to virus\r\nproducers, but a fraction also survives and proliferates. The\r\nsurviving fraction can be 20% of the population at input virus:cell\r\nratios of 500 pfu/cell. Resistance to the cytocidal action of the virus \r\nin mouse embryo cultures is due neither to interferon nor to genetically\r\nresistant cells; it appears to be due to a transient physiological state\r\nof the cells.
\r\n\r\nNo transformed cells have been found among the cells surviving\r\na brief exposure to high concentrations of virus. Cultures derived\r\nfrom these cells by growth in antiviral medium resemble uninfected\r\ncultures in cell morphology, growth pattern, and sensitivity to\r\nreinfection. Transformed cells arise only in cultures which are\r\nexposed to Py over a period of two to five weeks. It has been shown\r\nthat clonal cultures respond in the same way to Py infection as do un-\r\ncloned mouse embryo cultures; thus, transformation does not result\r\nfrom the infection of rare \"transformable variants\" preexisting in the\r\ncell population.
\r\n\r\nChanges similar to the transformation which takes place in\r\ninfected mouse embryo cultures also occur, and rapidly, in uninfected\r\ncultures. The occurrence of these changes complicates the analysis of \r\nPy induced transformation. It has been shown that \"spontaneous\" and\r\nvirus induced transformation are two different phenomena, since\r\ntransplantable cells arising in infected cultures differ antigenically\r\nfrom those arising in uninfected cultures. The relationship between\r\nalterations of cell lines observable in vitro and the ability of these\r\nlines produce tumors upon implantation have been studied; definite\r\ncorrelations have been demonstrated between these properties. These\r\nfacts have been discussed in the light of various theories of Py\r\ninduced transformations.
" }, { "name": "Guthrie, George Drake", "degree": "PhD", "year": "1962", "title": "Studies on the Interaction of Subviral Particles of the Bacteriophage \u03a6X174 with Protoplasts of Escherichia coli", "advisor": "Sinsheimer, Robert L.", "url": "https://resolver.caltech.edu/CaltechETD:etd-08092006-095212", "creators": [ { "name": { "family": "Guthrie", "given": "George Drake" }, "id": "Guthrie-George-Drake", "display_name": "Guthrie, George Drake" } ], "advisors": [ { "name": { "family": "Sinsheimer", "given": "Robert L." }, "id": "Sinsheimer-R-L", "role": "advisor", "display_name": "Sinsheimer, Robert L." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/197P-A064", "abstract": "Interaction of Subviral Particles of Bacteriophage [phi]X174 with Protoplasts of Escherichia coli. Part I: Infection of Protoplasts of Escherichia coli by Subviral Particles of Bacteriophage [phi]X174. G. D. Guthrie and R. L. Sinsheimer, J. Mol. Biol., (1960), 2, 297-305. Infection of protoplasts of E. coli leading to the production of mature phage can occur with subviral particles of phage [phi]X174, including the purified DNA of the phage. Methods for obtaining the proper conditions for infection are described. Results of experiments describing the characteristics of the infection and the nature of the subviral particles causing the infection are presented. Evidence is presented that net synthesis of mature [phi]X174 can be obtained from infection of protoplasts by purified [phi]X174 DNA.\r\n\r\nPart II: Initial Studies on Protoplasts and [phi]X-DNA as a system for investigating virus replication. G. D. Guthrie (unpublished thesis research). Further investigations on the interaction of [phi]X-DNA with protoplasts are described. General techniques to be employed in using the system for the study of the replication of virus are outlined.\r\n\r\nData from investigations on the initial \"infection\" process of [phi]X-DNA are presented. From these data, factors influencing the level of infection are discussed and a simple mathematical relationship relating these parameters to the infection is proposed." }, { "name": "Mendelson, Martin", "degree": "PhD", "year": "1962", "title": "Studies on the Activation of Crustacean Mechanoreceptors. The Movement Receptors of the Crab Pachygrapslls crassipes Randall and the Movement and Stretch Receptors of the Crayfish Procambarus clarkii (Girard)", "advisor": "Wiersma, Cornelius A. G", "url": "https://resolver.caltech.edu/CaltechTHESIS:08032011-135808240", "creators": [ { "name": { "family": "Mendelson", "given": "Martin" }, "id": "Mendelson-Martin", "display_name": "Mendelson, Martin" } ], "advisors": [ { "name": { "family": "Wiersma", "given": "Cornelius A. G" }, "id": "Wiersma-C-A-G", "role": "advisor", "display_name": "Wiersma, Cornelius A. G" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/8QY0-4C59", "abstract": "The activation of crab movement receptors and of crayfish movement and stretch receptors was studied. It was found that the adequate stimulus to movement receptors is a change of the length of the elastic strand into which they send their distal processes. One group of movement receptors responds to shortening of the strand; and another\r\ngroup to its lengthening. A hypothesis is presented that accounts for the differential sensitivity of these two groups of cells, and the difference between movement and position receptors in the same sense organ. It was found that the movement receptors may be activated by nicotine\r\nas well as by mechanical means; and that nicotine activation differs in a significant manner from mechanical activation. The unusual spiking pattern of movement receptors was examined using intracellular and extracellular recording. The mode of activation by nicotine and the intracellular recording data are combined to form a partial explanation of the movement receptors\u2019 discharge pattern. Caffeine was found to block the mechanical activation of movement receptors and it is concluded\r\nthat this is due to a direct effect on the mechanotransducer membrane. Several drugs which affect the crayfish stretch receptor were found to have no effect on movement receptors and conversely nicotine and caffeine were found to have no effect on stretch receptors. Stretch receptors were subjected to the same mechanical stimuli used on the movement receptors and the responses of the two types of end organs compared. The similarities and differences of movement and stretch receptors are discussed.\r\n" }, { "name": "Shreffler, Donald Cecil", "degree": "PhD", "year": "1962", "title": "I. Genetic Studies of Mouse Serum Protein Types. II. Molecular Hybridization of Sheep Hemoglobins", "advisor": "Owen, Ray David; Vinograd, Jerome Rubin", "url": "https://resolver.caltech.edu/CaltechETD:etd-06292004-095342", "creators": [ { "name": { "family": "Shreffler", "given": "Donald Cecil" }, "id": "Shreffler-Donald-Cecil", "display_name": "Shreffler, Donald Cecil" } ], "advisors": [ { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "advisor", "display_name": "Owen, Ray David" }, { "name": { "family": "Vinograd", "given": "Jerome Rubin" }, "id": "Vinograd-J-R", "role": "advisor", "display_name": "Vinograd, Jerome Rubin" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/PPYP-QM84", "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\r\n\r\nInvestigations were conducted to detect serum protein variant systems in mice and to define their genetic control. Three systems were detected. A pair of codominant alleles at a locus in the second linkage group controls electrophoretically different transferrin types. An electrophoretic difference among inbred lines in the presence or absence of a prealbumin is controlled by a single autosomal locus without dominance. A serologically detected system is controlled by a single locus closely linked or identical to the H-2 locus in the ninth linkage group. The variant component seems to be an [alpha]-globulin of high molecular weight. The principal difference between serum types is quantitative; qualitative differences are not excluded. The serum component is not detectably related serologically to H-2 erythrocyte antigens. The effects of development, pregnancy and stress upon these serum components were studied. Using these three serum variants as markers, the sera of homologous radiation chimeras were examined for donor type proteins. These occasionally appear transitorily, but long-term survivors having entirely donor erythrocytes have only host type serum proteins.\r\n\r\nMolecular hybridizations were performed with the two known sheep hemoglobin variants. Radioactive and ferriheme labels showed some exchange of subunits between variant types, but less than expected. Incomplete exchange is apparently due to inadequate asymmetric dissociation or to partial incompatibilities between both subunits of the two hemoglobins. The electrophoretic difference between the two types resides mainly, if not entirely, in one of the subunits." }, { "name": "Susman, Millard", "degree": "PhD", "year": "1962", "title": "The Size of the Mating Group in Bacteriophage T4", "advisor": "Delbruck, Max", "url": "https://resolver.caltech.edu/CaltechETD:etd-08092006-090416", "creators": [ { "name": { "family": "Susman", "given": "Millard" }, "id": "Susman-Millard", "display_name": "Susman, Millard" } ], "advisors": [ { "name": { "family": "Delbruck", "given": "Max" }, "id": "Delbr\u00fcck-M", "role": "advisor", "display_name": "Delbruck, Max" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/5M8C-EM53", "abstract": "In unequal-input, three-factor phage crosses, the kinetics of recombination should depend on the number of vegetative phages participating in each mating. Among a selected class of progeny recombinant for two closely linked markers, a third, freely-segregating marker will achieve genetic equilibrium immediately if the mating group is very large but will show a drift toward equilibrium if the mating group is small. Data are presented for T4 crosses which are in good agreement with the qualitative predictions for small group (pairwise) mating.\r\n\r\nMating parameters have been measured which permit calculation of the expected kinetics of recombination for various mating group sizes. The predictions are calculated using Steinberg and Stahl's theory of formal phage genetics. The data do not conform precisely to the theory, but it is argued that the disagreement cannot alter the conclusion that the T4 mating group is small.\r\n\r\nThe significance, or insignificance, of this finding is discussed in the light of recent discoveries concerning the geometry of recombination in T4." }, { "name": "Trevarthen, Colwyn Boyd", "degree": "PhD", "year": "1962", "title": "Studies on Visual Learning in Split-Brain Monkeys", "advisor": "Sperry, Roger Wolcott", "url": "https://resolver.caltech.edu/CaltechTHESIS:08192011-102232875", "creators": [ { "name": { "family": "Trevarthen", "given": "Colwyn Boyd" }, "id": "Trevarthen-Colwyn-Boyd", "display_name": "Trevarthen, Colwyn Boyd" } ], "advisors": [ { "name": { "family": "Sperry", "given": "Roger Wolcott" }, "id": "Sperry-R-W", "role": "advisor", "display_name": "Sperry, Roger Wolcott" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/YFG6-1968", "abstract": "The unity of perception and its divisibility were examined by a method of double learning. Polarized light and polarizing filters were used to present monkeys with two contradictory visual tasks simultaneously, one visible\r\nto each eye. Subjects were trained after surgical division of the visual pathways at the optic chiasm, and after the cerebral cortices were separated by cutting the corpus callosum. The distribution of learning between the two\r\nhalves of the brain gave information about the location of visual learning, and about the relationship between visual attention and the intention to respond with a particular limb. Two subjects learned conflicting tasks simultaneously.\r\nIn many tests, however, there remained some interaction between the two halves of the brain. This led to selective learning by one eye, the other eye remaining unretentive though it was open throughout training. In tasks involving brightness and color discriminations, there was significant interocular transfer of learning in spite of the surgery. It is concluded that the two surgically separated cerebral hemispheres may function independently in memorizing a visual pattern, but that there are also avenues for their communication. The motor system remains coordinated after split-brain surgery, although there is a tendency for preferential pairing of eye and hand of opposite sides of the body after surgery. Some visual tasks were found to involve interhemispheric processes to a higher degree than others. Visual recognition of comparative size, requiring interocular comparison, was found to survive chiasm and callosum section.\r\n" }, { "name": "Bernstein, Harris", "degree": "PhD", "year": "1961", "title": "I. Imadazole Compounds Accumulated by Purine Mutants of Neurospora crassa. II. Complementation Studies with Isoleucine-Valine Mutants of Neurospora crassa", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechETD:etd-03172006-084834", "creators": [ { "name": { "family": "Bernstein", "given": "Harris" }, "id": "Bernstein-Harris", "display_name": "Bernstein, Harris" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/S02K-EV24", "abstract": "PART I:\tImidazole Compounds Accumulated by Purine Mutants of Neurospora crassa:\r\n\r\nThe mycelis of mutants defective at seven adenine loci were investigated for imidazole accumulation. By the use of a paper chromatographic technique five imidazoles not present in wild type were discovered in the mutants. Two of these were isolated and characterized as the ribotide and riboside of 5-amino-4-imidazole-N-succinocarboxamide. The third compound was identified as 5-aminoimidazole riboside, and the fourth as 5-amino-4-imidazolecarboxamide riboside. The distribution of these accumulated compounds among the mutants allowed a correlation between the adenine loci and the steps of purine biosynthesis.\r\n\r\nPart II: Complementation Studies with Isoleucine-Valine\tMutants of Neurospora crassa:\r\n\r\nA total of 616 mutants capable of growth on a medium supplented with isoleucine and valine, but not on minimal medium, were obtained through the use of 9 different mutagens. On the basis of heterokaryon complementation tests all of the mutants could be allocated to five groups. These groups were designated val-1, val-2, iv-1, iv-2, and iv-3. Mutants in the val-1 and val-2 groups required valine as the sole supplement. Members of the iv-1 group were probably blocked in the dehydrase step of isoleucine-valine biosynthesis, and were characterized by slow growth between 4 and 6 days after inoculation on minimal medium. Evidence was also presented to indicate that iv-3 mutants were blocked in the condensing step of isoleucine-valine biosynthesis. An extensive program of complementation testing was performed among mutants within both the iv-2 and the iv-3 groups. The results of these tests allowed, in each case, the formulation of complementation map. However, three iv-3 mutants were found which could not be reconciled with any linear pattern. An interesting feature of the iv-3 studies, and to some extent of the iv-2 studies, was the finding that among the mutants found the same patterns of complementation interaction often reoccured. Mutants with the same complementation behavior constitute a complementation subgroup. Each of these subgroups usually contained mutants representative of several different mutagenic treatments. The hypothesis was considered that this observed clustering reflects an intrinsic property of the genetic locus --- namely that each locus has associated with it a unique pattern of discrete complementation subgroups. It was hoped that these genetc regularities would prove, in the course of further biochemical investigations, to relate in a direct way to the structural properties of the enzyme molecule." }, { "name": "Hardesty, Boyd Archer", "degree": "PhD", "year": "1961", "title": "The Biochemistry of Cytochrome C in Relation to Maternally Inherited Phenotypes of Neurospora", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechETD:etd-04032006-102552", "creators": [ { "name": { "family": "Hardesty", "given": "Boyd Archer" }, "id": "Hardesty-Boyd-Archer", "display_name": "Hardesty, Boyd Archer" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/NQE0-YY21", "abstract": "Cytochrome c from Neurospora was isolated and its properties studied with respect to its role as a protein accumulated in the extrachromasomal mutants, poky and mi 3. All samples of cytochrome isolated using several different procedures were electrophoretically heterogeneous. Further, electrophoretic differences were observed in the cytochrome c isolated from the different strains of Neurospora although cytochrome c from poky and wild type appeared homogenous during sedimentation and had very similar sedimentation coeffiecients. In contrast to wild type Neurospora, cytochrome c in homogenates of 2.5 day old poky was found to remain largely in the supernatant fraction after high speed centrifugation suggesting that the enzyme in poky is not bound to mitochondria or any other particulate, subcellular structure.\r\n\r\nLipid extracts from poky were found to contain up to 36 times the amount of total free fatty acids present in similar extracts from wild type although the chemically bound fatty acids of the two strains were similar. The interaction of free fatty acids with mammalian cytochrome c was studied in detail and it was suggested that they may cause an unfolding of the protein portion of the cytochrome c molecule therby exposing the heme to direct interaction with such compunds as hydrogen peroxide or molecular oxygen. Thus the large amounts of free fatty acid present in poky might allow its cytochrome c to function as the terminal oxidase in place of cytochromes a + a3 present in wild type Neurospora but not found in poky." }, { "name": "McCalla, Dennis Robert", "degree": "PhD", "year": "1961", "title": "I. Chemical Study of Necrotic Com Mutants. II. The Metabolism of a Kinin. III. The Chemical Nature of an Insect Gall Growth Factor", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechETD:etd-04102006-135936", "creators": [ { "name": { "family": "McCalla", "given": "Dennis Robert" }, "id": "McCalla-Dennis-Robert", "display_name": "McCalla, Dennis Robert" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/ZQ00-1H14", "abstract": "Part I:\r\n \r\nThe leaves of nec rd (a single gene necrotic maize mutant) become necrotic when they are exposed to light for a few days. Extensive analysis has failed to reveal any difference in chemical composition between leaves of normal plants and of homozygous nec rd plants before any necrotic symptoms are visible (prenecrotic leaves). Treatment of nec rd seedlings with various metabolites (B vitamins, purines, pyrimidines, amino acids, etc.) did not prevent the appearance of necrosis. The rate of photosynthesis of prenecrotic leaves is normal at low light intensities but 20 to 50% of normal at saturating light intensity. C14O2 feeding experiments indicate that the carbon fixing reactions function normally in the mutant. Hill reaction rates are also similar in mutant and normal plants, as is the metabolism of labelled inorganic phosphate. CMU, which specifically inhibits photosynthesis to the extent of about 90%, delays the onset of visible necrotic damage and reduces the severity of subsequent necrotic symptoms. It is suggested that the nec rd lesion is in some reaction associated with photosynthesis and that it causes the accumulation of one or more toxic substances. These lower rate of photosynthesis and damage cell membranes. The necrotic phenotype would appear to be the result of the breakdown of cell compartmentalization.\r\n\r\nPart II:\r\n\r\nThe kinin, 6-benzylaminopurine (benzyladenine), is converted to a number of low molecular weight materials by senescing leaves of Xanthium pensylvanicum Wall.(cocklebur). A major product is the riboside, benzyladenosine, which has been identified by comparison of its properties with those of benzyladenylic synthesized enzymatically using E. coli nucleoside phosphorylase and by degradative studies. The ribotide, benzyladenylic acid, also appears to be present. Labelled adenylic, guanylic and inosinic acids are produced, as are small amounts of adenine and guanine. Substantial amounts of label are also found in urea and in a ureide. Small amounts of labelled adenylic and guanylic acids are found in the RNA of the leaf, but benzyladenylic acid itself does not appear to be incorporated into RNA in measurable amounts.\r\n\r\nPart III:\r\n\r\nLow molecular weight material obtained from excised accessory glands of Pontania pacifica (gall-wasp) promotes the growth of Pontania galls on Salix alba (willow). Paper chromatographic analysis has indicated that uridine, uric acid and two unidentified adenine containing compounds are prominent constituents of this mixture. Uric acid and the two adenine containing compounds substantially increase the growth rate of small galls from which the larva has been removed while uridine has slight growth promoting activity. Various related compounds (e.g. adenosine, adenine and guanosine) also have growth-promoting activity. It appears likely that such compounds play an important role in the growth and development of Pontania galls." }, { "name": "Roller, Ann", "degree": "PhD", "year": "1961", "title": "Studies on the Replication and Transfer to Progeny of the DNA of the Bacteriophage T4", "advisor": "Meselson, Matthew S.", "url": "https://resolver.caltech.edu/CaltechETD:etd-08092006-124948", "creators": [ { "name": { "family": "Roller", "given": "Ann" }, "id": "Roller-Ann", "display_name": "Roller, Ann" } ], "advisors": [ { "name": { "family": "Meselson", "given": "Matthew S." }, "id": "Meselson-M-S", "role": "advisor", "display_name": "Meselson, Matthew S." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/DN8N-DF94", "abstract": "The purpose of the present research has been to learn more about the mechanism of replication and ultimate fate of the DNA of the phage T4. The distribution of parental material in DNA molecules in the replicating pool and in the DNA obtained from progeny phage has been examined. The association of parental atoms with the r genotype has also been examined.\r\n\r\nPhage in which the nitrogen and carbon we re replaced by the stable heavy isotopes N15 and C13 were used to infect unlabeled bacteria. At various times after infection, aliquots of the culture were removed and lysed; finally, progeny phage were harvested and lysed. DNA molecules containing heavy atoms show a corresponding increase in their density. Separation is achieved by density-gradient centrifugation.\r\n\r\nMost parental atoms transferred to progeny are found in DNA of light density. Following fragmentation, DNA containing parental atoms is found to have shifted to densities between those of light and half-heavy DNA. In the replicating pool, half-heavy DNA is also found. It is concluded that transferred DNA is incorporated into hybrid units which are attached to larger segments of completely new DNA. The parental atoms may comprise a part of one strand of a duplex DNA molecule.\r\n\r\nAn analysis was made of the density distribution of the viable progeny of a multiple infection with T4r and r+, one of which was labeled with N15 and C13. At densities greater than that of the average progeny, relatively more progeny of the genotype of the labeled parent are found. Thus, parental atoms tend to remain associated with parental genotype." }, { "name": "Steinberg, Charles Myron", "degree": "PhD", "year": "1961", "title": "Part One: Some Theoretical Problems Arising in the Genetics of Bacteriophage. Part Two: A Critical Test of a Current Theory of Genetic Recombination in Bacteriophage", "advisor": "Delbruck, Max", "url": "https://resolver.caltech.edu/CaltechETD:etd-08082006-084127", "creators": [ { "name": { "family": "Steinberg", "given": "Charles Myron" }, "id": "Steinberg-Charles-Myron", "display_name": "Steinberg, Charles Myron" } ], "advisors": [ { "name": { "family": "Delbruck", "given": "Max" }, "id": "Delbr\u00fcck-M", "role": "advisor", "display_name": "Delbruck, Max" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/YYKR-0Q08", "abstract": "ABSTRACT OF PART ONE:\r\n\r\nCalculations are presented giving the distribution of clone-sizes of mutants arising in an \"equilibrium pool\" of vegetative phage. It is assumed that replication and maturation are random-in-time processes. The equilibrium pool model is an idealization of current notions about a major portion of the phage life cycle. The calculations are compatible with experimental measurements of the mutant clone-size distribution. It is also shown that the model can account for the observed variance in total burst size.\r\n\r\nA generalized theory of the kinetics of genetic recombination in phage is presented. Essential assumptions of the theory are that recombination occurs as a result of discrete interactions or matings, and that the particles which participate in a mating are randomly chosen from the entire population of the vegetative pool. No restrictions are placed upon the number of particles that participate in the mating or upon the mechanism by which recombination occurs as a result of the mating. Experimental observations leading to the theory are discussed as well as the prospects for experimental determinations of the participation number and the mechanism of recombination.\r\n\r\nABSTRACT OF PART TWO:\r\n\r\nResults are reported concerning the segregation pattern of loosely linked outside markers within a selected class of particles which are recombinant for closely linked internal markers:\r\n\r\n(a) The outside markers maintain qualitatively normal linkage relations with the internal markers and with each other. This is true whether one or two exchanges are required to produce the selected recombinant class.\r\n\r\n(b) The region of HNI extends into the intervals adjacent to those in which the exchanges are selected but does not extend into a non-adjacent interval.\r\n\r\n(c) Multiple exchanges within the selected class occur with little, if any, additional interference.\r\n\r\n(d) A significant fraction of the selected recombinant class is heterozygous for at least the adjacent outside markers.\r\n\r\nIn summary, aside from the \"complications\" of negative interference and heterozygosis, the segregation pattern of outside markers is \"normal.\"\r\n\r\nIn view of current notions concerning the mechanism of recombination in phage, the \"complications\" are to be expected, while the \"normal\" result leads to a paradox. According to observations of other workers, recombinants for very closely linked markers arise as segregation products of phage heterozygotes, and furthermore, such heterozygotes are recombinant for outside markers. On the other hand, result (a) above means that recombinants for closely linked markers are not necessarily recombinant for outside markers. Some possible resolutions of this paradox are discussed." }, { "name": "Weiler, Ivan Jeanne Mayfield", "degree": "PhD", "year": "1961", "title": "Restoration of Visual Acuity after Optic Nerve Section and Regeneration, in Astronotus ocellatus Agassiz", "advisor": "Sperry, Roger Wolcott; Van Harreveld, Anthonie", "url": "https://resolver.caltech.edu/CaltechTHESIS:04122011-084330696", "creators": [ { "name": { "family": "Weiler", "given": "Ivan Jeanne Mayfield" }, "id": "Weiler-Ivan-Jeanne-Mayfield", "display_name": "Weiler, Ivan Jeanne Mayfield" } ], "advisors": [ { "name": { "family": "Sperry", "given": "Roger Wolcott" }, "id": "Sperry-R-W", "role": "advisor", "display_name": "Sperry, Roger Wolcott" }, { "name": { "family": "Van Harreveld", "given": "Anthonie" }, "id": "Van-Harreveld-A", "role": "co-advisor", "display_name": "Van Harreveld, Anthonie" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/KE2Y-0C22", "abstract": "The visual acuity of a cichlid fish, Astronotus ocellatus Agassiz, was measured by means of a conditioned visual discrimination response. The average minimum separable visual angle was 5.3\u2019, as measured in 16 fish with at least one normal eye; this corresponded closely to the fineness of grain of the retinal receptor mosaic.\r\nIn 12 fish, acuity of one experimental eye was measured after the optic nerve had been transected and allowed to regenerate. The value for postregenerative acuity was then compared with a previous value for acuity of one normal eye in one and the same fish, in each case. Restoration of acuity by regenerative processes averaged 78.4%. This high figure shows that formation of functional, specific synaptic contacts probably does not occur on a chance basis." }, { "name": "Maizel, Jacob Valentine", "degree": "PhD", "year": "1960", "title": "Isolation and Partial Characterization of Avenacin, an Antibiotic-Like Substance from Oats", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06162006-113638", "creators": [ { "name": { "family": "Maizel", "given": "Jacob Valentine" }, "id": "Maizel-Jacob-Valentine", "display_name": "Maizel, Jacob Valentine" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/972K-2K08", "abstract": "A procedure has been described for isolation and bioassay of a potent antifungal substance from the roots of oat seedlings. The purified substance has been named avenacin. Avenacin gives half-maximal inhibition of Neurospora at a concentration of 0.4 \u00b5g/ml and has been found to inhibit a number of plant pathogenic fungi and Mycobacterium tuberculosis at various concentratio a less than 50 \u00b5g/ml.\r\n\r\nMicroanalysis of avenacin has given the following composition: C, 59.28%; H, 7.75%; N, 1.41% and 0 by difference, 31.56%. The molecular weight is 1000 and the empirical formula is C44\u00b11 H78\u00b12 O20\u00b11 N. The ultraviolet absorption spectrum of avenacin has maxima at 353, 255 and 222 m\u00b5, in methanol.\r\n\r\nAlkaline hydrolysis of avenacin liberates the chromophore, N-methyl anthranilic acid, which is esterified to an aglycone in intact avenacin.\r\n\r\nBrief acid hydrolysis of avenacin liberates a mixture of oligo- and monosaccharides and a conjugate of N-methyl anthranilic acid with the aglycone. Subsequent basic hydrolysis of the conjugate frees the aglycone and N-methyl anthranilic acid. Further acid hydrolysis of the oligo- and monosaccharide fraction gives a hoxose-pentose mixture in the molar ratio of 2:l. Chromatography in methyl ethyl ketone-acetic acid water (30:3:10 v/v) did not separate the pentose from arabinose or the hexose from glucose and galactose. Evidence concerning the structure of avenacin is summarized and biological properties are discussed." }, { "name": "Simmons, John Robert", "degree": "PhD", "year": "1960", "title": "Studies on Amino Acid Incorporation in the Larvae of Drosophila Melanogaster", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechETD:etd-07122006-111435", "creators": [ { "name": { "family": "Simmons", "given": "John Robert" }, "id": "Simmons-John-Robert", "display_name": "Simmons, John Robert" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/EMPC-QM96", "abstract": "Amino acid incorporation in the larvae of Drosophila melanogaster was studied by injecting C14 labeled amino acids into the hemolymph of the animals. The resulting pattern of incorporation was investigated by direct paper chromatography of the injected larvae and by column fractionation of larval extracts.\r\n\r\nIt was found that injected amino acids were rapidly incorporated into a large number of components. Many of these were peptides, others contain non-amino acid constituents whose nature is yet unknown. As contrasted to previous reports, the present work indicates a major portion of the amino acids in insect hemolymph occur in bound form. It is felt that the difference is due to the higher degree of resolution of components achieved in the present experiments.\r\n\r\nIn the hope of obtaining larger amounts of amino acids in non-protein bound forms, attempts were made to inhibit protein synthesis. Of the treatments tested only Metrazol and chlorpromazine were effective in reducing protein synthesis and this reduction was accompanied by a decrease in amount in all radioactive metabolic product. The possible significance of this reduction is discussed.\r\n\r\nMaterial containing bound C14 glutamic acid was isolated from larval extracts and reinjected. It was found that radioactivity from the injected material was incorporated into the protein of the larvae but at a slower rate than was the free amino acid. The implications of this finding are discussed." }, { "name": "Simon, Edward Harvey", "degree": "PhD", "year": "1960", "title": "I. Transfer of DNA from Parent to Progeny Cell. II. A Study of the Possible Role of DNA in Viral RNA Synthesis During the Multiplication of an Animal Virus", "advisor": "Dulbecco, Renato", "url": "https://resolver.caltech.edu/CaltechETD:etd-08012006-111349", "creators": [ { "name": { "family": "Simon", "given": "Edward Harvey" }, "id": "Simon-Edward-Harvey", "display_name": "Simon, Edward Harvey" } ], "advisors": [ { "name": { "family": "Dulbecco", "given": "Renato" }, "id": "Dulbecco-R", "role": "advisor", "display_name": "Dulbecco, Renato" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/BXYZ-JC05", "abstract": "This work considered two approaches to the general problem of information transfer between molecules.\r\n\r\nThe first approach dealt with one aspect of the mechanism of deoxyribosenucleic acid (DNA) reproduction. In 1953 Watson and Crick proposed a structure of DNA which more recent work has essentially confirmed. The key postulate of the Watson-Crick structure is that DNA consists of two complementary chains of which either one may serve as a template for the formation of the other. This structure suggested three general models for DNA reproduction. In this work a test was devised to distinguish among these models by labeling cellular DNA with the thymine analog 5-bromouracil for one and/or two cell divisions. The results indicate that the semi-conservative model for the DNA replication originally proposed by Watson and Crick is valid for mammalian DNA.\r\n\r\nThe second approach considers the problem of whether in RNA-containing animal viruses the RNA is able to reproduce itself without interaction with cellular DNA. Various methods were used which either prevented the synthesis of DNA or led to the synthesis of abnormal DNA during the growth of the RNA-containing virus. These experiments led to the conclusion that viral RNA is able to multiply without interaction with newly synthesized DNA and probably without interacting with pre-existing cellular DNA." }, { "name": "Strohl, William Allen", "degree": "PhD", "year": "1960", "title": "Studies on the Modification, by Various Agents, of the Heat Sensitivity of Poliovirus", "advisor": "Dulbecco, Renato", "url": "https://resolver.caltech.edu/CaltechETD:etd-07242006-093842", "creators": [ { "name": { "family": "Strohl", "given": "William Allen" }, "id": "Strohl-William-Allen", "display_name": "Strohl, William Allen" } ], "advisors": [ { "name": { "family": "Dulbecco", "given": "Renato" }, "id": "Dulbecco-R", "role": "advisor", "display_name": "Dulbecco, Renato" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/CJ3R-X417", "abstract": "The decrease in heat sensitivity of Poliovirus, Type I, after incubation with cystine, has been studied. Characteristics of the inactivation by heat have also been studied. The results have led to an hypothesis which, while in no way completely satisfactory, provides a useful picture by which the data can be discussed, and also serves to indicate the areas to be clarified by future experiments. This hypothesis proposes the existence of two reactivity classes of viral sulfhydryl groups: -SH groups of the first class are oxidized by oxygen and by iodosobenzoic acid, probably to disulfides: -SH groups of the second class are so situated as to be unable to form disulfide bonds readily, and thus cannot be oxidized by oxygen, but can be oxidized to -SO2H or \u2013SO3H by iodosobenzoic acid.\r\n\r\nThe inactivation is postulated to consist of at least two steps: oxidation of sulfhydryl group(s), followed by denaturation of the viral protein. The existence of a denaturation step is suggested by the calculation of an Arrhenius constant for aerobic inactivation of 76,000 calories/mole. Evidence is presented to indicate that, at least in the case of oxidation by iodosobenzoate, the oxidation has a sensitizing effect; i.e., it increases the probability for the denaturation to occur.\r\n\r\nThe main requirement for stabilization appears to be the formation of a disulfide bond between a half cystine molecule and the viral -SH group. No compound has been found to produce stabilization as complete as L-cystine. The results suggest that the other compounds tested do not react with the viral -SH groups, and therefore one cannot decide whether formation of disulfide bonds with a stabilizing compound is in itself sufficient for stabilization. This does indicate, however, that the -SH groups must occur in an extremely stereo-specific environment" }, { "name": "Temin, Howard M.", "degree": "PhD", "year": "1960", "title": "The Interaction of Rous Sarcoma Virus and Cells in Vitro", "advisor": "Rubin, Harry; Dulbecco, Renato", "url": "https://resolver.caltech.edu/CaltechETD:etd-10222002-093114", "creators": [ { "name": { "family": "Temin", "given": "Howard M." }, "id": "Temin-Howard-M", "display_name": "Temin, Howard M." } ], "advisors": [ { "name": { "family": "Rubin", "given": "Harry" }, "id": "Rubin-H", "role": "advisor", "display_name": "Rubin, Harry" }, { "name": { "family": "Dulbecco", "given": "Renato" }, "id": "Dulbecco-R", "role": "advisor", "display_name": "Dulbecco, Renato" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/C1GF-9Y57", "abstract": "By the use of new methods for assaying in vitro Rous sarcoma virus (RSV) and Rous sarcoma cells and for isolating the progeny of single particles of RSV, it has been found that the morphology of Rous sarcoma cells is partially controlled by genetic factors in the virus. The influence of the state of the cell before infection, operating directly on the virus-cell complex has been demonstrated. A single particle of RSV is able to initiate infection, but viral genetic factors and genetic and physiological factors in the cell determine whether or not a cell does become infected. Host range mutants of the virus are described. It was shown that there exists a state in which resistant cells become competent to support the growth of RSV.
\r\n\r\nRSV rapidly adsorbs and penetrates into cells. After a 12 hour period new virus appears. The final rate of virus release of 10 focus-forming units per cell per 10 hours is reached about 40 hours after infection. The ability of a cell to produce RSV is transmitted to its progeny as an intracellular event, the number of Rous sarcoma cells doubling every 15 - 20 hours. Immediately after infection the X-ray resistance of the capacity of a cell to release virus is the same as its capacity to divide. By 15 - 20 hours after infection, the resistance of the capacity of the cells to release virus increases about 30 times.
\r\n\r\nDirect observation of isolated cells in microdrops shows that a cell can release virus and then divide. Interference caused by one particle of inactive virus, operating on some intracellular process after penetration of the virus into the cell was found.
\r\n\r\nThe relationship between RSV and temperate bacteriophage, the relevance of the findings to the cancer problem, and the relationship between RSV and other animal viruses are discussed.
\r\n\r\n" }, { "name": "Albersheim, Peter", "degree": "PhD", "year": "1959", "title": "Metabolism of the Pectic Substances", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechETD:etd-01312006-131728", "creators": [ { "name": { "family": "Albersheim", "given": "Peter" }, "id": "Albersheim-Peter", "display_name": "Albersheim, Peter" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "chair", "display_name": "Bonner, James Frederick" }, { "name": { "family": "Emerson", "given": "Sterling Howard" }, "id": "Emerson-S-H", "role": "member", "display_name": "Emerson, Sterling Howard" }, { "name": { "family": "Horowitz", "given": "Norman" }, "id": "Horowitz-N", "role": "member", "display_name": "Horowitz, Norman" }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "member", "display_name": "Mitchell, Herschel K." }, { "name": { "family": "Niemann", "given": "Carl G." }, "id": "Niemann-C-G", "role": "member", "display_name": "Niemann, Carl G." } ], "option_major": [ "bioch" ], "doi": "10.7907/K6BH-7R13", "abstract": "This thesis examines pectic metabolism by oat coleoptile sections. Pectin accounts for 5 per cent of the dry weight of such sections. Approximately 5 per cent of the pectin is cold water soluble--70 per cent alcohol insoluble. Approximately 50 per cent of the carboxyl groups of this fraction are methyl esterified. Hot water solubilizes from the cell wall a highly esterified pectin fraction which represents 15 per cent of the total. In the remaining hot water insoluble pectin, only 30 per cent of the pectic carboxyl groups are combined as esters.
\r\n\r\nPectins, once formed, are metabolized very slowly. There is little or no mixing of the various pectic fractions during a 15-hour period.
\r\n\r\nThe methyl ester group of pectin is supplied by the methyl group of methionine. Oat coleoptile sections, either intact or as homogenates, form S-methylmethionine and methionine sulfoxide from methionine. Both of these compounds are also active as methyl donors for the formation of pectic esters.
\r\n\r\nIndoleacetic acid accelerates both incorporation of the methyl of methionine into methyl ester moieties and the incorporation of glucose into galacturonic acid residues of water soluble pectins. This increase is a measure of an accelerated rate of pectin synthesis. Indoleacetic acid does not increase the rate of synthesis of the water insoluble pectins.
\r\n\r\nEvidence is presented which favors the supposition that the methyl ester groups are formed before polymerization of the galacturonic acid residues.
\r\n\r\nIn vitro incorporation of the methyl of methionine into methyl ester groups of pectin has not been achieved. Limited success has been obtained with the in vitro incorporation of glucose into galacturonic acid residues of water soluble pectins.
\r\n" }, { "name": "Davern, Cedric Inglis", "degree": "PhD", "year": "1959", "title": "I. Chemotherapy of High Temperature Inhibition of Plant Growth. II. Studies of the Relationship between Tobacco Mosaic Virus Infection and the DNA Metabolism of Tobacco Leaves. III. The Conservation of Microsomal RNA in Escherichia coli", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechETD:etd-01272006-132352", "creators": [ { "name": { "family": "Davern", "given": "Cedric Inglis" }, "id": "Davern-Cedric-Inglis", "display_name": "Davern, Cedric Inglis" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/1A8H-5F84", "abstract": "Part I. Chemotherapy of High Temperature Inhibition of Plant Growth:\r\n\r\nThe effects of biochemical supplements on the growth of five subterranean clover varieties grown under supra-optimal temperature conditions were studied. Some evidence of a chemotherapeutic effect of the high temperature growth inhibition was obtained.\r\n\r\nPart II. Studies on the Relationship between Tobacco Mosaic Virus Infection and the DNA Metabolism of Tobacco Leaves:\r\n\r\nA comparison of the DNA metabolisms of uninfected and TMV infected excised tobacco leaves, using p(32)-orthophosphate incorporation into the DNA as a measure of its metabolism, indicated that the DNA metabolism is not affected by TMV infection. This result was corroborated by the results of studies on the effect of 5-fluorouracil, a specific inhibitor of DNA synthesis, on the multiplication of TMV in tobacco-leaf discs. Although partial inhibition of TMV multiplication was observed, the absence of inhibition reversal thymidine, indicated that the mechanism of TMV inhibition probably did not involve a specific block of DNA synthesis. Finally unsuccessful attempts were made to see if intact host DNA was necessary for TMV infection by treating tobacco-leaf discs with DNAase.\r\n\r\nPart III. The Conservation of Microsomal RNA in Escherichia coli:\r\n\r\nA uniformly C13 N15 labeled exponentially growing culture of E. coli was transformed to light isotope substrates, and the metabolic fate of the pre-transfer synthesized heavy isotope microsomal RNA molecules followed by means of equilibrium sedimentation analysis of the RNA molecules in a density gradient. The results demonstrated complete conservation of the heavy isotopes by the pre-transfer RNA molecules remaining intact after transfer." }, { "name": "Sueoka, Noburu", "degree": "PhD", "year": "1959", "title": "Genetic and Biochemical Studies of Tyrosinase in Neurospora and Laccase in Neurospora", "advisor": "Horowitz, Norman Harold", "url": "https://resolver.caltech.edu/CaltechETD:etd-02172006-105349", "creators": [ { "name": { "family": "Sueoka", "given": "Noburu" }, "id": "Sueoka-Noburu", "display_name": "Sueoka, Noburu" } ], "advisors": [ { "name": { "family": "Horowitz", "given": "Norman Harold" }, "id": "Horowitz-N-H", "role": "advisor", "display_name": "Horowitz, Norman Harold" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/1G65-FS46", "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\r\n\r\nPart I: Genetic and Biochemical Studies of Tyrosinase in Neurospora
\r\n\r\n1) A new form of tyrosinase of Neurospora crassa (Sing-2) was found, which has different electrophoretic behavior and thermostability from the three previously known forms ([...], [...] and [...]).
\r\n\r\n2) The characteristics of the new form are determined by the single locus, T, which also controls the characteristics of the other forms.
\r\n\r\n3) The kinetics of thermal inactivation of the different tyrosinases were studied in detail at different temperatures.
\r\n\r\n4) Two tyrosinaseless genes (ty-l and ty-2) are independent from each other and from the T-locus, and both of them are epistatic to the T-locus.
\r\n\r\n5) Heterocaryons of the following genotypes were produced. [...]. It was found that a) the ty-1 allele is recessive to its normal form, [...], b) Het.B and Het.D produce a mixture of both forms of tyrosinase determined by their genotypes, and c) the ratio of the two enzyme forms produced corresponds to the ratio of the two component nuclei in the heterocaryons.
\r\n\r\n6) The significance of the present findings for the gene-enzyme relationship is discussed.
\r\n\r\nPart II: Laccase in Neurospora
\r\n\r\nThe \"second phenol oxidase\" in Neurospora reported by Horowitz and Fling (1953) was further studied.
\r\n\r\n1) The enzyme was purified, characterized as to substrate specificity, inhibitor spectrum, and pH optimum, and identified as a laccase.
\r\n\r\n2) Production of laccase shows wide variability among different strains of Neurospora and is influenced greatly by external factors, such as temperature, concentrations of sulfur and copper of the medium.
\r\n\r\n3) Immunological studies show that there is no serological similarity between laccase and tyrosinase of Neurospora.
\r\n\r\n4) Inducibility of laccase in Neurospora is a variable character, but seems to be strain specific.
" }, { "name": "Thompson, Guy Allen", "degree": "PhD", "year": "1959", "title": "The Biosynthesis of Carotenes in the Tomato", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechETD:etd-02232006-084220", "creators": [ { "name": { "family": "Thompson", "given": "Guy Allen" }, "id": "Thompson-Guy-Allen", "display_name": "Thompson, Guy Allen" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/SXP9-4527", "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\r\n\r\nRipening tomatoes were injected with 1C(14)-acetic acid or 2C(14)-mevalonic(3,5-dihydroxy-3-methylvaleric) acid and, after various incubation times, the carotenes were extracted and purified. The pattern of labeling among the carotenes leads to the conclusion that the more unsaturated pigments are not formed by the dehydrogenation of less unsaturated carotenes, but, rather, that most carotenes are synthesized by independent pathways, although from common precursors.\r\n\r\n2C(14)-Mevalonic acid was found to be incorporated into the carotene fraction with high efficiency. Most of the radioactivity was found by chromatographic separation of the polyenes to be associated with the phytoene fraction. Purification of this fraction yielded phytoene of little radioactivity and an unknown compound of specific activity higher than that of any purified carotene. This compound, designated as fraction II, has been shown to be a 20-carbon-atom isoprenoid hydrocarbon containing a pair of conjugated double bonds. Its probable structure is given below.\r\n\r\n[...]\r\n\r\nFraction II is rapidly synthesized from mevalonic acid in both ripening and immature tomatoes. A complete pathway for the formation of the carotenes from mevalonic acid is proposed. It is postulated that fraction II is formed throuhghout the life of the fruit and is continuously converted to an as yet uncharacterized polyene, which yields carotenes at the time of ripening. \r\n\r\nThe incorporation into carotenes of C(14)O(2), 1C(14)-glucose, uniformly C(14)-labeled-glucose, and uniformly C(14)-labeled leucine is described, and the patterns of labeling are compared with those obtained using mevalonate or acetate." }, { "name": "Burrows, Vernon Douglas", "degree": "PhD", "year": "1958", "title": "Studies on Translocation", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechETD:etd-10082004-111812", "creators": [ { "name": { "family": "Burrows", "given": "Vernon Douglas" }, "id": "Burrows-Vernon-Douglas", "display_name": "Burrows, Vernon Douglas" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/SFDB-W284", "abstract": "Studies have been made on the translocation of C14-labeled solutes (2,4-D, 2,4,6-T and sugar(s)) and labeled solvent (THO or H2O18) in the red kidney bean.\r\n\r\nTransport of 2,4-D can be controlled by regulating the supply of carbohydrate in the leaves. For the first six hours following treatment of leaves with 2,4-D, the amount of 2,4-D transported to the epicotyl increases linearly with time. Over short time intervals the amount of 2,4-D transported is linearly related to the concentration applied. Over longer time intervals high concentrations of 2,4-D tend to depress transport somewhat. Transport of 2,4-D does not however saturate at concentrations which saturate the growth process of the plant.\r\n\r\nEssentially the same amount of 2,4-D is transported to the epicotyl of plants grown under 1000 and 2000 f.c. of light. Growth of the epicotyls induced by equivalent amounts of 2,4-D is two to four times larger in plants grown under 2000 than in those grown under 1000 f.c. of light, however.\r\n\r\nThe compounds 2,4-D and 2,4,6-T are equally well absorbed by bean leaves and travel at the same speed in the phloem. The amount of 2,4,6-T which enters the phloem of the leaf, per unit time, is less than the amount of 2,4-D which so enters.\r\n\r\nTIBA applied as a pre-treatment to petioles inhibits the transport of C14-labeled 2,4-D, 2,4,6-T and sugars (predominantly sucrose). The inhibition of sugar movement may be used to interpret the inhibitory effect of TIBA on 2,4-D and 2,4,6-T transport.\r\n\r\nFoliarly applied tritium labeled (THO) and O18-labeled (H2O18) water are transported downward in bean seedlings. The carbohydrate status of the leaf does not govern the transport of labeled water in the same manner as it governs 2,4-D transport. The transport of THO takes place equally well or better in girdled as in normal plants. Movement of tritium apparently takes place in the xylem rather than in the phloem." }, { "name": "Clark, John Magruder", "degree": "PhD", "year": "1958", "title": "Studies on Amino Acid Activation and Protein Synthesis", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechETD:etd-10072004-105753", "creators": [ { "name": { "family": "Clark", "given": "John Magruder" }, "id": "Clark-John-Magruder", "display_name": "Clark, John Magruder" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/GVR0-N928", "abstract": "This thesis concerns the overall subject of protein synthesis in plants. Several different experimental approaches to the problem are described.\r\n\r\nThe initial studies concern the incorporation of amino acids into tissue sections and homogenates. Tissue sections show a constant rate of incorporation of labeled amino acids into protein for periods of time up to two hours. The rate of incorporation then decreases with time. Conversely, tissue homogenates show ever increasing rates of incorporation of labeled amino acids into protein. This incorporation is indicative of incorporation of labeled amino acids into bacteria within the homogenate rather than incorporation into plant protein. Sterile homogenate preparations do not show the kinetics of incorporation shown by other homogenate preparations. The incorporation of labeled amino acids into acid insoluble material obtained from sterile tissue homogenates is a function of the washing procedure used in the assay.\r\n\r\nAmino acid activation has been studied by a hydroxylamine trapping reaction and by pyrophosphate exchange. Plant tissues contain amino acid activating enzymes which may be detected by either method of assay. The pyrophosphate exchange assay is however more sensitive. The hydroxamate assay suffers from some added complications common to plant systems.\r\n\r\nThe specific nature of amino acid activation in plant preparations has been studied with enzymes isolated from spinach leaves. The activation reaction requires ATP and amino acids, the products being pyrophosphate and probably a mixed anhydride between the 5' phosphate of AMP and the carboxyl group of the amino acid. The enzymes occur in all plant tissues tested and appear to be a part of the soluble fraction of cells. Purification procedures have been developed which make it possible to remove the free amino acids from the preparations. The purified preparation is capable of activating all of the naturally occurring (in protein) amino acids except serine. The possible activation of serine is-not excluded. Preliminary evidence is presented which indicates that each amino acid is activated by its own specific amino acid activating enzyme.\r\n\r\nThe possibility of linking the process of amino acid activation to protein synthesis is considered. Crude particulate containing preparations of spinach exhibit an ATP and RNA dependent incorporation of leucine. Attempts to obtain an amino acid dependent exchange of AMP into ATP with spinach preparations were unsuccessful. Even so it has been possible to demonstrate an ATP dependent RNAase sensitive incorporation of labeled leucine into acid and alcohol washed material." }, { "name": "Dennison, David Severin", "degree": "PhD", "year": "1958", "title": "Studies on Phototropic Equilibrium and Phototropic-Geotropic Equilibrium in Phycomyces", "advisor": "Delbruck, Max", "url": "https://resolver.caltech.edu/CaltechETD:etd-10072004-133617", "creators": [ { "name": { "family": "Dennison", "given": "David Severin" }, "id": "Dennison-David-Severin", "display_name": "Dennison, David Severin" } ], "advisors": [ { "name": { "family": "Delbruck", "given": "Max" }, "id": "Delbr\u00fcck-M", "role": "advisor", "display_name": "Delbruck, Max" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/MBR0-0B22", "abstract": "Phototropic equilibrium was studied in Stage IV sporangiophores of Phycomyces blakesleeanus by illuminating specimens simultaneously from various directions with two beams of light. The dependence of the equilibrium position upon the angle between the light beams and upon their intensities was investigated and shown to be given by a simple empirical law.\r\n\r\nIf a sporangiaphore is illuminated by a single light beam, an equilibrium position is reached between the direction of the beam and the direction of gravity. The dependence of this position upon the angle between the beam and the vertical was investigated and found to be given by another simple empirical law. The equilibrium position was found to be unaffected by changes in the intensity of the light over the entire range of intensities to which the sporangiophore gives a normal phototropic response. Geotropism does not occur in the dark however.\r\n\r\nUnder certain specific conditions a sporangiophore in stable equilibrium shows regular oscillations around its equilibrium position. These oscillations are sinusoidal in form and often persist for 30 cycles, having a period of about 45 minutes and an amplitude of about 30 degrees. It is also possible for a sporangiophore, not necessarily in equilibrium, to show oscillations with a five minute period and a two degree amplitude. These two modes of oscillation appear to be independent of each other." }, { "name": "Drake, John Walter", "degree": "PhD", "year": "1958", "title": "Intracellular Interactions of Polioviruses: Interference and Multiplicity Reactivation", "advisor": "Dulbecco, Renato", "url": "https://resolver.caltech.edu/CaltechETD:etd-10072004-134911", "creators": [ { "name": { "family": "Drake", "given": "John Walter" }, "id": "Drake-John-Walter", "display_name": "Drake, John Walter" } ], "advisors": [ { "name": { "family": "Dulbecco", "given": "Renato" }, "id": "Dulbecco-R", "role": "advisor", "display_name": "Dulbecco, Renato" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/DRQ8-QH50", "abstract": "This study concerns interactions among polioviruses infecting the same cell, and can be divided into three parts. The first part consists of the detailed study of certain conditions of infection of HeLa cells by poliovirus. The second is concerned with the interfering activity of virus, either live or inactivated, with the multiplication of superinfecting live virus, either homotypic or heterotypic. It was found that live virus interferes with heterologous virus (homologous not tested), while UV-killed virus lacks interfering ability. It was also found that the virus released from cells infected by two heterotypic polioviruses, when interference does not occur, contains particles which are neutralized by both specific antisera. This phenomenon may be the result of a \"phenotypic mixing\" mechanism similar to that observed with phages.\r\n\r\nThe third part of the study deals with multiplicity reactivation among homotypic polioviruses inactivated by UV irradiation. When killed viruses are adsorbed to cells at multiplicities greater than one, more cells release live virus than can be accounted for on the basis of the input of UV-surviving viruses. This multiplicity reactivation is a function of the UV'd particles, not of non-viral agents in the lysate, heat-killed viruses, originally uninfectious particles, nor anomalies in adsorption. The fraction of cells releasing virus increases with multiplicities up to 40-80, when saturation appears to set in. At high doses the fraction of yielders decreases with the UV dose to the virus at a rate equal to that at which the virus itself is killed. The parameter n, representing the number of segments within a virus which can interact with segments from other particles, is calculated, and its significance is discussed." }, { "name": "Grell, Ellsworth Herman", "degree": "PhD", "year": "1958", "title": "Genetics and Biochemistry of \"Red Cells\" in Drosophila melanogaster", "advisor": "Lewis, Edward B.", "url": "https://resolver.caltech.edu/CaltechETD:etd-11012004-140047", "creators": [ { "name": { "family": "Grell", "given": "Ellsworth Herman" }, "id": "Grell-Ellsworth-Herman", "display_name": "Grell, Ellsworth Herman" } ], "advisors": [ { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "advisor", "display_name": "Lewis, Edward B." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/B5AB-S991", "abstract": "Studies were made of the \"red cells\" strain of Drosophila melanogaster. \"Red cells\" flies are characterized by the presence of reddish pigment in certain fat cells.\r\n\r\nA genetic analysis revealed that the fat cells are pigmented only when flies are homozygous for two mutant genes. The two genes are 4.4 crossover units apart on the second chromosome. They were separated, maintained in separate stocks and recombined. When they were recombined the \"red cells\" phenotype was again produced as it is in the original stock. The two mutant genes have been named red cells (rc) and lysine (lys). With or without rc, the mutant gene, lys, causes flies which are homozygous for it to contain a greater quantity of the amino acid, lysine, than normal flies. In experiments with injection of radioactive lysine, over a period of eight hours normal flies converted thirteen times more lysine into carbon dioxide than did lys flies.\r\n\r\nThe hypothesis was offered that lys is a mutation of a gene which is important in the degradation of lysine. The mutation causes an impairment in the processes by which lysine is degraded, therefore lysine tends to accumulate in the flies if they ingest more lysine than is required in protein synthesis.\r\n\r\nAttempts were made to localize the position of the step at which lys impairs the degradation of lysine." }, { "name": "Labouriau, Luiz Fernando Gouv\u00eaa", "degree": "PhD", "year": "1958", "title": "Studies on the Initiation of Sporangia in Ferns", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechETD:etd-10082004-105932", "creators": [ { "name": { "family": "Labouriau", "given": "Luiz Fernando Gouv\u00eaa" }, "id": "Labouriau-Luiz-Fernando-Gouv\u00eaa", "display_name": "Labouriau, Luiz Fernando Gouv\u00eaa" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/3BNE-T071", "abstract": "A survey of available data in the literature shows that initiation of sporangia in ferns is not strictly localized, but is possible in many areas of the fern organism, such as the gametophytes, the first leaves and several areas of the leaf of the adult plant, which do not have to be contiguous.
\r\n\r\nIn the usual sites of initiation, sporangia show correlations with the leaf veins and they may be replaced by a variety of alternative differentiations, such as cell proliferations, vegetative buds and aposporous prothalli.
\r\n\r\nInitiation and differentiation of sporangia may be arrested in several stages, both in natural conditions and experimentally. Some of the experimental procedures that produce this effect use changes in environmental factors.
\r\n\r\nExperimental results recorded in the literature indicate a day-neutral behavior in several species and a qualitative short-day behavior in one species (Salvinia natans).
\r\n\r\nThe results of experiments reported in this thesis show that Asplenium bulbiferum is a quantitative long-day plant with a critical night of 23 hours at 20\u00b0C. In this species adventitious buds are capable of producing sporangia on their leaves, while attached to the adult leaf, but this capacity is lost upon isolation of the buds.
\r\n\r\nIn Osmunda claytoniana determination of the sporophylls was found to be caused by processes that are independent of those that determine the cataphylls and to be enhanced by long days and high temperatures.
\r\n\r\nSalvinia rotundifolia was found to behave as a short-day plant at temperatures above 20\u00b0C and as a long-day plant at 17\u00b0C. Initiation of sporangia in this species responds also to daily thermoperiodicity.
\r\n\r\nRegnellidium diphyllum was found to develop sporocarps as a response to seasonal and to daily thermoperiodicity. This response is quantitatively modified by photoperiodism.
\r\n\r\nThese results are discussed in relation to the available data on the differentiation of vascular tissues, on heteroblastic leaf development, photoperiodism and thermoperiodicity.
" }, { "name": "Park, Roderic Bruce", "degree": "PhD", "year": "1958", "title": "I. The Biosynthesis of Open Chain Terpenes in Plants. II. Fractionation of the Stable Carbon Isotopes in Plants", "advisor": "Bonner, James Frederick; Epstein, Samuel", "url": "https://resolver.caltech.edu/CaltechETD:etd-01252006-084039", "creators": [ { "name": { "family": "Park", "given": "Roderic Bruce" }, "id": "Park-Roderic-Bruce", "display_name": "Park, Roderic Bruce" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" }, { "name": { "family": "Epstein", "given": "Samuel" }, "id": "Epstein-S", "role": "advisor", "display_name": "Epstein, Samuel" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/CJQ9-P816", "abstract": "I: Open chain terpene synthesis in plants was studied by measurement of the incorporation of potential intermediates into the rubber of the rubber producing plants Taraxacum kok saghyz and Hevea brasiliensis. Intact plants incorporate 1-C14-acetate and 2-C14-acetate into rubber without randomization of the label. [Beta]-Methylcrotonic acid was found to be an ineffective rubber precursor in intact plants.\r\n\r\nEnzymatic experiments were performed using Hevea latex as a source of enzyme. C14-acetate is rapidly incorporated into a volatile, non-acidic, non-polar substance in this system under anaerobic conditions. C14-acetate is not incorporated into rubber. Mevalonic acid is rapidly incorporated into rubber in this system. Partial degradation of the rubber indicates that no randomization occurs during incorporation. This result suggests that mevalonic acid is on the pathway of terpene synthesis in plants.\r\n\r\nII: The two stable carbon isotopes, C12 and C13, occur in nature in the ratio of about ninety to one. Various workers have shown that this ratio is not fixed, but may vary by as much as 5%. Interestingly enough, this variation is not random. Carbon reservoirs such as limestone, atmospheric CO2, land plants, algae and coal all exhibit characteristic C13/C12 ratios. This section of the dissertation is concerned with the differences between the C13/C12 ratios of plants and those of the carbon sources from which such plants have grown.\r\n\r\nBoth algae and terrestrial plants have smaller C13/C12 ratios than those of dissolved carbonates and atmospheric CO2 respectively. The magnitude of this fractionation was determined for tomato plants by growing the plants from seed in CO2 of known isotopic composition. Separation of the plant material into its component chemical constituents showed that only the lipid fraction differed markedly in C13/C12 ratio from that of the plant as a whole. The lipid fraction is enriched in C12 and possesses a C13/C12 ratio similar to that of petroleums derived from land plants. A similar relation was found to exist between marine algae, their lipids, and petroleums of marine origin. The CO2 evolved by plant respiration is slightly enriched in C13 as compared to the plant. This process apparently closely related to the C12 enrichment in lipid fractions.\r\n\r\nA possible mechanism for fractionation of C13 and C12 in photosynthesis is suggested. This suggestion is supported by observations of the C13/C12 ratio of CO2 dissolved in higher plants and by determination of the fractionation which occurs during fixation of CO2 by the photosynthetic carboxylation enzyme." }, { "name": "Baluda, Marcel Albert", "degree": "PhD", "year": "1957", "title": "Homologous Interference by Ultraviolet Inactivated Virus in Newcastle Disease Virus", "advisor": "Dulbecco, Renato", "url": "https://resolver.caltech.edu/CaltechETD:etd-06292004-111411", "creators": [ { "name": { "family": "Baluda", "given": "Marcel Albert" }, "id": "Baluda-Marcel-Albert", "display_name": "Baluda, Marcel Albert" } ], "advisors": [ { "name": { "family": "Dulbecco", "given": "Renato" }, "id": "Dulbecco-R", "role": "advisor", "display_name": "Dulbecco, Renato" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/S8Z4-1T83", "abstract": "The present study concerns a quantitative analysis of the interference between the irradiated and the active Newcastle disease virus.\r\n\r\nThe inactivated particles adsorb to the surface of the cells and do not proceed any further. This union induces surface changes which make it impossible for a superinfecting active particle to penetrate into the host cell and to initiate the production of new virus. The more UVI particles that are adsorbed, the faster the interfering reaction occurs.\r\n\r\nIn 50 per cent of the cells, however, interference is not complete; these cells can be superinfected provided the multiplicity of the superinfecting virus is high. The effect is equivalent to having on the average four per cent of the total surface of the cell unaffected by the changes induced by the inactivated virus.\r\n\r\nThe interfering reaction is dependent at all times upon the presence of the unmodified UYI particles at the critical sites - exposure of the interfered cells to specific anti-NDV serum eliminates interference. However, in 50 per cent of the cells interference becomes irreversible 30 minutes after the attachment of UVI virus. Whether this irreversible interference involves more profound cellular changes or depends upon the physiological state of the cells at the time of infection is at present unknown.\r\n\r\nEventually, the union between UVI virus and cellular site is broken with the subsequent return of the cell to susceptibility to infection. This loss of resistance occurs spontaneously from 26 to 60 hours after exclusion has been induced.\r\n\r\nThe superinfecting virus which does not initiate infection is destroyed after its adsorption to the lung cell. A cell where interference has been removed after superinfection with active virus must be infected a second time in order to yield progeny virus." }, { "name": "Berrian, James Henry", "degree": "PhD", "year": "1957", "title": "The Immune Response in Homologous Transplantation", "advisor": "Owen, Ray David", "url": "https://resolver.caltech.edu/CaltechETD:etd-07082004-134644", "creators": [ { "name": { "family": "Berrian", "given": "James Henry" }, "id": "Berrian-James-Henry", "display_name": "Berrian, James Henry" } ], "advisors": [ { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "advisor", "display_name": "Owen, Ray David" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "immun" ], "doi": "10.7907/2M3R-DW27", "abstract": "Part I discusses the immunogenetic basis of transplantation incompatibility and the nature of the immune response which adversely affects incompatible tissue transplants.\r\n\r\nPart II describes attempts to detect specific reactions between tissue extracts and antisera by means of light-scattering measurements. The few positive reactions obtained tend to support, rather than refute, previous claims made for individually specific serum antibodies formed during the rejection of transplants.\r\n\r\nPart III deals with an experimental demonstration of the destruction of mouse tissue by immune spleen cells, using [in vivo] cultured diffusion chambers. The results support the hypothesis that immune cells directly initiate the destruction of transplants.\r\n\r\nIn Part IV reactions between immune cells and isolated transplantation antigens are studied [in vitro]. It is possible to react immune cells as well as the transplantation antigens with fluorescent labels without abolishing their immunological effectiveness. With a biological test as the basis, evidence is described that transplantation antigens are removed from solution when suspended with immune cells [in vitro].\r\n\r\nIn Part V a method is presented for describing the uptake, distribution and binding of fluorescent labeled molecules using polarization of fluorescence as an indication of the motion of molecules and their interaction in the intracellular environment.\r\n\r\nPart VI describes a simplified test for mouse isohemagglutinating antibodies, which only requires purified hyaluronic acid and saline as the suspension medium.\r\n\r\nPart VII describes some details of the structure of elastin and attempts to explain the immunological tolerance of elastin and its soluble derivatives in terms of its mobile molecular structure." }, { "name": "Bertani, Lillian Elizabeth", "degree": "PhD", "year": "1957", "title": "Studies on the Establishment of Lysogeny by Bacteriophage P2", "advisor": "Delbruck, Max", "url": "https://resolver.caltech.edu/CaltechETD:etd-07012004-094123", "creators": [ { "name": { "family": "Bertani", "given": "Lillian Elizabeth" }, "id": "Bertani-Lillian-Elizabeth", "display_name": "Bertani, Lillian Elizabeth" } ], "advisors": [ { "name": { "family": "Delbruck", "given": "Max" }, "id": "Delbr\u00fcck-M", "role": "advisor", "display_name": "Delbruck, Max" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/BP9H-HS61", "abstract": "Some aspects of the processes leading to lysogenization of cells of Escherichia coli or Shigella dysenteriae by phage P2 have been studied with the following results. Treatment of infected cells with chloromycetin, amino acid analogues, or 5-OH-uridine, or starvation for a required amino acid, all increase the frequency of lysogenization, whereas treatment with cyanide, azide, dinitrophenol, or pretreatment with ultraviolet light have no effect. Treatment of infected cells with proflavine also increases the frequency of lysogenization, and chloromycetin and proflavine are most effective in this respect when added about halfway through the latent period. It is suggested that the primary action of these substances is to block processes, beginning about that time, that lead to the maturation of the phage. At about the same time, the infected cells that become lysogenic show an apparent resistance to ultraviolet light, higher than that of either phage P2 or established lysogenic cells." }, { "name": "Cleland, Robert Erskine", "degree": "PhD", "year": "1957", "title": "The Hormonal Control of Cell Wall Properties", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechETD:etd-07012004-102944", "creators": [ { "name": { "family": "Cleland", "given": "Robert Erskine" }, "id": "Cleland-Robert-Erskine", "display_name": "Cleland, Robert Erskine" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/2PSQ-9548", "abstract": "A technique has been developed for the separation of auxin action and cell elongation. Avena coleoptile sections are treated with auxin under conditions of nonexpansion. This is followed by expansion of the sections in water containing an inhibitor which blocks all auxin action. Under aerobic conditions, auxin initiates a loosening of the cell wall. The loosening can then persist over short periods of non-expansion under anaerobic conditions. The loosening can be abolished by general metabolic inhibitors such as KCN or counteracted by an auxin-independent stiffening process. The loosening is due to an increase in the plasticity of the cell wall. Ethionine administered during either phase of the process inhibits the residual auxin effect, suggesting that transmethylation is involved in the increase in plasticity.\r\n\r\nThe metabolism of the cell wall components has been investigated by the use of labeled glucose and labeled methionine. The only effect of auxin which was found was an increased rate of incorporation of label from both glucose and methionine into the methyl ester groups of pectin. This increase is abolished by the same inhibitors which abolish the auxin-induced cell wall loosening. It is proposed that the increase in rate of pectin methyl ester incorporation is an integral part of the cell wall loosening process.\r\n\r\nIncorporation of label from methionine C-14 into the pectin fraction has also been achieved with homogenates of Avena coleoptile sections although the rate is markedly decreased by homogenization. A procedure has been developed for obtaining reproducible results with such homogenates. Dialysis of the homogenate supernatant after solubilization of the pectin by boiling has proven satisfactory.\r\n\r\nAbout 85 percent of the label from methionine C-14 incorporated into the pectin of intact tissues is in the form of esterified methyl groups. Upon homogenization of the tissues, however, only 20 percent of the label is located in the methyl groups." }, { "name": "Herzenberg, Leonard Arthur", "degree": "PhD", "year": "1956", "title": "Studies on a Cytochrome Destroying System in Neurospora", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechETD:etd-03232004-140910", "creators": [ { "name": { "family": "Herzenberg", "given": "Leonard Arthur" }, "id": "Herzenberg-Leonard-Arthur", "display_name": "Herzenberg, Leonard Arthur" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/X9PN-W252", "abstract": "The results of investigations on a cytochrome destroying system in Neurospora are presented. It is shown that the destruction is complex and dependent on the action of several enzymes as well-as non-catalyzed reactions.\r\n\r\nPoky particles exhibit a rapid and extensive hydrolysis of their own and added proteins whereas fast poky and wild-type particles do so to a much smaller extent. When cytochrome c is added to poky particles or particle-derived preparations, heme-peptides are produced. Several heme-peptides are partially characterized. Evidence is presented which suggests that poky particles, succinate and cytochrome c, when incubated together in air, produce a verdohemochrome.\r\n\r\nAn ether soluble substance is found in poky and fast poky particles which combines with cytochrome c so that the iron is not reducible by ascorbate and the heme group is destroyed on reduction by hydrosulfite and reoxidation by oxygen. Purification of this substance and some of its chemical properties are described. It has been named \"pokonic acid.\"" }, { "name": "Hildemann, William Henry", "degree": "PhD", "year": "1956", "title": "Immunogenetic Studies of the Goldfish (Carassius auratus)", "advisor": "Owen, Ray David", "url": "https://resolver.caltech.edu/CaltechETD:etd-03242004-095231", "creators": [ { "name": { "family": "Hildemann", "given": "William Henry" }, "id": "Hildemann-William-Henry", "display_name": "Hildemann, William Henry" } ], "advisors": [ { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "advisor", "display_name": "Owen, Ray David" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/1011-D785", "abstract": "Transplantation experiments with goldfish scales showed that autografts became normally revascularized and persisted without any inflammation whatever. Homografts elicited an immune response, which was measured by determining median survival times and inflammatory reactions under various conditions. A plot of median survival times against temperature demonstrated that previous grafting from the same donor and higher water temperature both accelerated homograft destruction. The duration and intensity of the host's inflammatory reaction were closely associated with the rapidity of donor tissue destruction. The duration of the first inflammatory phase was dependent upon the rapidity of soft tissue destruction, while the second inflammatory phase was associated with slow digestion of the acellular scale plate. Both soft tissue and scale plate digestion showed the second-set phenomenon.\r\n\r\nReciprocal-homografting operations were undertaken within a pedigree in an effort to estimate the number of genetic loci concerned with scale transplant specificities. Complete crossgrafting of 23 F1 sibs and 23 F2 sibs, respectively, revealed no compatible combinations. Reciprocal parent-offspring homografts were also unsuccessful. It was determined that at least four independent histocompatibility loci were required to account for the mutual incompatibility observed.\r\n\r\nTechniques were developed for bleeding and isoimmunization of goldfish, together with methods for handling, preservation, and typing of goldfish erythrocytes. Experiments with isoimmune and rabbit antisera demonstrated the existence of numerous individual differences in the red cell antigens of goldfish." }, { "name": "Knudson, Alfred George", "degree": "PhD", "year": "1956", "title": "Histidine Metabolism in Liver", "advisor": "Borsook, Henry", "url": "https://resolver.caltech.edu/CaltechETD:etd-03252004-145831", "creators": [ { "name": { "family": "Knudson", "given": "Alfred George" }, "id": "Knudson-A-G", "display_name": "Knudson, Alfred George" } ], "advisors": [ { "name": { "family": "Borsook", "given": "Henry" }, "id": "Borsook-H", "role": "advisor", "display_name": "Borsook, Henry" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/SHM8-FB96", "abstract": "This is the report of an investigation of histidine metabolism in mammalian liver. Formiminoglutamic acid, reported to be the end-product of histidine metabolism in vitro, has been found to be degraded enzymatically to glutamic acid by a rat liver extract. Homogenates of spleen, kidney, and heart do not have such activity. A product of the metabolism of the formimino group is carbon dioxide. The effects of time, enzyme concentration, substrate concentration, temperature, and pH on the reaction are reported. Sulfhydryl groups are essential for activity. Inorganic phosphate is stimulatory, and arsenolysis occurs. On the basis of these findings and pertinent published information a scheme for the degradation of histidine to glutamic acid is proposed." }, { "name": "Lester, Robert Leonard", "degree": "PhD", "year": "1956", "title": "Incorporation of Amino Acids into the Proteins of Micrococcus Iysodeikticus", "advisor": "Borsook, Henry", "url": "https://resolver.caltech.edu/CaltechETD:etd-03262004-141353", "creators": [ { "name": { "family": "Lester", "given": "Robert Leonard" }, "id": "Lester-Robert-Leonard", "display_name": "Lester, Robert Leonard" } ], "advisors": [ { "name": { "family": "Borsook", "given": "Henry" }, "id": "Borsook-H", "role": "advisor", "display_name": "Borsook, Henry" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/V4K5-ZD08", "abstract": "Lysis of Micrococcus lysodeikticus cells with lysozyme in the presence of high concentrations of sucrose has yielded a particulate system, distinct from intact cells, that carries out the incorporation of amino acids into protein. All seven amino acids which have been tested have been incorporated.\r\n\r\nThe system was sedimentable, had a high endogenous respiration, and required the continued presence of high sucrose or KCl concentrations for activity. The importance of oxidative reactions for incorporation was indicated since anaerobiosis and 2,4-dinitrophenol treatment inhibited incorporation. Ribonuclease treatment diminished incorporation; desoxyribonuclease treatment accelerated incorporation. The activity of the particulate system was inversely related to the concentration at which these particles were assayed. Stimulation of incorporation was observed upon addition of unlabeled amino acids implying a net synthesis of protein molecules under these conditions. From data obtained on the composition of the free amino acid pool and the rates of amino acid incorporation it was concluded that in the absence of added amino acids, amino acid incorporation occurred either via an exchange reaction or that it reflected synthesis of new protein molecules with concomitant breakdown of pre-existing protein to amino acids.\r\n\r\nIt was shown that radioactive protein isolated after incubation with radioactive leucine, contained leucine as its sole radioactive constituent. The conversion of leucine to alpha-ketoisocaproic acid was also shown." }, { "name": "Metzenberg, Robert Lee", "degree": "PhD", "year": "1956", "title": "Studies on the Biosynthesis of Aromatic Compounds in Neurospora crassa", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechETD:etd-06102004-153533", "creators": [ { "name": { "family": "Metzenberg", "given": "Robert Lee" }, "id": "Metzenberg-Robert-Lee", "display_name": "Metzenberg, Robert Lee" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/WPDV-8S11", "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\t\t\t\t\t\r\n\r\nMutant strains of Neurospora have been isolated which exhibit nutritional requirements for aromatic compounds. Some of these mutants require a single nutrient in order to grow, for example, tryptophan, phenylalanine, or tyrosine. Others have been found to require all three of these amino acids, and in addition, [...] acid. It was desired to ascertain the number of gene changes from wild type which are responsible for the observed nutritional requirements. For this purpose, a number of the new strains were subjected to genetic analysis.\r\n\r\nThere was reason to believe that certain double and triple mutants would be found to accumulate compounds of interest which fail to accumulate in culture filtrates of strains containing only one altered gene. Therefore, these double and triple mutants were prepared by appropriate crosses, and were found to accumulate a number of compounds of biological interest. In addition, double and triple mutants have been of value in determining the order in which some of these genetic blocks occur in a biosynthetic sequence.\r\n\r\nCulture filtrates of the mutant strains have been examined for the presence of accumulated materials. Evidence is presented that the following compounds occur in filtrates of various mutants: protocatechuic acid, shikimic acid, 5-dehydroshikimic acid, vanillic acid, [...] acid, phenylpyruvic acid, anthranilic acid, prephenic acid, and [...].\r\n\r\nShikimic acid, although found to be accumulated in culture filtrates of one mutant, does not relieve the multiple requirement for aromatic compounds in other strains. This suggests that the biosynthesis of aromatic compounds in Neurospora may involve intermediates different from those elaborated by [...]. In the latter organism, 5-dehydroshikimic acid has been identified by Davis and co-workers as an \"early\" precursor of aromatic compound, whereas in Neurospora, it appears that 5-dehydroshikimic acid may be a \"late\" precursor, possibly the last compound that is a common precursor of all three aromatic amino acids.\r\n\r\nIt was felt that a method for the detection of vicinal diol compounds on paper chromatograms would aid the identification of such compounds as shikimic acid in culture filtrates of the mutants. Therefore, a method based on periodate oxidation was developed and found to be of use in the detection of various compounds of biological importance." }, { "name": "Sato, Gordon Hisashi", "degree": "PhD", "year": "1956", "title": "The Effect of Urea on Cofactor Requiring Bacteriophage", "advisor": "Delbruck, Max", "url": "https://resolver.caltech.edu/CaltechETD:etd-04292005-085816", "creators": [ { "name": { "family": "Sato", "given": "Gordon Hisashi" }, "id": "Sato-Gordon-Hisashi", "display_name": "Sato, Gordon Hisashi" } ], "advisors": [ { "name": { "family": "Delbruck", "given": "Max" }, "id": "Delbr\u00fcck-M", "role": "advisor", "display_name": "Delbruck, Max" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/ZQTH-RJ42", "abstract": "Some strains of bacteriophage must be activated by cofactor before they can adsorb to their bacterial hosts. Up to the present time, the only compounds shown to possess cofactor activity have been certain amino acids and amino acid analogues.\r\n\r\nIn the present study, it is shown that urea, a well known denaturing agent, is capable of activating cofactor requiring phage. By a process, statistically independent of the activation process, urea also kills the phage. Experiments are performed to characterize the properties of urea-activated phage, in regard to stability of urea induced adsorbability and in regard to adsorption rate. The kinetics of the urea activation process and of the urea killing process are studied in detail. Both processes are shown to depend on concentration of urea, temperature, and pH in a manner similar to denaturation of protein by urea.\r\n\r\nIt is concluded that urea effects the activation of phage by denaturing the protein of the phage adsorption organ. It is further postulated that the action of cofactors consists in a denaturation of phage protein. The biological implications of this hypothesis are discussed." }, { "name": "Terres, Geronimo Jr.", "degree": "PhD", "year": "1956", "title": "Part I. The Effect of Hydrogen Peroxide Oxidation on the Antigenicity of Ovalbumin, Bovine Serum Albumin, and Rabbit Gamma Globulin. Part II. Cortical Discontinuity and Propagation of Spreading Depression", "advisor": "Campbell, Dan Hampton", "url": "https://resolver.caltech.edu/CaltechETD:etd-06302004-112033", "creators": [ { "name": { "family": "Terres", "given": "Geronimo Jr." }, "id": "Terres-Geronimo", "display_name": "Terres, Geronimo Jr." } ], "advisors": [ { "name": { "family": "Campbell", "given": "Dan Hampton" }, "id": "Campbell-D-H", "role": "advisor", "display_name": "Campbell, Dan Hampton" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "immun" ], "doi": "10.7907/TPN0-8M67", "abstract": "PART ONE\r\n\r\nThree proteins were treated with solutions of hydrogen peroxide under a variety of conditions. The resulting products were soluble, heat stable, and showed some increase in heterogeneity of electrophoretic components. Osmotic pressure determinations indicated a marked reduction in number average molecular weights, and intrinsic viscosity studies showed increased frictional ratios for the treated proteins. UV absorption of the treated proteins indicated extensive oxidation of the tryptophane, tyrosine and phenylalanine residues in the protein. Chromatographic studies indicated that cystine and cysteine were oxidized to cysteic acid. Immunochemical investigation of the H2O2 treated proteins showed: (1) That treated ovalbumin had lost all its native specificity, and that treated bovine serum albumin and rabbit gamma globulin retained only traces; (2) Each protein apparently developed a new specificity as a result of treatment, but the antigenicity of such proteins was very low. Tests employed were the development of precipitins in rabbits and chickens, and Schultz-Dale and gross anaphylaxis in guinea pigs.\r\n\r\nPART TWO\r\n\r\nAn investigation into the possible mechanism underlying the propagation of Leao's spreading depression (S.D.) was conducted in rabbits by cutting the cortex in chronic experiments and thus destroying neuronal continuity. The-slow potential change (S.P.C.) concomitant with S.D. had been postulated as the agent of transmission in a manner similar to the nerve action potential associated with nerve conduction. It was found in this investigation that neither the S.D. or the S.P.C. crossed the cut even though in some cases the scar was only 0.1 mm thick and the cortical edges were well approximated. A small potential change was recorded as crossing the cut, but it was never instrumental in initiating a S.D. It was therefore concluded that either neuronal continuity or localized microfields smaller in radius than the scar are involved in the transmission of S.D. The possibility of a chemical agent being involved in the transmission of S.D. was not eliminated by these experiments." }, { "name": "Ts'o, Paul On Pong", "degree": "PhD", "year": "1956", "title": "On Plant Myosin", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechETD:etd-07152004-154314", "creators": [ { "name": { "family": "Ts'o", "given": "Paul On Pong" }, "id": "Ts'o-Paul-On-Pong", "display_name": "Ts'o, Paul On Pong" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/17XT-6E12", "abstract": "A systematic study has been carried out both on the cellular and the molecular level of the mechanism of protoplasmic streaming in the slime mold, Physarum polycephalum. This study is based on the hypothesis that ATP-protein interaction is an important feature of the mechanism.\r\n\r\nATP has been found to have a liquefying effect on the gel structure of the streaming plasmodium. Furthermore, when ATP is allowed to react with protoplasmic material extracted from plasmodia under proper conditions, the viscosity of the solution decreases immediately. This abrupt decrease in viscosity of the protoplasmic solution is followed by a slow recovery.\r\n\r\nThe active protein which is responsible for ATP induced viscosity lowering reaction has been identified by studies of viscosity, electrophoresis and ultracentrifuge. It has been purified to 75% by salt precipitation and differential centrifugation. It is proposed that this protein be termed myxomyosin because of its similarity to actomyosin.\r\n\r\nMyxomyosin is found to complex with RNA to ca. 9% of its own weight. The RNA does not appear to be essential for the ATP-protein interaction process. RNA does, however, exert a great influence on the physical states of myxomyosin in solution.\r\n\r\nMyxomyosin preparations possess an ATPase activity. The turnover number of this enzymatic activity is small, namely, 30. A refractory state of myxomyosin has been found. Immediately after myxomyosin reacts with a large excess of ATP, the system enters into a refractory state which persists during the recovery of the viscosity level. In this state, the protein is not sensitive to ATP.\r\n\r\nViscosity, sedimentation, flow birefringence and electron microscopy studies reveal that myxomyosin is a rod-like molecule with a weight average molecular weight of 6 millions [plus or minus] 10% and with a range of 4.9 to 10 millions. The molecule possesses a diameter of 50 A [plus or minus] 5 A and a most frequent length of 4000 A with a range of 3000 to 6000 A.\r\n\r\nThe effect of ATP on myxomyosin has been investigated by viscosity, sedimentation, flow birefringence, electron microscopy and electrophoresis studies. There is no indication that myxomyosin breaks down into small units or suffers an extensive change in shape when it reacts with ATP. The present data are best understood on the basis that in the absence of ATP, myxomyosin aggregates in a concentration-dependent manner. The binding of ATP to the myxomyosin moiety reduces the aggregation of the monomers.\r\n\r\nA proposed mechanism of protoplasmic streaming, suggested by the above observations is presented." }, { "name": "Brookbank, John Warren", "degree": "PhD", "year": "1955", "title": "The Effects of Specific Antisera on the Cleavage of the Sea Urchin Egg", "advisor": "Tyler, Albert", "url": "https://resolver.caltech.edu/CaltechETD:etd-11242003-103345", "creators": [ { "name": { "family": "Brookbank", "given": "John Warren" }, "id": "Brookbank-John-Warren", "display_name": "Brookbank, John Warren" } ], "advisors": [ { "name": { "family": "Tyler", "given": "Albert" }, "id": "Tyler-A", "role": "advisor", "display_name": "Tyler, Albert" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/44DN-JR12", "abstract": "Rabbit antisera against sea urchin unfertilized eggs (gelatinous coat removed), fertilized eggs and the gelatinous coat (fertilizin) of the unfertilized eggs have been found to inhibit the nuclear and cytoplasmic divisions of the fertilized egg. Antisera against whole sperm or sperm antifertilizin extracts were not effective in this regard, and absorption of the effective sera with sperm did not remove the blocking antibody of those sera.\r\n\r\nExtracts (calcium-free sea water) of whole fertilized eggs containing the ectoplasmic layer material were found to react with anti-egg sera after absorption with sperm, but not after absorption with eggs. This material in this extract has been tentatively described in this thesis as a carbohydrate. One such extract was found to agglutinate the sperm of the species, but not sand dollar sperm. Subsequent extracts have not shown this property.\r\n\r\nThe respiration of fertilized eggs, blocked by the effective sera, was found to increase over the controls. \r\n\r\nThe sodium content of fertilized eggs treated with immune blocking serum was not found to differ from the sodium content of eggs in normal serum.\r\n\r\nThe tension at the surface of unfertilized eggs increases in the presence of anti-unfertilized egg serum, as judged by centrifugation experiments." }, { "name": "Furshpan, Edwin Jean", "degree": "PhD", "year": "1955", "title": "Studies On Certain Sensory and Motor Systems of Decapod Crustaceans", "advisor": "Wiersma, Cornelius A. G", "url": "https://resolver.caltech.edu/CaltechETD:etd-11212003-113407", "creators": [ { "name": { "family": "Furshpan", "given": "Edwin Jean" }, "id": "Furshpan-Edwin-Jean", "display_name": "Furshpan, Edwin Jean" } ], "advisors": [ { "name": { "family": "Wiersma", "given": "Cornelius A. G" }, "id": "Wiersma-C-A-G", "role": "advisor", "display_name": "Wiersma, Cornelius A. G" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/JM6N-EC44", "abstract": "Part I. Muscle Receptor Organs of the Crayfish.
\r\n\r\nSome interesting structures were discovered in the abdomens of certain Crustacea by J. S. Alexandrowicz (Quart. J. Micr. Sci. (1951) 92: 163) and in the present study their function was investigated. From his histological observations, Alexandrowicz deduced that these structures would serve as stretch receptors and his assumption was confirmed by the present work. It was found possible to isolate the organs and remove them from the animal. When the organs were stretched, using a specially constructed apparatus, trains of impulses were recorded from the nerve supplying them. There are two types of receptors in each half of each abdominal segment and the responses of each were found to differ from those of the other. One type (RM1) exhibited a prolonged slowly adapting discharge in response to a constant stretch; while the other type (RM2) adapted rapidly. The organs each consist of a muscular strand on which end the dendrites of the nearby sensory cell. It was shown that the muscular parts are unnecessary for the evocation of the sensory discharge by stretch; but that the former, by their contraction, can initiate impulses. The reactions of the receptors to several drugs were tested. Acetylcholine (ACh) in concentrations above a certain low value (10-6 g/ml.) augmented or initiated discharge in RM1. The effect was abolished by previous administration of atropine and enhanced by eserine. The discharge accompanying stretch was not abolished by atropine and it was concluded that ACh probably did not serve as a mediator of the normal stretch-discharge.
\r\n\r\nPart II. Excitation in Decapod Crustacean Muscles.
\r\n\r\nSeveral muscles in a number of species of decapods were studied using a combination of two techniques. On the one hand was the procedure for isolating and stimulating single motor axons; and on the other, the microelectrode method for recording intracellularly from single muscle fibers. The existence of two classes of responses was corroborated. The first type, termed \"junctional potentials,\" constituted the first muscular response to nerve stimulation. These potentials were found to be distributed to all parts of the muscle fiber by the motor nerve, in corroboration of previous evidence. The junctional potentials could summate when repeated and upon exceeding a certain threshold of depolarization evoked the second type of response: the spike potential. The latter was found to have the unusual properties of being graded and local. The spike was, however, followed by refractoriness. The distribution of motor axons to the individual muscle fibers was also studied. Two types of muscles were studied in this regard: those receiving two motor axons and those receiving four. In the muscles innervated by two axons, most muscle fibers were found to receive each of the axons, although several exceptions were noted. Each axon evoked a response of different character in any particular fiber; and, in addition, the size of the response elicited by either one of the axons varied markedly from muscle fiber to muscle fiber. The situation was even more complicated in the muscles innervated by four motor axons. Fibers were found which received only one axon, some received two, others three, and a small number were innervated by all four. In this muscle, then, aside from heterogeneity of response size, a marked variation in the axon complement of individual fibers was found. The responses evoked by different axons in a muscle fiber were found to summate and could thus act in concert to evoke a spike. The crustacean neuromuscular apparatus is thus a very complex reaction system and has some of the attributes of central nervous systems.
" }, { "name": "Kaiser, Armin Dale", "degree": "PhD", "year": "1955", "title": "A Genetic Analysis of Bacteriophage Lambda", "advisor": "Weigle, Jean J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-12032003-105441", "creators": [ { "name": { "family": "Kaiser", "given": "Armin Dale" }, "id": "Kaiser-Armin-Dale", "display_name": "Kaiser, Armin Dale" } ], "advisors": [ { "name": { "family": "Weigle", "given": "Jean J." }, "id": "Weigle-J-J", "role": "advisor", "display_name": "Weigle, Jean J." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/RR9B-8753", "abstract": "The genetic structure of the temperate bacteriophage lambda was analysed by means of crosses. Mutations in lambda which affected the size or type of plaque initiated by single particles were induced by ultraviolet irradiation. Each of four mutants so obtained was found to differ by one factor from the reference type. Two factor crosses demonstrated the existence of linkage between the 4 mutant factors and suggested a linear sequence which was later confirmed by three factor crosses. In addition 5 other factors were found to be linked to the first 4, thus all 9 factors studied seem to lie on the same linkage group.\r\n\r\nA striking feature of three factor crosses was the high frequency of double crossover types. This \"apparent negative interference\" was traced to the yields of individual bacteria by means of single burst experiments.\r\n\r\nFinally, the compatibility of the results of the lambda crosses with two different theories of phage recombination was examined." }, { "name": "Mandell, Joseph David", "degree": "PhD", "year": "1955", "title": "Inactivation of Bacteriophage T4r by Specific Antiserum", "advisor": "Delbruck, Max", "url": "https://resolver.caltech.edu/CaltechETD:etd-01142004-140840", "creators": [ { "name": { "family": "Mandell", "given": "Joseph David" }, "id": "Mandell-Joseph-David", "display_name": "Mandell, Joseph David" } ], "advisors": [ { "name": { "family": "Delbruck", "given": "Max" }, "id": "Delbr\u00fcck-M", "role": "advisor", "display_name": "Delbruck, Max" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/FP11-W197", "abstract": "After a brief discussion of antigens, haptens, and antibodies, and a review of the literature on the phage antiphage reaction, a general theory of the inactivation of phage by antiserum is developed. The discovery by Jerne and Skovsted (38) that the rate of inactivation of phage T4r by specific antiserum is a thousand times faster in a medium of low ionic strength than in 0.1 M salt solution has made possible the study of the kinetics of the reaction at extremely low concentrations of antiserum. The use of low concentrations of antiserum has allowed the investigation of the reaction in conditions of phage excess at such low concentrations of phage that aggregation of phage does not take place. From a study of the kinetics of inactivation at low concentrations of phage and antibody in a low ionic strength medium quantitative estimates were made of the number of sites on a phage particle to which antibody molecules may attach (700), the number of these sites which, when occupied, result in the inactivation of the phage particle (10-30), the number of specific inactivating antibody molecules in antiserum (10[superscript 16]/ml), and the efficiency of killing of a phage particle per collision of antibody with the tip of the tail (0.8)." }, { "name": "Rogers, Bruce Joseph", "degree": "PhD", "year": "1955", "title": "Studies on the Amino Acid Metabolism of Higher Plants", "advisor": "Bonner, James Frederick; Went, Frits W.; Borsook, Henry", "url": "https://resolver.caltech.edu/CaltechETD:etd-01202004-155710", "creators": [ { "name": { "family": "Rogers", "given": "Bruce Joseph" }, "id": "Rogers-Bruce-Joseph", "display_name": "Rogers, Bruce Joseph" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" }, { "name": { "family": "Went", "given": "Frits W." }, "id": "Went-F-W", "role": "advisor", "display_name": "Went, Frits W." }, { "name": { "family": "Borsook", "given": "Henry" }, "id": "Borsook-H", "role": "advisor", "display_name": "Borsook, Henry" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/NB9G-Q853", "abstract": "It has been known for some time that the protein level of detached leaves decreases as a result of the excision. The possibility that detached leaves are unable to form certain amino acids has been advanced as a cause for the decrease in the protein level. This hypothesis was tested in the present work. The incorporation with time of carbon-14 from C14-carboxyl-labeled acetate and C14-uniformly-labeled sucrose into the free and protein-bound amino acids of excised organs of red kidney bean (Phaseolus vulgaris) was studied. Of sixteen amino acids investigated, all were found to have incorporated carbon atoms from both acetate and sucrose in the excised leaves, stems and roots. On the basis of this work, it is felt that the decrease in protein level in detached leaves is not due to an inability of leaves to synthesize amino acids.\r\n\r\nThe incorporation of radioactive amino acids (produced in vivo) into leaf, stem and root protein of red kidney bean was studied. It was found that radioactive amino acids are incorporated into root protein much more rapidly than into leaf or stem protein.\r\n\r\nThe enzymatic decarboxylation and oxidation of a number of amino acids was studied in a water extract prepared from acorn squash. Great variation in the relative rates of reaction for the amino acids was demonstrated for both the decarboxylative and oxidative reactions.\r\n\r\nThe partial N-terminal amino acid sequence of pancreatic trypsin inhibitor was found to be arginyl-phenylalanine. It is suggested that the system used would be applicable for the determination of the sequence of small peptides such as those discovered in the work with red kidney bean." }, { "name": "Sachs, Roy Monroe", "degree": "PhD", "year": "1955", "title": "Floral Initiation in Cestrum nocrurnum (the Nightblooming Jasmine)", "advisor": "Went, Frits W.", "url": "https://resolver.caltech.edu/CaltechETD:etd-12082003-113617", "creators": [ { "name": { "family": "Sachs", "given": "Roy Monroe" }, "id": "Sachs-Roy-Monroe", "display_name": "Sachs, Roy Monroe" } ], "advisors": [ { "name": { "family": "Went", "given": "Frits W." }, "id": "Went-F-W", "role": "advisor", "display_name": "Went, Frits W." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/13FE-YJ23", "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\r\n\r\n1. The requirement for floral initiation in Cestrum nocturnum is satisfied if plants receive long days followed by short days. Continuous short or long days or short days preceding long days are ineffective.\r\n\r\n2. A minimum of 5-7 long days followed by two short days is required for the production of flowers.\r\n\r\n3. The sequence of reactions necessary for floral initiation in Cestrum is as follows:\r\n\r\n[...]\r\n\r\na. \"L\" is a heat labile substance produced in leaves as a result of long-day induction and required for short-day induction. It is not translocated from the leaves.\r\n\r\nb. \"S\" is a heat labile substance, possibly identical with the floral stimulus, formed as a result of short-day induction. It is formed only in leaves which have previously received long-day induction and is translocated to the growing points.\r\n\r\nThe production of \"L\" in other short day plants is controlled by day length independent reactions, and the production of \"S\" is controlled by daylength independent reactions in other long day plants.\r\n\r\n4. For Cestrum the critical day and night lengths for long and short-day induction, respectively, overlap in the 11.5 to 12.5-hour photoperiod range; the low efficiency of the two processes in this range precludes the possibility of an intermediate daylength satisfying both requirements. Other plants, flowering only in intermediate daylengths, may be related to Cestrum in requiring long followed by short-day induction. However, the critical photo- and nyctoperiods, in these plants, overlap sufficiently to permit simultaneous satisfaction of both requirements in an intermediate daylength." }, { "name": "Salisbury, Frank Boyer", "degree": "PhD", "year": "1955", "title": "Kinetic Studies on the Physiology of Flowering", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechETD:etd-01202004-162406", "creators": [ { "name": { "family": "Salisbury", "given": "Frank Boyer" }, "id": "Salisbury-Frank-Boyer", "display_name": "Salisbury, Frank Boyer" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/VV4M-DG13", "abstract": "Xanthium pennsylvanicum is induced to flower by exposure to a single uninterrupted dark period equal to or exceeding some minimum duration (ca. 9 hours). A system of stages of floral bud development is described, which gives a quantitative measurement of the degree of induction caused by various treatments. A number of factors which may affect the degree of induction are investigated, and it is concluded that age of the leaf and light intensity before and after induction strongly influence the effects of a given inductive dark period, while other factors are less important in this respect. Methods in the use of auxin are also described.
\r\n\r\nAuxin is shown to inhibit induction in the leaf, but to promote floral bud development after induction is complete. Applied auxin will replace a requirement for the presence of active buds after and just before induction.
\r\n\r\nThe induced state is discussed, and it is proposed that this condition consists of a given concentration of florigen in the plant which is maintained at the level brought about by the act of induction.
\r\n\r\nConcentration curves indicate that auxin acts in floral inhibition such as it does in other auxin-induced phenomena. Applied auxin has little effect upon the critical night length, but inhibits the rate of florigen synthesis which follows. Auxin is most effective when applied two to three hours after the beginning of the dark period, and its effectiveness decreases up until translocation of florigen out of the leaf is complete. Light interruption of the dark period is most effective after 8 hours. Light interruption and auxin are additive in their inhibitory effect.
\r\n\r\nIt is suggested that the act of induction consists of at least three phases: transformation of photo-receptor pigment, preparatory reaction, and hormone synthesis. A possible mechanism of auxin action through destruction of florigen is discussed.
" }, { "name": "Fischer, Glenn Albert", "degree": "PhD", "year": "1954", "title": "Genetic and Biochemical Studies of the Cystine-Methionine Series of Mutants in Neurospora crassa", "advisor": "Horowitz, Norman Harold", "url": "https://resolver.caltech.edu/CaltechETD:etd-12102003-111246", "creators": [ { "name": { "family": "Fischer", "given": "Glenn Albert" }, "id": "Fischer-Glenn-Albert", "display_name": "Fischer, Glenn Albert" } ], "advisors": [ { "name": { "family": "Horowitz", "given": "Norman Harold" }, "id": "Horowitz-N-H", "role": "advisor", "display_name": "Horowitz, Norman Harold" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/FP61-BW75", "abstract": "NOTE: Text or symbols not renderale in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\r\n\r\n1. The following enzyme activities were studied in extracts of wild type and mutant Neurospora: [...]. Activity 2 was shown to be absent in a homocysteineless mutant and activity 3 was shown to be absent in a cystathionineless mutant. It was found that a suppressor, obtained by Giles, which causes these mutants to grow on minimal, returns enzyme activity to the mutants. Each activity is shown to be catalyzed by a different enzyme.\r\n\r\n2. Evidence is presented which indicates that a Neurospora cystathionineless mutant can synthesize cystathionine from methionine without the intervention of cysteine.\r\n\r\n3. Mutants which are blocked between thiosulfate and cysteine are shown to grow on elemental sulfur and H[subscript 2]S." }, { "name": "Gershowitz, Henry", "degree": "PhD", "year": "1954", "title": "Immunogenetic Studies of the Pigeon Columba livia", "advisor": "Owen, Ray David", "url": "https://resolver.caltech.edu/CaltechETD:etd-12022003-170229", "creators": [ { "name": { "family": "Gershowitz", "given": "Henry" }, "id": "Gershowitz-Henry", "display_name": "Gershowitz, Henry" } ], "advisors": [ { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "advisor", "display_name": "Owen, Ray David" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/KXNQ-CM57", "abstract": "Reagents made from rabbit anti-pigeon red blood cell sera detected differences in erythrocyte antigens among individual pigeons. The differences detected by several of the reagents are inherited in a regular Mendelian manner, the positive reaction always dominant to absence of a reaction.\r\n\r\nA graded series of reactivities in pigeon red blood cells was observed in the use of rabbit Reagent A. All the cells tested were found capable of absorbing all agglutinins from this reagent. Positive cells differing in intensity of reaction all absorbed activity at the same rate, indicating that the differences between them are quantitative rather than qualitative in nature. These quantitative differences are inherited in a reasonably straightforward manner.\r\n\r\nRabbit Reagent E was shown to be composed of several qualitatively distinct fractions. Three subtypes were detected by the use of several E sub-reagents.\r\n\r\nSix isoimmune sera were produced, two of which (RC and H) were analyzed in detail. Each serum was shown to be complex, but the different antigens detected by each were probably related. Matings of positives x negatives which had positive and negative offspring produced them in approximately equal numbers.\r\n\r\nThe E subtypes and the antigens recognized by the isoimmune sera were seen to be closely related in some as yet unexplained manner. The possibilities that these relationships consisted of linkage or allelism of the causative genes are discussed.\r\n\r\nPositivity to Reagent A was found in 14-day pigeon embryos. Antigen C was detected on the cells of some newly-hatched squabs, but the antigens recognized by Reagents E and H were first detected on the cells of 7-day-old squabs. Cells of positive squabs reached maximum intensities of reaction to all the reagents in one to three weeks after hatching." }, { "name": "Greene, Ronald Crundon", "degree": "PhD", "year": "1954", "title": "Studies on Uricase from Neurospora crassa", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechETD:etd-01072004-112743", "creators": [ { "name": { "family": "Greene", "given": "Ronald Crundon" }, "id": "Greene-Ronald-Crundon", "display_name": "Greene, Ronald Crundon" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/AWRN-5N12", "abstract": "A study has been made of the enzyme uricase from the mold Neurospora crassa.\r\n\r\nIt has been shown that the yield of the enzyme can be increased by growth of the mold in the presence of uric acid. It has also been observed that such addition of uric acid to the medium is stimulatory to growth.\r\n\r\nA procedure has been devised for purification of this enzyme which is not bound to particulate material in the mold and a product has been obtained which has a higher specific activity than that of any uricase previously prepared. Neurospora uricase has been found to differ in some physical properties, such as solubility, and pH optimum, from the uricases prepared from animal sources. However, inhibition and activation experiments have shown that the enzymes from both sources are functionally similar.\r\n\r\nResults of partial inhibition of uricase by p-chloromercuribenzoate and activation by reducing agents, such as glutathione and sulfide, have been obtained, which indicate involvement of sulfhydryl groups in at least an activating role.\r\n\r\nA boiled extract of whole Neurospora has been found to contain at least one activating substance, and reasons have been given for believing that this activation is different from that produced by reducing agents.\r\n\r\nThe question of the existence of a uricase cofactor has been discussed.\r\n\r\nSpectrophotometric evidence has been obtained for the existence of two or more transient intermediates during the course of uric acid oxidation. A product which has an absorption spectrum similar to those observed for one or more of the transient intermediates has been prepared by oxidation of uric acid with alkaline permanganate.\r\n\r\nThe question of whether uricase is a single enzyme or a complex of enzymes has been discussed." }, { "name": "Hinton, Claude Willey", "degree": "PhD", "year": "1954", "title": "The Behavior of an Unstable Ring Chromosome in Drosophila melanogaster", "advisor": "Sturtevant, Alfred Henry", "url": "https://resolver.caltech.edu/CaltechETD:etd-12152003-115148", "creators": [ { "name": { "family": "Hinton", "given": "Claude Willey" }, "id": "Hinton-Claude-Willey", "display_name": "Hinton, Claude Willey" } ], "advisors": [ { "name": { "family": "Sturtevant", "given": "Alfred Henry" }, "id": "Sturtevant-A-H", "role": "advisor", "display_name": "Sturtevant, Alfred Henry" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/KKEX-R511", "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document. \r\n\r\nThe behavior of \"Catcheside's ring,\" an unstable closed-X chromosome [...] of Drosophila melanogaster, has been analyzed with the purpose of identifying the factors controlling the variable-level of [...] instability and of determining the mechanism of [...] elimination. The Y chromosome and the structure of the homologous rod-X chromosome were shown to have no influence on the frequency of [...] elimination. Since no segregating autosomal or sex-linked modifiers could be detected, the primary control of [...] instability must be limited to the [...] chromosome itself. The behavior of unstable small duplications derive from the [...] chromosome suggests that the locus of [...] instability must be in or near the [...] centromere region.\r\n\r\nThe frequency of [...] elimination is directly related to developmental temperature and to age of maternal [...] parents. Certain preliminary results incurred the speculation that a cytoplasmic factor is operative in [...] elimination.\r\n\r\nEither anaphase lagging or the production of anaphase bridges by the [...] chromosome will account for its loss. The occurrence of [...] derivatives, deficient for either extensive or small euchromatic segments, suggests that anaphase bridges composed of continuous dicentric rings may be formed; however, such bridges do not necessarily constitute the exclusive means of elimination." }, { "name": "Jaffe, Lionel Francis", "degree": "PhD", "year": "1954", "title": "On a Carbon Dioxide Gradient in the Fucales Egg", "advisor": "Tyler, Albert", "url": "https://resolver.caltech.edu/CaltechETD:etd-01062004-120112", "creators": [ { "name": { "family": "Jaffe", "given": "Lionel Francis" }, "id": "Jaffe-Lionel-Francis", "display_name": "Jaffe, Lionel Francis" } ], "advisors": [ { "name": { "family": "Tyler", "given": "Albert" }, "id": "Tyler-A", "role": "advisor", "display_name": "Tyler, Albert" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/3H2M-P580", "abstract": "Previous investigations suggest that a CO2-pH gradient is involved in the development of polarity in the Fucales egg. To test this hypothesis, a method was developed to measure the relative CO2 output from the two hemispheres of this egg. The eggs are photosynthetically tagged with C14 and embedded in a thin jelly membrane in which the eggs develop. One measures separately the respiratory C14O2 emitted from the two surfaces of the membrane during each of a series of intervals before, during, and after the eggs are unilaterally illuminated to orient their future axes. New methods are described for obtaining eggs, for controlling and measuring the thicknesses of egg-bearing jelly membranes, and for measuring C14O2. Because of a drop in the specific radioactivity of the emitted carbon dioxide, the sum of the rates of respiratory C14O2 emission from the two faces of a membrane decreases rapidly with time; but the C14O2-ratio, or the ratio of the rates of C14O2 emission from the two faces, changes very slightly with time. That face which had been lower while the membrane gelled nearly always emits C14O2 more rapidly than the other face. This is attributed partly to a settling of the eggs in the membrane while it gelled and partly to a higher respiratory rate somehow induced in the hemispheres of the eggs which point toward the formerly lower face. These \"lower\" hemispheres also tend to become the rhizoid poles. The changes of the C14O2-ratios with time are attributed primarily to changes in the relative specific radioactivities in the carbon dioxide emitted from the two hemispheres of the eggs, rather than to further changes in the relative respiratory rates of these hemispheres with time." }, { "name": "Judd, Burke Haycock", "degree": "PhD", "year": "1954", "title": "Studies of Rearrangements Involving Heterochromatin in Drosophila melanogaster : l. A Proof of the Variegated-Type Position Effect at the White Locus. II. A Study of the Heterochromatin of Chromosome IV", "advisor": "Lewis, Edward B.", "url": "https://resolver.caltech.edu/CaltechETD:etd-01152004-095338", "creators": [ { "name": { "family": "Judd", "given": "Burke Haycock" }, "id": "Judd-Burke-Haycock", "display_name": "Judd, Burke Haycock" } ], "advisors": [ { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "advisor", "display_name": "Lewis, Edward B." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/4F9F-MV78", "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\r\n\r\nThe X-IV translocation, [...], is shown to contain the wild-type allele, [...], at the white locus. This [...] has been replaced with a mutant gene, w, and a comparison of R([...])/w with R(w)/w[...] shows the former to give a variegated white phenotype while the latter is completely wild-type. It is concluded that the white variegation is due to an instability in the action of [...] when it is located in the rearranged chromosome.\r\n\r\nCold temperature enhances variegation particularly when applied during the embryonic stages of development. A less sensitive period is found to exist during the pupal stages. These facts indicate the white gene is active during more than a single period of development.\r\n\r\nTwelve duplication-deficiency types have been obtained by combining the left and right parts of four X-IV translocations. Tests for survival of these combinations in the haplo-IV condition give somewhat contradictory results. These results are discussed and a possible order for the fourth chromosome translocation breaks is given." }, { "name": "Ames, Bruce Nathan", "degree": "PhD", "year": "1953", "title": "The Biosynthesis of Histidine in Neurospora crassa", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechETD:etd-04212003-092730", "creators": [ { "name": { "family": "Ames", "given": "Bruce Nathan" }, "id": "Ames-Bruce-Nathan", "display_name": "Ames, Bruce Nathan" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/R38C-Z453", "abstract": "The biosynthesis of histidine has been investigated using a series of histidine requiring mutants of Neurospora and Penicillium. A general method is given for the isolation of a number of imidazole derivatives that are accumulated by these mutants. Methods of degradation and chromatography have been used to identify with reasonable certainty three crystalline products as 4-(D-erythro-trihydroxypropyl)-imidazole, 4-(2-keto-3-hydroxypropyl)-imidazole and L-4-(2-amino-3-hydroxypropyl)-imidazole (L-histidinol). Evidence is presented to show that two other non-crystalline products are phosphate esters of the trihydroxy and the ketohydroxy compounds. Chromatographic evidence is presented which indicates two other compounds are imidazole acetic acid and 4-(2,3-dihydroxypropyl)-imidazole. A chemical synthesis of isomers of 4-(trihydroxypropyl)-imizadole is described.
\r\n\r\nThe trihydroxy, ketohydroxy, and aminohydroxy compounds have been placed in that order leading to histidine, on the basis of the sequence of the mutants and the accumulations by the double mutants. Evidence is discussed suggesting that the phosphate esters of these compounds are more likely to be the true precursors of histidine. A scheme is postulated for the relation of all these substances to histidine synthesis and the possibility that a pentose-5-phosphate may be an early precursor of histidine is discussed.
\r\n\r\nA method for the paper chromatography of imidazoles is presented and the Rf values of numerous synthetic derivatives are given.
" }, { "name": "Boroughs, Howard James", "degree": "PhD", "year": "1953", "title": "I. Studies on the Acid Phosphatases of Green Leaves. II. Studies on the Role of Indoleacetic Acid in Cell Elongation", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechETD:etd-04212003-115806", "creators": [ { "name": { "family": "Boroughs", "given": "Howard James" }, "id": "Boroughs-Howard-James", "display_name": "Boroughs, Howard James" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "role": "member", "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/H6ZH-WM47", "abstract": "The bulk protein of green leaves has been shown to be dissociable and probably distinct from the acid phosphatase previously associated with it. This conclusion is based on the differential sedimentation rates in the analytical ultracentrifuge of the bulk protein and of phosphatase, on the relative amount of enzyme present in the bulk protein prepared by salt-precipitation or by differential centrifugation, and on the comparative stability of the two proteins toward heat and acid.\r\n\r\nLeaf phosphatase is further shown to be a mixture of isodynamic enzymes with different pH optima and Michaelis constants. A separation was accomplished by adsorption, dialysis, and by antigen-antibody reactions.\r\n\r\nThe phosphatases are shown to lose enzymatic activity during dialysis, but to be capable of reactivation by certain metal ions. Copper ions are the most effective activators. Comparison of the spectrographic analysis of the ash of dialyzed phosphatase with enzymatic activity, however, disclosed a direct relation between activity and the amount of iron and manganese, as well as copper. Various comparative aspects of the enzymes were also studied.\r\n\r\nThe influence of auxin on metabolic pathways is studied by a new method, in which the rate of incorporation of isotopically labeled intermediates into varied components of living tissue is studied as a function of the auxin supplied.\r\n\r\nIt is found that auxin is without effect on the total protein amino nitrogen, or on the rate of incorporation of the C[superscript 14] label from glycine or leucine into tissue proteins of Avena and corn coleoptiles. Hence, auxin has no effect on protein metabolism during cell elongation.\r\n\r\nSimilarly, auxin exerted only small effects on the rate of incorporation of C[superscript 14] from acetate into the lipid constituents of Avena coleoptiles. Added auxin induced no important changes in the incorporation of the C[superscript 14] of acetate or sucrose into the cell wall components of Avena. It was shown, however, that absence of auxin favors incorporation of the isotopic label of acetate or sucrose into the pectates and soluble polyuronide hemicelluloses, while the presence of auxin favors incorporation into the non-cellulosic polysaccharides. These effects are of small magnitude in relation to gross increase in cell length." }, { "name": "Bowen, George Hamilton", "degree": "PhD", "year": "1953", "title": "Kinetic Studies on the Mechanical of Photoreactivation of Bacteriophage T2 Inactivated by Ultraviolet Light", "advisor": "Delbruck, Max", "url": "https://resolver.caltech.edu/CaltechETD:etd-04212003-134051", "creators": [ { "name": { "family": "Bowen", "given": "George Hamilton" }, "id": "Bowen-George-Hamilton", "display_name": "Bowen, George Hamilton" } ], "advisors": [ { "name": { "family": "Delbruck", "given": "Max" }, "id": "Delbr\u00fcck-M", "role": "advisor", "display_name": "Delbruck, Max" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/NCJW-ZP95", "abstract": "Bacteriophage particles are called active if they are capable of generating a plaque when plated on agar by a standard technique. Exposure of the particles to ultraviolet radiation of wave length 253.7 m[mu] inactivates them in this sense. Following adsorption of the inactive particles to sensitive host bacteria, exposure of the suspension to light of the violet and near ultraviolet region causes a fraction of the particles to regain their activity, a phenomenon called photoreactivation.\r\n\r\nThe kinetics of photoreactivation of bacteriophage T2 have been investigated for the purpose of studying the mechanism by which photoreactivation takes place. The presence of a dark reaction in addition to the light reaction has been demonstrated. The dark reaction precedes the other and has the function of supplying the light-absorbing material which enters into the light reaction. Both the light and the dark reactions follow first-order kinetics.\r\n\r\nThe amount of photoreactivation produced by a given light treatment is determined by the interaction of the light and dark reactions. This interaction can be described satisfactorily in terms of a simple model for the reaction mechanism." }, { "name": "Dubes, George Richard", "degree": "PhD", "year": "1953", "title": "Investigations of Some \"Unknown\" Mutants of Neurospora crassa", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechETD:etd-04222003-143216", "creators": [ { "name": { "family": "Dubes", "given": "George Richard" }, "id": "Dubes-George-Richard", "display_name": "Dubes, George Richard" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/WYR6-7736", "abstract": "A sample of 16 previously \"unknown\" mutants of Neurospora crassa was studied. Five of these were shown to be double mutants. The growth requirements of 19 of the 21 single-gene mutants available then from these \"unknowns\" were at least partially defined chemically. Of these 19, three have been classified as serineless, two as asparagineless, two as pyramidineless, two as succinicless, and one each as aceticless, arginineless, aromaticless, asparticless, homoserineless, leucineless, methionineless, methionine-adenine-cystineless, p-aminiobenzoicless, and slow-grower. These had been classified as \"unknowns\" apparently for various reasons, such as: (1) inhibitions from normal metabolites, (2) complex requirements for double-mutant \"unknowns,\" and (3) absence of active compound from chemically defined mixtures previously used. Some of these mutant genes have been located genetically. These results have been discussed briefly with reference to the hypothesis of the \"unifunctionality\" of the gene." }, { "name": "Eggman, William Luther", "degree": "PhD", "year": "1953", "title": "The Cytoplasmic Protein Component of Green Leaves", "advisor": "Wildman, Samuel G.; Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechETD:etd-05082003-154241", "creators": [ { "name": { "family": "Eggman", "given": "William Luther" }, "id": "Eggman-William-Luther", "display_name": "Eggman, William Luther" } ], "advisors": [ { "name": { "family": "Wildman", "given": "Samuel G." }, "id": "Wildman-Samuel-G", "role": "advisor", "display_name": "Wildman, Samuel G." }, { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/MQ5F-JT09", "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document. \r\n\r\nThe soluble cytoplasmic proteins of the leaves of dicotyledonous plant species have been characterized as to their chemical and physical chemical properties. Elephoretically, the proteins migrate as a single major component and from one to six minor components, the number depending upon the species. The principal component has a mobility of ca. ?5.0 to ?5.5 x 10[superscript ?5] [. . .] K-maleate buffer at pH 7.0, and constitutes ca. 50 to 80 per cent of the total protein. In the analytical ultracentrifuge, the proteins are resolved into two components; a high-molecular weight, apparently homogeneous component with a corrected sedimentation, constant of 19S which constitutes ca. 30 to 50 per cent of the total protein, and a low-molecular weight heterogeneous, fraction which contains the enzymatic activity.\r\n\r\nChemically, the cytoplasmic proteins contain 13 to 15 per cent nitrogen and from 0.1 to 0.8 per cent phosphorus. This protein-bound [phosphorus] was found to be associated with the 19S component of cytoplasm in the form of ribonucleic acid. The principal component of cytoplasm is, therefore, a nucleoprotein.\r\n\r\nThe nucleotide composition of the ribonucleic acid in the cytoplasms of leaves of different plant species and in leaves of different physiological ages but from the same plant species were studied. The composition varies with the plant species but not with physiological age.\r\n\r\nThe effects of pH, of temperatures from 0[degrees] C. to 37[degrees] C., of dialysis and of storage at ?20[degrees] C. upon the stability of whole cytoplasm preparations was studied by chemical and ultracentrifugal analysis. Acidity greater than pH 6.5 and storage at ?20[degrees] C. for extended periods preferentially denatures the protein moiety of the nucleoprotein component, while dialysis or incubation at room temperatures for short periods of time causes the loss, apparently by enzymatic degradation, of the ribonucleic acid moiety. No method of entirely preventing the loss of nucleic acid was found, although maintenance of a low temperature partially suppresses it.\r\n\r\nA differential sedimentation procedure capable of preparing high-molecular weight fractions of cytoplasm (Fraction I protein preparations) containing only 5 to 10 per cent of low-molecular weight contaminants was developed when classical methods of chemical fractionation proved unsuitable. Such preparations were used to determine the physical properties of the nucleoprotein component.\r\n\r\nThe nucleoprotein components of spinach and tobacco have been shown to be closely similar, although certain physical properties of the nucleoprotein, such as partial specific volume, sedimentation constant and molecular weight, are dependent on the ribonucleic acid content.\r\n\r\nThe molecular weight of the nucleoprotein component in a particular preparation containing 11 per cent nucleic acid is estimated to be 360,000, based on determination of the partial specific volume and the sedimentation constant of this preparation together with an estimation of the frictional coefficient from a deduced shape factor and an assumed hydration value.\r\n\r\nFraction I protein preparations always contain high-molecular weight components that are not initially present in whole cytoplasm. It is shown that these components probably have their origin in aggregation of the nucleoprotein component." }, { "name": "Goldacre, Peter Lionel", "degree": "PhD", "year": "1953", "title": "Destruction of Indole-3-Acetic Acid by Plant Tissues", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechETD:etd-05012003-110235", "creators": [ { "name": { "family": "Goldacre", "given": "Peter Lionel" }, "id": "Goldacre-Peter-Lionel", "display_name": "Goldacre, Peter Lionel" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/G9FF-M178", "abstract": "This paper concerns those systems present in the epicotyls of eliolated pea seedlings which inactivate the plant hormone indole-3-acetic acid (I.A.A.). It consists of two sections which deal with (a) the enzyme system collectively known as I.A.A. oxidase and (b) a group of dialyzable substances which sensitize the photodestruction of I.A.A.\r\n\r\nI.A.A. oxidase, which behaves as a flavoprotein coupled to a peroxidase is shown to have a partial cofactor requirement. Two alternative systems are postulated. The two systems are differentiated on the basis of their response to Mn++ and to 2,4-dichlorophenol (D.C.P.) and by their change in relative concentrations upon exposure of the seedlings to red light. D.C.P. is shown to increase the activity of I.A.A. oxidase at low concentrations. The mechanism of the effect is studied in detail. Although D.C.P. is a powerful inhibitor of catalase, which inhibits I.A.A. oxidase, it is concluded that the enhancement of I.A.A. oxidase by D.C.P. is not due to its inhibition of catalase. Probably D.C.P. is acting similarly to the native cofactor. D.C.P. increases the in vivo destruction of I.A.A. by some tissues, not by others. It is suggested as a useful tool for studying altered I.A.A. level in a tissue.\r\n\r\nThe dialyzate from epicotyl brei contains at least four components which sensitize the inactivation of I.A.A. in the light but not in the dark. Blue light is the most effective. The kinetics of the action of the dialyzate has been studied. The active material resides principally in the buds. Preliminary methods of purification have been explored. Some possible physiological roles of the I.A.A. oxidase and the dialyzate have been discussed" }, { "name": "Holloway, Bruce William", "degree": "PhD", "year": "1953", "title": "Heterocaryosis in Neurospora crassa", "advisor": "Beadle, George Wells; Emerson, Sterling", "url": "https://resolver.caltech.edu/CaltechETD:etd-04232003-113543", "creators": [ { "name": { "family": "Holloway", "given": "Bruce William" }, "id": "Holloway-Bruce-William", "display_name": "Holloway, Bruce William" } ], "advisors": [ { "name": { "family": "Beadle", "given": "George Wells" }, "id": "Beadle-G-W", "role": "advisor", "display_name": "Beadle, George Wells" }, { "name": { "family": "Emerson", "given": "Sterling" }, "id": "Emerson-S", "role": "advisor", "display_name": "Emerson, Sterling" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/VRRH-S044", "abstract": "It has been shown that heterocaryon formation between certain biochemical mutants of Neurospora crassa is controlled by a number of genes apart from the biochemical mutant genes concerned. Genetic control of heterocaryosis has been shown for several different combinations of mutants. A detailed investigation of the heterocaryon formed between a mutant requiring pantothenic acid and one requiring lysine has demonstrated four and possibly five genes to be concerned in the process of heterocaryon formation. These genes may not only prevent the formation of a heterocaryon but also modify the type of heterocaryotic growth. The characters so affected are the time at which heterocaryotic growth commences, the ability to maintain heterocaryotic growth and the vigor of the growth. The possible mode of action of such genes has been discussed." }, { "name": "Jansen, Leonard Leroy", "degree": "PhD", "year": "1953", "title": "Studies in Fruit Growth and in Vernalization", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechETD:etd-04232003-161523", "creators": [ { "name": { "family": "Jansen", "given": "Leonard Leroy" }, "id": "Jansen-Leonard-Leroy", "display_name": "Jansen, Leonard Leroy" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/9JPG-AE41", "abstract": "Growth and development of flowers and fruits of red currant tomato (Lycopersicon pimpinellifolium) have been studied both on the intact plant and with the excised flower cultivated in vitro. A new interpretation of development of the inflorescence has been presented. Two types of growth response of ovaries of pollinated flowers have been identified both on the plant and in culture. In one type growth is linear and in the other it is exponential. The latter type, on the plant, produces seeded fruits, and size of fruits is correlated with number of seeds. Development on the plant is apparently optimal at a night temperature of 17[degrees]C. Smog is detrimental to development.\r\n\r\nGrowth of the pollinated ovary in vitro can be supported on a minimal medium of mineral salts and sucrose. The two response types differ in carbohydrate requirements. A pollination-fertilization factor is postulated as the primary difference between them. Growth of the ovary in vitro can be influenced by temperature, auxin, casein hydrolysate, and concentration of sucrose. The temperature optimum for development in culture differs from that for development on the plant. Mechanisms of the responses have been discussed.\r\n\r\nThe cold-requiring processes which promote flower initiation in Petkus winter rye have been analyzed more critically for their oxygen and sugar requirements by vernalizing the excised embryos under atmospheres of air or nitrogen on media with or without sugar. The first partial process of vernalization requires sugar but does not require oxygen. The second process does not require sugar but is dependent upon oxygen. Separation of the partial processes now becomes possible." }, { "name": "McRae, Dougal Harold", "degree": "PhD", "year": "1953", "title": "Studies on the Kinetics of Auxin-Induced Growth", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechETD:etd-05082003-094406", "creators": [ { "name": { "family": "McRae", "given": "Dougal Harold" }, "id": "McRae-Dougal-Harold", "display_name": "McRae, Dougal Harold" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/R2GS-R439", "abstract": "The auxin-induced growth reaction of the Avena coleoptile has been treated by methods of classical enzyme kinetics. The kinetic treatment makes it possible to characterize the growth promoting activity of an auxin by two parameters, K[subscript s] and V[subscript max]. These express respectively the affinity of the auxin for the auxin-receptive site within the plant and the ability of the complex thus established to promote growth.\r\n \r\nTreatment of Avena section growth by the methods of enzyme kinetics has made it possible to determine rigorously whether or not inhibitors of auxin-induced growth are true antiauxins and act by competing with auxin for the auxin-receptive site within the plant. Certain auxin-inactive compounds have been shown to possess antiauxin activity. Among such substances are 2,4-dichloroanisole, 4,-chloro- and 2,4-dichlophenoxyisobutyric acids, and 2,6-dichloro-and 2,4,6-trichlorophenoxyacetic acids. Each of these substances can be considered as derived from an active auxin (2,4-dichlorophenoxyacetic acid) by elimination of one of the structural features essential to auxin activity and thereby capable of combining at one point of the auxin-receptive site but incapable of consumating the two-point attachment requisite for auxin activity.\r\n\r\nIt is shown that chemically different auxins compete with one another for the same receptive sites within the plant. Auxins of low v[subscript max] are capable of inhibiting or augmenting the activity of auxins of greater V[subscript max]. Whether inhibition or promotion result depend on the concentrations of the two substances and the differential in V[subscript max] exhibits apparent antiauxin activity. This activity is shown to be different from competitive inhibition of true antiauxins.\r\n\r\nThe relationship between Avena section growth rate and auxin concentration has been demonstrated to be predictable on the basis of a requirement for two-point attachment of the auxin molecule to some receptive entity within the plant.\r\n\r\nThe growth inhibition resulting from high auxin concentrations is not alleviated by antiauxins but rather the auxin inhibition is augmented by the presence of sufficiently high antiauxin concentrations. A necessary corollary to the single point attachment antiauxin concert and the bimolecular complex formation concept for inhibitory auxin concentrations is therefore confirmed.\r\n\r\nA preliminary investigation concerning herbicidal activity of mixtures of an antiauxin and an auxin on bean plants is presented. The data obtained do not unequivocally establish that inactive bimolecular auxin receptor complex formation at high auxin concentrations is a factor contributing to herbicidal action of 2,4-D, but the possibility that this is in fact so is considered.\r\n\r\nA cultural technique for obtaining isolated flax root clones is described and data for some experiments with an isolated flax root clone are presented. The inhibitory action of certain antimetabolites on the growth of young tomato plants is described." }, { "name": "Rappaport, Irving", "degree": "PhD", "year": "1953", "title": "An Immunological Study of Chicken Serum Albumin and Related Organ Antigens", "advisor": "Owen, Ray David", "url": "https://resolver.caltech.edu/CaltechETD:etd-05082003-163307", "creators": [ { "name": { "family": "Rappaport", "given": "Irving" }, "id": "Rappaport-Irving", "display_name": "Rappaport, Irving" } ], "advisors": [ { "name": { "family": "Owen", "given": "Ray David" }, "id": "Owen-R-D", "role": "advisor", "display_name": "Owen, Ray David" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "immun" ], "doi": "10.7907/CKW1-1Z19", "abstract": "1. The percentage of the proteins in the plasma of New Hampshire Red chickens was found to increase from 2.0g to 5.89g per 100 ml., as the chickens matured.\r\n\r\n2. The albumin/globulin ratio decreased from 0.75 to 0.25 with increasing age of the chickens.\r\n\r\n3. The mobility of serum albumin in veronal buffer at pH 8.6, u= 0.1 was found to be 6.8 ? 0.11 x 10[superscript -5] cm[superscript 2]/volt/sec.\r\n\r\n4. More than 95% of the soluble proteins from livers and kidneys migrated with mobilities different from serum albumin.\r\n\r\n5. Precipitin tests suggested that the soluble proteins of the liver include approximately one-half of one per cent homologous serum albumin, whereas the kidney does not include any serologically detectable amounts of serum albumin.\r\n\r\n6. No measurable cross reactions occurred between rabbit anti-chicken serum albumin and the soluble proteins from chicken livers and kidneys.\r\n\r\n7. More serum albumin was detected after the incubation of liver slices in KCl metabolite than before incubation. This suggested that a net synthesis may have occurred.\r\n\r\n8. More serum albumin was detected in the soluble proteins within the slices after incubation in KC1 metabolite to which different amounts of antiserum were added than in the controls where normal rabbit serum was present. This suggested that the antiserum inhibited the release of serum albumin from the cells.\r\n\r\n9. The significance of these findings has been discussed." }, { "name": "Ripley, Sherman Harvey", "degree": "PhD", "year": "1953", "title": "Studies on Myo-Neural Mechanisms in Arthropoda", "advisor": "Wiersma, Cornelius A. G; Van Harreveld, Anthonie", "url": "https://resolver.caltech.edu/CaltechETD:etd-05122003-120648", "creators": [ { "name": { "family": "Ripley", "given": "Sherman Harvey" }, "id": "Ripley-Sherman-Harvey", "display_name": "Ripley, Sherman Harvey" } ], "advisors": [ { "name": { "family": "Wiersma", "given": "Cornelius A. G" }, "id": "Wiersma-C-A-G", "role": "advisor", "display_name": "Wiersma, Cornelius A. G" }, { "name": { "family": "Van Harreveld", "given": "Anthonie" }, "id": "Van-Harreveld-A", "role": "advisor", "display_name": "Van Harreveld, Anthonie" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/S8TQ-7214", "abstract": "PART I. Innervation Patterns of Crustacean Limbs.\r\n\r\nIn contrast to the vertebrates where the skeletal muscle is built up of large numbers of motor units, the limb muscles of decapod Crustacea are innervated by a small number of motor and inhibitory axons. It is possible to determine the details of the innervation and of the muscular response elicited by each of the several axons by isolating them in the nerve trunk. By such techniques, the previously determined innervation patterns of the walking legs in the four tribes of the Decapoda Reptantia have been confirmed and extended. Incomplete innervation patterns for the Decapoda Natantia and Stomatopoda have also been determined. Many characteristic features of innervation and associated motor and inhibitory effects were found to be common to all the Decapoda and the Stomatopoda. These physiologically derived anatomical data support the classification of the Decapoda into Reptantia and Natantia with division of the former into four tribes. A tentative evolutionary hypothesis is presented to explain the divergence of the different innervation patterns.\r\n\r\nPART II. The Effect of Spaced Stimulation of Excitatory and Inhibitory Axons of the Cray-fish.\r\n\r\nIf a single motor axon to one of the limb muscles of a crustacean is stimulated with pairs of shocks repeated at regular intervals, the resulting contraction is greater than that elicited by the same number of regularly repeated single shocks. This effect was previously studied in the fast and slow contractions of muscles in various decapod Crustacea. The authors concluded that the effect was much more pronounced in the fast than in the slow system. In order to extend these observations and to determine whether or not it is a true myo-neural junctional phenomenon, the effect of unequally spaced stimulation of both the excitatory and inhibitory axons in the abductor of the dactylopodite was studied. The contractions resulting from such stimulation of the excitatory axon were more difficult to inhibit than were the normal contractions. Stimulation of the inhibitory axon with unequally spaced shocks resulted in a much greater reduction of the contraction than when it was stimulated with the same number of regularly recurring single shocks. It is concluded therefore, that the spacing effect occurs at the myo-neural junction and that it is not a mechanical effect at the level of the contraction.\r\n\r\nPART III. Neuromuscular Mechanisms in the Grasshopper, Romalea microptera (Beauv.).\r\n\r\nInsect myo-neural physiology has not been extensively studied but recently the existence of single and double innervation and of fast and slow contractions has been reported. The occurrence of peripheral inhibition has been claimed but the observations cannot be considered conclusive. In view of the phylogenetic relationship between the Crustacea and Insecta it might be expected that the two sub-phyla would have many characteristics of the myo-neural system in common. Both histological and physiological observations of the limb muscles of Romalea have confirmed this expectation. With one exception, these muscles are innervated by about six axons, each of which gives rise to a characteristic contraction. The majority of these contractions are of the fast type. True slow contractions have not been observed. There is no marked facilitation of either the contractions or the action potentials. The large 'jump' muscles of the third limb are specialized and exhibit only a single fast contraction type. No evidence for peripheral inhibition has been obtained." }, { "name": "Saltman, Paul David", "degree": "PhD", "year": "1953", "title": "Enzymatic Phosphate Transfer in Plant Systems", "advisor": "Lucas, Howard J.", "url": "https://resolver.caltech.edu/CaltechETD:etd-05092003-152156", "creators": [ { "name": { "family": "Saltman", "given": "Paul David" }, "id": "Saltman-Paul-David", "display_name": "Saltman, Paul David" } ], "advisors": [ { "name": { "family": "Lucas", "given": "Howard J." }, "id": "Lucas-H-J", "role": "advisor", "display_name": "Lucas, Howard J." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/P4DK-FW41", "abstract": "The experiments described in this thesis have established that the same phosphorylated intermediates of carbohydrate metabolism known to occur during the course of glycolysis in yeast and muscle tissue are involved in the carbohydrate metabolism of the pea seed. In the identification of these glycolytic intermediates use has been made of chromatographic and other techniques developed for the purpose.\r\n\r\nTwo enzymes of the glycolytic sequence not previously characterized in higher plants have been separated and studied. These are phosphofructokinase and hexokinase. Both of the enzymes are concerned with the direct phosphorylation of substrate with adenosine triphosphate (ATP) - a key compound in the energy metabolism of the cell.\r\n\r\nPhosphofructokinase, which catalyzes the reaction\r\n\r\nfructose-6-phosphate + ATP <-> fructose-1, 6-diphosphate + ADP\r\n \r\nhas been partially purified from pea seed meal and characterized with respect to its pH optimum, inhibitors, stability, substrate affinities, and substrate specificity. A method for the assay of the enzymatic activity has bean developed in which aldolase is employed for the direct determination of fructose diphosphate formed.\r\n\r\nHexokinase, which catalyzes the reaction\r\n\r\nhexose + ATP <-> hexose-6-phosphate + ADP\r\n\r\nhas, likewise, been partially purified and characterized with respect to several of its properties. Both enzymes have been found in a variety of higher plants, indicating the ubiquity of the glycolytic sequence.\r\n\r\nAlthough phosphofructokinase is a soluble cytoplasmic constituent, hexokinase appears to occur both in an insoluble and in an soluble form, the distribution being dependent upon the tissue studied and the method of preparation used. The fact that the insoluble or mitochondrial fraction seems to contain the major fraction of the hexokinase activity is of interest in light of the fundamental role of the mitochondria in the generation of ATP via oxidative phosphorylation." }, { "name": "Good, Norman Everett", "degree": "PhD", "year": "1952", "title": "Lysine Metabolism in Neurospora", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10062017-091942585", "creators": [ { "name": { "family": "Good", "given": "Norman Everett" }, "id": "Good-Norman-Everett", "display_name": "Good, Norman Everett" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/T8E4-N918", "abstract": "The lysine requiring mutants of Neurospora crass, when\r\nclassified by the criteria of symbiotic, specificity\r\nof growth requirements, intersterility and genetic recombination,\r\nfall into five groups representing at least five loci.\r\nMembers of two groups are capable of growth on a minimal\r\nmedium supplemented with either lysine, \u2208-hydroxynorleucine\r\nor \u03b1-aminoadipic acid. Members of a third group can utilize\r\nlysine or \u2208-hydroxynorleucine, while members of the fourth\r\nand fifth group are unable to grow in the absence of lysine \r\nitself.
\r\n\r\n\r\nThe D-isomers of all these amino acids stimulate growth \r\nin the presence of the natural isomers.\tNevertheless their\r\n\u03b1-keto analogues when added to the medium are without effect. \r\nSince it has been shown that Neurospora has enzymes cata\u00adlyzing \r\nthe interconversions of \u03b1-aminoadipic and \u03b1-keto\u00ad-adipic \r\nacids, the nonutilization of the exogenous keto acids\r\nis ascribed to a failure of assimilatory mechanisms.
\r\n\r\n\r\nInvestigations of the incorporation of isotopic nitrogen \r\ninto the mycelial lysine indicate that both the \u03b1 and \u2208-amino \r\ngroups are quite stable. Inasmuch as the nitrogen of \r\n\u03b1-aminoadipic acid is labile it is suggested that the de\u00adgradation \r\nof lysine via that acid is not quantitatively sig\u00adnificant.
\r\n\r\n\r\nNone of a number of other substances which might be \r\npostulated either as precursors of \u03b1-aminoadipic acid or\r\nas intermediates between that acid and lysine has any effect \r\non growth. Syntheses of several of these compounds are \r\ndescribed.
\r\n\r\n" }, { "name": "Hatcher, John Burton", "degree": "PhD", "year": "1952", "title": "The Course of Vitamin B\u2081 Metabolism in Man as Indicated by the Use of Radioactive Sulfur, a New Synthesis of 4-Methyl-5-\u03b2-Hudroxyethyl-Thiazole, and a Demonstration of the Use of Anti-Coincidence Method in Radioactivity Determinations", "advisor": "Unknown, Unknown", "url": "https://resolver.caltech.edu/CaltechTHESIS:08292017-161213519", "creators": [ { "name": { "family": "Hatcher", "given": "John Burton" }, "id": "Hatcher-John-Burton", "display_name": "Hatcher, John Burton" } ], "advisors": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/7V9W-6E30", "abstract": "Thiamin synthesized from radiosulfur S35 was injected\r\nintramuscularly in two series of experiments, using a human subject\r\non a normal and a B1-free diet. Determinations of the free B1 (by\r\nthe thiochrome method) in the uring, the radiosulfur in the inorganic,\r\nethereal, and neutral fractions of the urine, and the total radiosulfur\r\nin the feces were made. Rapid destruction of injected thiamin\r\nwas indicated in both experiments by the appearance of radiosulfur\r\nin the inorganic fraction of the urine. Rapid interaction of the\r\ninjected material with that pre-existing in the tissues was indicated\r\nby the appearance of free thiamin in the urine, without corresponding\r\nactive sulfur. Additional destruction of thiamin was indicated by\r\nan excess of the neutral radiosulfur activity over the free B1 after\r\nthe injections were stopped. After 36 days of the B1-free diet the\r\ninjection of 8 mg. over a period of 3 days resulted in the excretion\r\nof 0.8 mg. of pre-existing thiamin. On the normal diet the injection \r\nof 63 mg. over a period of 4 days resulted in the excretion of 16 mg.\r\nof pre-existing thiamin." }, { "name": "Kurtz, Edwin B.", "degree": "PhD", "year": "1952", "title": "Studies on the Metabolism of Lipids in Plants", "advisor": "Bonner, James Frederick; Went, Frits W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02062018-154306942", "creators": [ { "name": { "family": "Kurtz", "given": "Edwin B." }, "id": "Kurtz-Edwin-B", "display_name": "Kurtz, Edwin B." } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" }, { "name": { "family": "Went", "given": "Frits W." }, "id": "Went-F-W", "role": "co-advisor", "display_name": "Went, Frits W." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/RPCV-Y418", "abstract": "This work is concerned with some physiological and biochemical\r\nstudies of the lipids of higher plants, a subject in\r\nwhich only an extremely limited number of studies have\r\npreviously been made.
\r\n\r\n\r\nFrom a study of plants grown under controlled conditions,\r\nit was found that both character and amount of fat and wax\r\nproduced by a plant may be affected by a factor of two or three\r\nby day and night temperatures and soil moisture. The effect\r\nof increased day or night temperature on the yield of fat was\r\ndifferent for different species. Plants generally responded to\r\nincreased temperature by producing less wax. The fats and waxes\r\nfrom plants at high temperatures were of a higher melting point\r\nthan those from plants at low temperatures. Water stress plants\r\nalso produced large amounts of fat, or in Larrea, resin. Although\r\nthe wax content was only slightly affected by low soil moisture,\r\nin Nicotiana glauca an abundant formation of cuticle occurred\r\nunder this condition. These and other effects of climate on\r\nlipids were discussed.
\r\n\r\nWhereas changes in climate effect up to three-fold changes\r\nin lipid yield, a series of recessive genes in corn was found to\r\ncontrol ten-fold changes in wax yield. The genetic factors also\r\naffect the character and yield of the fats.
\r\n\r\n\r\nA system was obtained for studying the synthesis of fats in\r\na higher plant. Preliminary results show that short chain\r\ncompounds (ethyl alcohol, acetate, acetone, acetoacetate) may be\r\nrapidly utilized in the synthesis of fat. These substrates are\r\nreadily used only when an energy source such as sugar and the \r\nvitamin biotin are supplied. The effects of other substrates and\r\nvitamins on fat synthesis were also studied and found to be small\r\nor altogether absent.
" }, { "name": "Lindsley, Dan Leslie", "degree": "PhD", "year": "1952", "title": "Some Consequences of Spermatogonial Exchange in Long Inversions of the X Chromosome of Drosophila melanogaster", "advisor": "Sturtevant, Alfred Henry", "url": "https://resolver.caltech.edu/CaltechETD:etd-06072004-145444", "creators": [ { "name": { "family": "Lindsley", "given": "Dan Leslie" }, "id": "Lindsley-Dan-Leslie", "display_name": "Lindsley, Dan Leslie" } ], "advisors": [ { "name": { "family": "Sturtevant", "given": "Alfred Henry" }, "id": "Sturtevant-A-H", "role": "advisor", "display_name": "Sturtevant, Alfred Henry" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/1EMB-VT69", "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...].\r\nAbstract is included in .pdf document.\t\t\t\t\t\r\n\r\nAmong the progeny of males carrying the inverted I chromosome, [...], individuals are found which have lost the terminal euchromatic segment of the inversion. This has been interpreted in the past (Sidorov 1940, 1941) as the result of exchange between the distal heterochromatin of the inversion and the short arm of the Y chromosome, and, indeed, the reciprocal recombinant type may be recovered, i. e. a Y with its short arm replaced by the terminal euchromatin of [...]. The fact that such derivatives occur in clusters and represent recombination which has occurred in males indicate that the events in question are mitotic rather than meiotic in origin.\r\n\r\nAn analysis of 44 such recombinant X chromosomes recovered from 273,721 offspring of males carrying the distal heterochromatin of [...] reveals that previous notions represent an oversimplification of the actual case. When the chromosome carries large heterochromatic segments at its base, the distal heterochromatin many tend to pair with its own base in preference to pairing with the Y. Such pairing may occur in either direction, i.e., eucentrically or dyscentrically, as may pairing between the X and Y. Reversal of ordinary pairing relationships may result in the formation of dicentric bridges, and the evidence indicates that such bridges break at anaphase. It has been possible to demonstrate exchange between the distal heterochromatin of [...] and all other sex linked heterochromatic blocks except [...], and indications are that this event may not occur.\r\n\r\nNo evidence has been found favoring any sort of fusion circle subsequent to bridge breakage, but the experimental arrangement favored detection of only a chromatid and not a chromosome type of cycle." }, { "name": "Liverman, James Leslie", "degree": "PhD", "year": "1952", "title": "The Physiology and Biochemistry of Flowering", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechTHESIS:09272017-092747800", "creators": [ { "name": { "family": "Liverman", "given": "James Leslie" }, "id": "Liverman-James-Leslie", "display_name": "Liverman, James Leslie" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/6970-QP16", "abstract": "With the two SDP, Xanthium canadense and Chenopodium\r\nAmaranticolor, it has been possible to separate more clearly the\r\npartial reactions of the photoperiodic response of SDP and in some\r\ndegree to associate these processes with particular biochemical or\r\nphysiological processes of the plant. Thus it has been shown that\r\nsugars and Krebs cycle acids are able to replace the high intensity\r\nlight process. Further investigations have shown that substances\r\nformed during an inductive dark process are still susceptible to\r\nauxin inactivation even after exposure to several hours of high\r\nintensity light immediately following the dark period. It has been\r\nshown that the effect of a flash of light in inhibiting the dark\r\nprocess can be reversed by anti-auxins. Further experiments have \r\nconfirmed earlier work which show that LD leaves on SD plants\r\ninhibit flowering. Hypotheses have been advanced 1) to explain\r\nthe nature of this inhibition and 2) to explain the kinetics of the\r\ndark process.
\r\n\r\n\r\nExperiments with a newly discovered LDP, Silene\r\nArmeria, have shown that its critical day length is reduced by\r\nincreasing temperature. Studies on the auxin relations in the\r\nflowering of Silene and Hyoscyamus niger indicate that auxin\r\ncauses flowering of these LDP under conditions in which the\r\ncontrols remain vegetative.
" }, { "name": "Patterson, Earl Byron", "degree": "PhD", "year": "1952", "title": "Studies on Crossing Over in Homozygous and Heterozygous Chromosomes Rearrangements in Zea mays", "advisor": "Anderson, Ernest G.", "url": "https://resolver.caltech.edu/CaltechTHESIS:08302017-140323046", "creators": [ { "name": { "family": "Patterson", "given": "Earl Byron" }, "id": "Patterson-Earl-Byron", "display_name": "Patterson, Earl Byron" } ], "advisors": [ { "name": { "family": "Anderson", "given": "Ernest G." }, "id": "Anderson-Ernest-G", "role": "advisor", "display_name": "Anderson, Ernest G." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/MGE7-G829", "abstract": "The nature and behavior of chromosome inversions and translocations\r\nare discussed and the literature reviewed. Their uses in cytogenetic\r\nand biochemical studies are also outlined and applications are cited.
\r\n\r\nLinkage data are presented for the genetic positions of sixteen\r\ntranslocations in chromosomes 9 with reference to the marker genes C,\r\nsh1, and wx. Data are also presented for the map positions of fourteen\r\ntranslocations in chromosome 2, with reference to the genes lg1, gl2,\r\nB, sk1, and v4. The uses of duplicate-deficient gametes and pollen\r\ngrains in cytogenetic investigations are outlined and their applications\r\nto these studies are described. Techniques for the classification and\r\ntransmission of unbalanced chromosome complements are presented and the \r\nphenotypic effects of certain gene dosages are described. Available\r\ninformation on each of the thirty translocations mentioned above is\r\nsummarized and its bearing on previous information regarding\r\nchromosomes 2 and 9 is discussed.
\r\n\r\n\r\nStudies are also reported in which the lg1-gl2 and C-wx regions\r\nare moved to different positions in the chromosome complement. Changes\r\nof position were brought about by using chromosome rearrangements. In\r\neach case, the crossover value is measured in the homozygous rearrangement\r\nand is compared with the crossover value found when the region is\r\nin its standard position. In some cases recombination values from\r\nfemale and male transmission are also compared.
\r\n" }, { "name": "Reissig, Jos\u00e9 Luis", "degree": "PhD", "year": "1952", "title": "Studies on the Metabolism of Threonine and Related Substrates in Neurospora crassa", "advisor": "Emerson, Sterling", "url": "https://resolver.caltech.edu/CaltechTHESIS:02092018-110126859", "creators": [ { "name": { "family": "Reissig", "given": "Jos\u00e9 Luis" }, "id": "Reissig-Jos\u00e9-Luis", "display_name": "Reissig, Jos\u00e9 Luis" } ], "advisors": [ { "name": { "family": "Emerson", "given": "Sterling" }, "id": "Emerson-S", "role": "advisor", "display_name": "Emerson, Sterling" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/J2RA-YB37", "abstract": "We have investigated the following enzyme systems\r\nin extracts of a wild type strain of Neurospora:
\r\n\r\n1) Threonine deaminase. It catalyzes the reaction\r\ngiving rise to alpha-ketobutyric acid and ammonia from\r\nthreonine. It was concluded that alpha-ketobutyric acid,\r\nglutamic acid or a deaminated alpha-ketobutyric acid precursor\r\nin equilibrium with alpha-ketobutyric could not be\r\nintermediates in this reaction. Pyridoxal phosphate \r\nactivates the system, and a number of methods were tested in\r\norder to improve the resolution of enzyme and coenzyme in\r\nthe preparations.
\r\n\r\n\r\n2) Serine deaminase. Yields pyruvic acid and ammonia\r\nfrom serine, and is also activated by pyridoxal phosphate.\r\nThe responses of serine and threonine deaminases to\r\npyridoxal phosphate are at variance, suggesting that two\r\ndifferent enzymes are involved.
\r\n\r\n\r\nThe effect of pH and temperature on serine and \r\nthreonine deaminase was investigated.
\r\n\r\n3) Glutamic-alphaketobutyric transaminase, which is \r\nactivated by pyridoxal phosphate.
\r\n\r\n\r\n4) A system forming alpha-aminobutyric from threonine, \r\npossibly as a result of the summation of activities 1) \r\nand 3).
\r\n\r\n\r\n5) A system forming an unidentified blue fluorescent \r\nproduct by incubation with threonine, but not with any of\r\na number of related metabolites (serine included).
\r\n\r\n\r\n6) Alpha-ketobu tyric decarboxylase, which is acti\u00advated \r\nby cocarboxylase, and has a pH optimun1 of 5.5.
\r\n\r\n\r\nThe threonine deaminase activities of a number of \r\nthreonineless strains and of a B6-less strain were com\u00adpared \r\nwith those of wild type, using cultures grown under\r\ndifferent conditions. The significance of the variability \r\nin activity encountered is discussed. Mutant 35423, which \r\nrequires threonine for growth but is unable to use alpha\u00ad \r\nketobutyric or alpha-aminobutyric acid,has the ability of \r\nconverting threonine into those acids in vitro.
\r\n\r\n\r\nMutant 44104 cannot utilize alpha-ketobutyric acid \r\nin place of alpha-aminobutyric to initiate early growth, \r\nbut its glutamic-alpbaketobutyric transamtnase is as active \r\nin vitro as that of wild type.
\r\n\r\n\r\nA new scheme of threonine biosynthesis is presented\r\nto account for the information available.
\r\n\r\nAn attempt is made to find a common denominator to the \r\nmechanisms of the diverse coenzymatic activities of pyridoxal \r\nphosphate, and schemes for those mechanisms are proposed.
\r\n" }, { "name": "Thayer, Philip Standish", "degree": "PhD", "year": "1952", "title": "Studies on the L-Amino Acid Oxidase of Neurospora crassa", "advisor": "Horowitz, Norman Harold", "url": "https://resolver.caltech.edu/CaltechTHESIS:10052017-132646189", "creators": [ { "name": { "family": "Thayer", "given": "Philip Standish" }, "id": "Thayer-Philip-Standish", "display_name": "Thayer, Philip Standish" } ], "advisors": [ { "name": { "family": "Horowitz", "given": "Norman Harold" }, "id": "Horowitz-N-H", "role": "advisor", "display_name": "Horowitz, Norman Harold" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/JJN0-XC39", "abstract": "The L-amino acid oxidase of Neurospora is a \r\ngen\u00aderal amino acid oxidase attacking a wide range of L-amino \r\nacids at different rates. Activity of the enzyme is de\u00adpendent \r\non substrate concentration, oxygen tension and\r\npH. Different amino acids show different pH optima. \r\nActivity is significantly reduced by excess substrate, \r\nand competition is exhibited between mixed substrates.
\r\n\r\n\r\nL-oxidase production by mycelium is increased 4 to \r\n10 fold by biotin limitation. This effect is not produced \r\nby: changes in extractability of the enzyme; reduced level \r\nof growth; defective ammonia, aspartic acid, or riboflavin \r\nmetabolism; or production of an inhibitor of the enzyme.
\r\n\r\n\r\nThe enzyme is adaptively formed during growth in \r\nthe presence of substrate amino acids. Factors affecting \r\nthe degree of adaptation are biotin and substrate concen\u00adtrations, \r\npH and strain differences. Deadaptation is pro\u00adduced \r\nby excess biotin or the removal of substrate. The\r\nrelation of adaptation to the low biotin effect is discussed.
\r\n\r\n\r\nThe L-oxidase is involved in detoxification of cana\u00advanine. \r\nStrain differences in canavanine sensitivity are paralleled \r\nby certain qualitative differences in L-oxidase activity. \r\nCanavanine resistance is increased on low biotin. \r\nThe relation of these observations to canavanine sensiti\u00advity \r\nand its genetic control is discussed.
" }, { "name": "Wolfgram, Frederick John", "degree": "PhD", "year": "1952", "title": "On Saltatory Conduction in Peripheral Myelinated Nerve", "advisor": "Van Harreveld, Anthonie", "url": "https://resolver.caltech.edu/CaltechTHESIS:02062012-115321570", "creators": [ { "name": { "family": "Wolfgram", "given": "Frederick John" }, "id": "Wolfgram-Frederick-John", "display_name": "Wolfgram, Frederick John" } ], "advisors": [ { "name": { "family": "Van Harreveld", "given": "Anthonie" }, "id": "Van-Harreveld-A", "role": "advisor", "display_name": "Van Harreveld, Anthonie" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/9H5K-DZ62", "abstract": "Anatomically speaking, there are two types of nerve fibers in vertebrates: myelinated and unmyelinated fibers. In the former the axon is covered with a protein-lipid structure (myelin) which is interrupted at more or less regular intervals (Ranvier nodes). In unmyelinated fibers this myelin structure is absent. Myelinated fibers conduct impulses much faster than unmyelinated fibers. In order to account for the high conduction velocities of medullated nerves, Lillie has proposed that excitation takes place only at the nodes of Ranvier with the circuit between adjacent nodes being made through the axoplasm of the fiber and the medium external to the fiber. This is the theory of saltatory conduction.
\r\n\r\nThe literature on saltatory conduction is critically reviewed and it is concluded that only two experiments that have been done seem to indicate that saltatory conduction occurs. These are the narcosis experiments of Taskai and the air gap experiments of Huxley and Frankenhaeuser. These experiments have been carefully repeated and improved. From the results of these experiments it is concluded that medullated nerve can conduct in a situation where the conditions necessary for saltation do not exist. The possibility that saltation is the normal physiological process of conduction cannot be discounted.
" }, { "name": "Haskins, Francis Arthur", "degree": "PhD", "year": "1951", "title": "Biochemical Genetics of Neurospora Pertaining to Various Aromatic Metabolites", "advisor": "Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:10132017-112359126", "creators": [ { "name": { "family": "Haskins", "given": "Francis Arthur" }, "id": "Haskins-Francis-Arthur", "display_name": "Haskins, Francis Arthur" } ], "advisors": [ { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/2CKA-TG54", "abstract": "The following nine mutant strains of Neurospora crassa were \r\nused in these studies: E5212, C86, B1312, 39401, 10575, C83,\r\nE5029, 4540, and 3416.Each of these nine mutants is able to utilize \r\none or more of the following compounds: quinic acid, tyrosine, \r\nphenylalanine, anthranilic acid, indole, tryptophane, o-N-formylkynurenine, \r\nkynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic \r\nacid, and nicotinic acid.
\r\n\r\n\r\nMetabolic accumulations by four of the strains have been\r\nstudied. The accumulation of anthranilic acid by mutant 10575 has \r\nbeen recognized for some time. It is now clear that this strain \r\naccumulates other highly fluorescent substances and at least one\r\nother biologically active substance. Mutant C83 accumulates relatively \r\nlarge quantities of anthranilic acid, and an unidentified derivative of \r\nindole.\tStrain E5029 accumulates a compound, apparently anthranilic \r\nacid, with biological activity for strain B1312; unidentified 39401-active \r\nand E5212-active substances; and kynurenicacid, which is\r\nbiologically inactive. Mutant E5212 accumulates anthranilic acid, \r\nnicotinic acid, and also a substance which is active for E5212 itself.
\r\n\r\n\r\nEvidence has been obtained which indicates that in Neurospora \r\nantranilic acid is a precursor of tryptophane, with indole as an\r\nintermediate between the two compounds; and also that tryptophane is \r\ndegraded to anthranilic acid, with kynurenine as an intermediate.\r\nThus a metabolic cycle involving anthranilic acid, indole, tryptophane, \r\nand kynurenine is clearly indicated.
\r\n\r\n\r\nCrosses involving mutants 39401 and C86 have furnished evidence \r\nthat the mutation which prevents the growth of 39401 on minimal \r\nmedium is the same as that which prevents the growth of C86 on minimal, \r\nand that a number of modifiers affect this primary mutation in \r\nsuch a way that off spring are produced which differ qualitatively in\r\ngrowth requirements fr om both C86 and 39401. Six modifiers have\r\nbeen postulated to explain the observed ascus segregations. In addition \r\na suppressor of the primary mutation has been found. Possible \r\nmech\u00adanisms by which the modifiers exert their effects are considered \r\nbriefly.
\r\n" }, { "name": "Hull, Herbert Mitchell", "degree": "PhD", "year": "1951", "title": "The Effect of Temperature on Carbohydrate Translocation in the Tomato Plant", "advisor": "Went, Frits W.; Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechTHESIS:07292025-181002251", "creators": [ { "name": { "family": "Hull", "given": "Herbert Mitchell" }, "id": "Hull-Herbert-Mitchell", "display_name": "Hull, Herbert Mitchell" } ], "advisors": [ { "name": { "family": "Went", "given": "Frits W." }, "id": "Went-F-W", "role": "advisor", "display_name": "Went, Frits W." }, { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/d2g3-v157", "abstract": "The majority of experiments dealing with carbohydrate\r\ntranslocation in the higher plant have indicated a Q10 of\r\nmore than one, that is, a greater transport at higher temperature.\r\nCertain of the exper1ments reported in this work\r\nhave suggested that under certain conditions, in the tomato\r\nplant, a mechanism of translocat1on may be operative which\r\nacts almost independently of temperature, or even shows \r\nQ10 of less than one.
\r\n\r\nIn addition to different Q10's for carbohydrate translocation,\r\nvastly different rates of movement have been found,\r\nranging from less than 15 minutes to almost 48 hours, for\r\ntransport to take place from one leaf to the roots, the\r\ngrowing point, or to the leaves. These experiments have been\r\naccomplished by utilizing the valuable tools of radioactive\r\ntracers, end a bleeding technique, which is described in\r\nthe text.
\r\n\r\nJudging from the variety of results, concerning both\r\ntemperature effect and rate, it appears unlikely that one\r\nmechanism of transport is sufficiently versatile to account\r\nfor all data. On this basis, more precisely described in the\r\ntext, the possibility of two mechanisms being operative in\r\nthe same plant is suggested.
" }, { "name": "Nitsch, Jean Paul", "degree": "PhD", "year": "1951", "title": "The Role of Plant Hormones in Fruit Development", "advisor": "Went, Frits W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:04072017-091032690", "creators": [ { "name": { "family": "Nitsch", "given": "Jean Paul" }, "id": "Nitsch-Jean-Paul", "display_name": "Nitsch, Jean Paul" } ], "advisors": [ { "name": { "family": "Went", "given": "Frits W." }, "id": "Went-F-W", "role": "advisor", "display_name": "Went, Frits W." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/G2GR-7342", "abstract": "Starting with the initiation of the ovary primordium, a coordinate\r\nstudy of the hormonal relationships in fruit growth has been\r\nattempted with fruits attached on the plants and also by the new technique\r\nof in vitro culture. Several phases were recognized in the\r\nontogeny of a fruit. To be able to initiate flower primordia the plant\r\nhas first to enter a reproductive condition. The formation of ovary\r\nprimordia was studied in some cucurbits, in which it was found\r\nthat the environment profoundly influences the apparition of female\r\nvs. male flowers. It has been concluded that these environmental\r\nfactors regulate the length of successive phases in cucurbit flowering. \r\nOnce initiated, the ovary enlarges regularly, mainly by cell\r\ndivision until anthesis. If pollination is prevented, ovary growth\r\nceases at this stage, and there is indication that the auxin level of\r\nthe flower decreases. The ovary then shrinks or drops off the\r\nplant. Flower abscission is prevented and growth is stimulated by\r\nthe pollen which performs these effects mainly by increasing the\r\nauxin level of the ovary, partly at least through an enzymatic mechanism.\r\nAfter fertilization fruit growth is controlled by the developing \r\nseeds which release large quantities of auxin, the latter apparently\r\nmanufactured in the endosperm. Thus, auxin has been found to affect any stage of fruit development." }, { "name": "Paigen, Kenneth", "degree": "PhD", "year": "1951", "title": "Studies on the Source of Urea Carbon", "advisor": "Borsook, Henry", "url": "https://resolver.caltech.edu/CaltechTHESIS:09072017-142857992", "creators": [ { "name": { "family": "Paigen", "given": "Kenneth" }, "id": "Paigen-Kenneth", "display_name": "Paigen, Kenneth" } ], "advisors": [ { "name": { "family": "Borsook", "given": "Henry" }, "id": "Borsook-H", "role": "advisor", "display_name": "Borsook, Henry" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/WV7D-FW20", "abstract": "A method has been devised for the testing of any substance\r\nas an intermediate in a reaction sequence. Using this technique,\r\nthirty-three substances have been tested as precursors of the\r\nurea carbon atom. Of the compounds tested, only citrulline and\r\narginine were precursors.
\r\n\r\n\r\nGarbamyl L-glutamic acid was shown not to be a donor of\r\nthe urea carbon, although it does have some function in urea synthesis.
" }, { "name": "Robertson, Donald Sage", "degree": "PhD", "year": "1951", "title": "A Study of Viviparous Mutants of Maize", "advisor": "Anderson, Ernest G.", "url": "https://resolver.caltech.edu/CaltechTHESIS:07282025-160759217", "creators": [ { "name": { "family": "Robertson", "given": "Donald Sage" }, "id": "Robertson-Donald-Sage", "display_name": "Robertson, Donald Sage" } ], "advisors": [ { "name": { "family": "Anderson", "given": "Ernest G." }, "id": "Anderson-Ernest-G", "role": "advisor", "display_name": "Anderson, Ernest G." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/r5q0-4f92", "abstract": "The meaning and extent of vivipary among the\r\nhigher plants are considered. The nature of vivipary in\r\nmaize as reported by earlier workers is compared with\r\nobservations made in this study. The use of interchanges\r\namong chromosomes of the basic set (A-type chromosomes) \r\nand of interchanges between A and B-type chromosomes in\r\ngenetic studies is outlined. The results of genetic\r\nstudies with six viviparous mutants are reported. Experiments\r\ninvolving the use of embryo culture techniques\r\nto elucidate the cause of vivipary5 in maize are described.\r\nThese experiments revealed that viviparous embryos are\r\nmore tolerant of high carbon dioxide concentrations than\r\nnormal embryos. Experiments to check the role of the cob\r\nin preventing premature germination, as well as experiments \r\nto determine if acetaldehyde plays a similar role,\r\nyielded negative results. The total auxin contents of\r\nviviparous and normal seeds are reported. No obvious\r\ndifferences between the two classes of seeds with regard\r\nto this growth substance was round. The significance\r\nof the genetic and embryo culture studies as it relates\r\nto vivipary in maize is considered." }, { "name": "Shen, San-Chiun", "degree": "PhD", "year": "1951", "title": "Genetics and Biochemistry of the Cysteine-Tyrosine Relationship in Neurospora crassa", "advisor": "Horowitz, Norman Harold", "url": "https://resolver.caltech.edu/CaltechETD:etd-06282004-093704", "creators": [ { "name": { "family": "Shen", "given": "San-Chiun" }, "id": "Shen-San-Chiun", "display_name": "Shen, San-Chiun" } ], "advisors": [ { "name": { "family": "Horowitz", "given": "Norman Harold" }, "id": "Horowitz-N-H", "role": "advisor", "display_name": "Horowitz, Norman Harold" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/BE55-1F07", "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\r\n\r\nMutant 84605 which was obtained from wild type Neurospora crassa following X-ray treatment differs from wild type by a single gene located 4.8 units from the centromere of the second chromosome.
\r\n\r\nAt 25 [degrees], the mutant requires both cysteine and tyrosine for normal growth. At 35[degrees] only cysteine is required. The block in cysteine synthesis is the step sulfite [...] thiosulfate.
\r\n\r\nHigh tyrosinase activity was found in the mutant grown at 25[degrees], but not when grown at 35[degrees]. Under the same conditions, wild type shows little or no tyrosinase activity.
\r\n\r\nThe addition of cysteine to the medium causes an inhibition of the growth of wild type, and at the same time a marked increase in the tyrosinase activity occurs. The inhibition can be overcome by adding tyrosine to the medium, or by culturing at 35[degrees].
\r\n\r\nIt is suggested that the tyrosine requirement is caused by the high tyrosinase activity and that the latter, in turn, is caused by the defect in sulfur metabolism.
\r\n\r\nTwo natural inhibitors of tyrosinase have been found in Neurospora.
\r\n\r\nA powerful inhibitor of the growth of wild type accumulates in cultures of the mutant.
\r\n\r\nExperiments designed to test whether sulfide can serve as a sulfur source for Neurospora indicate that sulfide is utilized slightly, if at all.
\r\n\r\nExperiments with a double mutant have indicated that the production of cysteine from methionine by Neurospora does not involve simple reversal of the step homocysteine [...] methionine.
" }, { "name": "Siegel, Albert", "degree": "PhD", "year": "1951", "title": "I. Irradiation Experiments with Neurospora crassa. II. An Electrophoretic Comparison of the Soluble Proteins of Normal and Virus-Infected Escherichia coli", "advisor": "Beadle, George Wells", "url": "https://resolver.caltech.edu/CaltechETD:etd-08092006-104758", "creators": [ { "name": { "family": "Siegel", "given": "Albert" }, "id": "Siegel-Albert", "display_name": "Siegel, Albert" } ], "advisors": [ { "name": { "family": "Beadle", "given": "George Wells" }, "id": "Beadle-G-W", "role": "advisor", "display_name": "Beadle, George Wells" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/MAKD-DR63", "abstract": "Part I: \r\n\r\n1. A case is described and discussed in which a mutant strain of Neurospora which spontaneously reverts to wild type cannot be induced to revert by mutagenic agents.\r\n\r\n2. The action of ultraviolet light on macroconidia of Neurospora is described.\r\n\r\n3. The effect of heat and light post-treatment of ultra-violet irradiated macroconidia has been studied.\r\n\r\n4. Data have been obtained which suggest that the killing effect of UV is more easily reversed by heat or light post-treatment than the mutagenic effect.\r\n\r\nPart II: \r\n\r\n1. The soluble proteins of Escherichia coli were extracted by disrupting the bacteria in a colloid mill and centrifuging all particulate matter out of solution.\r\n\r\n2. The soluble proteins so obtained, were resolved by electrophoresis.\r\n\r\n3. A comparison was made of the electrophoretic scanning patterns of the soluble proteins of non-infected bacteria, bacteria infected with bacteriophage T2 during the \"eclipse\" stage of infection, and bacteria infected for longer than the \"eclipse\" with, the following findings:\r\n\r\na) The amount of free-moving desoxyribonucleic acid (DNA) present in the extracts of uninfected bacteria decreases during the \"eclipse\" stage of infection.\r\n\r\nb) The amount of free-moving DNA increases strikingly after the end of the \"eclipse.\"\r\n\r\n4. Bacteriophage particles were disrupted in the colloid mill. The increase in free moving DNA in the extracts of bacteria infected for longer than the \"eclipse\" was found to be due to disruption of intracellular bacteriophage particles.\r\n\r\n5. DNA was extracted both from bacteriophage particles and from Escherichia coli. A comparison of the two DIVAS by electrophoretical and ultracentrifugal methods reveals a close similarity between them.\r\n\r\n6. The experimental findings are discussed and several suggestions are made for further elucidating the course of virus multiplication." }, { "name": "Windsor, Emanuel", "degree": "PhD", "year": "1951", "title": "\u03b1-Aminoadipic Acid as a Constituent of a Natural Protein", "advisor": "Borsook, Henry", "url": "https://resolver.caltech.edu/CaltechTHESIS:09292017-114236162", "creators": [ { "name": { "family": "Windsor", "given": "Emanuel" }, "id": "Windsor-Emanuel", "display_name": "Windsor, Emanuel" } ], "advisors": [ { "name": { "family": "Borsook", "given": "Henry" }, "id": "Borsook-H", "role": "advisor", "display_name": "Borsook, Henry" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/0T5Z-M233", "abstract": "Thirty-five proteins or protein-containing materials\r\nwere analyzed for \u03b1-aminoadipic acid by means of chromatography\r\non starch columns. Of these materials corn and\r\nTaka-diastase (from Aspergillus oryzae) were shown to \r\ncontain free \u03b1-aminoadipic acid.
\r\n\r\n\r\nAs a protein constituent aminoadipic acid was found\r\nin a water-soluble corn protein. Corn steepwater concentrate\r\nwas an adequate source of this protein. \u03b1-Aminoadipic acid\r\nwas shown to be a constituent of the protein by\r\nits isolation from hydrolysate. Its identity with the\r\nsynthetic amino acid was proved by elementary analysis,\r\nmixed melting point, chromatographic behavior on both starch\r\nand Dowex-50 columns, use as a growth substance for a \r\nNeurospora crassa mutant which requires it for growth, and\r\ninhibition of growth by a specific inhibitor.
\r\n\r\n\r\nThe ionization constants for aminoadipic acid in\r\nwater were determined.
\r\n\r\n\r\nThe conversion of radioactive \u03b1-aminoadipic acid into\r\nradioactive lysine in a mutant strain of Neurospora\r\ncrassa was shown.
" }, { "name": "Pao, Wen Kwe", "degree": "PhD", "year": "1950", "title": "Investigations of the Thermophobic Character in Neurospora crassa, Especially of the Relationships Between Temperature and Carbohydrate Utilization", "advisor": "Emerson, Sterling; Borsook, Henry", "url": "https://resolver.caltech.edu/CaltechTHESIS:12122012-103207950", "creators": [ { "name": { "family": "Pao", "given": "Wen Kwe" }, "id": "Pao-Wen-Kwe", "display_name": "Pao, Wen Kwe" } ], "advisors": [ { "name": { "family": "Emerson", "given": "Sterling" }, "id": "Emerson-S", "role": "advisor", "display_name": "Emerson, Sterling" }, { "name": { "family": "Borsook", "given": "Henry" }, "id": "Borsook-H", "role": "advisor", "display_name": "Borsook, Henry" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/7M5A-FG46", "abstract": "The thermophobic character is a temperature mutant which is unable to grow at\r\n35\u00b0C on certain sugars in tube culture. This character is controlled by a single\r\ngene located far away from the centromere on the chromosome of Paba linkage group.
\r\n\r\nA limited growth of thermophobic strain on lactose minimal medium can be\r\nforced at 35\u00b0 after activation at 25\u00b0 for as little as 2 hours. The experiments\r\nshow that the inhibition of growth at higher temperature is not due to nutritional\r\ndeficiency, change of permeability of cell membrane to certain sugars or inhibition\r\nof hydrolytic enzymes of oligosaccharides. At 40\u00b0 the thermophobic strain can\r\nnot grow on any sugar, while the normal strain can grow on certain sugars but not\r\nothers, just as the thermophobic strain did at 35\u00b0. The difference of effective\r\ntemperature between the strains is only 5\u00b0C. This led to the studies of the\r\ncommon step of carbohydrate degradation, e.g., glycolysis and Krebs' cycle. All\r\nKrebs' acids were found to have a strong thermophobic effect at 35\u00b0 on the thermophobic\r\nstrain on sucrose. But the manometric studies of certain of the enzyme\r\nsystems in Krebs' cycle reveal no significant differences between the strains.
\r\n\r\nIn liquid culture on lactose at 35\u00b0 there appears a morphological difference\r\nbetween the strains, i.e., semicolonial for the thermophobic strain and usual\r\nfilamentous for the normal. This led to the studies of cell wall material in\r\nNeurospora, and it was found that there is a difference in response to chlorozinc\r\niodine staining. The staining reaction is quantitatively related to the length of\r\nunacetylated end group of chitin. Estimates show that the chitin of the thermophobic\r\nstrain has a length of half molecule only about 1/5 that of normal. A length of\r\nchitin half mol ecule less than 100 acetylglucosamine units will induce a growth\r\nof semicolonial form in liquid culture, and will have very little measurable growth\r\nin tube culture. The length of chitin molecule is influenced by glucose-acetate\r\nratio. Temperature and type of sugar will influence the ratio greatly, and will\r\nthus change the habit of growth of both strains, but different quantitatively as\r\nthe thermophobic gene causes a shortness of chitin molecule from the start.
" }, { "name": "Strauss, Bernard Samuel", "degree": "PhD", "year": "1950", "title": "Studies on Vitamin B6 - Requiring Mutants of Neurospora crassa", "advisor": "Horowitz, Norman Harold; Mitchell, Herschel K.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06242025-173859797", "creators": [ { "name": { "family": "Strauss", "given": "Bernard Samuel" }, "id": "Strauss-Bernard-Samuel", "display_name": "Strauss, Bernard Samuel" } ], "advisors": [ { "name": { "family": "Horowitz", "given": "Norman Harold" }, "id": "Horowitz-N-H", "role": "advisor", "display_name": "Horowitz, Norman Harold" }, { "name": { "family": "Mitchell", "given": "Herschel K." }, "id": "Mitchell-H-K", "role": "advisor", "display_name": "Mitchell, Herschel K." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/1nem-3155", "abstract": "1. Mutants of Neurospora crassa requiring vitamin B6 have been investigated.
\r\n\r\n2. As reported by Stokes et al. (1943) certain of these mutants\r\nwill grow in the absence of added vitamin B6 if the pH of the \r\nmedium is above 6 and if ammonium ion is present.
\r\n\r\n3. The requirement for high pH and ammonium ion has been found to\r\nbe due to a requirement for free ammonia in the medium. The\r\nrole of pH is to determine the concentration of free ammonia.
\r\n\r\n4. Ammonia is specific for the initiation of growth of the pH\r\nsensitive mutants in the absence of added vitamin B6.
\r\n\r\n5. When grown in the presence of sufficient ammonia the pH\r\nsensitive mutants are able to synthesize vitamin B6.
\r\n\r\n6. The net rate of vitamin B6 synthesis is lower in the pH\r\nsensitive mutants than in wild type.
\r\n\r\n7. A glutamic-alanine transaminase has been found in Neurospora.\r\nThe activity of this enzyme is lower in the mycelium of the\r\npH sensitive mutants grown without added vitamin B6 than in wild type \r\nmycelium.
\r\n\r\n8. Resting mycelium of a pH sensitive mutant will destroy more\r\nvitamin B6 in a given period than resting mycelium of a\r\nmutant which requires vitamin B6 under all conditions tested.
\r\n\r\n9. The pH sensitive mutant is inhibited by methionine. This\r\ninhibition is overcome by added ammonia, vitamin B6 or\r\nsulfanilamide and is competitively overcome by threonine.
\r\n\r\n10. When either mutant or wild type is grown on a mixture of\r\nnitrate and ammonium as a nitrogen source at pH 7 ammonium\r\nis assimilated before nitrate.
\r\n\r\n11. Nitrate can be reduced by pH sensitive mutants grown in the\r\nabsence of vitamin B6 but the product of reduction cannot be\r\nused for growth.
\r\n\r\n12. It is likely that the lower net rate of B6 synthesis in the\r\npH sensitive mutants is due to a higher rate of vitamin B6\r\ndestruction than is shown by wild type.
\r\n\r\n13. A hypothesis to explain the specificity of ammonia has been\r\ndeveloped. According to this idea ammonia in high concentration\r\n(relative to wild type requirements) prevents the destruction of \r\nvitamin B6.
" }, { "name": "Ycas, Martynas Freelandas", "degree": "PhD", "year": "1950", "title": "Studies on the Respiratory Enzymes of Sea Urchin Eggs", "advisor": "Tyler, Albert", "url": "https://resolver.caltech.edu/CaltechTHESIS:09232025-213823820", "creators": [ { "name": { "family": "Ycas", "given": "Martynas Freelandas" }, "id": "Ycas-Martynas-Freelandas", "display_name": "Ycas, Martynas Freelandas" } ], "advisors": [ { "name": { "family": "Tyler", "given": "Albert" }, "id": "Tyler-A", "role": "advisor", "display_name": "Tyler, Albert" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/ajnr-pa79", "abstract": "1. The literature pertaining to the respiratory metabolism of sea urchin eggs is reviewed.
\r\n\r\n2. Homogenates prepared from the unfertilized eggs of Lythechinus pictus and Strongylocentrotus purpuratus were found to possess enzymatic activity corresponding to the enzymes phosphoglucomutase, oxo isomerase, aldolase, triose phosphate dehydrogenase, lactic dehydrogenase and enolase. Lactic acid accumulates anaerobically in intact eggs. Respiration of homogenates in inhibited by fluoride.
\r\n\r\n3. Citric, alpha ketoglutaric, succinic and malic acids are metabolized. Respiration is inhibited by malonate and increased by cytochrome o.
\r\n\r\n4, Spectroscopic evidence is presented for the existence of cytochromes a and b. A method of preparing an insoluble fraction containing these cytochromes is described.
\r\n\r\n5. It was found that the increased respiration obtainable with dinitrophenol increases faster than the normal respiration during the first eight hours of development.
\r\n\r\n6. It is concluded that the respiratory pathways of the sea urchin egg are similar to those known to exist in adult organisms.
\r\n\r\n7. The chemical basis for the similarity or the glycolytic mechanism in most forms of life is discussed.
" }, { "name": "Krauss, Max", "degree": "PhD", "year": "1949", "title": "Lytic Agents of the Sperm of Some Marine Animals", "advisor": "Tyler, Albert", "url": "https://resolver.caltech.edu/CaltechTHESIS:06082025-130151751", "creators": [ { "name": { "family": "Krauss", "given": "Max" }, "id": "Krauss-Max", "display_name": "Krauss, Max" } ], "advisors": [ { "name": { "family": "Tyler", "given": "Albert" }, "id": "Tyler-A", "role": "advisor", "display_name": "Tyler, Albert" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/dp79-k562", "abstract": "This thesis consists primarily of investigations of lytic agents\r\nand related substances that are extracted from sperm of echinoderms,\r\nmolluscs and mammals.
\r\n\r\nSea-urchin fertilizin is found to give a mucin clot reaction with\r\nserumprotein to give a stable turbidity. Hyaluronidase, from mammalian\r\nsperm, has no dissolving action on sea-urchin egg jelly, nor could\r\na hyaluronidase-like substance, capable of dissolving sea-urchin jelly,\r\nacting on purified fertilizin preparations or on hyaluronic acid, be\r\nextracted from sea-urchin sperm.
\r\n\r\nThe presence in sea-urchin sperm extracts of egg surface lysin\r\nactivity is confirmed.
\r\n\r\nThe egg membrane lysin of sperm of the giant keyhole limpet is\r\nfound to be separable from antifertilizin. Lysin is released from living\r\nsperm in sea water at neutral and alkaline pH. Antifertilizin does\r\nnot appear to be released under these conditions. Release of lysin seems\r\nto be independent of metabolic activity. The lysin does not seem to act\r\ncatalytically. It is inactivated by treatments that denature proteins,\r\nand by proteolytic enzymes. The egg membrane is dissolved by SH compounds\r\nand is slightly digested by trypsin. It is thought to be of\r\nkeratin-like nature. It is shown that complete dissolution of the egg\r\nmembrane is not necessary for fertilization.
\r\n\r\nAn egg membrane lysin is demonstrated in sea-urchin sperm extracts.\r\nIt dissolves the keyhole limpet egg membrane.
\r\n\r\nCross reactions are shown to occur between sperm extracts and egg\r\nmembranes of the keyhole limpet, abalone and mussel.
" }, { "name": "Yu, Sien-Chiue", "degree": "PhD", "year": "1949", "title": "A Genetic and Cytological Study of Some X-Ray Induced Mutations and Reverse Mutations in Drosophila melanogaster", "advisor": "Sturtevant, Alfred Henry; Lewis, Edward B.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06112025-172549461", "creators": [ { "name": { "family": "Yu", "given": "Sien-Chiue" }, "id": "Yu-Sien-Chiue", "display_name": "Yu, Sien-Chiue" } ], "advisors": [ { "name": { "family": "Sturtevant", "given": "Alfred Henry" }, "id": "Sturtevant-A-H", "role": "advisor", "display_name": "Sturtevant, Alfred Henry" }, { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-E-B", "role": "advisor", "display_name": "Lewis, Edward B." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/422e-d558", "abstract": "The literature pertaining to the problem of mutations,\r\nwith special reference to reverse mutations and position effect,\r\nis reviewed. Data are presented from genetic and cytological\r\nstudies of several reversions at the Dichaete and Glued loci, of\r\nforty-six cases of position effect at the white locus, thirty-six\r\nmutations at the light locus and twenty-five mutations at the\r\nstraw locus. Three of the mutations at the light locus and one\r\nof the mutations at the straw locus are shown to be position effects.\r\nOne case of position effect at the Bar locus and two new mutants,\r\nAntennapedia and Scarred, are described. These reversions and\r\nmutations were induced by irradiation with X-rays. The effects\r\nof adding and subtracting Y-chromosomes, and of temperature upon\r\nthe light-mutant character of mutants resulting from a position\r\neffect at the light locus are demonstrated." }, { "name": "Feigen, George Alexander", "degree": "PhD", "year": "1948", "title": "Part I. Physiological Behavior of Oxypolygelatin. Part II. Relative Sensitivity of the Mucosal and Peritoneal Surfaces of Guinea Pig Ileum to Histamine Acetylcholine and Specific Antigens", "advisor": "Campbell, Dan Hampton; Van Harreveld, Anthonie", "url": "https://resolver.caltech.edu/CaltechTHESIS:06062025-145842225", "creators": [ { "name": { "family": "Feigen", "given": "George Alexander" }, "id": "Feigen-George-Alexander", "display_name": "Feigen, George Alexander" } ], "advisors": [ { "name": { "family": "Campbell", "given": "Dan Hampton" }, "id": "Campbell-D-H", "role": "advisor", "display_name": "Campbell, Dan Hampton" }, { "name": { "family": "Van Harreveld", "given": "Anthonie" }, "id": "Van-Harreveld-A", "role": "advisor", "display_name": "Van Harreveld, Anthonie" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "immun" ], "doi": "10.7907/k39p-ss37", "abstract": "No abstract." }, { "name": "Gray, Reed Alden", "degree": "PhD", "year": "1948", "title": "Inhibitors of Plant Growth From the Leaves of Encelia farinosa", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechTHESIS:09102025-020008158", "creators": [ { "name": { "family": "Gray", "given": "Reed Alden" }, "id": "Gray-Reed-Alden", "display_name": "Gray, Reed Alden" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/109n-z614", "abstract": "It has been shown that the leaves of Encelia farinose when applied to tomato and other plants in sand cultures cause a striking growth inhibition. Water and ether extracts of the leaves when fed to tomato seedlings in solution culture cause the death of the plants within one day. Fractionation of the leaf extracts yielded a crystalline compound which was isolated in pure form and is toxic to tomato seedlings in solution culture.
\r\n\r\nThe structure determination and synthesis of this new compound, 3-acetyl-6-methoxybenzaldehyde (AMB), has been successfully worked out and is given in detail. The inhibitory activity of related compounds on the growth of tomato seedlings is demonstrated.
\r\n\r\nAnother crystalline toxic compound has been isolated from leaves of Encelia farinose gathered in a different geographical location. Its toxic action is even more pronounced than that of AMB. This compound has been partly characterized and shown to be an aliphatic unsaturated lactone containing one hydroxyl group, three double bonds, and having a molecular formula of C16 H20 O4.
\r\nThe presence of these growth inhibitors found in the leaves of Encelia farinose is offered as an explanation of why so few desert annuals are found growing in close relationship with the Encelia shrub on the desert.
" }, { "name": "Singleton, Jesse Robertson", "degree": "PhD", "year": "1948", "title": "Cytogenetic Studies of Neurospora crassa", "advisor": "Unknown, Unknown", "url": "https://resolver.caltech.edu/CaltechETD:etd-08042006-130929", "creators": [ { "name": { "family": "Singleton", "given": "Jesse Robertson" }, "id": "Singleton-Jesse-Robertson", "display_name": "Singleton, Jesse Robertson" } ], "advisors": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/SK7K-FX44", "abstract": "The literature pertinent to the nuclear cycle in the life history of Neurospora is reviewed. A description of methods used in the cytological studies is presented, and the most useful are summarized as schedules of treatment. The morphology of the mitotic and pachytene chromosomes of N. crassa is described and diagrammed. The chromosome cycle in the developing ascus is described in detail, and special points of interest are discussed. The cytological basis of spore abortion caused by the presence in the ascus, in the homozygous condition, of genes from the Abbott strains is discussed briefly. Five translocations, three previously found by McClintock (1945) and two others found by the writer, are described and tentatively identified as to the chromosomes involved. From a consideration of the consequences of crossing-over and disjunction in the translocation heterozygote, equations are derived by means of which the frequencies of the various types of exchange and disjunction can be calculated or estimated. Non-random segregation in T4637 following an effective exchange in a single interstitial segment is described, and a possible mechanism suggested." }, { "name": "Regnery, David Cook", "degree": "PhD", "year": "1947", "title": "A Study of the Leucineless Mutants of Neurospora crassa", "advisor": "Beadle, George Wells", "url": "https://resolver.caltech.edu/CaltechTHESIS:04252025-202844428", "creators": [ { "name": { "family": "Regnery", "given": "David Cook" }, "id": "Regnery-David-Cook", "display_name": "Regnery, David Cook" } ], "advisors": [ { "name": { "family": "Beadle", "given": "George Wells" }, "id": "Beadle-G-W", "role": "advisor", "display_name": "Beadle, George Wells" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/kt75-xm57", "abstract": "No abstract." }, { "name": "Teas, Howard Jones", "degree": "PhD", "year": "1947", "title": "The Biochemistry and Genetics of Threonine-Requiring Mutants of Neurospora crassa", "advisor": "Beadle, George Wells; Horowitz, Norman Harold", "url": "https://resolver.caltech.edu/CaltechTHESIS:04012025-202225347", "creators": [ { "name": { "family": "Teas", "given": "Howard Jones" }, "id": "Teas-Howard-Jones", "display_name": "Teas, Howard Jones" } ], "advisors": [ { "name": { "family": "Beadle", "given": "George Wells" }, "id": "Beadle-G-W", "role": "advisor", "display_name": "Beadle, George Wells" }, { "name": { "family": "Horowitz", "given": "Norman Harold" }, "id": "Horowitz-N-H", "role": "advisor", "display_name": "Horowitz, Norman Harold" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/5jev-5168", "abstract": "No abstract." }, { "name": "Arregu\u00edn-Lozano, Barbar\u00edn", "degree": "PhD", "year": "1946", "title": "Carbohydrate Metabolism in Potato Tubers as Influenced by Temperatures", "advisor": "Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechTHESIS:06012025-072136808", "creators": [ { "name": { "family": "Arregu\u00edn-Lozano", "given": "Barbar\u00edn" }, "id": "Arregu\u00edn-Lozano-Barbar\u00edn", "display_name": "Arregu\u00edn-Lozano, Barbar\u00edn" } ], "advisors": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/4k89-yf97", "abstract": "No abstract." }, { "name": "Bonner, Walter Daniel, Jr.", "degree": "PhD", "year": "1946", "title": "Studies on the Organic Acids of Plants", "advisor": "Haagen-Smit, Arie Jan", "url": "https://resolver.caltech.edu/CaltechTHESIS:05022025-202915526", "creators": [ { "name": { "family": "Bonner", "given": "Walter Daniel, Jr." }, "id": "Bonner-Walter-Daniel", "display_name": "Bonner, Walter Daniel, Jr." } ], "advisors": [ { "name": { "family": "Haagen-Smit", "given": "Arie Jan" }, "id": "Haagen-Smit-A-J", "role": "advisor", "display_name": "Haagen-Smit, Arie Jan" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/mx8n-4513", "abstract": "No abstract." }, { "name": "Dubbs, Clyde Andrew", "degree": "PhD", "year": "1946", "title": "Chemical and Physiological Investigations on Guayule Essential Oil", "advisor": "Haagen-Smit, Arie Jan", "url": "https://resolver.caltech.edu/CaltechTHESIS:06012025-093758946", "creators": [ { "name": { "family": "Dubbs", "given": "Clyde Andrew" }, "id": "Dubbs-Clyde-Andrew", "display_name": "Dubbs, Clyde Andrew" } ], "advisors": [ { "name": { "family": "Haagen-Smit", "given": "Arie Jan" }, "id": "Haagen-Smit-A-J", "role": "advisor", "display_name": "Haagen-Smit, Arie Jan" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/8vy8-kz07", "abstract": "No abstract." }, { "name": "Fong, Conrad Tuck Onn", "degree": "PhD", "year": "1946", "title": "Investigation on Partheniol, a Sesquiterpene Alcohol from Guayule", "advisor": "Haagen-Smit, Arie Jan", "url": "https://resolver.caltech.edu/CaltechTHESIS:06082025-133401537", "creators": [ { "name": { "family": "Fong", "given": "Conrad Tuck Onn" }, "id": "Fong-Conrad-Tuck-Onn", "display_name": "Fong, Conrad Tuck Onn" } ], "advisors": [ { "name": { "family": "Haagen-Smit", "given": "Arie Jan" }, "id": "Haagen-Smit-A-J", "role": "advisor", "display_name": "Haagen-Smit, Arie Jan" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/7g5v-4r45", "abstract": "No abstract." }, { "name": "Dandliker, Walter Beach", "degree": "PhD", "year": "1945", "title": "The Isolation of 3-Indole Acetic Acid from Immature Corn", "advisor": "Haagen-Smit, Arie Jan", "url": "https://resolver.caltech.edu/CaltechTHESIS:03282025-223427071", "creators": [ { "name": { "family": "Dandliker", "given": "Walter Beach" }, "id": "Dandliker-Walter-Beach", "display_name": "Dandliker, Walter Beach" } ], "advisors": [ { "name": { "family": "Haagen-Smit", "given": "Arie Jan" }, "id": "Haagen-Smit-A-J", "role": "advisor", "display_name": "Haagen-Smit, Arie Jan" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/kxfg-5c11", "abstract": "No abstract." }, { "name": "Wright, Ernest Bevier", "degree": "PhD", "year": "1945", "title": "The Effects of Oxygen Lack on Peripheral Nerves", "advisor": "Van Harreveld, Anthonie; Wiersma, Cornelius A. G; Went, Frits W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:06092025-160037685", "creators": [ { "name": { "family": "Wright", "given": "Ernest Bevier" }, "id": "Wright-Ernest-Bevier", "display_name": "Wright, Ernest Bevier" } ], "advisors": [ { "name": { "family": "Van Harreveld", "given": "Anthonie" }, "id": "Van-Harreveld-A", "role": "advisor", "display_name": "Van Harreveld, Anthonie" }, { "name": { "family": "Wiersma", "given": "Cornelius A. G" }, "id": "Wiersma-C-A-G", "role": "advisor", "display_name": "Wiersma, Cornelius A. G" }, { "name": { "family": "Went", "given": "Frits W." }, "id": "Went-F-W", "role": "advisor", "display_name": "Went, Frits W." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/6kv4-r626", "abstract": "No abstract." }, { "name": "Dubnoff, Jacob William", "degree": "PhD", "year": "1944", "title": "I. Biological Synthesis of Creatine. II. Corneal Vascularity and Ariboflavinosis. III. Root \"Bleeding\"", "advisor": "Borsook, Henry", "url": "https://resolver.caltech.edu/CaltechTHESIS:04012025-230418750", "creators": [ { "name": { "family": "Dubnoff", "given": "Jacob William" }, "id": "Dubnoff-Jacob-William", "display_name": "Dubnoff, Jacob William" } ], "advisors": [ { "name": { "family": "Borsook", "given": "Henry" }, "id": "Borsook-H", "role": "advisor", "display_name": "Borsook, Henry" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/t2ay-dg34", "abstract": "No abstract." }, { "name": "Keighley, Geoffrey Lorrimer", "degree": "PhD", "year": "1944", "title": "I. Studies on Vibration Sensation. II. Heat Production of Slow and Fast Contractions of a Crustacean Muscle with Double Motor Innervation", "advisor": "Unknown, Unknown", "url": "https://resolver.caltech.edu/CaltechTHESIS:04052025-001958471", "creators": [ { "name": { "family": "Keighley", "given": "Geoffrey Lorrimer" }, "id": "Keighley-Geoffrey-Lorrimer", "display_name": "Keighley, Geoffrey Lorrimer" } ], "advisors": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/qqpz-1d24", "abstract": "Apparatus and a method are described for testing the responses in men to mechanical vibrations of different\r\nfrequencies. This method has been applied to over 600 men. In the whole group the thresholds at all frequencies increase with the age of the subjects. Similarly the upper frequency limit to which there is a response falls off with age. Half of the men received vitamins for a period of approximately one year, half did not. In the older age group, those receiving the vitamins were found to have lower thresholds than those who did not, but the difference is below the level of statistical significance.
\r\nA method has been developed for measuring the heat produced during sustained contractions, of both the slow and fast types, of the closer of the cheliped of the crayfish Cambarus clarkii. The contractions occur in the same muscle fibers. Any heat produced by, or accompanying, the muscle action current alone is less than can be measured by the method used. On repetition, both types of contraction become more efficient. The slow contractions are more efficient than the fast, both when the mechanical effects of the contraction are dissimilar and when they are made as alike as possible.
" }, { "name": "Cushing, John Eldridge, Jr.", "degree": "PhD", "year": "1943", "title": "A Comparative Study of Complement in the Vertebrates", "advisor": "Emerson, Sterling", "url": "https://resolver.caltech.edu/CaltechTHESIS:04182025-185601299", "creators": [ { "name": { "family": "Cushing", "given": "John Eldridge, Jr." }, "id": "Cushing-John-Eldridge", "display_name": "Cushing, John Eldridge, Jr." } ], "advisors": [ { "name": { "family": "Emerson", "given": "Sterling" }, "id": "Emerson-S", "role": "advisor", "display_name": "Emerson, Sterling" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/aqzb-s631", "abstract": "No abstract" }, { "name": "Ellis, Charles Herbert", "degree": "PhD", "year": "1943", "title": "I. The Effect of Electronarcosis on the Secretory Activity of the Pituitary Gland. II. Studies on the Extensor Mechanism of the Spider Leg. III. A Comparative Study of Peripheral Inhibition in Decapod Crustaceans", "advisor": "Wiersma, Cornelius A. G; Van Harreveld, Anthonie", "url": "https://resolver.caltech.edu/CaltechTHESIS:01302017-153658359", "creators": [ { "name": { "family": "Ellis", "given": "Charles Herbert" }, "id": "Ellis-Charles-Herbert", "display_name": "Ellis, Charles Herbert" } ], "advisors": [ { "name": { "family": "Wiersma", "given": "Cornelius A. G" }, "id": "Wiersma-C-A-G", "role": "advisor", "display_name": "Wiersma, Cornelius A. G" }, { "name": { "family": "Van Harreveld", "given": "Anthonie" }, "id": "Van-Harreveld-A", "role": "advisor", "display_name": "Van Harreveld, Anthonie" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/3VVT-6H32", "abstract": "Part I
\r\n\r\nIn studying the effect of electronarcosis on the\r\nsecretory activity of the pituitary gland, electronarcosis\r\nhas been applied to guinea pigs and dogs.
\r\n\r\nIncreased secretion of the thyrotropic, adrenocorticotropic\r\nand gonadotropic hormones has been shown to result\r\nfrom this passage of electric current through the head of\r\nthe animal. These increases have been shown to be reversible\r\nupon discontinuing the electronarcosis series.
\r\n\r\nThe \"tropic\" activity following electronarcosis has\r\nbeen shown to be endocrine in nature by demonstrating that in\r\ndogs the blood serum shows a marked increase in its ability\r\nto produce hypertrophy in the thyroids, adrenals, and testes\r\nof day-old chicks.
\r\n\r\nIn guinea pigs a marked elevation of the basal\r\nmetabolic rate occurs, subsequent to electronarcosis. In view\r\nof the short latency of this response it has been suggested\r\nthat the increased B.M.R. results from an increased production\r\nof a substance such as the specific metabolic principle\r\ndescribed by O'Donovan and Collip.
\r\n\r\nThe symptoms observed during electronarcosis in\r\nguinea pigs have been described.
\r\n\r\nPart II
\r\n\r\nThe musculature of the legs of spiders of several\r\nspecies has been studied. For the most part the work of\r\nPetrunkevitch has been confirmed by anatomical observation\r\nand physiological experimentation; however, one and possibly\r\ntwo heretofore undescribed muscles are reported. The function\r\nof these muscles has not been demonstrated physiologically,\r\nbut from their structural arrangement they may have a role\r\nin raising the chitinous horseshoe-shaped plate in the femoro-patellar\r\nand tibia-metatarsal joints respectively.
\r\n\r\nAn histological study of the leg muscles in spiders\r\nhas shown them to consist of long, striated, multinucleate\r\nfibers loosely associated in parallel groups to form the\r\nrespective muscles; that is to say, the fibers run parallel\r\nfrom origin to insertion for the full length of the muscle.
\r\n\r\nDistribution of the arterial supply in the legs has\r\nbeen found to be quite extensive. Branching and re-branching\r\nof the main artery results in an elaborate arborization\r\nintimately distributed throughout the muscles.
\r\n\r\nA discussion is presented which indicates that in\r\nthose joints which characteristically lack extensor muscles\r\nextension is carried out by means of an hydraulic mechanism.\r\nTwo possible mechanisms are suggested; the one considered\r\nmost probable involves partially closing off the main\r\narterial stem, thereby diverting a greater amount of blood\r\ninto the membranous pocket formed by the thin flexible\r\ninterarticular membrane of the ventral surface of the joint.\r\nExtension is thus a purely mechanical result of the pressure\r\nexerted by the balooning-out of the membrane.
\r\n\r\nPart III
\r\n\r\nThe effectiveness with which different contractions in a number of muscles\r\ncan be inhibited was investigated. As a measure of this effectiveness the frequency\r\nof inhibition which can just inhibit a contraction with a given frequency of excitation\r\nwas determined. It was found that in all system the ratio (Rc) of such inhibitory\r\nfrequencies to that of the excitatory frequencies they can suppress was constant\r\nfor a wide range of frequencies.
\r\n\r\nAt high frequencies either the inhibition or the excitation may become less\r\neffective. This is explained by failure of the respective system to function normally\r\nat such a frequency.
\r\n\r\nThe effectiveness of inhibition of different systems was determined. Some\r\nsystems show a very constant Rc value; in a second group Rc varies within wider \r\nlimits; and a third group shows two distinct Rc's sometimes in the same preparation\r\nat different times.
\r\n\r\nRc values have been found to vary widely. For instance, in the bender inhibitor slow\r\nbender system of Paehygrapsus three excitatory impulses are suppressed by\r\none inhibitory impulse; in the closer inhibitor-slow closer system of Cambarus\r\none excitatory impulse needs five inhibitory impulses to counteract its effect. The\r\nfast closer contraction of Cambarus and the fast closer and fast bender contraction\r\nof Paehygrapsus were found to be uninhibitable; i.e. no effect of inhibition whatsoever\r\nwas noticed on any of these contractions. All three systems are distinguished\r\nby giving a mechanical response to a single stimulus in contrast with all the\r\ninhibitable systems which do not respond to single impulses.
\r\n\r\nReduction of the action potentials during inhibition is obtainable in only a few\r\nsystems, namely, the opener inhibitor-opener and the stretcher inhibitor-stretcher\r\nsystems of Cambarus and the crabs. (In the crabs this applies only to the 'true'\r\ninhibitors.) In all other systems, including every system of Panulirus, no reduction\r\nof the muscle action potential is obtained.
" }, { "name": "Gorham, Paul Raymond", "degree": "PhD", "year": "1943", "title": "Growth Factor Studies with Spirodela polyrrhiza (L.) Schlied", "advisor": "Went, Frits W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:01242017-135027892", "creators": [ { "name": { "family": "Gorham", "given": "Paul Raymond" }, "id": "Gorham-Paul-Raymond", "display_name": "Gorham, Paul Raymond" } ], "advisors": [ { "name": { "family": "Went", "given": "Frits W." }, "id": "Went-F-W", "role": "advisor", "display_name": "Went, Frits W." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/H9KZ-FX44", "abstract": "Leeuwenhoek was one of the first men to record observations\r\nupon the peculiar growth habits of members of the family Lemnaceae.\r\nTaxonomists and morphologists have long argued about the proper classification\r\nand anatomical interpretation of these, most modified of\r\nhigher plants, while physiologists from the time of Sachs have seen in\r\nLemna and Spirodela a type of plant peculiarly suited for experimental\r\ninvestigations in the field of nutrition. This has been because of\r\ntheir small size and aquatic habit which make them ideal for culture \r\nin nutrient solution.
\r\n\r\nIn recent times there has been considerable controversy over\r\nthe question as to whether organic manures, humus, peat, and soil supply\r\nsmall amounts of organic materials which promote the growth of\r\ngreen plants. Are the beneficial effects often observed upon treatment\r\nwith these substances simply the result of correcting recognized or\r\nunrecognized inorganic deficiencies, or are they the result of accessory\r\ngrowth factors?
\r\n\r\nIn undertaking to answer this question, Spirodela polyrrhiza\r\n(L.) Schleid. was chosen as the test plant since careful control of\r\nits growth environment is readily achieved. The possibility of freeing\r\nthe plants of microorganisms and employing sterile culture technique\r\nmake its use particularly desirable in a study concerned with the\r\neffects of organic materials.
\r\n\r\nThe primary aim in the investigation was to demonstrate that\r\norganic additions to a medium of inorganic salts balanced for optimal\r\ngrowth can produce a significant increase in growth. Also, that manures,\r\nhumus, peat, and soil are sources of these growth promoting substances,\r\nand to find out as much about their nature as possible. Growing out\r\not this line of study was a broader one, namely, an attempt to gain\r\nsome insight into the mechanism of the action and interaction of the\r\nmany different factors, such as light, carbon dioxide, carbohydrate\r\nsupply, etc., which are known to affect the growth of Spirodela. Finally,\r\nunsuccessful attempts were made to induce flowering of Spirodela, which\r\noccurs only rarely in nature and has never been induced experimentally.
" }, { "name": "Gottlieb, Sidney", "degree": "PhD", "year": "1943", "title": "Studies on a Growth Inhibitor in Guayule", "advisor": "Haagen-Smit, Arie Jan; Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechTHESIS:01242017-111743193", "creators": [ { "name": { "family": "Gottlieb", "given": "Sidney" }, "id": "Gottlieb-Sidney", "display_name": "Gottlieb, Sidney" } ], "advisors": [ { "name": { "family": "Haagen-Smit", "given": "Arie Jan" }, "id": "Haagen-Smit-A-J", "role": "advisor", "display_name": "Haagen-Smit, Arie Jan" }, { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/TBXH-5W50", "abstract": "1. Evidence is presented to show that when guayule plants (6\r\nmonths to 2 years of age) are grown in gravel culture, an influence\r\nis exerted which is inhibitory to the growth of guayule seedlings.
\r\n\r\n2. The development of two types of semi-quantitative assay for\r\nthe detection of the inhibiting principle is described.
\r\n\r\n3. A method is described for the collection of large quantities\r\nof nutrient solution containing the inhibiting principle, and for the\r\nsubsequent concentration of this material to small volume. It is\r\npointed out that the production of the inhibiting principle decreases\r\nsharply during the winter months.
\r\n\r\n4. Evidence is presented to show that the inhibiting principle\r\nis an organic substance. The critical evidence consists of the disappearance\r\nof activity on ashing and of the extractability of the\r\ninhibitor by organic solvents.
\r\n\r\n5. It is estimated that a guayule plant (6 months to 2 years of\r\nage), growing in gravel culture gives up to the nutrient medium, on\r\nthe average, a minimum of 200 gammas per day of organic matter.
\r\n\r\n6. Various chemical properties of the inhibitor are given. It\r\nis shown to be relatively stable to heat, stable to drying, non-volatile,\r\nnon-adsorbable by charcoal, and probably non-precipitable by lead.
\r\n\r\n7. An experiment is described in which a crude crystalline material\r\nwas obtained which was very active.
\r\n\r\n8. Evidence is presented to show that the effect of the inhibitor\r\nis not due to an influence on the pH of the nutrient solution.
\r\n\r\n9. The occurance of growth stimulatory organic substances in\r\nthe guayule leachate is described. These stimulatory fractions were\r\nactive at concentrations as low as 2 mg. per liter.
\r\n\r\n10. A number of pure synthetic organic acids were tested for\r\ninhibitory activity, of these only one, cis-9,10 dihydroxy stearic\r\nacid was found to be active. This compound caused marked inhibition\r\nin concentrations as low as 3.5 mg. per liter.
\r\n\r\n11. The chemical fractionation of a soil on which guayule plants\r\nhad grown is described and it is shown to contain inhibitory organic\r\nsubstances having some chemical properties similar to those of the\r\nsubstances in the guayule leachates.
" }, { "name": "Hovanitz, William", "degree": "PhD", "year": "1943", "title": "Investigations on Racial Biology Especially in Lepidoptera", "advisor": "Morgan, Thomas Hunt; Sturtevant, Alfred Henry", "url": "https://resolver.caltech.edu/CaltechTHESIS:06192025-015323034", "creators": [ { "name": { "family": "Hovanitz", "given": "William" }, "id": "Hovanitz-William", "display_name": "Hovanitz, William" } ], "advisors": [ { "name": { "family": "Morgan", "given": "Thomas Hunt" }, "id": "Morgan-Thomas Hunt", "role": "advisor", "display_name": "Morgan, Thomas Hunt" }, { "name": { "family": "Sturtevant", "given": "Alfred Henry" }, "id": "Sturtevant-A-H", "role": "advisor", "display_name": "Sturtevant, Alfred Henry" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/898x-s884", "abstract": "Genetic and wild population studies have shown that the two races of Colias chrysotheme are different on a multiple-factor basis, these including physiological differences as well as a color difference. Sterility in the crosses may be largely related to diet differences between the races though evidence is presented to show that the hybrid segregants are less viable than the parental types. A wild population where intercrossing occurs shows about 10% of intermediates - mostly fertile.
\r\nA dominant autosomal gene for white female color is found in wild populations of both races. Genetic results indicate that it is probably homologous in the two races and is interchangeable between them. The gene may be either lethal or semi-lethal with certain modifiers when homozygous dominant. Within each race the gene is most abundant in the northern populations as compared with the southern.
\r\nThe history of the recent establishment of the orange race in the area east of the Mississippi River is reviewed; the evidence shows that the planting of alfalfa in that area has been the primary factor which has encouraged the extension of range of the butterfly.
\r\nThe causes of genetic alterations in wild populations has been studied by means of the white females. Genetic population changes are described which have been caused by migration, environmental selection, random fluctuations and differential development rate of the genotypes.
" }, { "name": "Mampell, Klaus", "degree": "PhD", "year": "1943", "title": "Genetic Studies in Drosophila Pseudoobscura", "advisor": "Unknown, Unknown", "url": "https://resolver.caltech.edu/CaltechTHESIS:12142016-110541139", "creators": [ { "name": { "family": "Mampell", "given": "Klaus" }, "id": "Mampell-Klaus", "display_name": "Mampell, Klaus" } ], "advisors": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/EQ1V-FY29", "abstract": "No abstract." }, { "name": "Saunders, Paul Rome", "degree": "PhD", "year": "1943", "title": "I. Studies on the Mescal Alkaloids and Related Compounds. II. Preparation of Several Phospho-Organic Compounds", "advisor": "Haagen-Smit, Arie Jan", "url": "https://resolver.caltech.edu/CaltechTHESIS:03132025-173850426", "creators": [ { "name": { "family": "Saunders", "given": "Paul Rome" }, "id": "Saunders-Paul-Rome", "display_name": "Saunders, Paul Rome" } ], "advisors": [ { "name": { "family": "Haagen-Smit", "given": "Arie Jan" }, "id": "Haagen-Smit-A-J", "role": "advisor", "display_name": "Haagen-Smit, Arie Jan" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/1jdk-2j68", "abstract": "No abstract." }, { "name": "Siu, Ralph Gun-Hoy", "degree": "PhD", "year": "1943", "title": "Chemical Studies on the Development of Plant Embryos", "advisor": "Haagen-Smit, Arie Jan", "url": "https://resolver.caltech.edu/CaltechTHESIS:03142025-221133774", "creators": [ { "name": { "family": "Siu", "given": "Ralph Gun-Hoy" }, "id": "Siu-Ralph-Gun-Hoy", "display_name": "Siu, Ralph Gun-Hoy" } ], "advisors": [ { "name": { "family": "Haagen-Smit", "given": "Arie Jan" }, "id": "Haagen-Smit-A-J", "role": "advisor", "display_name": "Haagen-Smit, Arie Jan" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/y6p0-4b68", "abstract": "No abstract." }, { "name": "Lewis, Edward B.", "degree": "PhD", "year": "1942", "title": "A Genetic and Cytological Analysis of a Tandem Duplication and its Included Loci in Drosophila melanogaster", "advisor": "Sturtevant, Alfred Henry", "url": "https://resolver.caltech.edu/CaltechETD:etd-10172002-112502", "creators": [ { "name": { "family": "Lewis", "given": "Edward B." }, "id": "Lewis-Edward-B", "display_name": "Lewis, Edward B." } ], "advisors": [ { "name": { "family": "Sturtevant", "given": "Alfred Henry" }, "id": "Sturtevant-A-H", "role": "advisor", "display_name": "Sturtevant, Alfred Henry" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/7W60-4740", "abstract": "Summary
\r\n\r\nI.
\r\nTwo loci, Star and asteroid, in the second chromosome of D. melanogaster were found to be extremely closely linked with an estimated standard map distance of 0.01 to 0.04 unit. It was demonstrated that these two loci are included in the 21 E. 1-2 doublet structure of the salivary gland chromosomes. Not only do the Star and asteroid mutants affect the eye in a similar way, but they also show position effects depending on how they are distributed between the two chromosomes. An interpretation of the observed phenomena is made in terms of a naturally occurring repeat in the chromosomes.
II.
\r\nThe Star Duplication is a tandem repeat in direct order for the four bands, 21 D 3, 4 and 21 E 1-2. It includes the Star and asteroid loci and with the use of mutants at these loci as markers. it has been possible to study unequal crossing over in females heterozygous as well as homozygous for the duplication. The genetic composition of the Star Duplication has been varied in ten distinct ways; the results indicate that there is probably a position effect exerted on the asteroid locus 1n the left section of the duplication causing the originally present asteroid change to act like a reversion to normal. It is likely that there is little or no position effect exerted by the Star and asteroid 1oci in one section on those loci in the other section.
Introduction
\r\n\r\nThe genetic evidence available at present indicates that certain groups of genes tend to remain linked in the various members of the genus Drosophila (Donald 1936; Sturtevant and Tan, 1937; Sturtevant, 1938a, 1940; Sturtevant and Novitaki., 1941). These elements are comparable to the arms of the chromosomes of melanogaster and have accordingly been lettered A, B, C, D, E, and F for X, IIL, IIR, IIIL, IIIR, and IV, respectively. In view of the extensive cytological studies on the various members of the affinis group (Dobzhansky and Socolov, 1939; Miller, 1939; Novitski, unpublished; Sturtevant, unpublished), it has seemed pertinent to extend the homologies with the elements to the salivary gland chromosomes of this group. The high degree of similarity of the salivary gland chromosome arms of the various affinis group species examined renders it possible to make such a correlation within one species, in this case affinis, with the reasonable certainty that the results obtained will apply to other members of the group (athabasca, algonquin, and azteca).
\r\n\r\nSturtevant (1939) has homologized a number of mutant genes in affinis with those of melanogaster and pseudoobseura, applying to the linkage groups of affinis the same designations as those of their homologs in pseudoobsoura. In terms of the above mentioned elements, consequently, the affinis linkage groups, XL, XR, II, III, IV, and V correspond to A, D, E, C, B, and F. respectively. Miller (1939), in a study of the salivary gland chromosomes of algonquin, has arbitrarily lettered the units found therein: a two-armed X-chromosome (XL and XS), a single armed autosome (A), two two-armed autosomes (B and C), and a microchromosome (D). In order to avoid confusion, the symbols to be used until the complete correlation has been established will be those of Sturtevant for the linkage groups and those of Miller for the salivary gland chromosomes.
\r\n\r\nThe metaphase chromosomes of males of the affinis group include a V-shaped X-chromosome, a J-shaped Y-chromosome, two pairs of J-shaped autosomes, a pair of rods and a pair of dots (Metz, 1916; Sturtevant and Dobzhansky, 1936; Dobzhansky and Socolov, 1939; Miller, 1939). It seems clear that there is a correspondence between the number of large chromosome arms in the metaphase plate and the euchromatic arms In the salivary gland nucleus (Miller, 1939, for algonquin; Sturtevant, unpublished, for afinis; Novitski, unpublished, for athabasca; Novitski, in Sturtevant, 1940, for azteca.
" }, { "name": "Bergren, William Raymond", "degree": "PhD", "year": "1941", "title": "Studies Upon the Isolation and Identification of Auxins in Plant Materials", "advisor": "Haagen-Smit, Arie Jan", "url": "https://resolver.caltech.edu/CaltechTHESIS:03072025-015358294", "creators": [ { "name": { "family": "Bergren", "given": "William Raymond" }, "id": "Bergren-William-Raymond", "display_name": "Bergren, William Raymond" } ], "advisors": [ { "name": { "family": "Haagen-Smit", "given": "Arie Jan" }, "id": "Haagen-Smit-A-J", "role": "advisor", "display_name": "Haagen-Smit, Arie Jan" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/hmqp-2k82", "abstract": "1. A survey was made of plant materials in search of a suitable new source material for auxin isolation.
\r\n2. The existing methods of isolation were considered as to their usefulness and point of application in the procedure. An alkali acetone purification and an exhaustive ligroin extraction were introduced.
\r\n3. It was found that indole-3-acetic acid rendered uncertain the interpretations of separations made in the final isolation steps. Heretofore, it has been considered that all of the indole-3-acetic acid is completely destroyed by the esterification procedure preliminary to high-vacuum distillation. This has been shown not to be the case.
\r\n4. The phenomenon of the increased yield of auxin from wheat and corn by alkali treatment was investigated by isolation. Both pseudo-auxin-a and indole-3-acetic acid were isolated and identified, which marks the first time that this last substance has been found in a higher plant material. The evidence obtained indicates that about 90% of the increased amount of auxin obtained by the alkali hydrolysis is due to indole-3-acetic acid.
\r\n5. A substance giving a positive curvature in the Avena test was discovered. This substance is a growth inhibitor.
" }, { "name": "Bonner, David Mahlon", "degree": "PhD", "year": "1940", "title": "Leaf Growth Factors", "advisor": "Haagen-Smit, Arie Jan; Went, Frits W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02132025-222534811", "creators": [ { "name": { "family": "Bonner", "given": "David Mahlon" }, "id": "Bonner-David-Mahlon", "display_name": "Bonner, David Mahlon" } ], "advisors": [ { "name": { "family": "Haagen-Smit", "given": "Arie Jan" }, "id": "Haagen-Smit-A-J", "role": "advisor", "display_name": "Haagen-Smit, Arie Jan" }, { "name": { "family": "Went", "given": "Frits W." }, "id": "Went-F-W", "role": "advisor", "display_name": "Went, Frits W." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/cp01-0h46", "abstract": "No abstract." }, { "name": "Fox, Sidney Walter", "degree": "PhD", "year": "1940", "title": "I. The Energy Relationships in the Ornithine-Citrulline-Arginine-Urea Cycle. II. Thermal Data for other Biochemicals. III. A Study of Sperm Agglutination", "advisor": "Unknown, Unknown", "url": "https://resolver.caltech.edu/CaltechTHESIS:01302025-014752334", "creators": [ { "name": { "family": "Fox", "given": "Sidney Walter" }, "id": "Fox-Sidney-Walter", "display_name": "Fox, Sidney Walter" } ], "advisors": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/sz3g-8t03", "abstract": "I. Methods for preparation and purification of arginine, citrulline, and ornithine were developed. The necessary entropies, heats of formation, solubilities, dissociation constants, activities, and vapor pressures were evaluated from the appropriate physical measurements of these compounds. These and other data were combined to calculate the free energy changes in each step of the ornithine-citrulline-arginine-urea cycle.
\r\nII. Methods of preparation, and purification, for other thermal data, of many biochemicals were developed. The entropies of creatine hydrate, glycyl glycine, hippuric acid, hippuryl glycine, 1-proline, and taurine were evaluated from specific heat measurements. Heats of combustion of fumaric and maleic acids were measured.
\r\nIII. The biological properties of egg-borne marine sperm agglutinins were studied. Bio-assays were developed and active extracts were prepared. These extracts were shown to be of a protein nature. Activation and agglutination of marine sperm by various substances were studied. Correlations of the relation of molecular structure to agglutinating activity were made and discussed.
" }, { "name": "Helfer, Robert George", "degree": "PhD", "year": "1940", "title": "A Comparison of X-ray Induced and Naturally Occurring Chromosomal Variations in Drosophila Pseudoobscura", "advisor": "Dobzhansky, Theodosius Grigorievich", "url": "https://resolver.caltech.edu/CaltechTHESIS:05212024-230410309", "creators": [ { "name": { "family": "Helfer", "given": "Robert George" }, "id": "Helfer-Robert-George", "display_name": "Helfer, Robert George" } ], "advisors": [ { "name": { "family": "Dobzhansky", "given": "Theodosius Grigorievich" }, "id": "Dobzhansky-Th", "role": "advisor", "display_name": "Dobzhansky, Theodosius Grigorievich" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/jreq-0n39", "abstract": "Summary:
\r\n\r\n1. The distribution of 347 breaks induced by X-ray treatment\r\nin the chromosomes of Drosophila pseudoobscura was studied.\r\nThe frequencies of the breaks in the different chromosomes\r\nare in the proportion to the lengths of the latter.
\r\n\r\n2. The induced breaks in the third chromosome are not distributed\r\nentirely at random. The frequency of the breaks in the\r\nheterochromatin as compared with those in the euchromatin,\r\nis much greater than would be expected on the basis of the\r\nlengths of the heterochromatic portions in the salivary gland\r\ncells, but probably smaller than would be expected on the\r\nbasis of its length in the mitotic chromosomes. Within the\r\neuchromatic portions the frequency of breaks increases\r\nslightly from the proximal to the distal end.
\r\n\r\n3. Aside from the regularity mentioned in the preceding paragraph,\r\nthe breaks in the third chromosome show no tendency to be\r\nconcentrated around any \"weak points.\" In any case, a\r\ncomparison of the induced breaks with those observed in\r\nthe naturally occurring chromosomal aberrations shows very\r\nfew coincidences. None of the inversions induced by X-ray\r\ntreatment proved similar to any of the naturally occurring\r\ninversions.
\r\n\r\n4. The reunion of the chromosome fragments produced by X-ray\r\ntreatment is not at random, inversions being more and translocations\r\nless frequent than expected.
\r\n\r\n5. Several mosaic chromosomal observations are described. An\r\nanalysis of these aberrations seems to argue in favor of the\r\n\"breakage first,\" rather than the \"contact\" hypothesis of\r\nthe origin of chromosomal aberrations.
\r\n\r\n6. The \"breakage first\" hypothesis is also favored by the\r\nobserved aberration in which paternal as well as maternal\r\nchromosomes seem to be involved.
\r\n\r\n7. An aberration involving a terminal attachment, and another\r\nshowing what appears to be a branched chromosome, are\r\ndescribed.
" }, { "name": "Hlynka, Isydore", "degree": "PhD", "year": "1940", "title": "Free Energy and Other Data for Eight Compounds of Physiological Interest", "advisor": "Unknown, Unknown", "url": "https://resolver.caltech.edu/CaltechTHESIS:01162025-230014323", "creators": [ { "name": { "family": "Hlynka", "given": "Isydore" }, "id": "Hlynka-Isydore", "display_name": "Hlynka, Isydore" } ], "advisors": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/x32s-0h08", "abstract": "No abstract." }, { "name": "Marmont, George Heinemann", "degree": "PhD", "year": "1940", "title": "The Factors Concerned in the Leading Off of Action Potentials from Single Crustacean Nerve Fibers", "advisor": "Wiersma, Cornelius A. G; Van Harreveld, Anthonie", "url": "https://resolver.caltech.edu/CaltechTHESIS:12132024-181609716", "creators": [ { "name": { "family": "Marmont", "given": "George Heinemann" }, "id": "Marmont-George-Heinemann", "display_name": "Marmont, George Heinemann" } ], "advisors": [ { "name": { "family": "Wiersma", "given": "Cornelius A. G" }, "id": "Wiersma-C-A-G", "role": "advisor", "display_name": "Wiersma, Cornelius A. G" }, { "name": { "family": "Van Harreveld", "given": "Anthonie" }, "id": "Van-Harreveld-A", "role": "advisor", "display_name": "Van Harreveld, Anthonie" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/gksk-4k11", "abstract": "No abstract." }, { "name": "McRary, Willard Lee", "degree": "PhD", "year": "1940", "title": "Studies on the Biochemical Synthesis of Asparagine", "advisor": "Borsook, Henry", "url": "https://resolver.caltech.edu/CaltechTHESIS:04062017-095011293", "creators": [ { "name": { "family": "McRary", "given": "Willard Lee" }, "id": "McRary-Willard-Lee", "display_name": "McRary, Willard Lee" } ], "advisors": [ { "name": { "family": "Borsook", "given": "Henry" }, "id": "Borsook-H", "role": "advisor", "display_name": "Borsook, Henry" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/2Z2E-PV49", "abstract": "Methods are described for the in vitro cultivation of the\r\nexcised lupin embryo on a synthetic medium.
\r\n\r\nThe postulate that asparagine synthesis is not conditioned\r\nprimarily by the presence of excess free ammonia and hence is not a\r\ndetoxication mechanism has been experimentally confirmed in the lupin.
\r\n\r\nIsolated embryos, when supplied with adequate carbohydrate and\r\nnitrogen in the medium, are unable to absorb and convert succinic,\r\nmalic, pyruvic, lactic, and glyceric acids, and glycerol and succinmonamide\r\nto asparagine.
\r\n\r\nUnder similar conditions of carbohydrate and nitrogen supply\r\nfumaric, maleic, aspartic, exalacetic, and glutamic acids, and\r\nsuccindiamide are effective in stimulating the synthesis of asparagine.\r\nOf these compounds; glutamic adid is the most effective.\r\nMetabolic products of glutamic acid such as ketoglutaric, glutaric,\r\nand succinic acids are not effective. Combinations of glutamic acid\r\nwith aspartic and oxalacetic acids in the absence of ammonia are\r\nlikewise not effective in the stimulation of asparagine synthesis.
\r\n\r\nAn extract of embryo tissue is able to oxidize glutamic acid\r\nin the presence of boiled yeast juice. Amide formation has been\r\ndemonstrated in such an extract to which was added glutamic and\r\naspartic acids and ammonia.
\r\n\r\n\r\nAn attempt has been made to unify the above observations, and\r\nto explain the role of glutamate as an energy source in asparagine\r\nsynthesis, with the energy transfer possibly taking place through\r\nthe intermediation of a pyridine coenzyme.
\r\n\r\nThe unsatisfactory status of the postulated conversion of\r\naspartic acid to asparagine has been pointed out and a scheme proposed\r\nto explain the introduction of energy into the system.
\r\n\r\n" }, { "name": "Miller, Dwight Dana", "degree": "PhD", "year": "1940", "title": "Cytological and Genetic Studies on Drosophila algonquin and Related Species", "advisor": "Sturtevant, Alfred Henry; Dobzhansky, Theodosius Grigorievich", "url": "https://resolver.caltech.edu/CaltechTHESIS:03032025-224902902", "creators": [ { "name": { "family": "Miller", "given": "Dwight Dana" }, "id": "Miller-Dwight-Dana", "display_name": "Miller, Dwight Dana" } ], "advisors": [ { "name": { "family": "Sturtevant", "given": "Alfred Henry" }, "id": "Sturtevant-A-H", "role": "advisor", "display_name": "Sturtevant, Alfred Henry" }, { "name": { "family": "Dobzhansky", "given": "Theodosius Grigorievich" }, "id": "Dobzhansky-Th", "role": "advisor", "display_name": "Dobzhansky, Theodosius Grigorievich" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/adsk-sq92", "abstract": "No abstract." }, { "name": "Webb, John Leyden", "degree": "PhD", "year": "1940", "title": "Magnetic Properties of Hemocyanin", "advisor": "Pauling, Linus", "url": "https://resolver.caltech.edu/CaltechETD:etd-02012005-110219", "creators": [ { "name": { "family": "Webb", "given": "John Leyden" }, "id": "Webb-John-Leyden", "display_name": "Webb, John Leyden" } ], "advisors": [ { "name": { "family": "Pauling", "given": "Linus" }, "id": "Pauling-L", "role": "advisor", "display_name": "Pauling, Linus" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/RZKK-QM20", "abstract": "The primary purpose of this investigation was to determine the magnetic properties of hemocyanin in order to elucidate the oxygenation reaction. However, a number of other properties were examined as a preliminary basis for this work. These experiments were also of value in demonstrating the similarity of the hemocyanin used to the other hemocyanins and hence of the applicability of the magnetic work to these other proteins. As the hemocyanins are a class of proteins well adapted to studies from a comparative point of view, these results are of interest in themselves as the animal used was of a group not yet investigated. In order to increase the facility with which these comparative deductions are made I have devoted some space to results on other hemocyanins.\r\n\r\nAs no review of the hemocyanins has appeared for eight years, and much important work has been done meanwhile, I have felt justified in including some of the results of the recent work that bear on the final interpretation of the magnetic experiments.\r\n\r\nThe animal chosen for use was the giant key-hole limpet, Megathura crenulata, a Streptoneuran of the class Gasteropoda, found on the rocks in several places along the Pacific coast.\r\n\r\nFor the convenience of those who are not acquainted with all of the species mentioned I have included a table of common names at the end of the paper." }, { "name": "Addicott, Fredrick Taylor", "degree": "PhD", "year": "1939", "title": "Experiments on the Nutrition of Isolated Roots", "advisor": "Morgan, Thomas Hunt", "url": "https://resolver.caltech.edu/CaltechTHESIS:01232018-095827563", "creators": [ { "name": { "family": "Addicott", "given": "Fredrick Taylor" }, "id": "Addicott-Frederick-Taylor", "display_name": "Addicott, Fredrick Taylor" } ], "advisors": [ { "name": { "family": "Morgan", "given": "Thomas Hunt" }, "id": "Morgan-Thomas Hunt", "role": "advisor", "display_name": "Morgan, Thomas Hunt" } ], "committee": [ { "name": { "family": "Morgan", "given": "Thomas Hunt" }, "id": "Morgan-Thomas Hunt", "role": "chair", "display_name": "Morgan, Thomas Hunt" }, { "name": { "family": "Anderson", "given": "E. G." }, "id": "Anderson-E-G", "role": "member", "display_name": "Anderson, E. G." }, { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "member", "display_name": "Bonner, James Frederick" }, { "name": { "family": "Emerson", "given": "Robert" }, "id": "Emerson-Robert", "role": "member", "display_name": "Emerson, Robert" }, { "name": { "family": "Haagen-Smit", "given": "Arie Jan" }, "id": "Haagen-Smit-A-J", "role": "member", "display_name": "Haagen-Smit, Arie Jan" }, { "name": { "family": "Sturtevant", "given": "Alfred H." }, "id": "Sturtevant-Alfred-H", "role": "member", "display_name": "Sturtevant, Alfred H." }, { "name": { "family": "van Overbeek", "given": "Johannes" }, "id": "van-Overbeek-Johannes", "role": "member", "display_name": "van Overbeek, Johannes" }, { "name": { "family": "Went", "given": "Frits W." }, "id": "Went-F-W", "role": "member", "display_name": "Went, Frits W." } ], "option_major": [ "biology" ], "doi": "10.7907/F2N6-4H73", "abstract": "1. The history of plant tissue culture is outlined.
\r\n\r\n2. The technique of root cultures is reinvestigated from several aspects and some changes are incorporated for the present work.
\r\n\r\n3. The section of vitamin B1 as a root growth hormone is examined, and its presence is found to be closely correlated with the growth rate, rate of cell divisions, and extent of meristem in isolated roots.
\r\n\r\n4. A second growth factor of pea roots present in yeast extract is found not to be among the micorelements of plant nutrition nor among the amino acids.
\r\n\r\n5. Nicotinic acid is found to be the second growth factor of pea roots. With vitamin B1 it is capable of supporting a higher rate of growth for pea roots than can yeast extract.
\r\n\r\n6. Evidence is presented to show that inositol, phosphate, and optimum hydrogen ion concentration may have promotice effects on the growth of pea roots.
\r\n\r\n7. A number of experiments are described which indicate that it may be impossible to establish a clone of pea roots.
\r\n\r\n8. Preliminary experiments on the culture of excised anthers are described, and the high activity of embryo extracts on anther growth is noted.
" }, { "name": "Davenport, Horace Willard", "degree": "PhD", "year": "1939", "title": "Carbonic Anhydrase in the Gastric Mucosa", "advisor": "Unknown, Unknown", "url": "https://resolver.caltech.edu/CaltechETD:etd-04202004-105923", "creators": [ { "name": { "family": "Davenport", "given": "Horace Willard" }, "id": "Davenport-Horace-Willard", "display_name": "Davenport, Horace Willard" } ], "advisors": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/8JKG-2J47", "abstract": "Summary
\r\n\r\nThe evidence on the site of formation of hydrochloric acid in the gastric mucosa of mammals is reviewed. The evidence points to the participation of the parietal cells in the formation and secretion of the acid, but it is insufficient to prove that the acid is or is not formed directly in or by the parietal cells.
\r\n\r\nThe discovery of large amounts of carbonic anhydrase in the gastric mucosa is described. A technique of analysis for the enzyme in the gastric mucosa of cats and rats and for determining histologically the number of different kinds of cells in the material analysed is described. A strong positive correlation between the enzyme concentration and the number of parietal cells is taken as proof that carbonic anhydrase is present in the parietal cells in concentration higher than in the red blood cells. Similar evidence is given that carbonic anhydrase is present in small amounts in the cells of the surface epithelium. Evidence is also given that carbonic anhydrase may be present in small amounts in the gastric juice of cats, rats and humans. The enzyme in human gastric juice is different from that in red blood cells.
\r\n\r\nThe bearing of these facts on the theory of the formation of hydrochloric acid is discussed, and it is concluded that the hydration of carbon dioxide to carbonic acid and the subsequent ionization of the acid may be the means by which hydrogen ions are provided. The energy necessary for the formation of hydrochloric acid from blood is estimated, and the prevailing theories of secretion are reviewed. All are found inadequate.
" }, { "name": "Horowitz, Norman Harold", "degree": "PhD", "year": "1939", "title": "The Rising Rate of Respiration in Developing Eggs", "advisor": "Tyler, Albert; Borsook, Henry", "url": "https://resolver.caltech.edu/CaltechETD:etd-05082003-155023", "creators": [ { "name": { "family": "Horowitz", "given": "Norman Harold" }, "id": "Horowitz-Norman-Harold", "display_name": "Horowitz, Norman Harold" } ], "advisors": [ { "name": { "family": "Tyler", "given": "Albert" }, "id": "Tyler-A", "role": "advisor", "display_name": "Tyler, Albert" }, { "name": { "family": "Borsook", "given": "Henry" }, "id": "Borsook-H", "role": "advisor", "display_name": "Borsook, Henry" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/0GNC-2P03", "abstract": "No abstract submitted" }, { "name": "Stewart, William Sheldon", "degree": "PhD", "year": "1939", "title": "I. A Plant Growth Inhibitor and Plant Growth Inhibition. II. Extensibility of Cell Wall Material in Indole-3-Acetic Acid", "advisor": "Unknown, Unknown", "url": "https://resolver.caltech.edu/CaltechTHESIS:12182024-183614320", "creators": [ { "name": { "family": "Stewart", "given": "William Sheldon" }, "id": "Stewart-William-Sheldon", "display_name": "Stewart, William Sheldon" } ], "advisors": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/jgkt-9a09", "abstract": "No abstract." }, { "name": "Boche, Robert DeVore", "degree": "PhD", "year": "1938", "title": "Studies on Hymenopteran Parasitism of Drosophila", "advisor": "Unknown, Unknown", "url": "https://resolver.caltech.edu/CaltechTHESIS:04072014-112057299", "creators": [ { "name": { "family": "Boche", "given": "Robert DeVore" }, "id": "Boche-Robert-DeVore", "display_name": "Boche, Robert DeVore" } ], "advisors": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/M75F-ND48", "abstract": "Flies of the genus Drosophila are subject to attack by a\r\nnumber of parasitic forms. Sturtevant (1921) has listed records\r\nof parasitism by protozoa (Leptomonas), fungi (Muiaria and\r\nStigmatomyces), nematodes, mites and v~rious hymenoptera.\r\nAccording to Sturtevant, Perkins (1913) has bred at least\r\nfive species of hymenoptera, belonging to the proctotrupoid,\r\ncynipoid and chalcidoid groups, upon Drosophiline flies.\r\nH.S. Smith has bred an unidentified proctotrupoid and a\r\nchalcidoid, Pachy crepoideus dubius Ashmead* from both\r\nDrosophila melanogaster ani D. hydei. Kieffer ( 1913) has\r\ndescribed three species of hymenoptera from Africa collected \r\nby Silvestri and stated by him to be parasitic on Drosophila,\r\nspecies not given. They are Trichopria (Planopria) rhopalica\r\n(Diapriidae), Ashmeadopria drosophilae (Diapriidae), and the\r\ninsect which forms the subject matter of the present investigation,\r\nEucoila drosophilae (Figitidae).
\r\n\r\nThere are in addition a number of predacious enemies\r\namong wasps, spiders, flies and beetles.
\r\n\r\nThe present account is concerned with parasitism of various\r\nspecies of Drosophila by Eucoila drosophilae Kieff. The wasps\r\nwere found b y Dr. w. P. Spencer who exposed traps in an\r\neffort to collect Drosophila at Long Lake, Ohio, in Sept. 1934 .\r\nDrosophila larvae from the trap gave a large number of pupae\r\nfrom which wasps emerged in considerable proportions. Since\r\nthat time stock s have been maintained in culture on Drosophila\r\nmelanogaster.
" }, { "name": "Cooper, William Cecil", "degree": "PhD", "year": "1938", "title": "Hormones and Root Formation", "advisor": "Went, Frits W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:11262024-225357787", "creators": [ { "name": { "family": "Cooper", "given": "William Cecil" }, "id": "Cooper-William-Cecil", "display_name": "Cooper, William Cecil" } ], "advisors": [ { "name": { "family": "Went", "given": "Frits W." }, "id": "Went-F-W", "role": "advisor", "display_name": "Went, Frits W." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/mvqk-nd12", "abstract": "No abstract." }, { "name": "Ives, Philip Truman", "degree": "PhD", "year": "1938", "title": "Temperature Effects on Scute in Drosophila melanogaster", "advisor": "Sturtevant, Alfred Henry", "url": "https://resolver.caltech.edu/CaltechTHESIS:11262024-211809036", "creators": [ { "name": { "family": "Ives", "given": "Philip Truman" }, "id": "Ives-Philip-Truman", "display_name": "Ives, Philip Truman" } ], "advisors": [ { "name": { "family": "Sturtevant", "given": "Alfred Henry" }, "id": "Sturtevant-A-H", "role": "advisor", "display_name": "Sturtevant, Alfred Henry" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/wn1t-db75", "abstract": "No abstract." }, { "name": "Clark, William Gilbert", "degree": "PhD", "year": "1937", "title": "Bioelectric Properties of Plants and Polar Transport of the Plant Hormone, Auxin", "advisor": "Went, Frits W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:02212024-230307835", "creators": [ { "name": { "family": "Clark", "given": "William Gilbert" }, "id": "Clark-William-Gilbert", "display_name": "Clark, William Gilbert" } ], "advisors": [ { "name": { "family": "Went", "given": "Frits W." }, "id": "Went-F-W", "role": "advisor", "display_name": "Went, Frits W." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/5w63-na35", "abstract": "[Introduction] The polar basal transport of the growth substances (auxins, growth hormones) in plants is a well known phenomenon, demonstrated\r\nfirst by Went (1928), and studied in detail by van der Weij (1932, 1934). These investigators used the Avena (oat) coleoptile. That the phenomenon is more or less general is indicated by the polar transport of auxin in roots ( Cholodny, 1934), (Nagao, 1936); in hypocotyls of Raphanus (van Overbeek,1933), Pisum (Skoog,1936); in leaves (Avery,1935); in the Avena coleoptile (Went, 1928), (Laibach and Kornmann,1933), (van der Weij, 1932,1934), and (Skoog,1936); in corn coleoptiles (van Overbeek,1936); in Elaeagnus ( woody cutting) ( van der VVeij, 1933); in stems of Coleus, Vicia, and Phaseolus; and in hypocotyls of Vicia, Phaseolus, and Lupinus (Mai,1934)." }, { "name": "Michener, Harold David", "degree": "PhD", "year": "1937", "title": "The Relation of Plant Growth Hormones to the Action of Ethylene Upon Plants", "advisor": "Went, Frits W.; Thimann, Kenneth V.; Bonner, James Frederick", "url": "https://resolver.caltech.edu/CaltechTHESIS:01292025-041902678", "creators": [ { "name": { "family": "Michener", "given": "Harold David" }, "id": "Michener-Harold-David", "display_name": "Michener, Harold David" } ], "advisors": [ { "name": { "family": "Went", "given": "Frits W." }, "id": "Went-F-W", "role": "advisor", "display_name": "Went, Frits W." }, { "name": { "family": "Thimann", "given": "Kenneth V." }, "id": "Thimann-Kenneth-V", "role": "advisor", "display_name": "Thimann, Kenneth V." }, { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-J-F", "role": "advisor", "display_name": "Bonner, James Frederick" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/2g89-8b32", "abstract": "The attention of investigators was first brought to this subject by the damage done to street trees by illuminating gas. This was investigated by Girardin (1864) and Virchow (1870). Further studies along this line were ma.de by Stone (1907, 1913) and Wilcox (1911). Among the effects described\r\nwere wilting, yellowing, and. falling of the leaves. and injury to the cambium.
\r\nKny (1871) found variation among different species in their sensitivity to illuminating gas. Spath and Meyer (1873) found gas most damaging to plants which were actively growing. Wiesner (1878)\r\ndid experiments on phototropism with seedlings of Vicia, Pisum, and Phaseolus. As a light source he used a gas light. He noticed that gas effected geotropism, and termed the effect 11undulierende\r\nnutation\". He considered this to be due to the disappearance of geotropism under the influence of ethylene. Molisch (1884) observed that ethylene affects geotropism in roots. He also investigated the toxicity to plants of tobacco smoke, and found that it is not due to nicotine. It was later shown (Knight and Crocker, 1913) to be due to ethylene.
" }, { "name": "Yin, Hongzhang", "degree": "PhD", "year": "1937", "title": "Effect of Auxin on Chlorella vulgaris and Studies on the Movement of Leaves", "advisor": "Went, Frits W.", "url": "https://resolver.caltech.edu/CaltechETD:etd-07202004-142333", "creators": [ { "name": { "family": "Yin", "given": "Hongzhang" }, "id": "Yin-Hongzhang", "display_name": "Yin, Hongzhang" } ], "advisors": [ { "name": { "family": "Went", "given": "Frits W." }, "id": "Went-F-W", "role": "advisor", "display_name": "Went, Frits W." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/5QC4-X325", "abstract": "[Part 1] Chlorella vulgaris of a re-isolated clone was cultured in Knop's solution to which different amounts of pure heteroauxin were added. The results show that auxin promotes the enlargement of the individual cells. Particularly in the young cultures the cell volume is proportional to the logarithm of the auxin concentration. In older cultures the matter is complicated by the shortage of another factor, presumably food. At high concentrations auxin retards cell division. This probably due to a decreased amount of chlorophyll and a decreased photosynthetic activity.
\r\n\r\nIn agar medium heteroauxin in very low concentrations stimulates growth, but at high concentrations inhibits grown of the alga.
\r\n\r\nAuxin does not affect respiration but it markedly affects the rate of photosynthesis. The effect is indirect through the changes in the chlorophyll content and the cell size. In the young cultures the rate of photosynthesis is proportional to the chlorophyll concentration, while in the old cultures it is proportional to both the chlorophyll concentration and the extent of the cell surface.
\r\n\r\n[Part 2] Malva leaves are diaphototropically sensitive, i.e., the leaves orient themselves transversely to the sun's rays. They follow the sun's course during the day and return to their original position at night. No such movement can be observed in diffuse light or in darkness. Light is perceived by the lamina. The movement consists of a curvature of the laminar joint which is capable of reversible contraction and extension. A difference in osmotic pressures on the two sides of the petiole is found to be responsible for the curvature.
\r\n\r\nThe leaves of Carica papaya assume a horizontal position in the day and droop abaxially at night. The movement has a normal periodicity of twenty four hours due to some internal cuase (autonomic).
\r\n\r\nThe mechanism of the movement has been shown to be of the following sequence:
\r\n\r\n(1) There is a differential distribution (or production) of auxin on the apical and basal lobes of the leaf blade.
\r\n\r\n(2) Because of its special vasicular structure the auxin from the apical lobe goes to the lower (abaxial) side of the petiole, while that from the basal lobe goes to the upper (adaxial) side of the petiole.
\r\n\r\n(3) Because of the differential supply of auxin, the two sides of the petiole grow unequally and give rise to nyctinastic curvature.
The progress of genetics has been so rapid in a number of respects that many fields of inquiry have been entirely neglected or left for a future in which fields now so fertile may be exhausted. Much of this progress has been concerned with the relations between genes or groups of genes and the chromosomes bearing them, i.e., with the mechanism of transmission of characters. The problems of what the gene itself does in development and the manner in which it acts have received recent attention (Schultz 1935), but are still completely open.
\r\n\r\nThe relative roles of gene and cytoplasm have long been known. An egg is incapable of further development without a nucleus, although some division is not impossible in the absence of the latter. That at least one set of chromosomes is required for development and two for complete normal development was found in early experiments with Echinoderm eggs. That the character of the embryo is determined by the genes carried in its chromosomes has also long been known. A\r\ncertain amount of information is available concerning what happens when extra chromosomes or sets of chromosomes are added to the normal diploid number - upset genic balance. Likewise the exaggeration effects of heterozygous small deficiencies have been studied. One approach has only recently been made use of: the behavior of lethal factors and of homozygous deficiencies. Lethal types have been studied in some forms, e. g., the mouse and the fowl. But in only one case in the mouse is it very definitely known that the lethal is a deficiency (Snell and others - 1934). Nearly all homozygous deficiencies have been shown to be lethal at earlier or later stages. The most favorable material for such studies exists in Drosophila melanogaster. The behavior of many small deficiencies has been studied there by Demerec (1934) using stocks of flies which give a high frequency of chromosome elimination and thus patches\u00b7 of \u201cdeficient tissue.\u201d No studies have been made on the whole organism homozygous for deficiencies. It was proposed, therefore, to determine what happens to such \u201cdeficient\u201d eggs. This seemed not impossible, as techniques for observing living eggs throughout development had just been introduced by Huettner and his students. Such studies proved not too difficult in cases where normal development ceased very early, but in cases where development became abnormal in the later stages, the situation was more complex. This was principally because the embryology of Drosophila beyond the time of inclusion of the pole cells was unknown. In the developmental studies which had been made, especially those by Sturtevant (1929) on gynandromorphs, the processes had been assumed to be similar to those of the related Muscids. Although the validity of this assumption has since been shown, it was essential that a careful study of embryonic development be made not only as a standard of comparison for deficient types, but also to give detailed information concerning the origin of the various larval organs and the anlagen of the adult, the imaginal discs.
\r\n\r\nThe results of these investigations are embodied in the present work, the first section of which consists of a survey of the literature on insect embryology, particularly that of the Diptera. This is essential as a background for the account of Drosophila embryology to follow. Later sections deal with the literature on deficiencies and the behavior of a number of the deficiencies studied.
" }, { "name": "Skoog, Folke Karl", "degree": "PhD", "year": "1936", "title": "Some Physiological Functions of the Growth Hormone in Higher Plants", "advisor": "Went, Frits W.", "url": "https://resolver.caltech.edu/CaltechTHESIS:05172018-102043322", "creators": [ { "name": { "family": "Skoog", "given": "Folke Karl" }, "id": "Skoog-Folke-Karl", "display_name": "Skoog, Folke Karl" } ], "advisors": [ { "name": { "family": "Went", "given": "Frits W." }, "id": "Went-F-W", "role": "advisor", "display_name": "Went, Frits W." } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/PBH5-Q260", "abstract": "The results of the experiments are included in the conclusions at the end of different chapters and in the last chapter of each Part. It is therefore only necessary to summarize the main results.
\r\n\r\nPart I.
\r\n\r\n1. The inhibition of lateral bud development in Vicia and in Pisum is controlled by the growth hormone produced by the terminal bud.
\r\n\r\n2. The inhibiting effect produced by the terminal bud can be completely substituted by a continuous application of auxin in relatively high concentrations to the stem of decapitated plants.
\r\n\r\n3. The inhibiting action of auxin is due to its prevention of synthesis of hormone in the buds and is independent of the action of the hormone in promoting growth of the stem.
\r\n\r\n4. In Vicia older than seedlings auxin is produced only in the presence of light, but the growth response of the plant to auxin is higher in the dark.
\r\n\r\n5. Auxin is an essential factor for the growth of the stem.
\r\n\r\nPart II.
\r\n\r\n1. Auxin is destroyed by x-irradiation both in solution and in the plant.
\r\n\r\n2. Inactivation of auxin in solution is indirectly through the oxidation by strong oxidizing agents formed by irradiation.
\r\n\r\n3. In the Avena coleoptile X-rays cause only a temporary decrease in auxin.
\r\n\r\n4. In green plants (Vicia and Pisum) grown in the light the mechanism of formation of auxin is additionally destroyed so that a gradual permanent decrease in auxin is produced by irradiation.
\r\n\r\n5. The destruction of auxin and the mechanism of its formation in the plant is a major factor in the immediate inhibition of growth of plants caused by irradiation.
\r\n\r\n6. The development of lateral buds in irradiated plants is due to the removal of auxin and is not due to a direct stimulation of growth by the X-rays.
\r\n\r\nPart III.
\r\n\r\n1. A quantitative \"deseeded\" Avena test method for small amounts of auxin and precursors of auxin has been described.
\r\n\r\n2. The presence in the Avena coleoptile of a precursor of auxin transported from the seed has been demonstrated.
\r\n\r\n3. Tryptophane and \u03b2-indol-ethyl amine, which lack auxin activity, have been shown to be changed chemically by the action of the plant into hetero auxin.
\r\n\r\n4. The physiological behavior of these substances in distinction to auxin has been studied.
\r\n\r\n\r\n" }, { "name": "Tan, Jiazhen", "degree": "PhD", "year": "1936", "title": "Genetic and Cytological Maps of the Autosomes in Drosophila pseudoobscura", "advisor": "Dobzhansky, Theodosius Grigorievich", "url": "https://resolver.caltech.edu/CaltechETD:etd-06302004-101131", "creators": [ { "name": { "family": "Tan", "given": "Jiazhen" }, "id": "Tan-Jiazhen", "display_name": "Tan, Jiazhen" } ], "advisors": [ { "name": { "family": "Dobzhansky", "given": "Theodosius Grigorievich" }, "id": "Dobzhansky-Th", "role": "advisor", "display_name": "Dobzhansky, Theodosius Grigorievich" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/72KD-6D58", "abstract": "No Abstract." }, { "name": "Bonner, James Frederick", "degree": "PhD", "year": "1934", "title": "Growth Substance and Cell Elongation", "advisor": "Unknown, Unknown", "url": "https://resolver.caltech.edu/CaltechETD:etd-11252003-083628", "creators": [ { "name": { "family": "Bonner", "given": "James Frederick" }, "id": "Bonner-James-Frederick", "display_name": "Bonner, James Frederick" } ], "advisors": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/86ND-SX71", "abstract": "No abstract." }, { "name": "Ellis, Emory Leon", "degree": "PhD", "year": "1934", "title": "The Free Energy of the Sulfhydryl-Disulfide Oxidation-Reduction System and its Physiological Significance", "advisor": "Unknown, Unknown", "url": "https://resolver.caltech.edu/CaltechTHESIS:03132024-205158064", "creators": [ { "name": { "family": "Ellis", "given": "Emory Leon" }, "id": "Ellis-Emory-Leon", "display_name": "Ellis, Emory Leon" } ], "advisors": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/b5h3-tw60", "abstract": "[Summary]
\r\nComplete thermodynamic data are presented concerning two\r\norganic sulfhydryl-disulfide oxidation-reduction systems (cysteine:\r\ncystine and \u03b2-thiolactic acid: \u03b2\u03b2'-dithiodilactic acid). These data\r\ninclude heat capacity measurements on the crystalline substances from\r\n90\u00b0 to 298\u00b0 K, heat of combustion determinations, solubility measurements\r\nand determinations of the ionization constants of dissociable\r\ngroups in the molecules.
\r\n\r\nThe method of calculating from these data the reduction\r\npotential for any conditions of concentration, pH, and temperature\r\nis discussed. The results of such calculations for several sets\r\nof conditions are submitted.
\r\n\r\nThe relationship of the sulfhydryl-disulfide system to the\r\nprocess of cell division is discussed and experimental evidence is\r\nsubmitted which shows that sulfhydryl substances have no direct\r\nconnection with the process of cell division.
\r\n\r\nThe role of oxidation-reduction systems in intracellular\r\nmetabolism is discussed. From the available experimental data relating\r\nto the function of -SH substances in living tissue, an hypothesis\r\nof the general function of these substances is developed\r\nand discussed.
" }, { "name": "Sargent, Marston Cleaves", "degree": "PhD", "year": "1934", "title": "Aspects of the Physiology of the Blue-Green Algae", "advisor": "Unknown, Unknown", "url": "https://resolver.caltech.edu/CaltechTHESIS:05102018-090737965", "creators": [ { "name": { "family": "Sargent", "given": "Marston Cleaves" }, "id": "Sargent-Marston-Cleaves", "display_name": "Sargent, Marston Cleaves" } ], "advisors": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/8pp8-k146", "abstract": "Summary:Two coccus-form species of blue-green algae have\r\nbeen isolated in species-pure culture and cultivated on\r\na number of solid and liquid media. For one species, a\r\nmedium of strictly-known composition has been developed.
\r\n\r\nThe principal characteristics of the process of\r\nassimilation in these algae have been studied by the\r\nmanometric method. The process is essentially like that\r\nin other green plants. The shape of the curve relating\r\nassimilation to light intensity gives an indication that\r\nthe blue-pigment of the Cyanophyceae does not take part \r\nin photosynthesis.
\r\n\r\nA study of the relation of the color of a blue-green\r\nalga to environmental conditions shows that the color\r\ndepends principally on the chemical constitution of the\r\nmedium and the intensity of incident light. Temperature\r\nand light color may have minor effects.
" }, { "name": "Schott, Hermann Franz", "degree": "PhD", "year": "1933", "title": "Reversibility in Biological Oxidations", "advisor": "Unknown, Unknown", "url": "https://resolver.caltech.edu/CaltechTHESIS:01312017-154929894", "creators": [ { "name": { "family": "Schott", "given": "Hermann Franz" }, "id": "Schott-Hermann-Franz", "display_name": "Schott, Hermann Franz" } ], "advisors": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/XKVV-TR72", "abstract": "I. The molal electrode potential for the succinate-enzyme-\r\nfumarate equilibrium was determined for a series of\r\nvariations of all the constituents, at 25\u00b0.
\r\n\r\nThe free energy and the heat of the reaction, determined\r\nelectrometrically, are compared to the values calculated\r\nfrom known physico-chemical properties of succinic and fumaric\r\nacids. The values agree within the limits of experimental\r\nerror. This, together with the lack of dependence of the\r\nfree energy on the source of the enzyme, is taken to indicate\r\nthat the enzyme behaves as a perfect catalyst.
\r\n\r\nII. From the equilibrium constant for the reaction of\r\nfumarate and water to form 1-malate in the presence of the\r\nenzyme fumarase, the free energy of the reaction was calculated.\r\nFrom this value and the known physico-chemical properties of\r\nfumaric acid, the free energy of the formation of 1-malic acid\r\nwas estimated.
\r\n\r\nIII. It was demonstrated that in the presence of toluene-treated\r\nB. coli, lactate is oxidized to pyruvate, and that\r\nfurthermore, pyruvate may also be reduced to lactate.
\r\n\r\nIV. It was shown that in the presence of toluene-treated\r\nB. coli electron or hydrogen transfer from one\r\nmetabolite to another occurs only thru the mediation of a\r\nreversible oxidizing agent.
\r\n\r\nV. The implications of these findings for the theory\r\nof biological oxidations are discussed.
" }, { "name": "Biddle, Russell Lee", "degree": "PhD", "year": "1931", "title": "The Number of Bristles and the Pairing of the Chromosomes in Hybrids Between Drosophila melanogaster and Drosophila simulans", "advisor": "Unknown, Unknown", "url": "https://resolver.caltech.edu/CaltechTHESIS:08262024-202647332", "creators": [ { "name": { "family": "Biddle", "given": "Russell Lee" }, "id": "Biddle-Russell-Lee", "display_name": "Biddle, Russell Lee" } ], "advisors": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/zeg9-v823", "abstract": "No abstract." }, { "name": "Lindegren, Carl Clarence", "degree": "PhD", "year": "1931", "title": "Genetic Study of Sex and Cultural Characters in Neurospora crassa", "advisor": "Morgan, Thomas Hunt", "url": "https://resolver.caltech.edu/CaltechETD:etd-12202005-084117", "creators": [ { "name": { "family": "Lindegren", "given": "Carl Clarence" }, "id": "Lindegren-Carl-Clarence", "display_name": "Lindegren, Carl Clarence" } ], "advisors": [ { "name": { "family": "Morgan", "given": "Thomas Hunt" }, "id": "Morgan-Thomas Hunt", "role": "advisor", "display_name": "Morgan, Thomas Hunt" } ], "option_major": [ "biology" ], "doi": "10.7907/HR2E-X838", "abstract": "No abstract." }, { "name": "Winegarden, Howard Merlin", "degree": "PhD", "year": "1931", "title": "An Application of Thermodynamics to Physiology. 1. On the Free Energy of Glucose and of Tripalmitin. 2. The Work of the Kidney in the Production of Urine. 3. The Energy Cost of the Excretion of Urine. 4. On the Specific Dynamic Action of Protein", "advisor": "Borsook, Henry", "url": "https://resolver.caltech.edu/CaltechTHESIS:09112024-211500747", "creators": [ { "name": { "family": "Winegarden", "given": "Howard Merlin" }, "id": "Winegarden-Howard-Merlin", "display_name": "Winegarden, Howard Merlin" } ], "advisors": [ { "name": { "family": "Borsook", "given": "Henry" }, "id": "Borsook-H", "role": "advisor", "display_name": "Borsook, Henry" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "bioch" ], "doi": "10.7907/417b-z477", "abstract": "No abstract." }, { "name": "Titlebaum (Tyler), Albert", "degree": "PhD", "year": "1929", "title": "Experimental Production of Double Embryos", "advisor": "Unknown, Unknown", "url": "https://resolver.caltech.edu/CaltechETD:etd-09112007-080349", "creators": [ { "name": { "family": "Titlebaum (Tyler)", "given": "Albert" }, "id": "Titlebaum-(Tyler)-Albert", "display_name": "Titlebaum (Tyler), Albert" } ], "advisors": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "committee": [ { "name": { "family": "Unknown", "given": "Unknown" }, "display_name": "Unknown, Unknown" } ], "option_major": [ "biology" ], "doi": "10.7907/JGJW-D344", "abstract": "[Introduction] The egg of the polychaet, Chaetopterus, has never been observed normally to produce double-embryos. It has been found, however, by experimental treatment, such as pressure and low temperature, that double-embryos can be produced.\r\n\r\n" } ]