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.
", "doi": "10.7907/1ksn-sb30", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:17771", "collection": "thesis", "collection_id": "17771", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11242025-191228061", "type": "thesis", "title": "Naturally-Inspired Circuits for Microbial Composition Control and Biosensing", "author": [ { "family_name": "Kratz", "given_name": "Matthieu Francois", "clpid": "Kratz-Matthieu-Francois" } ], "thesis_advisor": [ { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "thesis_committee": [ { "family_name": "Demirer", "given_name": "G\u00f6zde S.", "orcid": "0000-0002-3007-1489", "clpid": "Demirer-G\u00f6zde-S" }, { "family_name": "Hay", "given_name": "Bruce A.", "orcid": "0000-0002-5486-0482", "clpid": "Hay-B-A" }, { "family_name": "Bois", "given_name": "Justin S.", "orcid": "0000-0001-7137-8746", "clpid": "Bois-J-S" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "local_group": [ { "literal": "div_bbe" } ], "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", "doi": "10.7907/d42b-jh46", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:17435", "collection": "thesis", "collection_id": "17435", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06092025-211524859", "primary_object_url": { "basename": "2025-Andrew-Lu-Caltech-PhD-Thesis.pdf", "content": "final", "filesize": 9969754, "license": "other", "mime_type": "application/pdf", "url": "/17435/21/2025-Andrew-Lu-Caltech-PhD-Thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Engineered Protein Circuits for Cancer Therapy", "author": [ { "family_name": "Lu", "given_name": "Andrew C.", "orcid": "0000-0002-7594-6445", "clpid": "Lu-Andrew-C" } ], "thesis_advisor": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Voorhees", "given_name": "Rebecca M.", "orcid": "0000-0003-1640-2293", "clpid": "Voorhees-R-M" }, { "family_name": "Dawson", "given_name": "David", "orcid": "0000-0002-0215-5861", "clpid": "Dawson, David" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "local_group": [ { "literal": "div_bbe" } ], "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.", "doi": "10.7907/kxvr-r291", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:16911", "collection": "thesis", "collection_id": "16911", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12092024-181948039", "primary_object_url": { "basename": "DDH_2024_Thesis.pdf", "content": "final", "filesize": 5078904, "license": "other", "mime_type": "application/pdf", "url": "/16911/1/DDH_2024_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Development and Application of Proteomic and Genomic Methods in RNA Biology", "author": [ { "family_name": "Honson", "given_name": "Drew Daniel", "orcid": "0000-0002-4654-8974", "clpid": "Honson-Drew-Daniel" } ], "thesis_advisor": [ { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Fejes Toth", "given_name": "Katalin", "orcid": "0000-0001-6558-2636", "clpid": "Fejes-Toth-K" }, { "family_name": "Zernicka-Goetz", "given_name": "Magdalena", "orcid": "0000-0002-7004-2471", "clpid": "Zernicka-Goetz-M" }, { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" } ], "local_group": [ { "literal": "div_bbe" } ], "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.", "doi": "10.7907/08dq-1q63", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17354", "collection": "thesis", "collection_id": "17354", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022025-044636280", "primary_object_url": { "basename": "Caltech-Thesis-Shirin-Shivaei-final.pdf", "content": "final", "filesize": 19237378, "license": "other", "mime_type": "application/pdf", "url": "/17354/6/Caltech-Thesis-Shirin-Shivaei-final.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "A Viral Toolkit for Ultrasound Imaging of Cellular Activity and Gene Expression", "author": [ { "family_name": "Shivaei", "given_name": "Shirin", "orcid": "0000-0002-6894-3289", "clpid": "Shivaei-Shirin" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Oka", "given_name": "Yuki", "orcid": "0000-0003-2686-0677", "clpid": "Oka-Yuki" }, { "family_name": "Tanter", "given_name": "Mickael", "orcid": "0000-0001-7739-8051", "clpid": "Tanter-Mickael" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/r9gz-1482", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17219", "collection": "thesis", "collection_id": "17219", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05112025-035044867", "type": "thesis", "title": "Bridging Space and Time: Resolving the Temporal Dynamics of the Seminiferous Epithelial Cycle Using Spatial Transcriptomics", "author": [ { "family_name": "Chakravorty", "given_name": "Arun", "orcid": "0000-0003-2890-0855", "clpid": "Chakravorty-Arun" } ], "thesis_advisor": [ { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "thesis_committee": [ { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "local_group": [ { "literal": "div_bbe" } ], "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.", "doi": "10.7907/2rcd-0v79", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:16147", "collection": "thesis", "collection_id": "16147", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07272023-175309910", "primary_object_url": { "basename": "Goronzy_CalTechThesis_Final2.pdf", "content": "final", "filesize": 5387002, "license": "other", "mime_type": "application/pdf", "url": "/16147/2/Goronzy_CalTechThesis_Final2.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Higher-Order Chromatin States and Nuclear Structures Regulating Gene Expression", "author": [ { "family_name": "Goronzy", "given_name": "Isabel Nadine", "orcid": "0000-0002-6713-9192", "clpid": "Goronzy-Isabel-Nadine" } ], "thesis_advisor": [ { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" }, { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Pachter", "given_name": "Lior S.", "orcid": "0000-0002-9164-6231", "clpid": "Pachter-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Although the same genome is present in every cell, each cell type orchestrates a distinct gene expression program, which can be rapidly adapted in response to stimuli. Accordingly, gene regulation is a highly complex, context-specific process that involves the dynamic interplay between numerous regulatory factors. Most methods to study these regulatory factors only measure pairwise interactions between molecules and are limited to mapping one regulatory protein at a time. Consequently, the combinatorial complexity of gene regulation at individual genomic loci and the functional consequence of many regulatory factors remain underexplored. To address this, we have developed new sequencing-based approaches and computational analyses to comprehensively profile, at unprecedented scale, the diverse gene regulatory landscape and directly establish the link between regulatory factors and transcriptional outcomes. In Chapter 2, we present Chromatin Immunoprecipitation Done-In-Parallel (ChIP-DIP), a highly multiplexed method for mapping hundreds of proteins to DNA within a single sample. ChIP-DIP increases the throughput of existing methods by > 100-fold and enables the production of consortium-scale, cell type-specific data within a single lab. Capitalizing on the scale and diversity provided by ChIP-DIP, we uncover unique quantitative combinations of histone modifications that define distinctive classes of regulatory elements. Specifically, we find features distinguishing classes of promoters that correspond to different polymerase activity, transcriptional levels, and gene types and find acetylation patterns distinguishing classes of enhancers that exhibit distinct activity states, induction potential, and regulatory potential. Next, in Chapter 3, we apply RNA-DNA SPRITE (RD-SPRITE), a method for simultaneous measurement of RNA and DNA organization, to investigate the functional relationship between genome structure and transcription. We demonstrate that RD-SPRITE precisely detects individual, nascent pre-mRNAs at their transcriptional locus and, as a result, can be used to assess the 3D genome structure present during active transcription. We find that RNA polymerase II transcription occurs within genomic structures previously thought to be inactive, such as the B compartment and DNA regions near the nucleolus. This suggests that active transcription can occur throughout the nucleus and argues against structural domains that preclude transcription. Overall, our findings highlight the ability of RD-SPRITE to establish a structure-function link. Finally, in Chapter 4, we apply RD-SPRITE to study the transcriptional dependence of nuclear organization. We demonstrate that transcriptional inhibition leads to the loss of high-order structure around multiple RNA-processing bodies \u2014 the nucleolus, the scaRNA hub and the histone locus body \u2014 that are responsible for essential nuclear functions such as RNA processing and gene regulation. These findings suggest a role for RNA and nascent transcription in the formation and maintenance of long-range 3D contacts and critical nuclear compartments. In summary, we have developed new approaches to explore epigenomic and organizational complexity within the mammalian nucleus and have uncovered genome-wide principles of gene regulation.
", "doi": "10.7907/8gm2-jn84", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16454", "collection": "thesis", "collection_id": "16454", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05312024-185303498", "type": "thesis", "title": "Customized and Modular Control of Gene Expression for Precision Gene Therapies", "author": [ { "family_name": "Mayfield", "given_name": "Acacia Michelle Hori", "orcid": "0000-0001-7308-6480", "clpid": "Mayfield-Acacia-Michelle-Hori" } ], "thesis_advisor": [ { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "thesis_committee": [ { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "local_group": [ { "literal": "div_bbe" } ], "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.", "doi": "10.7907/d05v-0t68", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16473", "collection": "thesis", "collection_id": "16473", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06032024-011201674", "primary_object_url": { "basename": "Mengtong Duan 2024 Jun 14.pdf", "content": "final", "filesize": 35875270, "license": "other", "mime_type": "application/pdf", "url": "/16473/1/Mengtong Duan 2024 Jun 14.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Expanding Frontiers in Biomedical Imaging and Synthetic Biology: Dynamic Acoustic Reporter Gene Imaging and Ratio-Tuning of Mammalian mRNA Polycistronic Expression", "author": [ { "family_name": "Duan", "given_name": "Mengtong (Tom)", "orcid": "0000-0002-1601-8876", "clpid": "Duan-Mengtong-Tom" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-Shu-ou" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/avgk-yc71", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16486", "collection": "thesis", "collection_id": "16486", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06032024-182223499", "primary_object_url": { "basename": "Thesis_Draft_final_final.pdf", "content": "final", "filesize": 21944874, "license": "other", "mime_type": "application/pdf", "url": "/16486/1/Thesis_Draft_final_final.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Revealing Regulatory Network Organization Through Single-Cell Perturbation Profiling and Maximum Entropy Models", "author": [ { "family_name": "Jiang", "given_name": "Jialong", "orcid": "0000-0001-8560-8397", "clpid": "Jiang-Jialong" } ], "thesis_advisor": [ { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Pachter", "given_name": "Lior S.", "orcid": "0000-0002-9164-6231", "clpid": "Pachter-L" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" } ], "local_group": [ { "literal": "div_bbe" } ], "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.", "doi": "10.7907/5zta-9818", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16503", "collection": "thesis", "collection_id": "16503", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06042024-163846436", "type": "thesis", "title": "Implementing and Modeling Gene Drives for Population Modification and Suppression", "author": [ { "family_name": "Ivy", "given_name": "Tobin William", "orcid": "0000-0002-9116-3854", "clpid": "Ivy-Tobin-William" } ], "thesis_advisor": [ { "family_name": "Hay", "given_name": "Bruce A.", "orcid": "0000-0002-5486-0482", "clpid": "Hay-B-A" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Bois", "given_name": "Justin S.", "orcid": "0000-0001-7137-8746", "clpid": "Bois-J-S" }, { "family_name": "Hay", "given_name": "Bruce A.", "orcid": "0000-0002-5486-0482", "clpid": "Hay-B-A" } ], "local_group": [ { "literal": "div_bbe" } ], "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.", "doi": "10.7907/10pa-x574", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16459", "collection": "thesis", "collection_id": "16459", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06012024-054725051", "primary_object_url": { "basename": "240531_PB_thesis_final.pdf", "content": "final", "filesize": 44817586, "license": "other", "mime_type": "application/pdf", "url": "/16459/1/240531_PB_thesis_final.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Modeling and Design of Synthetic Biochemical Circuits for Biological Phenotypes", "author": [ { "family_name": "Bhamidipati", "given_name": "Pranav Subramanyam", "orcid": "0000-0002-6199-6505", "clpid": "Bhamidipati-Pranav-Subramanyam" } ], "thesis_advisor": [ { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Bois", "given_name": "Justin S.", "orcid": "0000-0001-7137-8746", "clpid": "Bois-J-S" }, { "family_name": "Barr", "given_name": "Alan H.", "clpid": "Barr-A-H" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/gpc6-hb40", "publication_date": "2024-06-14", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:15225", "collection": "thesis", "collection_id": "15225", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292023-181810775", "primary_object_url": { "basename": "Marken_Thesis_Final.pdf", "content": "final", "filesize": 4502877, "license": "other", "mime_type": "application/pdf", "url": "/15225/1/Marken_Thesis_Final.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Experimental and Theoretical Frameworks for Enabling Environmental Synthetic Biology", "author": [ { "family_name": "Marken", "given_name": "John Paul", "orcid": "0000-0001-9696-088X", "clpid": "Marken-John-Paul" } ], "thesis_advisor": [ { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Hay", "given_name": "Bruce A.", "orcid": "0000-0002-5486-0482", "clpid": "Hay-B-A" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/h50w-p058", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15229", "collection": "thesis", "collection_id": "15229", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05302023-040856946", "primary_object_url": { "basename": "P.Bhat.MergedThesis.Final.pdf", "content": "final", "filesize": 18273524, "license": "other", "mime_type": "application/pdf", "url": "/15229/1/P.Bhat.MergedThesis.Final.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "On the Role of Three-Dimensional Genome Organization in Gene Regulation and mRNA Splicing", "author": [ { "family_name": "Bhat", "given_name": "Prashant", "orcid": "0000-0003-3832-4871", "clpid": "Bhat-Prashant" } ], "thesis_advisor": [ { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" }, { "family_name": "Chang", "given_name": "Howard Y.", "orcid": "0000-0002-9459-4393", "clpid": "Chang-Howard-Y" }, { "family_name": "Black", "given_name": "Douglas L.", "orcid": "0000-0002-2705-8187", "clpid": "Black-Douglas-L" } ], "local_group": [ { "literal": "div_bbe" } ], "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.", "doi": "10.7907/vg8a-y851", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15086", "collection": "thesis", "collection_id": "15086", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01172023-195222304", "type": "thesis", "title": "Applications of Dynamic Nucleic Acid Nanotechnology in Closed-Loop Genetic Circuits and Detection of Viral Pathogens", "author": [ { "family_name": "Huang", "given_name": "Jining", "orcid": "0000-0002-3798-4790", "clpid": "Huang-Jining" } ], "thesis_advisor": [ { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" } ], "thesis_committee": [ { "family_name": "Winfree", "given_name": "Erik", "orcid": "0000-0002-5899-7523", "clpid": "Winfree-E" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/54tw-ym95", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:16061", "collection": "thesis", "collection_id": "16061", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022023-192547723", "primary_object_url": { "basename": "ShengThesis_CaltechTHESIS.pdf", "content": "final", "filesize": 8747307, "license": "other", "mime_type": "application/pdf", "url": "/16061/1/ShengThesis_CaltechTHESIS.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Synthetic Circuits for Multicellular Spatial Patterning", "author": [ { "family_name": "Wang", "given_name": "Sheng", "orcid": "0000-0002-4070-7313", "clpid": "Wang-Sheng" } ], "thesis_advisor": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "thesis_committee": [ { "family_name": "Goentoro", "given_name": "Lea A.", "orcid": "0000-0002-3904-0195", "clpid": "Goentoro-L-A" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Meister", "given_name": "Markus", "orcid": "0000-0003-2136-6506", "clpid": "Meister-M" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/3rbk-g805", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15235", "collection": "thesis", "collection_id": "15235", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05302023-215054202", "type": "thesis", "title": "Diversity in Notch Ligand-Receptor Signaling Interactions", "author": [ { "family_name": "Kuintzle", "given_name": "Rachael Christine", "orcid": "0000-0002-1035-4983", "clpid": "Kuintzle-Rachael-Christine" } ], "thesis_advisor": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "thesis_committee": [ { "family_name": "Pachter", "given_name": "Lior S.", "orcid": "0000-0002-9164-6231", "clpid": "Pachter-L" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Bronner", "given_name": "Marianne E.", "orcid": "0000-0003-4274-1862", "clpid": "Bronner-M-E" }, { "family_name": "Hay", "given_name": "Bruce A.", "orcid": "0000-0002-5486-0482", "clpid": "Hay-B-A" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The ability to understand and predict signaling between different cell types is a major challenge in biology. The Notch pathway enables direct signaling through membrane-bound ligands and receptors, and is used in diverse contexts. While its canonical molecular signaling mechanism is well characterized, its many-to-many interacting pathway components, the complexity of their expression patterns, and the presence of same-cell (cis) as well as inter-cellular (trans) receptor-ligand interactions, have made it difficult to predict how a given cell will signal to others. Here, we use a cell-based approach, with Chinese hamster ovary (CHO-K1) cells and C2C12 mouse myoblasts, to systematically characterize trans-activation, cis-inhibition, and cis-activation efficiencies for the essential receptors (Notch1 and Notch2) and activating ligands (Dll1, Dll4, Jag1, and Jag2), in the presence of Lunatic Fringe (Lfng) or the enzymatically dead Lfng D289E mutant. All ligands trans-activate Notch1 and Notch2, except for Jag1, which competitively inhibits Notch1 signaling, and whose Notch1 binding strength is potentiated by Lfng. For Notch1, cis-activation is generally weaker than trans-activation, but for Notch2, cis-activation by Delta ligands is much stronger than trans-activation, and Notch2 cis-activation by Jag1 is similar in strength to trans-activation. Cis-inhibition is associated with weak cis-activation, as Dll1 and Dll4 do not cis-inhibit Notch2. Lfng expression potentiates trans-activation of both Notch1 and Notch2 by the Delta ligands and weakens trans-activation of both receptors by the Jagged ligands. The map of receptor-ligand-Fringe interaction outcomes revealed here should help guide rational perturbation and control of the Notch pathway.", "doi": "10.7907/w8gj-jb92", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15132", "collection": "thesis", "collection_id": "15132", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04132023-015900885", "primary_object_url": { "basename": "Ma_Yitong_2023.pdf", "content": "final", "filesize": 17632710, "license": "other", "mime_type": "application/pdf", "url": "/15132/1/Ma_Yitong_2023.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Multicellular Synthetic Biology in Mammalian Systems", "author": [ { "family_name": "Ma", "given_name": "Yitong", "orcid": "0000-0003-4446-7326", "clpid": "Ma-Yitong" } ], "thesis_advisor": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "thesis_committee": [ { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/w0q1-7s17", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:14986", "collection": "thesis", "collection_id": "14986", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07252022-061122576", "primary_object_url": { "basename": "Thesis Ronghui Zhu.pdf", "content": "final", "filesize": 31903908, "license": "other", "mime_type": "application/pdf", "url": "/14986/1/Thesis Ronghui Zhu.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Multicellular Circuit Design in Mammalian Cells", "author": [ { "family_name": "Zhu", "given_name": "Ronghui", "orcid": "0000-0001-8171-482X", "clpid": "Zhu-Ronghui" } ], "thesis_advisor": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "thesis_committee": [ { "family_name": "Hay", "given_name": "Bruce A.", "orcid": "0000-0002-5486-0482", "clpid": "Hay-B-A" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/p0fn-qa56", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:14455", "collection": "thesis", "collection_id": "14455", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12212021-193826426", "primary_object_url": { "basename": "Shur-Andrey-2021-thesis-final.pdf", "content": "final", "filesize": 4080541, "license": "other", "mime_type": "application/pdf", "url": "/14455/3/Shur-Andrey-2021-thesis-final.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Serine Integrase-Based Event Recording in E. coli", "author": [ { "family_name": "Shur", "given_name": "Andrey Sergeyvich", "orcid": "0000-0001-9372-6713", "clpid": "Shur-Andrey-Sergeyvich" } ], "thesis_advisor": [ { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/x4q0-nx18", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14399", "collection": "thesis", "collection_id": "14399", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10172021-215439860", "primary_object_url": { "basename": "Dobreva_Tatyana_2021_v7.pdf", "content": "final", "filesize": 10892340, "license": "other", "mime_type": "application/pdf", "url": "/14399/1/Dobreva_Tatyana_2021_v7.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Engineering Tools to Probe and Manipulate the Immune System at Single-Cell Resolution", "author": [ { "family_name": "Dobreva", "given_name": "Tatyana", "orcid": "0000-0002-2625-8873", "clpid": "Dobreva-Tatyana" } ], "thesis_advisor": [ { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "thesis_committee": [ { "family_name": "Gao", "given_name": "Wei", "orcid": "0000-0002-8503-4562", "clpid": "Gao-Wei" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "My thesis focuses on developing experimental and computational tools to probe and manipulate cellular transcriptomes in the context of human health and disease. Chapter 1 and 2 focus on published work where we leverage single-cell RNA sequencing (scRNA-seq) to understand human immune variability, characterize cell-type specific biases of multiple viral variants within an animal, and assess temporal immune response in the brain to delivery of genetic cargo via an adeno-associated virus (AAV). Chapter 3 and 4 present progress I have made on tools for exporting RNA extracellularly and engineering of a transcription factor for modulating macrophage state.
\r\n\r\nFor probing cellular transcriptome states, we have developed a platform using multiplexed single-cell sequencing and out-of-clinic capillary blood extraction to understand temporal and inter-individual variability of gene expression within immune cell types. Our platform enables simplified, cost-effective profiling of the human immune system across subjects and time at single-cell resolution. To demonstrate the power of our platform, we performed a three day time-of-day study of four healthy individuals, generating gene expression data for 24,087 cells across 22 samples. We detected genes with cell type-specific time-of-day expression and identified robust genes and pathways particular to each individual, all of which could have been missed if analyzed with bulk RNA-sequencing. Also, using scRNA-seq, we have developed a method to screen and characterize cellular tropism of multiple AAV variants. Additionally, I have looked at AAV-mediated transcriptomic changes in animals injected with AAV-PHP.eB three days and twenty-five days post-injection. I have found that there is an upregulation of genes involved in p53 signaling in endothelial cells three days post-injection.
\r\n\r\nIn the context of manipulating cellular transcriptomic states, I demonstrate that a fusion between RNA targeting enzyme, dCas13, and capsid-forming neuronal protein, Arc, is able to form a capsid-like structure capable of encapsulating RNA. I also present methods and preliminary data for tuning macrophage states through mutations in transcription factor EB (TFEB) using scRNA-seq as a readout.
", "doi": "10.7907/n3rs-ft69", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14465", "collection": "thesis", "collection_id": "14465", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01032022-225455509", "primary_object_url": { "basename": "Mengsha_thesis_final.pdf", "content": "final", "filesize": 36868015, "license": "other", "mime_type": "application/pdf", "url": "/14465/18/Mengsha_thesis_final.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Remodeling Jellyfish", "author": [ { "family_name": "Gong", "given_name": "Mengsha", "orcid": "0000-0003-3940-9869", "clpid": "Gong-Mengsha" } ], "thesis_advisor": [ { "family_name": "Goentoro", "given_name": "Lea A.", "orcid": "0000-0002-3904-0195", "clpid": "Goentoro-L-A" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Dickinson", "given_name": "Michael H.", "orcid": "0000-0002-8587-9936", "clpid": "Dickinson-M-H" }, { "family_name": "Goentoro", "given_name": "Lea A.", "orcid": "0000-0002-3904-0195", "clpid": "Goentoro-L-A" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/pfn8-aw15", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14434", "collection": "thesis", "collection_id": "14434", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11272021-193744399", "primary_object_url": { "basename": "PhD_Thesis_KeHuan_Edmonds.pdf", "content": "final", "filesize": 11827016, "license": "other", "mime_type": "application/pdf", "url": "/14434/1/PhD_Thesis_KeHuan_Edmonds.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Imaging Cell Lineage with a Synthetic Digital Recording System", "author": [ { "family_name": "Edmonds", "given_name": "KeHuan Kuo", "orcid": "0000-0002-7317-2669", "clpid": "Edmonds-KeHuan-Kuo" } ], "thesis_advisor": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "thesis_committee": [ { "family_name": "Hay", "given_name": "Bruce A.", "orcid": "0000-0002-5486-0482", "clpid": "Hay-B-A" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Lois", "given_name": "Carlos", "orcid": "0000-0002-7305-2317", "clpid": "Lois-Carlos" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "local_group": [ { "literal": "div_bbe" } ], "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.", "doi": "10.7907/a4m3-m603", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14497", "collection": "thesis", "collection_id": "14497", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02152022-081613451", "primary_object_url": { "basename": "Meirelles_LucasAndrade_2022_thesis.pdf", "content": "final", "filesize": 24486462, "license": "other", "mime_type": "application/pdf", "url": "/14497/1/Meirelles_LucasAndrade_2022_thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "The Nuanced Effects of Redox-Active Metabolites on Bacterial Physiology and Antibiotic Susceptibility", "author": [ { "family_name": "Andrade Meirelles", "given_name": "Lucas", "orcid": "0000-0003-3194-7136", "clpid": "Andrade-Meirelles-Lucas" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Bronner", "given_name": "Marianne E.", "orcid": "0000-0003-4274-1862", "clpid": "Bronner-M-E" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/67p2-q992", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14934", "collection": "thesis", "collection_id": "14934", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022022-032024376", "primary_object_url": { "basename": "Thesis_ChristinaSu_2022.pdf", "content": "final", "filesize": 5427101, "license": "other", "mime_type": "application/pdf", "url": "/14934/1/Thesis_ChristinaSu_2022.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Principles of Addressing Specificity in Promiscuous Ligand-Receptor Systems", "author": [ { "family_name": "Su", "given_name": "Christina Janet", "orcid": "0000-0002-9223-9777", "clpid": "Su-Christina-Janet" } ], "thesis_advisor": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "thesis_committee": [ { "family_name": "Chan", "given_name": "David C.", "orcid": "0000-0002-0191-2154", "clpid": "Chan-D-C" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Goentoro", "given_name": "Lea A.", "orcid": "0000-0002-3904-0195", "clpid": "Goentoro-L-A" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/z7dv-m192", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14227", "collection": "thesis", "collection_id": "14227", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022021-012404326", "primary_object_url": { "basename": "Thesis-YodaiTakei-final.pdf", "content": "final", "filesize": 50506093, "license": "other", "mime_type": "application/pdf", "url": "/14227/1/Thesis-YodaiTakei-final.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Integrated Spatial Genomics Reveals Organizational Principles of Single-Cell Nuclear Architecture", "author": [ { "family_name": "Takei", "given_name": "Yodai", "orcid": "0000-0002-7226-5185", "clpid": "Takei-Yodai" } ], "thesis_advisor": [ { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/4ces-zm75", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:14212", "collection": "thesis", "collection_id": "14212", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05312021-221223611", "type": "thesis", "title": "Context-Dependent, Combinatorial Logic of BMP Signaling", "author": [ { "family_name": "Klumpe", "given_name": "Heidi Elizabeth", "orcid": "0000-0001-8938-2006", "clpid": "klumpe-Heidi-Elizabeth" } ], "thesis_advisor": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Goentoro", "given_name": "Lea A.", "orcid": "0000-0002-3904-0195", "clpid": "Goentoro-L-A" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Evolution generated diverse signaling proteins for the control of multicellular patterns and organ- isms. These include the proteins of the Bone Morphogenetic Protein (BMP) pathway. Nearly a dozen BMPs activate the BMP pathway to promote the formation of tissues as diverse as bone, cartilage, blood vessels, and the kidney, making them attractive therapeutics for regenerating those tissues in adults. During development, the response to a given BMP depends heavily on context, such as which other BMPs are present and which BMP receptors are expressed on the cell being ac- tivated. However, despite knowing that context matters, the overall logic of this context-dependent signal processing, including the roles of specific ligands and receptors in shaping context and how this logic arises from biochemical features of specific pathway components, remains unclear. Inspired by maps of gene epistasis and drug interactions that functionally classify members of complex biological systems, we comprehensively measured responses to all pairs of ten BMP homodimers (BMP2, BMP4, BMP5, BMP6, BMP7, BMP9, BMP10, GDF5, GDF6, and GDF7), combining robotic liquid handling with a high-throughput fluorescent reporter of pathway activa- tion. These data functionally classify ligands into \"equivalence groups,\" or ligands that combine in the same way with all other ligands across combinations. Surprisingly, the functional groupings do not correlate with similarity of ligand sequence and can change with cell context. Together, the context-dependent equivalence groups summarize the diverse responses to combinations of BMP ligands and their dependence on specific BMP receptors. The experimentally observed pairwise responses are also consistent with a mathematical model where BMP ligands compete for limited BMP receptors with different affinities and then produce outputs with different ligand-specific activ- ities. Ultimately, these results provide a useful reference for explaining the unique effects of BMP combinations in different tissues or time points in development, as well as highlighting counter- intuitive mechanisms for this complex signal processing. Chapter 1 provides an introduction to how and why we study cell-cell signaling. Chapter 2 provides a summary of the determination of equivalence groups, their dependence on receptor context, and fitting the mathematical model of receptor competition. Chapter 3 provides suggestions for future work, including recommendations for improved model fitting as well as crucial extensions to the definitions of BMP \"combinations\" and \"context\" to deepen our understanding and control of this critical pathway.", "doi": "10.7907/3vv4-bk06", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:14257", "collection": "thesis", "collection_id": "14257", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06072021-211522720", "type": "thesis", "title": "Engineering and Delivery of Programmable Protein Circuits as Potential Therapeutic Devices", "author": [ { "family_name": "Chong", "given_name": "Lucy Shin", "orcid": "0000-0002-5858-9984", "clpid": "Chong-Lucy-Shin" } ], "thesis_advisor": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "thesis_committee": [ { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Hay", "given_name": "Bruce A.", "orcid": "0000-0002-5486-0482", "clpid": "Hay-B-A" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/jdhe-by95", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:14073", "collection": "thesis", "collection_id": "14073", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02032021-180657616", "type": "thesis", "title": "Diverse Roles of RNA-protein Interactions: From Viral Antagonism to Mammalian Development", "author": [ { "family_name": "Banerjee", "given_name": "Abhik Kumar", "orcid": "0000-0002-9797-0104", "clpid": "Banerjee-Abhik-Kumar" } ], "thesis_advisor": [ { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" } ], "local_group": [ { "literal": "COVID-19" }, { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/tfb9-n887", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:14175", "collection": "thesis", "collection_id": "14175", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05262021-072231177", "primary_object_url": { "basename": "Ren_Caltech_Thesis_v2.pdf", "content": "final", "filesize": 7116984, "license": "other", "mime_type": "application/pdf", "url": "/14175/1/Ren_Caltech_Thesis_v2.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Principles for Designing Robust and Stable Synthetic Microbial Consortia", "author": [ { "family_name": "Ren", "given_name": "Xinying (Cindy)", "orcid": "0000-0002-8852-6722", "clpid": "Ren-Xinying-Cindy" } ], "thesis_advisor": [ { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" } ], "thesis_committee": [ { "family_name": "Doyle", "given_name": "John Comstock", "orcid": "0000-0002-1828-2486", "clpid": "Doyle-J-C" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" }, { "family_name": "Franco", "given_name": "Elisa", "clpid": "Franco-Elisa" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Engineering stable microbial consortia with robust functions are useful in many areas, including bioproduction and human health. Robust and stable properties depend on proper control of dynamics ranging from single cell-level to population-environment interactions. In this thesis, I discuss principles of building microbial consortia with synthetic circuits in two design scenarios.
\r\n\r\nFirst, for one microbial population, strong disturbances in environments often severely perturb cell states and lead to heterogeneous responses. Single cell-level design of control circuits may fail to induce a uniform response as needed. I demonstrate that cell-cell signaling systems can facilitate coordination among cells and achieve robust population-level behaviors. Moreover, I show that heterogeneity can be harnessed for robust adaptation at population-level via a bistable state switch.
\r\n\r\nSecond, multi-pecies consortia are intrinsically unstable due to competitive exclusion. Previous theoretical investigations based on models of pairwise interactions mainly explored what interaction network topology ensures stable coexistence. Yet neglecting detailed interaction mechanisms and spatial context results in contradictory predictions. Focusing on chemical-mediated interaction, I show that detailed mechanisms of chemical consumption/accumulation and chemical-induced growth/death, interaction network topology and spatial structures of environments all are critical factors to maintain stable coexistence. With a two population-system, I demonstrate that the same interaction network topology can exhibit qualitatively different or even opposite behaviors due to interaction mechanisms and spatial conditions.
", "doi": "10.7907/hc8x-3280", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:11888", "collection": "thesis", "collection_id": "11888", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11042019-135312842", "primary_object_url": { "basename": "Hanewich-Hollatz_Mikhail_2020.pdf", "content": "final", "filesize": 16901753, "license": "other", "mime_type": "application/pdf", "url": "/11888/2/Hanewich-Hollatz_Mikhail_2020.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function via Dynamic RNA Nanotechnology", "author": [ { "family_name": "Hanewich-Hollatz", "given_name": "Mikhail Henning", "orcid": "0000-0002-5369-3846", "clpid": "Hanewich-Hollatz-Mikhail-Henning" } ], "thesis_advisor": [ { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" } ], "thesis_committee": [ { "family_name": "Winfree", "given_name": "Erik", "orcid": "0000-0002-5899-7523", "clpid": "Winfree-E" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/NS2B-DJ96", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13767", "collection": "thesis", "collection_id": "13767", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022020-102020436", "primary_object_url": { "basename": "GriffinChure_Thesis.pdf", "content": "final", "filesize": 96078878, "license": "cc_by", "mime_type": "application/pdf", "url": "/13767/2/GriffinChure_Thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "The Molecular Biophysics of Evolutionary and Physiological Adaptation", "author": [ { "family_name": "Chure", "given_name": "Griffin Daniel", "orcid": "0000-0002-2216-2057", "clpid": "Chure-Griffin-Daniel" } ], "thesis_advisor": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "thesis_committee": [ { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/q8h6-xr92", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:11805", "collection": "thesis", "collection_id": "11805", "cite_using_url": "http://resolver.caltech.edu/CaltechTHESIS:10012019-095132591", "type": "thesis", "title": "Development of Analytical Tools and Animal Models for Studies of Small-Intestine Dysbiosis", "author": [ { "family_name": "Bogatyrev", "given_name": "Said R.", "orcid": "0000-0003-0486-9451", "clpid": "Bogatyrev-Said-R" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/VJDZ-7B52", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13742", "collection": "thesis", "collection_id": "13742", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05302020-111741817", "primary_object_url": { "basename": "Silverman_thesis_submission_200530.pdf", "content": "final", "filesize": 4442708, "license": "other", "mime_type": "application/pdf", "url": "/13742/3/Silverman_thesis_submission_200530.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Protein-Mediated Colloidal Assembly", "author": [ { "family_name": "Silverman", "given_name": "Bradley Ross", "orcid": "0000-0002-9256-8941", "clpid": "Silverman-Bradley-Ross" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "clpid": "Ismagilov-R-F" }, { "family_name": "Brady", "given_name": "John F.", "clpid": "Brady-J-F" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "Rosen Bioengineering Center" }, { "literal": "div_chem" } ], "abstract": "The assembly of colloidal-sized particles into larger structures by the manipulation of inter-particle forces has been a subject of significant research towards applications in materials science, soft matter physics, and synthetic biology. To date, much of this work has utilized manipulation of electrostatic or depletion interactions to drive the aggregation of the particles. More recently, specific (bio)-chemical interactions have been harnessed, particularly the use of deoxyribonucleic acid (DNA) linkers to program particle interactions by Watson-Crick base-pairing. In this thesis, we will demonstrate the use of an alternative set of biochemical interactions, protein-protein interactions, which have useful properties (in particular, their ability to be completely genetically-programmable).
\r\n\r\nIn Chapter 2, we discuss the development of a model system for the protein-mediated assembly of colloidal micro-particles. Associative proteins are grafted onto the surface of polystyrene micro-particles, enabling their assembly into aggregates either through reversible coiled-coil interactions or by irreversible isopeptide linkages. The sizes of the resulting aggregates are tunable and can be controlled by the concentration of the immobilized associative proteins on their surface. Further, we show that particles grafted with different protein pairs show excellent self-sorting into separate aggregates. Finally, we demonstrate that these protein-protein interactions can be used to assemble complex core-shell aggregates. The principles of protein-mediated colloidal assembly learned in this chapter will be instructive as we attempt the more complex assembly of living microbial cells.
\r\n\r\nIn Chapter 3, we discuss the implementation of a protein-driven aggregation system in living bacterial cells. Similarly to Chapter 2, we demonstrate that we can drive the aggregation of bacteria by the surface display of proteins enabling reversible coiled-coil interactions or irreversible isopeptide bonds. The sizes of these aggregates are tunable by titration of surface expression levels by standard synthetic biology techniques. Finally, we show that this programmable aggregation of bacteria may have physiological consequences for the cells, in particular, the activation of a quorum sensing circuit due to a higher local concentration of bacteria.
\r\n\r\nIn Chapter 4, we further investigate how the properties of the aggregates described in Chapter 3 can be controlled and how these relate to the underlying properties of the associative proteins and shear field. we demonstrate control of the assembly kinetics and equilibrium sizes of the resulting flocs over several orders of magnitude using different associating proteins and expression levels. Finally, we show that a single point mutation in the associative protein leads to an unexpected ultra-sensitive pH-responsive coil, demonstrating the importance of molecular-scale interactions on the macro-scale properties of the aggregates.
\r\n\r\nIn Chapter 5, we discuss the ability of the bacterial aggregates described in Chapters 3 and 4 to enable substrate channeling between bacterial strains, leading to enhancement of titers in multi-step biosynthetic pathways. When biosynthetic pathways are split into separate bacterial strains, dilution of the intermediate compound into the bulk media may decrease reaction flux. By aggregating the bacteria, the intermediate compound is able to rapidly diffuse into the downstream cell without being diluted, enabling higher reaction fluxes. we demonstrate through the model flavonoid synthesis pathway that aggregation can lead to substantially higher titers of the desired compound without pathway re-engineering, and develop a mathematical model by which this result can be understood.
", "doi": "10.7907/x3ya-fq67", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:11570", "collection": "thesis", "collection_id": "11570", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05302019-145256322", "type": "thesis", "title": "Biological Responses to Therapeutic Treatments of Human Vascular Diseases", "author": [ { "family_name": "Shao", "given_name": "Zixuan", "orcid": "0000-0002-4676-6023", "clpid": "Shao-Zixuan" } ], "thesis_advisor": [ { "family_name": "Kornfield", "given_name": "Julia A.", "clpid": "Kornfield-J-A" } ], "thesis_committee": [ { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Kornfield", "given_name": "Julia A.", "clpid": "Kornfield-J-A" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/P2S4-AY40", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11334", "collection": "thesis", "collection_id": "11334", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01062019-215120039", "primary_object_url": { "basename": "Caltech-thesis-PeiYinShih complete.pdf", "content": "final", "filesize": 136482147, "license": "other", "mime_type": "application/pdf", "url": "/11334/40/Caltech-thesis-PeiYinShih complete.pdf", "version": "v9.0.0" }, "type": "thesis", "title": "The Ethology of Stress in Nematodes \r ", "author": [ { "family_name": "Shih", "given_name": "Pei-Yin", "orcid": "0000-0003-3082-9242", "clpid": "Shih-Pei-Yin" } ], "thesis_advisor": [ { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "thesis_committee": [ { "family_name": "Prober", "given_name": "David A.", "clpid": "Prober-D-A" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/1E7G-K373", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11353", "collection": "thesis", "collection_id": "11353", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01222019-133152032", "type": "thesis", "title": "Linearity in Cell Signaling Pathways", "author": [ { "family_name": "Nunns", "given_name": "Harry James Rogan", "orcid": "0000-0002-9669-0039", "clpid": "Nunns-Harry- James-Rogan" } ], "thesis_advisor": [ { "family_name": "Goentoro", "given_name": "Lea A.", "orcid": "0000-0002-3904-0195", "clpid": "Goentoro-L-A" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Goentoro", "given_name": "Lea A.", "orcid": "0000-0002-3904-0195", "clpid": "Goentoro-L-A" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/WP0J-E945", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11532", "collection": "thesis", "collection_id": "11532", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05222019-160829503", "primary_object_url": { "basename": "frick_chris_2019_thesis.pdf", "content": "final", "filesize": 14657328, "license": "other", "mime_type": "application/pdf", "url": "/11532/11/frick_chris_2019_thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "How Single Cells Sense Smad3 Signal", "author": [ { "family_name": "Frick", "given_name": "Christopher Lee", "orcid": "0000-0001-6823-5920", "clpid": "Frick-Christopher-Lee" } ], "thesis_advisor": [ { "family_name": "Goentoro", "given_name": "Lea A.", "orcid": "0000-0002-3904-0195", "clpid": "Goentoro-L-A" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Hay", "given_name": "Bruce A.", "orcid": "0000-0002-5486-0482", "clpid": "Hay-B-A" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Goentoro", "given_name": "Lea A.", "orcid": "0000-0002-3904-0195", "clpid": "Goentoro-L-A" } ], "local_group": [ { "literal": "div_bbe" } ], "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.", "doi": "10.7907/WNM4-4R58", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11687", "collection": "thesis", "collection_id": "11687", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06052019-122355847", "primary_object_url": { "basename": "Frankiw_Luke_2019_Thesis_FINAL.pdf", "content": "final", "filesize": 6138653, "license": "other", "mime_type": "application/pdf", "url": "/11687/1/Frankiw_Luke_2019_Thesis_FINAL.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "mRNA Splicing-Mediated Gene Expression Regulation in Innate Immunity", "author": [ { "family_name": "Frankiw", "given_name": "Luke Steven", "clpid": "Frankiw-Luke-Steven" } ], "thesis_advisor": [ { "family_name": "Baltimore", "given_name": "David L.", "clpid": "Baltimore-D-L" } ], "thesis_committee": [ { "family_name": "Guttman", "given_name": "Mitchell", "clpid": "Guttman-M" }, { "family_name": "Baltimore", "given_name": "David L.", "clpid": "Baltimore-D-L" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/NBQG-BS67", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11730", "collection": "thesis", "collection_id": "11730", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06092019-220453694", "type": "thesis", "title": "Memory and Decoding in Signaling Transduction Pathways", "author": [ { "family_name": "Kim", "given_name": "Kibeom", "orcid": "0000-0002-0764-4875", "clpid": "Kim-Kibeom" } ], "thesis_advisor": [ { "family_name": "Goentoro", "given_name": "Lea A.", "orcid": "0000-0002-3904-0195", "clpid": "Goentoro-L-A" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Hay", "given_name": "Bruce A.", "orcid": "0000-0002-5486-0482", "clpid": "Hay-B-A" }, { "family_name": "Goentoro", "given_name": "Lea A.", "orcid": "0000-0002-3904-0195", "clpid": "Goentoro-L-A" }, { "family_name": "Zinn", "given_name": "Kai George", "orcid": "0000-0002-6706-5605", "clpid": "Zinn-K-G" } ], "local_group": [ { "literal": "div_bbe" } ], "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", "doi": "10.7907/BJDQ-JX45", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:10551", "collection": "thesis", "collection_id": "10551", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11032017-102218314", "primary_object_url": { "basename": "ThesisMergeAll_PR.pdf", "content": "final", "filesize": 4913908, "license": "other", "mime_type": "application/pdf", "url": "/10551/1/ThesisMergeAll_PR.pdf", "version": "v9.0.0" }, "type": "thesis", "title": "Revealing the Mechanism of Xist-mediated Silencing", "author": [ { "family_name": "Chen", "given_name": "Chun-Kan", "orcid": "0000-0002-1194-9137", "clpid": "Chen-Chun-Kan" } ], "thesis_advisor": [ { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0002-1221-0967", "clpid": "Guttman-M" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Aravin", "given_name": "Alexei A.", "orcid": "0000-0002-6956-8257", "clpid": "Aravin-A-A" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/Z94J0C9J", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10623", "collection": "thesis", "collection_id": "10623", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01052018-221609680", "type": "thesis", "title": "Quantitative Dissection of the Allosteric and Sequence-Dependent Regulatory Genome in E. coli", "author": [ { "family_name": "Belliveau", "given_name": "Nathan Maurice", "orcid": "0000-0002-1536-1963", "clpid": "Belliveau-Nathan-Maurice" } ], "thesis_advisor": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "thesis_committee": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Van Valen", "given_name": "David A.", "orcid": "0000-0001-7534-7621", "clpid": "Van-Valen-D" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/Z9DN438T", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10622", "collection": "thesis", "collection_id": "10622", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12312017-234413682", "primary_object_url": { "basename": "park_jin_thesis_composed_5.pdf", "content": "final", "filesize": 4771190, "license": "other", "mime_type": "application/pdf", "url": "/10622/1/park_jin_thesis_composed_5.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Circuits of Dynamically Interacting Sigma Factors in Single Cells", "author": [ { "family_name": "Park", "given_name": "Jin", "clpid": "Park-Jin" } ], "thesis_advisor": [ { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" }, { "family_name": "Goentoro", "given_name": "Lea A.", "clpid": "Goentoro-L-A" }, { "family_name": "Murray", "given_name": "Richard M.", "clpid": "Murray-R-M" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/CT0B-Q853", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10620", "collection": "thesis", "collection_id": "10620", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12282017-140713708", "type": "thesis", "title": "New Capabilities of the Notch Signaling Pathway", "author": [ { "family_name": "Nandagopal", "given_name": "Nagarajan", "orcid": "0000-0002-0469-6549", "clpid": "Nandagopal-Nagarajan" } ], "thesis_advisor": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "thesis_committee": [ { "family_name": "Goentoro", "given_name": "Lea A.", "orcid": "0000-0002-3904-0195", "clpid": "Goentoro-L-A" }, { "family_name": "Bronner", "given_name": "Marianne E.", "orcid": "0000-0003-4274-1862", "clpid": "Bronner-M-E" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/Z98050TB", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10606", "collection": "thesis", "collection_id": "10606", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12132017-121914940", "type": "thesis", "title": "Application, Computation, and Theory for Synthetic Gene Circuits", "author": [ { "family_name": "Swaminathan", "given_name": "Anandh", "orcid": "0000-0001-9935-6530", "clpid": "Swaminathan-Anandh" } ], "thesis_advisor": [ { "family_name": "Murray", "given_name": "Richard M.", "clpid": "Murray-R-M" } ], "thesis_committee": [ { "family_name": "Murray", "given_name": "Richard M.", "clpid": "Murray-R-M" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Goentoro", "given_name": "Lea A.", "clpid": "Goentoro-L-A" }, { "family_name": "Beck", "given_name": "James L.", "clpid": "Beck-J-L" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "The field of synthetic gene circuits is concerned with engineering novel gene expression dynamics into organisms. This field, a subset of synthetic biology, was started almost two decades ago with the creation of two synthetic circuits: a bistable toggle switch and an oscillator. From the very outset, modeling has played a role in the development of synthetic circuits. However, modeling has been used to gain qualitative insight into dynamics, and actual quantitative modeling has been lagging behind.
\r\n\r\nParameters for quantitative models for biological systems often cannot be adequately estimated from measured data, because far too many sets of parameters can produce the same set of limited measured outputs. Additionally, models for synthetic gene circuits are often not correct the first time, and iterating on cycles of modeling and parameter estimation is difficult. Finally, there is a gap between development of modeling and system identification tools and their application to experiments on actual synthetic gene circuits.
\r\n\r\nThis thesis attempts to work towards addressing these issues with quantitative modeling for synthetic gene circuits. First, we derive theoretical conditions for identifiability of stochastic linear systems from heterogenous types of measurement data. These identifiability conditions can provide insight into what type of model to use and what measurements to collect in order to ensure that the resulting model can be identified.
\r\n\r\nSecond, we develop a software package for fast and flexible modeling and parameter estimation for synthetic gene circuits. The user can input models into our software using a simple text format and perform simulations of all types at optimized speeds. By inputting measured experimental data along with the model, the software can be used to perform Bayesian parameter estimation in an automated manner. To bridge the gap between computation and application, we apply this software to parameter estimation of DNA recombinase dynamics using real experimental data collected in an in vitro cell extract.
\r\n\r\nFinally, we use modeling to guide the design of an improved single gene synthetic oscillator. While the original synthetic genetic oscillator contained three genes, we show that a simple circuit with a single gene can produce robust and synchronized oscillations across a population.
\r\n\r\nOur results constitute an additional step towards the incorporation of quantitative modeling and parameter inference as part of the design-build-test cycle for synthetic gene circuits.
", "doi": "10.7907/Z9833Q67", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10958", "collection": "thesis", "collection_id": "10958", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292018-133205686", "primary_object_url": { "basename": "barnes_stephanie_2018.pdf", "content": "final", "filesize": 61047841, "license": "other", "mime_type": "application/pdf", "url": "/10958/1/barnes_stephanie_2018.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Decoding the Regulatory Genome: Quantitative Analysis of Transcriptional Regulation in Escherichia coli", "author": [ { "family_name": "Barnes", "given_name": "Stephanie Loos", "orcid": "0000-0002-5237-603X", "clpid": "Barnes-Stephanie-Loos" } ], "thesis_advisor": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "thesis_committee": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/D13T-7868", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10146", "collection": "thesis", "collection_id": "10146", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04262017-114441886", "type": "thesis", "title": "Compact Microscope System for Biomedical Applications", "author": [ { "family_name": "Kim", "given_name": "Jinho", "clpid": "Kim-Jinho" } ], "thesis_advisor": [ { "family_name": "Yang", "given_name": "Changhuei", "clpid": "Yang-Changhuei" } ], "thesis_committee": [ { "family_name": "Yang", "given_name": "Changhuei", "clpid": "Yang-Changhuei" }, { "family_name": "Tai", "given_name": "Yu-Chong", "clpid": "Tai-Yu-Chong" }, { "family_name": "Vaidyanathan", "given_name": "P. P.", "clpid": "Vaidyanathan-P-P" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Demands for an imaging system which has high space-bandwidth product (SBP) are increasing in modern biomedical research as the amount of information to be dealt with is increasing. However, conventional microscopy has a limited SBP of about 10 mega pixels, and as such if a user wants an image in high resolution, the field of view (FOV) of the image is reduced, or if a wide FOV is necessary, the user needs to give up the resolution of image. A common way of overcoming this SBP limit in the conventional microscopy is to use mechanical moving stages and scan through wide sample area, however, it is time consuming to image large area using a high numerical aperture (NA) objective lens. This thesis presents compact imaging systems based on Fourier ptychographic microscopy for biomedical applications which are able to increase SBP without having any mechanical moving parts: one imaging system for an incubator embedded imaging system to be used in in-vitro cell culture monitoring, and the other for a high throughput 96 well plate imaging system for fast drug screening.
", "doi": "10.7907/Z9H9937R", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:10091", "collection": "thesis", "collection_id": "10091", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03082017-163613964", "primary_object_url": { "basename": "Guo_Shaobin_2017_thesis.pdf", "content": "final", "filesize": 27841341, "license": "other", "mime_type": "application/pdf", "url": "/10091/1/Guo_Shaobin_2017_thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Prototyping Diverse Synthetic Biological Circuits in a Cell-Free Transcription-Translation System", "author": [ { "family_name": "Guo", "given_name": "Shaobin", "orcid": "0000-0001-9736-4078", "clpid": "Guo-Shaobin" } ], "thesis_advisor": [ { "family_name": "Murray", "given_name": "Richard M.", "clpid": "Murray-R-M" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Murray", "given_name": "Richard M.", "clpid": "Murray-R-M" }, { "family_name": "Goentoro", "given_name": "Lea A.", "clpid": "Goentoro-L-A" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Synthetic biological circuits are the foundation for the ultimate goals of controlling cells and building artificial cells from the ground up. To get closer to these goals in a more efficient way, we utilize a cell-free transcription-translation system to help perfect biological circuits for the simplicity, freedom, and convenience that the system offers. In this thesis, we demonstrate three distinct aspects of biological circuits in a cell-free transcription-translation system: circuit dynamics, phosphorylation, and membrane proteins. We start with a simple feedforward circuit, which shows dynamic responses to the input. We first prototype the feedforward circuit in the cell-free system with the aid of mathematical modeling. Then, based on the knowledge learned from prototyping, we successfully implement the circuit in cells. Not only do we show that a circuit with dynamics can be prototyped in the cell- free system, but we also test a more complicated circuit involving a phosphorylation cycle. The phosphorylation-based insulator circuit is prototyped and then a model created for the circuit is shown to be identifiable in the cell-free system. To further expand the capability of the cell-free system, we demonstrate that biologically active membrane proteins can be generated in the cell-free system with engineering, suggesting that even biological circuits requiring membrane proteins can be prototyped in the system. These results help advance our knowledge of both biological circuits and the cell-free transcription-translation system, and bring us one step closer to our ultimate goals of implementing control theory in synthetic biology.", "doi": "10.7907/Z9CR5RDK", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:9928", "collection": "thesis", "collection_id": "9928", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09302016-110627143", "type": "thesis", "title": "Chromatin Topology and Transcription in Myogenesis", "author": [ { "family_name": "Fisher-Aylor", "given_name": "Katherine Irene", "orcid": "0000-0003-3371-2947", "clpid": "Fisher-Aylor-Katherine-Irene" } ], "thesis_advisor": [ { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" } ], "thesis_committee": [ { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" }, { "family_name": "Stathopoulos", "given_name": "Angelike", "clpid": "Stathopoulos-A" }, { "family_name": "Bronner", "given_name": "Marianne E.", "clpid": "Bronner-M-E" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" } ], "local_group": [ { "literal": "div_bbe" } ], "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.", "doi": "10.7907/Z9K07290", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:9995", "collection": "thesis", "collection_id": "9995", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12152016-144548062", "type": "thesis", "title": "Highly Multiplexed Single Cell In Situ RNA Detection", "author": [ { "family_name": "Shah", "given_name": "Sheel Mukesh", "orcid": "0000-0002-6321-4669", "clpid": "Shah-Sheel-Mukesh" } ], "thesis_advisor": [ { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" } ], "thesis_committee": [ { "family_name": "Gradinaru", "given_name": "Viviana", "clpid": "Gradinaru-V" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Allman", "given_name": "John Morgan", "clpid": "Allman-J-M" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/Z9X63JXH", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:9589", "collection": "thesis", "collection_id": "9589", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02262016-115004310", "primary_object_url": { "basename": "elubeck_thesis_sub2.pdf", "content": "final", "filesize": 27206724, "license": "other", "mime_type": "application/pdf", "url": "/9589/1/elubeck_thesis_sub2.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Towards in situ Single Cell Systems Biology", "author": [ { "family_name": "Lubeck", "given_name": "Eric", "orcid": "0000-0002-5457-0258", "clpid": "Lubeck-Eric" } ], "thesis_advisor": [ { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Gradinaru", "given_name": "Viviana", "clpid": "Gradinaru-V" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Systems-level studies of biological systems rely on observations taken at a resolution lower than\r\nthe essential unit of biology, the cell. Recent technical advances in DNA sequencing have enabled\r\nmeasurements of the transcriptomes in single cells excised from their environment, but it remains a\r\ndaunting technical problem to reconstruct in situ gene expression patterns from sequencing data. In\r\nthis thesis I develop methods for the routine, quantitative in situ measurement of gene expression\r\nusing fluorescence microscopy.
\r\n\r\nThe number of molecular species that can be measured simultaneously by fluorescence microscopy\r\nis limited by the pallet of spectrally distinct fluorophores. Thus, fluorescence microscopy is traditionally\r\nlimited to the simultaneous measurement of only five labeled biomolecules at a time. The\r\ntwo methods described in this thesis, super-resolution barcoding and temporal barcoding, represent\r\nstrategies for overcoming this limitation to monitor expression of many genes in a single cell.\r\nSuper-resolution barcoding employs optical super-resolution microscopy (SRM) and combinatorial\r\nlabeling via-smFISH (single molecule fluorescence in situ hybridization) to uniquely label individual\r\nmRNA species with distinct barcodes resolvable at nanometer resolution. This method dramatically\r\nincreases the optical space in a cell, allowing a large numbers of barcodes to be visualized\r\nsimultaneously. As a proof of principle this technology was used to study the S. cerevisiae calcium\r\nstress response. The second method, sequential barcoding, reads out a temporal barcode through\r\nmultiple rounds of oligonucleotide hybridization to the same mRNA. The multiplexing capacity of\r\nsequential barcoding increases exponentially with the number of rounds of hybridization, allowing\r\nover a hundred genes to be profiled in only a few rounds of hybridization.
\r\n\r\nThe utility of sequential barcoding was further demonstrated by adapting this method to study\r\ngene expression in mammalian tissues. Mammalian tissues suffer both from a large amount of\r\nauto-fluorescence and light scattering, making detection of smFISH probes on mRNA difficult. An\r\namplified single molecule detection technology, smHCR (single molecule hairpin chain reaction),\r\nwas developed to allow for the quantification of mRNA in tissue. This technology is demonstrated\r\nin combination with light sheet microscopy and background reducing tissue clearing technology,\r\nenabling whole-organ sequential barcoding to monitor in situ gene expression directly in intact\r\nmammalian tissue.
\r\n\r\nThe methods presented in this thesis, specifically sequential barcoding and smHCR, enable multiplexed\r\ntranscriptional observations in any tissue of interest. These technologies will serve as a\r\ngeneral platform for future transcriptomic studies of complex tissues.
", "doi": "10.7907/Z9BK1999", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9835", "collection": "thesis", "collection_id": "9835", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06032016-143422651", "primary_object_url": { "basename": "Thesis Final v2.pdf", "content": "final", "filesize": 75628401, "license": "other", "mime_type": "application/pdf", "url": "/9835/1/Thesis Final v2.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Multiplexed Analysis of Diverse RNA Classes via Hybridization Chain Reaction", "author": [ { "family_name": "Acharya", "given_name": "Aneesh", "orcid": "0000-0002-4402-7147", "clpid": "Acharya-Aneesh" } ], "thesis_advisor": [ { "family_name": "Pierce", "given_name": "Niles A.", "clpid": "Pierce-N-A" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Pierce", "given_name": "Niles A.", "clpid": "Pierce-N-A" }, { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" } ], "local_group": [ { "literal": "div_bbe" } ], "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.", "doi": "10.7907/Z95M63N0", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9724", "collection": "thesis", "collection_id": "9724", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05172016-133516099", "primary_object_url": { "basename": "RiosGustavo2016thesis.pdf", "content": "final", "filesize": 49831621, "license": "other", "mime_type": "application/pdf", "url": "/9724/1/RiosGustavo2016thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Nanofabricated Neural Probe System for Dense 3-D Recordings of Brain Activity", "author": [ { "family_name": "Rios", "given_name": "Gustavo", "orcid": "0000-0003-1411-4933", "clpid": "Rios-Gustavo" } ], "thesis_advisor": [ { "family_name": "Siapas", "given_name": "Athanassios G.", "clpid": "Siapas-A-G" } ], "thesis_committee": [ { "family_name": "Dickinson", "given_name": "Michael H.", "clpid": "Dickinson-M-H" }, { "family_name": "Siapas", "given_name": "Athanassios G.", "clpid": "Siapas-A-G" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Lubenov", "given_name": "Evgueniy V.", "clpid": "Lubenov-E-V" } ], "local_group": [ { "literal": "Kavli Nanoscience Institute" }, { "literal": "div_bbe" } ], "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.", "doi": "10.7907/Z9BG2M0B", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9705", "collection": "thesis", "collection_id": "9705", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05082016-170628018", "primary_object_url": { "basename": "20160509_Hsiao_Victoria_2016.pdf", "content": "final", "filesize": 67580263, "license": "other", "mime_type": "application/pdf", "url": "/9705/1/20160509_Hsiao_Victoria_2016.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Synthetic Circuits for Feedback and Detection in Bacteria", "author": [ { "family_name": "Hsiao", "given_name": "Victoria", "orcid": "0000-0001-9297-1522", "clpid": "Hsiao-Victoria" } ], "thesis_advisor": [ { "family_name": "Murray", "given_name": "Richard M.", "clpid": "Murray-R-M" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Murray", "given_name": "Richard M.", "clpid": "Murray-R-M" }, { "family_name": "Pierce", "given_name": "Niles A.", "clpid": "Pierce-N-A" }, { "family_name": "Rothemund", "given_name": "Paul W. K.", "clpid": "Rothemund-P-W-K" } ], "local_group": [ { "literal": "div_bbe" } ], "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.", "doi": "10.7907/Z9WD3XJW", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9191", "collection": "thesis", "collection_id": "9191", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10012015-221355676", "primary_object_url": { "basename": "SunZacharyZhipeng2016thesis.pdf", "content": "final", "filesize": 45522801, "license": "other", "mime_type": "application/pdf", "url": "/9191/1/SunZacharyZhipeng2016thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "An in vitro Biomolecular Breadboard for Prototyping Synthetic Biological Circuits", "author": [ { "family_name": "Sun", "given_name": "Zachary Zhipeng", "orcid": "0000-0002-9425-2924", "clpid": "Sun-Zachary-Zhipeng" } ], "thesis_advisor": [ { "family_name": "Murray", "given_name": "Richard M.", "clpid": "Murray-R-M" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Baltimore", "given_name": "David L.", "clpid": "Baltimore-D-L" }, { "family_name": "Murray", "given_name": "Richard M.", "clpid": "Murray-R-M" }, { "family_name": "Noireaux", "given_name": "Vincent", "clpid": "Noireaux-V" } ], "local_group": [ { "literal": "div_bbe" } ], "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.", "doi": "10.7907/Z9TB14TW", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:8890", "collection": "thesis", "collection_id": "8890", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05262015-152206802", "primary_object_url": { "basename": "20150513_ZSSinger_Thesis.pdf", "content": "final", "filesize": 5536349, "license": "other", "mime_type": "application/pdf", "url": "/8890/1/20150513_ZSSinger_Thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Metastability and Dynamics of Stem Cells: From Direct Observations to Inference at the Single Cell Level", "author": [ { "family_name": "Singer", "given_name": "Zakary Sean", "clpid": "Singer-Zakary-Sean" } ], "thesis_advisor": [ { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" } ], "thesis_committee": [ { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Hay", "given_name": "Bruce A.", "clpid": "Hay-B-A" }, { "family_name": "Guttman", "given_name": "Mitchell", "clpid": "Guttman-M" }, { "family_name": "Plath", "given_name": "Kathrin", "clpid": "Plath-K" } ], "local_group": [ { "literal": "div_bbe" } ], "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.
", "doi": "10.7907/Z9ST7MS1", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8959", "collection": "thesis", "collection_id": "8959", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06012015-171243593", "primary_object_url": { "basename": "NiranjanSrinivas_2015_thesis.pdf", "content": "final", "filesize": 15297188, "license": "other", "mime_type": "application/pdf", "url": "/8959/1/NiranjanSrinivas_2015_thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Programming Chemical Kinetics: Engineering Dynamic Reaction Networks with DNA Strand Displacement", "author": [ { "family_name": "Srinivas", "given_name": "Niranjan", "clpid": "Srinivas-Niranjan" } ], "thesis_advisor": [ { "family_name": "Winfree", "given_name": "Erik", "clpid": "Winfree-E" } ], "thesis_committee": [ { "family_name": "Murray", "given_name": "Richard M.", "clpid": "Murray-R-M" }, { "family_name": "Winfree", "given_name": "Erik", "clpid": "Winfree-E" }, { "family_name": "Pierce", "given_name": "Niles A.", "clpid": "Pierce-N-A" }, { "family_name": "Rothemund", "given_name": "Paul W. K.", "clpid": "Rothemund-P-W-K" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Seelig", "given_name": "Georg", "clpid": "Seelig-G" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Over the last century, the silicon revolution has enabled us to build faster, smaller and more sophisticated computers. Today, these computers control phones, cars, satellites, assembly lines, and other electromechanical devices. Just as electrical wiring controls electromechanical devices, living organisms employ \"chemical wiring\" to make decisions about their environment and control physical processes. Currently, the big difference between these two substrates is that while we have the abstractions, design principles, verification and fabrication techniques in place for programming with silicon, we have no comparable understanding or expertise for programming chemistry.
\r\n\r\nIn this thesis we take a small step towards the goal of learning how to systematically engineer prescribed non-equilibrium dynamical behaviors in chemical systems. We use the formalism of chemical reaction networks (CRNs), combined with mass-action kinetics, as our programming language for specifying dynamical behaviors. Leveraging the tools of nucleic acid nanotechnology (introduced in Chapter 1), we employ synthetic DNA molecules as our molecular architecture and toehold-mediated DNA strand displacement as our reaction primitive.
\r\n\r\nAbstraction, modular design and systematic fabrication can work only with well-understood and quantitatively characterized tools. Therefore, we embark on a detailed study of the \"device physics\" of DNA strand displacement (Chapter 2). We present a unified view of strand displacement biophysics and kinetics by studying the process at multiple levels of detail, using an intuitive model of a random walk on a 1-dimensional energy landscape, a secondary structure kinetics model with single base-pair steps, and a coarse-grained molecular model that incorporates three-dimensional geometric and steric effects. Further, we experimentally investigate the thermodynamics of three-way branch migration. Our findings are consistent with previously measured or inferred rates for hybridization, fraying, and branch migration, and provide a biophysical explanation of strand displacement kinetics. Our work paves the way for accurate modeling of strand displacement cascades, which would facilitate the simulation and construction of more complex molecular systems.
\r\n\r\nIn Chapters 3 and 4, we identify and overcome the crucial experimental challenges involved in using our general DNA-based technology for engineering dynamical behaviors in the test tube. In this process, we identify important design rules that inform our choice of molecular motifs and our algorithms for designing and verifying DNA sequences for our molecular implementation. We also develop flexible molecular strategies for \"tuning\" our reaction rates and stoichiometries in order to compensate for unavoidable non-idealities in the molecular implementation, such as imperfectly synthesized molecules and spurious \"leak\" pathways that compete with desired pathways.
\r\n\r\nWe successfully implement three distinct autocatalytic reactions, which we then combine into a de novo chemical oscillator. Unlike biological networks, which use sophisticated evolved molecules (like proteins) to realize such behavior, our test tube realization is the first to demonstrate that Watson-Crick base pairing interactions alone suffice for oscillatory dynamics. Since our design pipeline is general and applicable to any CRN, our experimental demonstration of a de novo chemical oscillator could enable the systematic construction of CRNs with other dynamic behaviors.
", "doi": "10.7907/Z9KD1VVJ", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8865", "collection": "thesis", "collection_id": "8865", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05182015-163708506", "primary_object_url": { "basename": "delossantos_elc_2015_thesis.pdf", "content": "final", "filesize": 11163927, "license": "other", "mime_type": "application/pdf", "url": "/8865/1/delossantos_elc_2015_thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Expanding the Toolkit for Synthetic Biology: Frameworks for Native-like Non-natural Gene Circuits", "author": [ { "family_name": "Cornejo de los Santos", "given_name": "Emmanuel Lorenzo", "clpid": "Cornejo-de-los-Santos-Emmanuel-Lorenzo-Cornejo" } ], "thesis_advisor": [ { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_bbe" } ], "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.", "doi": "10.7907/Z9M61H64", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8839", "collection": "thesis", "collection_id": "8839", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04292015-153123844", "primary_object_url": { "basename": "John_Yong_thesis_deposited 20150601.pdf", "content": "final", "filesize": 13676844, "license": "other", "mime_type": "application/pdf", "url": "/8839/1/John_Yong_thesis_deposited 20150601.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Dynamics and Heterogeneity of Gene Expression and Epigenetic Regulation at the Single-Cell Level", "author": [ { "family_name": "Yong", "given_name": "John", "orcid": "0000-0002-4914-2259", "clpid": "Yong-John" } ], "thesis_advisor": [ { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" } ], "thesis_committee": [ { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Guttman", "given_name": "Mitchell", "clpid": "Guttman-M" }, { "family_name": "Siapas", "given_name": "Athanassios G.", "clpid": "Siapas-A-G" }, { "family_name": "Plath", "given_name": "Kathrin", "clpid": "Plath-K" } ], "local_group": [ { "literal": "div_bbe" } ], "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
", "doi": "10.7907/Z9ZC80VX", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8971", "collection": "thesis", "collection_id": "8971", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022015-150554177", "type": "thesis", "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", "author": [ { "family_name": "Goldberg", "given_name": "Mark David", "clpid": "Goldberg-Mark-David" } ], "thesis_advisor": [ { "family_name": "Scherer", "given_name": "Axel", "clpid": "Scherer-A" } ], "thesis_committee": [ { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" }, { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Kartalov", "given_name": "Emil P.", "clpid": "Kartalov-E-P" }, { "family_name": "Scherer", "given_name": "Axel", "clpid": "Scherer-A" } ], "local_group": [ { "literal": "Kavli Nanoscience Institute" }, { "literal": "div_bbe" } ], "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. ", "doi": "10.7907/Z9P848V6", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8737", "collection": "thesis", "collection_id": "8737", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12052014-154140797", "primary_object_url": { "basename": "AlborzThesisFinal.pdf", "content": "final", "filesize": 11354462, "license": "other", "mime_type": "application/pdf", "url": "/8737/1/AlborzThesisFinal.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Synthetic Biology Tools for Targeted Incorporation of Non-Canonical Amino Acids into Cellular Proteins", "author": [ { "family_name": "Mahdavi", "given_name": "Alborz", "orcid": "0000-0002-8790-8112", "clpid": "Mahdavi-Alborz" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Hajimiri", "given_name": "Ali", "orcid": "0000-0001-6736-8019", "clpid": "Hajimiri-A" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Proteins mediate many essential functions in cells, and methods to profile cellular proteins are of great interest for biological discovery. Whereas all of the cells in an organism share the same genome, the landscape of proteins (the proteome) varies between different cell types and over the lifetime of the organism. Rapid progress in mass spectrometers is enabling the detailed analysis of cellular proteomes. Whereas better instruments increase coverage, throughput, and measurement precision, new chemical reporters, metabolic tags, and synthetic biology techniques are required to enhance the specificity and spatiotemporal resolution of protein labeling and detection. This work introduces methods for cell-selective proteome analysis through the incorporation of non-canonical amino acids into newly synthesized proteins.
\r\n \r\nChapter I provides an overview of current technologies for translational profiling and proteomic analysis in cells. Strategies for the residue-specific incorporation of non-canonical amino acids and bioorthogonal non-canonical amino acid tagging are discussed. Chapter II introduces a new approach for the identification of secreted bacterial proteins from infected host cells using non-canonical amino acid labeling. This work demonstrates an application of cell-selective proteome labeling. Selectivity is achieved through controlled expression of a mutant aminoacyl tRNA synthetase (aaRS) enzyme that enables the metabolic incorporation of a non-canonical amino acid.
\r\n \r\nIdeally, the activity of multiple genes should be used to genetically control the extent of proteome labeling in cells. This is useful because many cell states are characterized by the activity of multiple genes and identified based on the expression of several proteins. Therefore chapter III introduces a novel approach to control proteome labeling as a function of multiple promoters using a genetically encoded AND gate based on a bisected methionyl-tRNA synthetase, a class I aaRS. Cellular protein labeling occurs only upon activation of two different promoters that drive expression of the N- and C-terminal fragments of this bisected aaRS. The utility of this tool is demonstrated by the selective labeling of proteins in subpopulations of bacterial cells in a laminar-flow microfluidic channel.
\r\n \r\nChapter IV extends the cell-selective incorporation of non-canonical amino acids from bacterial systems to mammalian cells by introducing a mutant mammalian methionyl-tRNA synthetase for cell-targeted proteome labeling. This enzyme is genetically encoded and can be conditionally activated for time-resolved and cell-targeted proteome analysis in a variety of different mammalian cell types. Chapter V uses this enzyme for lineage-specific proteomic analysis of mouse embryonic stem cells during differentiation to cardiac and mesoderm lineages. This approach for lineage-specific protein labeling enables the unbiased and comprehensive analysis of proteomic changes that occur during stem cell differentiation and cell-fate commitment.
\r\n \r\nAppendices A-G provide brief summaries of publications and research efforts during my PhD that are not directly related to this thesis. These publications are the result of a number of collaborations that I have been fortunate to be involved with during my graduate research.
\r\n\r\nThe technologies and methods introduced in this thesis provide versatile tools for the comprehensive and unbiased detection and identification of newly synthesized proteins in complex multicellular systems. Time-resolved, genetically encoded, and spatially defined non-canonical amino acid incorporation enables the identification of proteins involved in cell-cell interactions and the proteins made during specific cell states.
\r\n", "doi": "10.7907/Z9W66HS4", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8724", "collection": "thesis", "collection_id": "8724", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11172014-000718184", "type": "thesis", "title": "The Structure of Hippocampal Activity During REM Sleep", "author": [ { "family_name": "Hoenselaar", "given_name": "Andreas", "clpid": "Hoenselaar-Andreas" } ], "thesis_advisor": [ { "family_name": "Siapas", "given_name": "Athanassios G.", "clpid": "Siapas-A-G" } ], "thesis_committee": [ { "family_name": "Perona", "given_name": "Pietro", "clpid": "Perona-P" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Roukes", "given_name": "Michael Lee", "clpid": "Roukes-M-L" }, { "family_name": "Dickinson", "given_name": "Michael H.", "clpid": "Dickinson-M-H" }, { "family_name": "Siapas", "given_name": "Athanassios G.", "clpid": "Siapas-A-G" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "The hippocampus is a brain structure critical for the formation of long-term episodic memories. The current predominant theory is that memories are gradually established across neocortical networks under the influence of hippocampal activity. This process of memory consolidation is conjectured to occur during sleep, which is characterized by two different modes of activation: slow-wave sleep (SWS) and rapid eye movement (REM) sleep. The functional roles of these two different sleep states remain unknown. Paradoxically, REM sleep exhibits the main features of awake activity, and is the stage of sleep when most dreams occur. Despite decades of study, the organization and function of REM sleep activity remains poorly understood. The goal of this thesis is to achieve a deeper quantitative understanding of the patterns of firing in area CA1 of the hippocampus during REM sleep using chronic multi-tetrode recordings from freely behaving and naturally sleeping rats. Our analysis shows that CA1 neurons significantly elevate their firing rate for periods that are short in relation to the duration of the REM sleep episode. Furthermore, for the majority of neurons, there is exactly one such burst per REM episode. This leads to lower overall firing rates and sparser population activity in CA1 compared to SWS. The time of onset of these bursts defines a natural order of firing across the population of recorded neurons within each REM episode. We demonstrate that this order does not repeat across REM episodes. Our results suggest that CA1 neurons are activated in random sequences across REM episodes, resulting in sparse patterns with only a small fraction of neurons active at any given time.", "doi": "10.7907/Z98W3B8H", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8021", "collection": "thesis", "collection_id": "8021", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10312013-102814054", "type": "thesis", "title": "Experimental and Theoretical Studies of Notch Signaling-Mediated Spatial Pattern", "author": [ { "family_name": "Lakhanpal", "given_name": "Amit", "clpid": "Lakhanpal-Amit" } ], "thesis_advisor": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Hay", "given_name": "Bruce A.", "orcid": "0000-0002-5486-0482", "clpid": "Hay-B-A" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_bbe" } ], "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.", "doi": "10.7907/RGPT-RS80", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8068", "collection": "thesis", "collection_id": "8068", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02112014-133121063", "primary_object_url": { "basename": "Thesis_HidehikoInagaki.pdf", "content": "final", "filesize": 21946509, "license": "other", "mime_type": "application/pdf", "url": "/8068/19/Thesis_HidehikoInagaki.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Neuronal Mechanism of State Control in Drosophila melanogaster", "author": [ { "family_name": "Inagaki", "given_name": "Hidehiko K.", "clpid": "Inagaki-Hidehiko-K" } ], "thesis_advisor": [ { "family_name": "Anderson", "given_name": "David J.", "clpid": "Anderson-D-J" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Zinn", "given_name": "Kai George", "clpid": "Zinn-K-G" }, { "family_name": "Prober", "given_name": "David A.", "clpid": "Prober-D-A" }, { "family_name": "Anderson", "given_name": "David J.", "clpid": "Anderson-D-J" } ], "local_group": [ { "literal": "div_bbe" } ], "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", "doi": "10.7907/MPZX-TN59", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8225", "collection": "thesis", "collection_id": "8225", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05072014-160207088", "primary_object_url": { "basename": "OC Loson Thesis 2014.pdf", "content": "final", "filesize": 6805019, "license": "other", "mime_type": "application/pdf", "url": "/8225/1/OC Loson Thesis 2014.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Regulation of Mitochondrial Division by the Drp1 Receptors", "author": [ { "family_name": "Loson", "given_name": "Oliver Calvin", "clpid": "Loson-Oliver-Calvin" } ], "thesis_advisor": [ { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" } ], "local_group": [ { "literal": "div_bbe" } ], "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", "doi": "10.7907/J23G-KQ18", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8252", "collection": "thesis", "collection_id": "8252", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05212014-094412950", "primary_object_url": { "basename": "2014-05-09_LeBon.pdf", "content": "final", "filesize": 12937954, "license": "other", "mime_type": "application/pdf", "url": "/8252/1/2014-05-09_LeBon.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "The Logic of Receptor-Ligand Interactions in the Notch Signaling Pathway", "author": [ { "family_name": "LeBon", "given_name": "Lauren E.", "clpid": "LeBon-Lauren-E" } ], "thesis_advisor": [ { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" } ], "thesis_committee": [ { "family_name": "Hay", "given_name": "Bruce A.", "clpid": "Hay-B-A" }, { "family_name": "Weinmaster", "given_name": "Geraldine", "clpid": "Weinmaster-G" }, { "family_name": "Anderson", "given_name": "David J.", "clpid": "Anderson-D-J" }, { "family_name": "Goentoro", "given_name": "Lea A.", "clpid": "Goentoro-L-A" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "The Notch signaling pathway enables neighboring cells to coordinate developmental fates in diverse processes such as angiogenesis, neuronal differentiation, and immune system development. Although key components and interactions in the Notch pathway are known, it remains unclear how they work together to determine a cell's signaling state, defined as its quantitative ability to send and receive signals using particular Notch receptors and ligands. Recent work suggests that several aspects of the system can lead to complex signaling behaviors: First, receptors and ligands interact in two distinct ways, inhibiting each other in the same cell (in cis) while productively interacting between cells (in trans) to signal. The ability of a cell to send or receive signals depends strongly on both types of interactions. Second, mammals have multiple types of receptors and ligands, which interact with different strengths, and are frequently co-expressed in natural systems. Third, the three mammalian Fringe proteins can modify receptor-ligand interaction strengths in distinct and ligand-specific ways. Consequently, cells can exhibit non-intuitive signaling states even with relatively few components.
\r\n\r\nIn order to understand what signaling states occur in natural processes, and what types of signaling behaviors they enable, this thesis puts forward a quantitative and predictive model of how the Notch signaling state is determined by the expression levels of receptors, ligands, and Fringe proteins. To specify the parameters of the model, we constructed a set of cell lines that allow control of ligand and Fringe expression level, and readout of the resulting Notch activity. We subjected these cell lines to an assay to quantitatively assess the levels of Notch ligands and receptors on the surface of individual cells. We further analyzed the dependence of these interactions on the level and type of Fringe expression. We developed a mathematical modeling framework that uses these data to predict the signaling states of individual cells from component expression levels. These methods allow us to reconstitute and analyze a diverse set of Notch signaling configurations from the bottom up, and provide a comprehensive view of the signaling repertoire of this major signaling pathway.
", "doi": "10.7907/83PA-9833", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8434", "collection": "thesis", "collection_id": "8434", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292014-234047875", "primary_object_url": { "basename": "TanFEH-2014-Thesis 2.pdf", "content": "final", "filesize": 2288705, "license": "other", "mime_type": "application/pdf", "url": "/8434/1/TanFEH-2014-Thesis 2.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Brfl Post-Transcriptionally Regulates Pluripotency and Differentiation Responses Downstream of Erk MAP Kinase", "author": [ { "family_name": "Tan", "given_name": "Frederick Eng How", "clpid": "Tan-Frederick-Eng-How" } ], "thesis_advisor": [ { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" } ], "thesis_committee": [ { "family_name": "Stathopoulos", "given_name": "Angelike", "clpid": "Stathopoulos-A" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" }, { "family_name": "Plath", "given_name": "Kathrin", "clpid": "Plath-K" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "FGF/Erk MAP Kinase Signaling is a central regulator of mouse embryonic stem cell (mESC) self-renewal, pluripotency and differentiation. However, the mechanistic connection between this signaling pathway activity and the gene circuits stabilizing mESCs in vitro remain unclear. Here we show that FGF signaling post-transcriptionally regulates the mESC transcription factor network by controlling the expression of Brf1 (zfp36l1), an AU-rich element mRNA binding protein. Changes in Brf1 level disrupts the expression of core pluripotency-associated genes and attenuates mESC self-renewal without inducing differentiation. These regulatory effects are mediated by rapid and direct destabilization of Brf1 targets, such as Nanog mRNA. Interestingly, enhancing Brf1 expression does not compromise mESC pluripotency, but does preferentially regulate differentiation to mesendoderm by accelerating the expression of primitive streak markers. Together, these studies demonstrate that FGF signals utilize targeted mRNA degradation by Brf1 to enable rapid post-transcriptional control of gene expression.", "doi": "10.7907/MM53-XC23", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8467", "collection": "thesis", "collection_id": "8467", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022014-165211576", "primary_object_url": { "basename": "thesis.pdf", "content": "final", "filesize": 22394380, "license": "other", "mime_type": "application/pdf", "url": "/8467/1/thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Sequence-Function Relationships in E. coli Transcriptional Regulation", "author": [ { "family_name": "Jones", "given_name": "Daniel Lawson III", "clpid": "Jones-Daniel-Lawson-III" } ], "thesis_advisor": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "thesis_committee": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Roukes", "given_name": "Michael Lee", "orcid": "0000-0002-2916-6026", "clpid": "Roukes-M-L" }, { "family_name": "Winfree", "given_name": "Erik", "orcid": "0000-0002-5899-7523", "clpid": "Winfree-E" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Understanding how transcriptional regulatory sequence maps to regulatory function remains a difficult problem in regulatory biology. Given a particular DNA sequence for a bacterial promoter region, we would like to be able to say which transcription factors bind there, how strongly they bind, and whether they interact with each other and/or RNA polymerase, with the ultimate objective of integrating knowledge of these parameters into a prediction of gene expression levels. The theoretical framework of statistical thermodynamics provides a useful framework for doing so, enabling us to predict how gene expression levels depend on transcription factor binding energies and concentrations. We used thermodynamic models, coupled with models of the sequence-dependent binding energies of transcription factors and RNAP, to construct a genotype to phenotype map for the level of repression exhibited by the lac promoter, and tested it experimentally using a set of promoter variants from E. coli strains isolated from different natural environments. For this work, we sought to ``reverse engineer'' naturally occurring promoter sequences to understand how variations in promoter sequence affects gene expression. The natural inverse of this approach is to ``forward engineer'' promoter sequences to obtain targeted levels of gene expression. We used a high precision model of RNAP-DNA sequence dependent binding energy, coupled with a thermodynamic model relating binding energy to gene expression, to predictively design and verify a suite of synthetic E. coli promoters whose expression varied over nearly three orders of magnitude.
\r\n\r\nHowever, although thermodynamic models enable predictions of mean levels of gene expression, it has become evident that cell-to-cell variability or ``noise'' in gene expression can also play a biologically important role. In order to address this aspect of gene regulation, we developed models based on the chemical master equation framework and used them to explore the noise properties of a number of common E. coli regulatory motifs; these properties included the dependence of the noise on parameters such as transcription factor binding strength and copy number. We then performed experiments in which these parameters were systematically varied and measured the level of variability using mRNA FISH. The results showed a clear dependence of the noise on these parameters, in accord with model predictions.
\r\n\r\nFinally, one shortcoming of the preceding modeling frameworks is that their applicability is largely limited to systems that are already well-characterized, such as the lac promoter. Motivated by this fact, we used a high throughput promoter mutagenesis assay called Sort-Seq to explore the completely uncharacterized transcriptional regulatory DNA of the E. coli mechanosensitive channel of large conductance (MscL). We identified several candidate transcription factor binding sites, and work is continuing to identify the associated proteins.
", "doi": "10.7907/4J7V-WD59", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:7234", "collection": "thesis", "collection_id": "7234", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10102012-101657790", "primary_object_url": { "basename": "Zheng_Guoan_Final_Thesis_12_17_2012.pdf", "content": "final", "filesize": 20784687, "license": "other", "mime_type": "application/pdf", "url": "/7234/4/Zheng_Guoan_Final_Thesis_12_17_2012.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Innovations in Imaging System Design: Gigapixel, Chip-Scale and MultiFunctional Microscopy", "author": [ { "family_name": "Zheng", "given_name": "Guoan", "clpid": "Zheng-Guoan" } ], "thesis_advisor": [ { "family_name": "Yang", "given_name": "Changhuei", "clpid": "Yang-Changhuei" } ], "thesis_committee": [ { "family_name": "Yang", "given_name": "Changhuei", "clpid": "Yang-Changhuei" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" }, { "family_name": "Choo", "given_name": "Hyuck", "clpid": "Choo-Hyuck" }, { "family_name": "Tai", "given_name": "Yu-Chong", "clpid": "Tai-Yu-Chong" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Microscopy imaging is of fundamental importance in diverse disciplines of science and technology. In a typical microscopy imaging platform, the light path can be generalized to the following steps: photons leave the light source, interact with the sample, and finally are detected by the image sensor. Based on such a light path, this thesis presents several new microscopy imaging techniques from three aspects: illumination design, sample manipulation, and imager modification.
\r\n \r\nThe first design strategy involves the active control of the illumination sources. Based on this strategy, we demonstrate a simple and cost-effective imaging method, termed Non-interferometric Aperture-synthesizing Microscopy (NAM), for breaking the spatial-bandwidth product barrier of a conventional microscope. We show that the NAM method is capable of providing two orders of magnitude higher throughput for most existing bright-field microscopes without involving any mechanical scanning. Based on NAM, we report the implementation of a 1.6 gigapixel microscope with a maximum numerical aperture of 0.5, a field-of-view of 120 mm2, and a resolution-invariant imaging depth of 0.3 mm. This platform is fast (acquisition time of ~ 3 minutes), free from chromatic aberration, capable for phase imaging, and, most importantly, compatible with most existing microscopes. High quality color images of histology slides were acquired by using such a platform for demonstration. The proposed NAM method provides a robust way to transform the problem of high-throughput microscopy from one that is tied to physical limitations of the optics to one that is computationally solvable. The active control of illumination sources can also be adapted for chip-scale microscopy imaging. To this end, we present a lensless microscopy solution termed ePetri-dish. This ePetri-dish platform can automatically perform high resolution (~ 0.66 micron) microscopy imaging over a large field-of-view (6 mm \u00d7 4 mm). This new approach is fully capable of working with cell cultures or any samples in which cells/bacteria may be contiguously connected, and thus, it can significantly improve Petri-dish-based cell/bacteria culture experiments. With this approach providing a low-cost and disposable microscopy solution, we can start to transit Petri-dish-based experiments from the traditionally labor-intensive process to an automated and streamlined process.
\r\n\r\nThe second strategy in design considerations is to manipulate the sample. We present a fully on-chip, lensless, sub-pixel resolving optofluidic microscope (SROFM). This device utilizes microfluidic flow to deliver specimens directly across an image sensor to generate a sequence of low-resolution projection images, where resolution is limited by the sensor\u2019s pixel size. This image sequence is then processed to reconstruct a single high-resolution image, where features beyond the Nyquist rate of the LR images are resolved. We demonstrate the device\u2019s capabilities by imaging microspheres, protist Euglena gracilis, and Entamoeba invadens cysts with sub-cellular resolution.
\r\n\r\nThe third accessing point in design considerations is the image sensor. Imager modification is an emerging technique that performs pre-detection light field manipulation. We present two novel optical structure designs: surface-wave-enabled darkfield aperture (SWEDA) and light field sensor. These structures can be directly incorporated onto optical sensors to accomplish pre-detection background suppression and wavefront sensing. We further demonstrate SWEDA\u2019s ability to boost the detection sensitivity, with a contrast enhancement of 27 dB.
\r\n", "doi": "10.7907/SF6E-S775", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7773", "collection": "thesis", "collection_id": "7773", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05302013-135915010", "primary_object_url": { "basename": "Final_submitted_thesis.pdf", "content": "final", "filesize": 41849563, "license": "other", "mime_type": "application/pdf", "url": "/7773/1/Final_submitted_thesis.pdf", "version": "v11.0.0" }, "type": "thesis", "title": "Chemical and Neural Regulation of Embryonic Branching Morphogenesis", "author": [ { "family_name": "Bower", "given_name": "Danielle Vera Brown", "clpid": "Bower-Danielle-Vera-Brown" } ], "thesis_advisor": [ { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" } ], "thesis_committee": [ { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "family_name": "Warburton", "given_name": "David", "clpid": "Warburton-D" }, { "family_name": "Zinn", "given_name": "Kai George", "clpid": "Zinn-K-G" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" } ], "local_group": [ { "literal": "div_biol" } ], "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.
", "doi": "10.7907/Z9SX6B6R", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7874", "collection": "thesis", "collection_id": "7874", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06072013-170244348", "primary_object_url": { "basename": "Sternberg_Jonathan_2013_thesis.pdf", "content": "final", "filesize": 63369791, "license": "other", "mime_type": "application/pdf", "url": "/7874/1/Sternberg_Jonathan_2013_thesis.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Signal Transduction with Hybridization Chain Reactions", "author": [ { "family_name": "Sternberg", "given_name": "Jonathan Ben-Zion", "clpid": "Sternberg-Jonathan-Ben-Zion" } ], "thesis_advisor": [ { "family_name": "Pierce", "given_name": "Niles/ A", "clpid": "Pierce-N-A" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Pierce", "given_name": "Niles A.", "clpid": "Pierce-N-A" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Some of the most exciting developments in the field of nucleic acid engineering include the utilization of synthetic nucleic acid molecular devices as gene regulators, as disease marker detectors, and most recently, as therapeutic agents. The common thread between these technologies is their reliance on the detection of specific nucleic acid input markers to generate some desirable output, such as a change in the copy number of an mRNA (for gene regulation), a change in the emitted light intensity (for some diagnostics), and a change in cell state within an organism (for therapeutics). The research presented in this thesis likewise focuses on engineering molecular tools that detect specific nucleic acid inputs, and respond with useful outputs.
\r\n\r\nFour contributions to the field of nucleic acid engineering are presented: (1) the construction of a single nucleotide polymorphism (SNP) detector based on the mechanism of hybridization chain reaction (HCR); (2) the utilization of a single-stranded oligonucleotide molecular Scavenger as a means of enhancing HCR selectivity; (3) the implementation of Quenched HCR, a technique that facilitates transduction of a nucleic acid chemical input into an optical (light) output, and (4) the engineering of conditional probes that function as sequence transducers, receiving target signal as input and providing a sequence of choice as output. These programmable molecular systems are conceptually well-suited for performing wash-free, highly selective rapid genotyping and expression profiling in vitro, in situ, and potentially in living cells.
", "doi": "10.7907/Z90Z719Z", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7863", "collection": "thesis", "collection_id": "7863", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06072013-095239958", "primary_object_url": { "basename": "Venturelli-O-S-2013-thesis.pdf", "content": "final", "filesize": 15828338, "license": "other", "mime_type": "application/pdf", "url": "/7863/13/Venturelli-O-S-2013-thesis.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Role of Feedback and Dynamics of a Gene Regulatory Network", "author": [ { "family_name": "Venturelli", "given_name": "Ophelia Shalini", "clpid": "Venturelli-Ophelia-Shalini" } ], "thesis_advisor": [ { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" } ], "thesis_committee": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Deshaies", "given_name": "Raymond Joseph", "orcid": "0000-0002-3671-9354", "clpid": "Deshaies-R-J" }, { "family_name": "El-Samad", "given_name": "Hana", "clpid": "El-Samad-H" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" } ], "local_group": [ { "literal": "div_biol" } ], "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", "doi": "10.7907/WGK3-Y839", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7088", "collection": "thesis", "collection_id": "7088", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292012-120719001", "primary_object_url": { "basename": "young_jw.pdf", "content": "final", "filesize": 7339570, "license": "other", "mime_type": "application/pdf", "url": "/7088/1/young_jw.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Architecture, Dynamics, and Function of the General Stress Response System in B. subtilis", "author": [ { "family_name": "Young", "given_name": "Jonathan Wan", "clpid": "Young-Jonathan-Wan" } ], "thesis_advisor": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Fraser", "given_name": "Scott E.", "orcid": "0000-0002-5377-0223", "clpid": "Fraser-S-E" } ], "local_group": [ { "literal": "div_biol" } ], "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.", "doi": "10.7907/93MF-Q808", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:7146", "collection": "thesis", "collection_id": "7146", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06072012-150636005", "primary_object_url": { "basename": "Fullthesis-KEGALLOWAY-FINAL.pdf", "content": "final", "filesize": 12596632, "license": "other", "mime_type": "application/pdf", "url": "/7146/20/Fullthesis-KEGALLOWAY-FINAL.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "The Development of RNA-based Control Systems to Regulate Signaling and Dictate Cell Fate in a Model MAPK Pathway", "author": [ { "family_name": "Galloway", "given_name": "Kate Elizabeth", "clpid": "Galloway-Kate-Elizabeth" } ], "thesis_advisor": [ { "family_name": "Smolke", "given_name": "Christina D.", "clpid": "Smolke-C-D" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Murray", "given_name": "Richard M.", "clpid": "Murray-R-M" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Smolke", "given_name": "Christina D.", "clpid": "Smolke-C-D" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Cells integrate extracellular information via native signaling pathways to spatially and temporally coordinate complex tasks such as development and the immune response. Cellular programming holds the potential of harnessing the sophisticated and complex biological processes of living cells for diverse applications. In the last decade, cellular reprogramming has emerged as a viable therapeutic strategy. In large, reprogramming strategies have relied on statically programmed levels of gene expression to alter cellular behaviors. To construct more sophisticated programs requires dynamic control of expression and strategies for the facile construction of complex control architectures. Additionally, the application of synthetic programs to the control of native regulatory pathways requires the development of tools for interfacing with these pathways, as well as the construction of stringent controllers. Further, control systems composed of modular and tunable elements will facilitate the expansion of synthetic circuitry to a wide array of natural networks with varying system properties.
\r\n \r\nHere we describe the development of RNA-based control systems to regulate signaling and dictate cell fate in a model mitogen-activated protein kinase (MAPK) pathway. We construct networks of RNA-based control systems that interface with the Saccharomyces cerevisiae mating pathway to dictate entry into one of three programmed alternative fates dependent on environmental stimuli. We present a readily translatable method for identifying control points within natural networks that enable the construction of a modular interface between synthetic circuitry and native networks. In building these networks, we demonstrate the rational tuning of circuit performance via the exchange of well-defined parts to compose networks capable of actuating changes in cellular behavior in response to environmental cues. Further, we construct network architectures which facilitate reduced interference from simultaneously integrated opposing programs and identified sensitive parameters for engineering robust circuit performance. Finally, we present the development of a novel RNA-based control element for the regulation of both synthetic and endogenous transcripts. This work provides a model for engineering systems that regulate signaling and direct cell fate which may be applied to additional decision-making pathways to advance tissue engineering strategies, treat diseases, and study the behavior of natural regulatory networks.
\r\n", "doi": "10.7907/CBBN-GX03", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:7096", "collection": "thesis", "collection_id": "7096", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05302012-122930768", "primary_object_url": { "basename": "master.pdf", "content": "final", "filesize": 10961399, "license": "other", "mime_type": "application/pdf", "url": "/7096/1/master.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "I. Quantal Effects in Biochemical Cooperativity and a Proposed Mechanism for the Differentiation of Calcium Signaling in Synaptic Plasticity. II. Evolutionary Algorithms for the Optimization of Methods in Computational Chemistry", "author": [ { "family_name": "Ford", "given_name": "William Chastang", "clpid": "Ford-William-Chastang" } ], "thesis_advisor": [ { "family_name": "Hoffmann", "given_name": "Michael R.", "clpid": "Hoffmann-M-R" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Abrol", "given_name": "Ravinder", "clpid": "Abrol-R" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" }, { "family_name": "Hoffmann", "given_name": "Michael R.", "clpid": "Hoffmann-M-R" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "In Part 1 of this thesis, we propose that biochemical cooperativity is a fundamentally non-ideal process. We show quantal effects underlying biochemical cooperativity and highlight apparent ergodic breaking at small volumes. The apparent ergodic breaking manifests itself in a divergence of deterministic and stochastic models. We further predict that this divergence of deterministic and stochastic results is a failure of the deterministic methods rather than an issue of stochastic simulations.
\r\n\r\nErgodic breaking at small volumes may allow these molecular complexes to function as switches to a greater degree than has previously been shown. We propose that this ergodic breaking is a phenomenon that the synapse might exploit to differentiate Ca2+ signaling that would lead to either the strengthening or weakening of a synapse. Techniques such as lattice-based statistics and rule-based modeling are tools that allow us to directly confront this non-ideality. A natural next step to understanding the chemical physics that underlies these processes is to consider in silico specifically atomistic simulation methods that might augment our modeling efforts.
\r\n\r\nIn the second part of this thesis, we use evolutionary algorithms to optimize in silico methods that might be used to describe biochemical processes at the subcellular and molecular levels. While we have applied evolutionary algorithms to several methods, this thesis will focus on the optimization of charge equilibration methods. Accurate charges are essential to understanding the electrostatic interactions that are involved in ligand binding, as frequently discussed in the first part of this thesis.
", "doi": "10.7907/Z9HH6H1Z", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:7135", "collection": "thesis", "collection_id": "7135", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06052012-121432503", "primary_object_url": { "basename": "jhl_thesis.pdf", "content": "final", "filesize": 19139044, "license": "other", "mime_type": "application/pdf", "url": "/7135/1/jhl_thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Genetic Regulatory Circuit Dynamics: Analysis and Synthesis", "author": [ { "family_name": "Levine", "given_name": "Joseph H.", "clpid": "Levine-Joseph-H" } ], "thesis_advisor": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "thesis_committee": [ { "family_name": "Winfree", "given_name": "Erik", "orcid": "0000-0002-5899-7523", "clpid": "Winfree-E" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Fraser", "given_name": "Scott E.", "orcid": "0000-0002-5377-0223", "clpid": "Fraser-S-E" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "Kavli Nanoscience Institute" }, { "literal": "div_eng" } ], "abstract": "How can cells shape and utilize dynamic gene regulation to enable complex cellular behaviors? I study this question in natural and synthetic contexts.
\r\n\r\nThe first project studies how a natural genetic network can imbue cells with a sense of \u2018time\u2019. It has long been known that environmental signals induce diverse cellular differentiation programs. In certain systems, cells defer differentiation for extended time periods after the signal appears, proliferating through multiple rounds of cell division before committing to a new fate. How can cells set a deferral time much longer than the cell cycle? Here we study Bacillus subtilis cells that respond to sudden nutrient limitation with multiple rounds of growth and division before differentiating into spores. A well characterized genetic circuit controls the concentration and phosphorylation of the master regulator Spo0A, which rises to a critical concentration to initiate sporulation. However, it remains unclear how this circuit enables cells to defer sporulation for multiple cell cycles. Using quantitative time-lapse fluorescence microscopy of Spo0A dynamics in individual cells, we observed pulses of Spo0A phosphorylation at a characteristic cell cycle phase. Pulse amplitudes grew systematically and cell-autonomously over multiple cell cycles leading up to sporulation. This pulse growth required a key positive feedback loop involving the sporulation kinases, without which the deferral of sporulation became ultrasensitive to kinase expression. Thus, deferral is controlled by a pulsed positive feedback loop in which kinase expression is activated by pulses of Spo0A phosphorylation. This pulsed positive feedback architecture provides a more robust mechanism for setting deferral times than constitutive kinase expression. Finally, using mathematical modeling, we show how pulsing and time delays together enable \u2018polyphasic\u2019 positive feedback, in which different parts of a feedback loop are active at different times. Polyphasic feedback can enable more accurate tuning of long deferral times. Together, these results suggest that Bacillus subtilis uses a pulsed positive feedback loop to implement a timer that operates over time scales much longer than a cell cycle.
\r\n\r\nThe second project proposes a method to rapidly generate and test complex genetic network dynamics in living cells. Existing microorganisms have evolved genetic circuitry to meet diverse challenges and maximize their survival and fitness. These challenges arise from external environmental pressures, or internal evolved constraints. Furthermore, these challenges may be either static or dynamic in nature. While existing circuits have likely evolved to be \u2018good enough\u2019 to respond to historical challenges, it remains unclear if they can be improved upon, and whether they respond well to novel situations. Synthetic biology seeks to engineer organisms with complex novel phenotypes, both to harness these novel organisms for a function and to understand their underlying biology. Dynamic gene expression strategies may be necessary to successfully generate these phenotypes. Unfortunately, generating novel dynamic gene expression patterns with conventional genetic engineering remains a challenge. Here I propose and describe progress towards a computerized feedback control setup to enable the programming and rapid testing of dynamic gene regulatory patterns in living cells. Small sets of genes will be regulated optogenetically based on programmed control laws, and past and present cellular state. This setup will enable us to explore the functions and limits of engineered dynamic gene regulation, while hopefully, in the process, providing lessons about the underlying biology.
", "doi": "10.7907/35A7-K421", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:6502", "collection": "thesis", "collection_id": "6502", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06062011-122322347", "type": "thesis", "title": "Non-Equilibrium Dynamics of DNA Nanotubes ", "author": [ { "family_name": "Hariadi", "given_name": "Rizal Fajar", "clpid": "Hariadi-Rizal-Fajar" } ], "thesis_advisor": [ { "family_name": "Winfree", "given_name": "Erik", "orcid": "0000-0002-5899-7523", "clpid": "Winfree-E" } ], "thesis_committee": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Fraser", "given_name": "Scott E.", "orcid": "0000-0002-5377-0223", "clpid": "Fraser-S-E" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Yurke", "given_name": "Bernard", "clpid": "Yurke-B" }, { "family_name": "Winfree", "given_name": "Erik", "orcid": "0000-0002-5899-7523", "clpid": "Winfree-E" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Can the fundamental processes that underlie molecular biology be understood and simulated by DNA nanotechnology? The early development of DNA nanotechnology by Ned Seeman was driven by the desire to find a solution to the protein crystallization problem. Much of the later development of the field was also driven by envisioned applications in computing and nanofabrication. While the DNA nanotechnology community has assembled a versatile tool kit with which DNA nanostructures of considerable complexity can be assembled, the application of this tool kit to other areas of science and technology is still in its infancy. This dissertation reports on the construction of non-equilibrium DNA nanotube dynamic to probe molecular processes in the areas of hydrodynamics and cytoskeletal behavior.
\r\n\r\nAs the first example, we used DNA nanotubes as a molecular probe for elongational flow measurement in different micro-scale flow settings. The hydrodynamic flow in the vicinity of simple geometrical objects, such as a rigid DNA nanotube, is amenable to rigorous theoretical investigation. We measured the distribution of elongational flows produced in progressively more complex settings, ranging from the vicinity of an orifice in a microfluidic chamber to within a bursting bubble of Pacific ocean water. This information can be used to constrain theories on the origin of life in which replication involves a hydrodynamically driven fission process, such as the coacervate fission proposed by Oparin.
\r\n\r\nA second theme of this dissertation is the bottom-up construction of a de novo artificial cytoskeleton with DNA nanotubes. The work reported here encompasses structural, locomotion, and control aspects of non-equilibrium cytoskeletal behavior. We first measured the kinetic parameters of DNA nanotube assembly and tested the accuracy of the existing polymerization models in the literature. Toward recapitulation of non-equilibrium cytoskeletal dynamics, we coupled the polymerization of DNA nanotubes with an irreversible energy consumption reaction, analogous to nucleotide hydrolysis in actin and microtubule polymerization. Finally, we integrated the DNA strand displacement circuits with DNA nanotube polymerization to achieve programmable kinetic control of behavior within artificial cytoskeleton. Our synthetic approach may provide insights into natural cytoskeleton dynamics, such as minimal architectural or reaction mechanism requirements for non-equilibrium behaviors including treadmilling and dynamic instability.
\r\n\r\nThe outgrowth of DNA nanotechnology beyond its own boundaries, serving as a general model system for biomolecular dynamics, can lead to an understanding of molecular processes that advances both basic and applied sciences.
", "doi": "10.7907/6GQW-YG26", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:6153", "collection": "thesis", "collection_id": "6153", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10212010-102536869", "primary_object_url": { "basename": "KuntzThesis.pdf", "content": "final", "filesize": 10515789, "license": "other", "mime_type": "application/pdf", "url": "/6153/1/KuntzThesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "hlh-1 and the C. elegans Body Wall Muscle Transcriptional Differentiation Network", "author": [ { "family_name": "Kuntz", "given_name": "Steven Gregory", "clpid": "Kuntz-Steven-Gregory" } ], "thesis_advisor": [ { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" }, { "family_name": "Stathopoulos", "given_name": "Angelike", "clpid": "Stathopoulos-A" }, { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "local_group": [ { "literal": "div_biol" } ], "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. ", "doi": "10.7907/18XS-YM65", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:6443", "collection": "thesis", "collection_id": "6443", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05252011-222115269", "primary_object_url": { "basename": "ssen_complete_thesis.pdf", "content": "final", "filesize": 2949050, "license": "other", "mime_type": "application/pdf", "url": "/6443/1/ssen_complete_thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Regulatory Consequences of Bandpass Feedback in a Bacterial Phosphorelay", "author": [ { "family_name": "Sen", "given_name": "Shaunak", "orcid": "0000-0002-1412-8633", "clpid": "Sen-Shaunak" } ], "thesis_advisor": [ { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Murray", "given_name": "Richard M.", "clpid": "Murray-R-M" }, { "family_name": "Doyle", "given_name": "John Comstock", "clpid": "Doyle-J-C" }, { "family_name": "Garcia-Ojalvo", "given_name": "Jordi", "clpid": "Garcia-Ojalvo-J" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Under conditions of nutrient limitation, Bacillus subtilis cells terminally differentiate into a dormant spore state. Progression to sporulation is controlled by a genetic circuit structured as a phosphorelay embedded in multiple transcriptional feedback loops, and which is used to activate the master regulator Spo0A by phosphorylation. These transcriptional regulatory interactions are 'bandpass'-like, in the sense that activation occurs within a limited band of Spo0A~P concentrations, and have recently been shown to pulse in a cell-cycle-dependent fashion. Additionally, the core phosphorelay is an architectural variant of the canonical two-component signaling system, which allows signal integration from a larger number of inputs, including two types of phosphatases that act on different protein components. However, the impact of these pulsed bandpass interactions on the circuit dynamics preceding sporulation and the utility of two types of phosphatases remains unclear. In order to address these questions, we measured key features of the bandpass interactions at the single-cell level and analyzed them in the context of a simple mathematical model. The model predicted the emergence of a delayed phase shift between the pulsing activity of the different sporulation genes, as well as the existence of a stable state, with elevated Spo0A activity but no sporulation, embedded within the dynamical structure of the system. To test the model, we used time-lapse fluorescence microscopy to measure dynamics of single cells initiating sporulation. We observed the delayed phase shift emerging during the progression to sporulation, while a re-engineering of the sporulation circuit revealed behavior resembling the predicted additional state. The core phosphorelay model also showed a post-translational bandpass response, and we find that the two types of phosphatases can independently tune the two bandpass thresholds. These results show that periodically-driven bandpass feedback loops can give rise to complex dynamics in the progression towards sporulation, and that similar inputs can tune different response features. ", "doi": "10.7907/NPZD-G382", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:11763", "collection": "thesis", "collection_id": "11763", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05312011-123357339", "primary_object_url": { "basename": "CEWard-ThesisDone.pdf", "content": "final", "filesize": 3810119, "license": "other", "mime_type": "application/pdf", "url": "/11763/1/CEWard-ThesisDone.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Medea Selfish Genetic Elements as Tools for Altering Traits of Wild Populations: A Theoretical Analysis", "author": [ { "family_name": "Ward", "given_name": "Catherine Marie", "clpid": "Ward-Catherine-Marie" } ], "thesis_advisor": [ { "family_name": "Hay", "given_name": "Bruce", "clpid": "Hay-B-A" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Murray", "given_name": "Richard M.", "clpid": "Murray-R-M" }, { "family_name": "Hay", "given_name": "Bruce A.", "clpid": "Hay-B-A" } ], "local_group": [ { "literal": "div_biol" } ], "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.", "doi": "10.7907/T656-ZN91", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:6356", "collection": "thesis", "collection_id": "6356", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04272011-021639361", "primary_object_url": { "basename": "Corrected_thesis_complete.pdf", "content": "updated", "filesize": 4943557, "license": "other", "mime_type": "application/pdf", "url": "/6356/9/Corrected_thesis_complete.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Dynamic Regulation of the Dauer Decision", "author": [ { "family_name": "Schaedel", "given_name": "Oren Noah", "clpid": "Schaedel-Oren-Noah" } ], "thesis_advisor": [ { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" }, { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" }, { "family_name": "Guo", "given_name": "Chin-Lin", "clpid": "Guo-Chin-Lin" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "local_group": [ { "literal": "div_biol" } ], "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", "doi": "10.7907/JZ9E-AD68", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:6212", "collection": "thesis", "collection_id": "6212", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12162010-123122193", "primary_object_url": { "basename": "HernanGarciaThesis2010-12-15HG.pdf", "content": "final", "filesize": 53906433, "license": "other", "mime_type": "application/pdf", "url": "/6212/1/HernanGarciaThesis2010-12-15HG.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Transcriptional Regulation by the Numbers", "author": [ { "family_name": "Garcia", "given_name": "Hernan G.", "clpid": "Garcia-Hernan-G" } ], "thesis_advisor": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "thesis_committee": [ { "family_name": "Politzer", "given_name": "Hugh David", "orcid": "0000-0002-4983-6621", "clpid": "Politzer-H-D" }, { "family_name": "Roukes", "given_name": "Michael Lee", "orcid": "0000-0002-2916-6026", "clpid": "Roukes-M-L" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Fraser", "given_name": "Scott E.", "orcid": "0000-0002-5377-0223", "clpid": "Fraser-S-E" }, { "family_name": "Widom", "given_name": "Jonathan", "clpid": "Widom-Jonathan" }, { "family_name": "Weiss", "given_name": "Shimon", "clpid": "Weiss-Simon" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_pma" } ], "abstract": "Recent decades have seen dramatic advances in our ability to make quantitative measurements of the level of gene expression in organisms of all types. The data resulting from these experiments has raised the need for quantitative models that go beyond the verbal and cartoon-level descriptions that have been so useful in developing a qualitative picture of the nature of gene expression. The improvement in our quantitative description of regulatory networks and our corresponding ability to rewire these networks at will has led many to argue for an analogy between biological regulatory networks and their electronic counterparts. In the electronic setting, we can predict the output current given knowledge of the input voltage and the parameters characterizing the circuit. However, this has so far been nothing more than a hopeful analogy since the input-output functions of most quantitative models of transcriptional regulation are based on phenomenological fits with little-to-no connection to the microscopic parameters of the system. This thesis sharpens this analogy by presenting an integrated approach to understanding transcriptional regulation in bacteria in terms of the microscopic parameters involved in the decision-making processes. This is achieved by a three-pronged approach consisting of theoretical models, in vivo measurements and single-molecule experiments in vitro.
\r\n\r\nThe theoretical analysis is based upon two different families of models aimed at describing the output of several regulatory architectures as a function of their input parameters. Thermodynamic models of transcriptional regulation are used to predict the mean level of gene expression of several bacterial promoter architectures as a function of the concentration of the intervening regulatory proteins and their binding energies to DNA and to the associated transcriptional machinery. In recent years, however, an increasing body of work has been performed where levels of gene expression are quantified in single cells and sometimes even at the single molecule level. These measurements have revealed that \"noise\" in gene expression can play a significant role in decision-making processes in systems ranging from bacteria to mammalian cells. Stochastic models of transcriptional regulation predict this variability in gene expression as a function of the microscopic parameter of the system. Unlike thermodynamic models, however, the predictions from stochastic models are dependent on the rate constants describing the regulatory circuit of interest. A complete set of models that predict input-output functions of regulatory systems in bacteria as a function of not only equilibrium parameters, but also probabilities of transition between different regulatory states is presented.
\r\n\r\nThe second half of the thesis complements the theoretical analyses by presenting several experiments aimed at testing the various predictions generated by these models. One of the experiments is carried out in vivo and aims to test the theoretical predictions for the input-output function of simple repression in terms of its microscopic parameters such as the concentration of repressor inside the cell and its binding energy to DNA. By quantifying the output level of gene expression as a function of the intracellular absolute concentration of repressor it is shown that our models can account for the level of gene expression as a function of the input parameters over several orders of magnitude. The simple repression motif is also explored experimentally using a second method based upon evaluating fluctuations in the partitioning of regulatory proteins during the cell division process. A third set of experiments performed at the single-molecule level in vitro show how a particular repressor protein binds to DNA at two different sites and loops the intervening DNA.
\r\n", "doi": "10.7907/63RG-8P84", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:5488", "collection": "thesis", "collection_id": "5488", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12292009-170615072", "primary_object_url": { "basename": "HarryChoi_Thesis_submit.pdf", "content": "final", "filesize": 70867126, "license": "other", "mime_type": "application/pdf", "url": "/5488/1/HarryChoi_Thesis_submit.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Programmable In Situ Amplification for Multiplexed Bioimaging", "author": [ { "family_name": "Choi", "given_name": "Harry Ming Tak", "orcid": "0000-0002-1530-0773", "clpid": "Choi-Harry-Ming-Tak" } ], "thesis_advisor": [ { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" } ], "thesis_committee": [ { "family_name": "Fraser", "given_name": "Scott E.", "orcid": "0000-0002-5377-0223", "clpid": "Fraser-S-E" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "In situ hybridization methods enable the mapping of mRNA expression within intact biological samples. With current approaches, it is challenging to simultaneously detect multiple target mRNAs in vertebrate embryos and tissue sections \u2013 a significant limitation in attempting to study interacting regulatory elements in systems most relevant to human development and disease. This thesis presents a multiplexed fluorescent in situ hybridization method based on orthogonal amplification with hybridization chain reaction (HCR). Using this approach, RNA probes complementary to mRNA targets trigger chain reactions in which fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. Robust performance and high signal-to-background are achieved when imaging five target mRNAs at the same time in fixed whole-mount zebrafish embryos. The programmability and sequence specificity of these amplification cascades enable all five amplifiers to operate orthogonally at the same time in the same sample. The fact that amplification polymers are triggered to self-assemble in situ results in excellent sample penetration and high signal-to-background. These properties suggest the broad applicability of fluorescent in situ HCR amplification to multiplexed imaging of mRNA expression in normal and pathological cells, embryos, and tissue sections.\r\n", "doi": "10.7907/K4A3-1K41", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5588", "collection": "thesis", "collection_id": "5588", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03102010-232405639", "primary_object_url": { "basename": "thesis_MING_GU.pdf", "content": "final", "filesize": 12058507, "license": "other", "mime_type": "application/pdf", "url": "/5588/1/thesis_MING_GU.pdf", "version": "v9.0.0" }, "type": "thesis", "title": "Interactions Between Hippocampal Areas CA3 and CA1 During Slow-Wave Sleep\r ", "author": [ { "family_name": "Gu", "given_name": "Ming", "clpid": "Gu-Ming" } ], "thesis_advisor": [ { "family_name": "Siapas", "given_name": "Athanassios G.", "orcid": "0000-0001-8837-678X", "clpid": "Siapas-A-G" } ], "thesis_committee": [ { "family_name": "Laurent", "given_name": "Gilles J.", "orcid": "0000-0002-2296-114X", "clpid": "Laurent-G-J" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" }, { "family_name": "Siapas", "given_name": "Athanassios G.", "orcid": "0000-0001-8837-678X", "clpid": "Siapas-A-G" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Many lines of evidence suggest that the hippocampus plays a critical role in memory formation. The predominant hypothesis is that new memories are initially stored within hippocampal circuits during awake behavior, and are subsequently consolidated across neocortical networks under the influence of hippocampal activity during sleep. The hippocampal memory trace is conjectured to reside within the recurrent circuits of area CA3, which is believed to function as an autoassociative memory. Area CA3 projects almost exclusively to area CA1, one of the major output stages of the hippocampus. How is CA3 activity transformed in CA1, and what is the function of the CA1 subfield that intermediates between CA3 and the neocortex, the presumed long-term storage site of memories?
\r\n\r\nHere we characterize the relationships between CA3 and CA1 activity during slow-wave sleep (SWS), a stage of sleep conjectured to be important in memory consolidation. Activity in SWS is marked by the presence of short-lived (~100 ms) population bursts that are believed to be spontaneously generated within CA3 and that cooccur with high-frequency oscillations (~200 Hz ripples) in area CA1. We demonstrate that:
\r\n\r\n1. CA1 amplifies transient increases in CA3 activity levels, while attenuating background fluctuations.\r\n2. The fraction of co-active neurons is higher in CA1 than in CA3, while the firing intensity of active neurons is higher in CA3 than in CA1. \r\n3. The above dichotomy is particularly pronounced during the population bursts associated with ripples. \r\n4. In comparison to isolated spikes, bursts of action potentials by CA3 neurons are particularly effective at triggering large CA1 responses and predicting the onset of CA1 ripples.
\r\n\r\nThese results show that CA1 acts as a selective filter and amplifier of CA3 activity patterns, and that bursting of individual CA3 neurons plays a special role in this CA3-CA1 transformation. We hypothesize that coordinated bursts in CA3 reflect convergence to attractors, each representing a stored pattern in the auto-associative network. Our observations suggest that these stored patterns are preferentially amplified by CA1 and transmitted to downstream targets, while activity representing intermediate states in-between attractors are less likely to be transmitted.
\r\n", "doi": "10.7907/Z9BV7DMM", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5667", "collection": "thesis", "collection_id": "5667", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03312010-150904525", "primary_object_url": { "basename": "thesis_final.pdf", "content": "final", "filesize": 2477730, "license": "other", "mime_type": "application/pdf", "url": "/5667/21/thesis_final.pdf", "version": "v9.0.0" }, "type": "thesis", "title": "Causes and Consequences of Gene Expression Noise", "author": [ { "family_name": "Dalal", "given_name": "Chiraj Kiran", "clpid": "Dalal-Chiraj-Kiran" } ], "thesis_advisor": [ { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" }, { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" }, { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" } ], "local_group": [ { "literal": "div_biol" } ], "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.", "doi": "10.7907/MHSR-9875", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:1589", "collection": "thesis", "collection_id": "1589", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05022009-103703", "primary_object_url": { "basename": "GordonThesis.pdf", "content": "final", "filesize": 4285643, "license": "other", "mime_type": "application/pdf", "url": "/1589/1/GordonThesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Hormone and Gene Feedback during Development and Regeneration in Arabidopsis thaliana", "author": [ { "family_name": "Gordon", "given_name": "Sean Patrick", "orcid": "0000-0003-3431-5804", "clpid": "Gordon-Sean-Patrick" } ], "thesis_advisor": [ { "family_name": "Meyerowitz", "given_name": "Elliot M.", "clpid": "Meyerowitz-E-M" } ], "thesis_committee": [ { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "clpid": "Meyerowitz-E-M" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" }, { "family_name": "Deshaies", "given_name": "Raymond Joseph", "clpid": "Deshaies-R-J" } ], "local_group": [ { "literal": "div_biol" } ], "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", "doi": "10.7907/T3CR-TC54", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:1444", "collection": "thesis", "collection_id": "1444", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-04212009-104756", "primary_object_url": { "basename": "Brenner-CompleteThesis.pdf", "content": "final", "filesize": 1967823, "license": "other", "mime_type": "application/pdf", "url": "/1444/10/Brenner-CompleteThesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Engineering Synthetic Biofilm-Forming Microbial Consortia", "author": [ { "family_name": "Brenner", "given_name": "Sarah Katherine", "clpid": "Brenner-Sarah-Katherine" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Most bacteria on earth live in heterogeneous surface-bound congregations called biofilms, and vast reaches of the earth are coated in these living films. In many cases, the microorganisms comprising this ubiquitous coating form complex, interactive communities called consortia. Microbial consortia are implicated in processes of great importance to humans, from environmental remediation and wastewater treatment to assistance in food digestion. Synthetic biologists are honing their ability to program the behavior of individual microbial populations, forcing the microbes to focus on specific applications, such as the production of drugs and fuels. Given that microbial consortia can perform even more complicated tasks and endure more changeable environments than monocultures can, they represent an important new frontier for synthetic biology. This thesis describes two engineered microbial consortia that live and perform their designed functions in biofilms. The biofilm consortium elucidated in Chapter 2 serves as a proof of concept for the development of the symbiotic biofilm consortium of Chapter 3. To provide a context for these two consortia, the first chapter highlights the salient features of microbial consortia that are of interest to synthetic biologists and reviews recent efforts to engineer synthetic microbial consortia, while the final chapter suggests challenges associated with and future directions for engineering microbial consortia.\r\n", "doi": "10.7907/PMZ3-4Z96", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:4118", "collection": "thesis", "collection_id": "4118", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10162008-131654", "primary_object_url": { "basename": "full_thesis.pdf", "content": "final", "filesize": 12975030, "license": "other", "mime_type": "application/pdf", "url": "/4118/7/full_thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Synthetic Regulation and Genetic Control of an Ecological Strategy", "author": [ { "family_name": "Bayer", "given_name": "Travis Scott", "clpid": "Bayer-Travis-Scott" } ], "thesis_advisor": [ { "family_name": "Smolke", "given_name": "Christina D.", "clpid": "Smolke-C-D" } ], "thesis_committee": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Smolke", "given_name": "Christina D.", "clpid": "Smolke-C-D" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The construction of synthetic gene regulatory circuits inside living cells has illuminated how organisms process environmental signals, and has suggested that biological systems can be engineered for useful purposes. However, these lines of inquiry are limited by a lack of technologies for programming gene expression and an understanding of the adaptive or ecological consequences of manipulating gene expression. Here, I describe the design of noncoding RNA regulators of gene expression in Saccharomyces cerevisiae. These regulators are able to regulate gene expression in response to a small molecule ligand, which offers the ability to tailor control devices for a variety of applications. In light of this, an open question is the dependence of organism fitness on the levels of a regulator, which has seldom been measured. I found that the expression level of a transcriptional regulator of nitrogen metabolism mediates a trade-off between growth in resource abundant and resource limited environments in S. cerevisiae. Redundancy in the metabolic pathways of ammonia assimilation allowed noise, or random fluctuations in the amount of protein present, to dictate whether cells specialized in maximizing fitness in abundant or limiting environments. These results show how gene expression may be programmed via noncoding RNA regulators, and that the manipulation of regulator levels can affect the strategy by which organisms adapt to their environments.\r\n", "doi": "10.7907/3GDB-C409", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:4118", "collection": "thesis", "collection_id": "4118", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10162008-131654", "primary_object_url": { "basename": "full_thesis.pdf", "content": "final", "filesize": 12975030, "license": "other", "mime_type": "application/pdf", "url": "/4118/7/full_thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Synthetic Regulation and Genetic Control of an Ecological Strategy", "author": [ { "family_name": "Bayer", "given_name": "Travis Scott", "clpid": "Bayer-Travis-Scott" } ], "thesis_advisor": [ { "family_name": "Smolke", "given_name": "Christina D.", "clpid": "Smolke-C-D" } ], "thesis_committee": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Smolke", "given_name": "Christina D.", "clpid": "Smolke-C-D" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The construction of synthetic gene regulatory circuits inside living cells has illuminated how organisms process environmental signals, and has suggested that biological systems can be engineered for useful purposes. However, these lines of inquiry are limited by a lack of technologies for programming gene expression and an understanding of the adaptive or ecological consequences of manipulating gene expression. Here, I describe the design of noncoding RNA regulators of gene expression in Saccharomyces cerevisiae. These regulators are able to regulate gene expression in response to a small molecule ligand, which offers the ability to tailor control devices for a variety of applications. In light of this, an open question is the dependence of organism fitness on the levels of a regulator, which has seldom been measured. I found that the expression level of a transcriptional regulator of nitrogen metabolism mediates a trade-off between growth in resource abundant and resource limited environments in S. cerevisiae. Redundancy in the metabolic pathways of ammonia assimilation allowed noise, or random fluctuations in the amount of protein present, to dictate whether cells specialized in maximizing fitness in abundant or limiting environments. These results show how gene expression may be programmed via noncoding RNA regulators, and that the manipulation of regulator levels can affect the strategy by which organisms adapt to their environments.\r\n", "doi": "10.7907/3GDB-C409", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:2485", "collection": "thesis", "collection_id": "2485", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06062009-131454", "primary_object_url": { "basename": "Ursell_Thesis_2009.pdf", "content": "final", "filesize": 26146411, "license": "other", "mime_type": "application/pdf", "url": "/2485/1/Ursell_Thesis_2009.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Stretching the Definition of a Lipid Bilayer: Elasticity's Role in Protein and Lipid Organization", "author": [ { "family_name": "Ursell", "given_name": "Tristan Scott", "orcid": "0000-0001-9273-8413", "clpid": "Ursell-Tristan-Scott" } ], "thesis_advisor": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "thesis_committee": [ { "family_name": "Roukes", "given_name": "Michael Lee", "orcid": "0000-0002-2916-6026", "clpid": "Roukes-M-L" }, { "family_name": "Klug", "given_name": "William", "clpid": "Klug-W" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Huang", "given_name": "Kerwyn", "clpid": "Huang-Kerwyn" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "The Central Dogma forms the foundation of molecular biology couched in polymer language; all the key players are there \u2014 DNA, RNA, protein \u2014 or so it would seem. Yet one class of biologically synthesized molecules, crucial for life, is often over looked: lipids. These amphiphilic molecules exhibit a number of strange properties, integral to the cells ability to separate self from non-self in a chemically diverse environment. Lipids self-assemble into two-dimensional bi-layered fluids with aspect ratios of a thousand to one or more, capable of self-healing and bending into extraordinarily complex shapes. Within the cell, membranes allow for numerous chemically-distinct compartments, essential for metabolism, protein assembly, genome management, and cell division. With literally hundreds of different kinds of lipids and proteins interacting on a given membrane, we have much to learn about how membranes regulate the flow of materials into and out of cells. Clearly, molecular level detail is integral to our understanding of these systems, however, on the mesoscopic level membranes exhibit certain mechanical effects that serve to organize lipids and proteins, the study of which forms the bulk of this dissertation. We start by building an elastic model of bilayers, where embedded proteins deform the surrounding membrane and incur a free energy cost. This allows the mechanical attributes of the bilayer to influence the conformation of embedded proteins. We explore this connection in the context of mechanosensation in bacteria, as well as developing methods that allow bilayer mechanics to comment on the structure of classically voltage-gated ion channels. In addition to affecting conformational preferences, these same deformations have a finite length-scale that results in interactions between embedded proteins. Depending on the protein shape, these interactions can be attractive or repulsive, may exert torques on proteins, provide for a mechanism of shape-specific oligomerization, and importantly allow proteins to utilize the bilayer as a generic communicator of conformational information. The effects of these elastic interactions are discussed in the context of mean protein spacing, dimerization, conformational cooperativity, and likely pathways to multi-mer protein assembly, with the bacterial mechanosensitive channel MscL as a structural example. In subsequent chapters, bilayer elasticity is used to shed light on the large-scale organization of lipids themselves. Biological membranes likely have multiple fluid, lipid phases, where sequestration of saturated lipids and cholesterol form lipid domains. We found that formation of domains above a certain critical size induces morphological transitions to a \u2018dimpled\u2019 phase which turns on repulsive, elastic interactions that serve to spatially organize domains as well as severely inhibit domain coalescence. This provides a mechanism for the maintenance of lipid lateral heterogeneity on relatively short length-scales and long time scales. We further observed discrete transitions to a \u2018budded\u2019 domain morphology and developed a set of interpretive energetic transition rules between flat, dimpled and budded domains. We demonstrate that these morphologies and their attendant transitions lead to a unique form of domain-size-dependent transport in membranes. Further, we employ the mechanics of vesicles to model osmoregulation via channel proteins, and in the setting of conserved surface area and volume to develop a theoretical and experimental framework to study membrane adhesion in the context of the homophilic protein binding.\r\n", "doi": "10.7907/Q0R5-K353", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:2485", "collection": "thesis", "collection_id": "2485", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06062009-131454", "primary_object_url": { "basename": "Ursell_Thesis_2009.pdf", "content": "final", "filesize": 26146411, "license": "other", "mime_type": "application/pdf", "url": "/2485/1/Ursell_Thesis_2009.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Stretching the Definition of a Lipid Bilayer: Elasticity's Role in Protein and Lipid Organization", "author": [ { "family_name": "Ursell", "given_name": "Tristan Scott", "orcid": "0000-0001-9273-8413", "clpid": "Ursell-Tristan-Scott" } ], "thesis_advisor": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "thesis_committee": [ { "family_name": "Roukes", "given_name": "Michael Lee", "orcid": "0000-0002-2916-6026", "clpid": "Roukes-M-L" }, { "family_name": "Klug", "given_name": "William", "clpid": "Klug-W" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Huang", "given_name": "Kerwyn", "clpid": "Huang-Kerwyn" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "The Central Dogma forms the foundation of molecular biology couched in polymer language; all the key players are there \u2014 DNA, RNA, protein \u2014 or so it would seem. Yet one class of biologically synthesized molecules, crucial for life, is often over looked: lipids. These amphiphilic molecules exhibit a number of strange properties, integral to the cells ability to separate self from non-self in a chemically diverse environment. Lipids self-assemble into two-dimensional bi-layered fluids with aspect ratios of a thousand to one or more, capable of self-healing and bending into extraordinarily complex shapes. Within the cell, membranes allow for numerous chemically-distinct compartments, essential for metabolism, protein assembly, genome management, and cell division. With literally hundreds of different kinds of lipids and proteins interacting on a given membrane, we have much to learn about how membranes regulate the flow of materials into and out of cells. Clearly, molecular level detail is integral to our understanding of these systems, however, on the mesoscopic level membranes exhibit certain mechanical effects that serve to organize lipids and proteins, the study of which forms the bulk of this dissertation. We start by building an elastic model of bilayers, where embedded proteins deform the surrounding membrane and incur a free energy cost. This allows the mechanical attributes of the bilayer to influence the conformation of embedded proteins. We explore this connection in the context of mechanosensation in bacteria, as well as developing methods that allow bilayer mechanics to comment on the structure of classically voltage-gated ion channels. In addition to affecting conformational preferences, these same deformations have a finite length-scale that results in interactions between embedded proteins. Depending on the protein shape, these interactions can be attractive or repulsive, may exert torques on proteins, provide for a mechanism of shape-specific oligomerization, and importantly allow proteins to utilize the bilayer as a generic communicator of conformational information. The effects of these elastic interactions are discussed in the context of mean protein spacing, dimerization, conformational cooperativity, and likely pathways to multi-mer protein assembly, with the bacterial mechanosensitive channel MscL as a structural example. In subsequent chapters, bilayer elasticity is used to shed light on the large-scale organization of lipids themselves. Biological membranes likely have multiple fluid, lipid phases, where sequestration of saturated lipids and cholesterol form lipid domains. We found that formation of domains above a certain critical size induces morphological transitions to a \u2018dimpled\u2019 phase which turns on repulsive, elastic interactions that serve to spatially organize domains as well as severely inhibit domain coalescence. This provides a mechanism for the maintenance of lipid lateral heterogeneity on relatively short length-scales and long time scales. We further observed discrete transitions to a \u2018budded\u2019 domain morphology and developed a set of interpretive energetic transition rules between flat, dimpled and budded domains. We demonstrate that these morphologies and their attendant transitions lead to a unique form of domain-size-dependent transport in membranes. Further, we employ the mechanics of vesicles to model osmoregulation via channel proteins, and in the setting of conserved surface area and volume to develop a theoretical and experimental framework to study membrane adhesion in the context of the homophilic protein binding.\r\n", "doi": "10.7907/Q0R5-K353", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:5180", "collection": "thesis", "collection_id": "5180", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-04082009-102110", "primary_object_url": { "basename": "AxelrodBWThesisFinal.pdf", "content": "final", "filesize": 3781779, "license": "other", "mime_type": "application/pdf", "url": "/5180/1/AxelrodBWThesisFinal.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Single Cell Pico Force Microscopy: A Novel Tool for High Resolution Measurement of Cell Forces", "author": [ { "family_name": "Axelrod", "given_name": "Blake Waters", "clpid": "Axelrod-Blake-Waters" } ], "thesis_advisor": [ { "family_name": "Roukes", "given_name": "Michael Lee", "orcid": "0000-0002-2916-6026", "clpid": "Roukes-M-L" } ], "thesis_committee": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Roukes", "given_name": "Michael Lee", "orcid": "0000-0002-2916-6026", "clpid": "Roukes-M-L" }, { "family_name": "Fraser", "given_name": "Scott E.", "orcid": "0000-0002-5377-0223", "clpid": "Fraser-S-E" } ], "local_group": [ { "literal": "Kavli Nanoscience Institute" }, { "literal": "div_eng" } ], "abstract": "Nearly all eukaryotic cells exert forces on their surroundings to generate and maintain tension within their cytoskeleton which is critical for normal cell function. In addition, cells exert forces on their surroundings to orient themselves within an organism, thus gaining information that influences cell fate and behavior, a process called mechanotransduction. In order to study mechanotransduction, a tool is needed that can observe the molecular level sensing events that trigger a decision by a cell as well as the ultimate response that occurs on the whole cell level. There are a number of optical techniques that are used to measure forces from adherent cells at the single cell level; some are good for measuring whole cell forces and some for measuring single molecule level forces, but none have the dynamic range necessary to span both regimes, which is critical for understanding mechanotransduction. To address this need, I have developed a Nano-ElectroMechanical Systems (NEMS) based tool, Single-Cell-Pico-Force-Microscopy (SCPFM), that measures forces exerted by adherent cells with macro-molecular level force sensitivity and sufficient dynamic range to monitor whole cell responses to stimuli with macro-molecular resolution. I have used SCPFM to measure force versus time data from a NIH-3T3 fibroblast as it is perturbed with Cytochalasin D (CD) and allowed to recover in growth media. Within the data there are three excellent examples of previously inaccessible molecular-mechanical processes that illustrate the immense potential of SCPFM to significantly enhance resolution of cell biology at the single cell level: 1) an initial contraction upon exposure to CD followed by the expected force drop, 2) small force oscillations, roughly 400pN peak-to-peak, with frequency that is monotonically dependent on the force being exerted by the lamellipodia, and 3) large, stable, quantized force steps of order 1nN are manifested when a cell\u2019s cytoskeleton is perturbed with CD and allowed to recover in growth media. I propose two complimentary experimental efforts to undertake: a systematic effort to build a library of molecular-mechanical force signatures of various common cytoskeleton reactions and an effort to stimulate and observe compliance sensing and response in adherent cells.\r\n", "doi": "10.7907/ESGS-Z942", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:390", "collection": "thesis", "collection_id": "390", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-01282008-150852", "primary_object_url": { "basename": "LinHan.pdf", "content": "final", "filesize": 15479140, "license": "other", "mime_type": "application/pdf", "url": "/390/1/LinHan.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "In vitro DNA Mechanics in Gene Regulation: One Molecule at a Time", "author": [ { "family_name": "Han", "given_name": "Lin", "clpid": "Han-Lin" } ], "thesis_advisor": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Guo", "given_name": "Chin-Lin", "clpid": "Guo-Chin-Lin" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "The biological significance of DNA is primarily attributed to its sequence information. On the other hand, the mechanical properties of DNA can play a critical role in a wide variety of biological processes. One prime example is DNA looping in the context of transcriptional regulation. The emergence of single molecule tracking techniques in the last two decades presents an unprecedented opportunity for studying looping kinetics. One such powerful technique, tethered particle motion (TPM), harnesses the Brownian motion of a microsphere as a means of reporting on the excursion of its tethered molecule, such as DNA. The present work focuses on a looping system found in Escherichia coli, which is mediated by the Lac repressor (LacI) protein. TPM is used to measure individual, real-time looping/unlooping events in DNA of various length and sequence characteristics. By monitoring the magnitude, frequency, and time interval of these features while tuning different parameters, such as LacI concentration, DNA length and DNA sequence, one can survey a host of important information about looping kinetics. A measurement of the LacI concentration dependence of looping probability was found to be in quantitative agreement with a simple thermodynamic model, which also led to the measurement of free energy of LacI-mediated looping, the first such measurement in a single molecule, in vitro setting. A quantitative characterization of free energy was obtained under conditions of different inter-operator spacing, systematically varied from 300 to 310 base pairs in one-base-pair increments. An important conclusion from this study is that free energy is modulated by DNA\u2019s helical structure, yet the energy difference between the aligned and unaligned operator configurations is small compared to expectation from simple polymer physics. TPM measurements also revealed an additional looped state, lending support to the hypothesis that two distinct conformations of LacI, the closed and open forms, can coexist. This study also confirmed that the presence of certain DNA sequences, particularly TA pairs in the minor groove of the nucleosomal positioning sequence, makes DNA substantially softer than a corresponding random sequence. This provides direct support for the notion of sequence-dependent DNA elasticity. Finally, a surprising result is that loops as short as 100 base pairs-only two-thirds the persistence length of DNA-can form by LacI-DNA binding. Classical elasticity theory almost forbids this, suggesting that LacI itself plays a more direct role in the bending process, or classical understanding of DNA elasticity breaks down at length scales comparable to its persistence length.", "doi": "10.7907/7573-A922", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:2320", "collection": "thesis", "collection_id": "2320", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05302008-141602", "primary_object_url": { "basename": "00_mjdunlop_complete_thesis.pdf", "content": "final", "filesize": 4934029, "license": "other", "mime_type": "application/pdf", "url": "/2320/1/00_mjdunlop_complete_thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Dynamics and Correlated Noise in Gene Regulation", "author": [ { "family_name": "Dunlop", "given_name": "Mary Julia", "orcid": "0000-0002-9261-8216", "clpid": "Dunlop-Mary-Julia" } ], "thesis_advisor": [ { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" } ], "thesis_committee": [ { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Winfree", "given_name": "Erik", "orcid": "0000-0002-5899-7523", "clpid": "Winfree-E" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Gene regulatory interactions are context dependent, active in some cell types or cellular states but not in others. In this thesis we present a method for determining when a regulatory link is active given temporal measurements of gene expression. Correlations in time-series data are used to determine how genes influence each other and their causal relationships. Natural stochastic noise is shown to aid in the process of network identification by perturbing the expression of genes; the speed and direction at which the noisy signal propagates shows how the network is connected. Cross correlation functions are used to reveal time-delayed correlations.
\r\n\r\nWe develop a stochastic model of gene expression and show that by measuring correlations in cellular noise, it is possible to infer network activity and temporal properties of gene regulation. Using a linearized version of the model, we introduce a method for analytically deriving cross correlation functions for arbitrary networks. These results are validated experimentally using a synthetic gene circuit in E. coli bacteria. Single-cell time-lapse microscopy is used to measure noisy expression of multiple genes over time. Extending this work to natural systems, we study feed-forward loops and determine that certain classes of feed-forward loops are more robust to noise and parameter variations that others. Noise in two naturally occurring feed-forward loops involved in galactose utilization is measured experimentally and it is shown that neither is actively regulating its target in the conditions tested.
", "doi": "10.7907/AC8V-6S05", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:5194", "collection": "thesis", "collection_id": "5194", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05222007-141119", "primary_object_url": { "basename": "thesis.pdf", "content": "final", "filesize": 104843840, "license": "other", "mime_type": "application/pdf", "url": "/5194/1/thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Microfluidic Large Scale Integration and its Application to Systems Biology", "author": [ { "family_name": "Maerkl", "given_name": "Sebastian Josef", "orcid": "0000-0003-1917-5268", "clpid": "Maerkl-Sebastian-Josef" } ], "thesis_advisor": [ { "family_name": "Quake", "given_name": "Stephen R.", "orcid": "0000-0002-1613-0809", "clpid": "Quake-S-R" } ], "thesis_committee": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Fraser", "given_name": "Scott E.", "orcid": "0000-0002-5377-0223", "clpid": "Fraser-S-E" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Roukes", "given_name": "Michael Lee", "orcid": "0000-0002-2916-6026", "clpid": "Roukes-M-L" }, { "family_name": "Quake", "given_name": "Stephen R.", "orcid": "0000-0002-1613-0809", "clpid": "Quake-S-R" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "The goal of biology is to understand how complex systems such as cells and entire organisms function. Systems Biology attempts to quantitatively characterize all components comprising these systems. A considerable task. Microfluidics provides a powerful tool for undertaking this endeavor. This thesis describes the development of Microfluidic Large Scale Integration (MLSI) using devices fabricated by Multilayer Soft Lithography (MSL). MLSI and fluidic components, such as multiplexers and free-standing membranes, serve as the infrastructure for performing large-scale biophysical measurements of biological systems. Transcription factor binding energy landscapes were determined using MLSI and MITOMI, a novel method for measuring molecular interactions. The biophysical characterization of transcription factors described herein were the first comprehensive measurements of its kind, and answered basic questions regarding how transcription factors recognize DNA. Furthermore, it was possible to predict the in vivo function of transcription factors using only the measured binding topographies and a genome sequence, indicating that biological processes can be predicted with high accuracy. More generally, the methods described in this thesis are generally applicable to understanding the properties of any biological system and should find broad usage in the field of Systems Biology.\r\n", "doi": "10.7907/5CMX-CV08", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:871", "collection": "thesis", "collection_id": "871", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-03042008-130011", "primary_object_url": { "basename": "cox_thesis_final_cover.pdf", "content": "final", "filesize": 13529144, "license": "other", "mime_type": "application/pdf", "url": "/871/1/cox_thesis_final_cover.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Transcriptional Regulation and Combinatorial Genetic Logic in Synthetic Bacterial Circuits", "author": [ { "family_name": "Cox", "given_name": "Robert Sidney, III", "orcid": "0000-0002-2009-6236", "clpid": "Cox-Robert-Sidney-III" } ], "thesis_advisor": [ { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" }, { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" }, { "family_name": "Smolke", "given_name": "Christina D.", "clpid": "Smolke-C-D" }, { "family_name": "Winfree", "given_name": "Erik", "clpid": "Winfree-E" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" } ], "local_group": [ { "literal": "div_biol" } ], "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", "doi": "10.7907/KRW6-DH88", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:5210", "collection": "thesis", "collection_id": "5210", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05282008-163501", "primary_object_url": { "basename": "MSM_Thesis_7May2008.pdf", "content": "final", "filesize": 4241288, "license": "other", "mime_type": "application/pdf", "url": "/5210/1/MSM_Thesis_7May2008.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Understanding and Treating Eye Diseases: Mechanical Characterization and Photochemical Modification of the Cornea and Sclera", "author": [ { "family_name": "Mattson", "given_name": "Matthew Sanford", "clpid": "Mattson-Matthew-Sanford" } ], "thesis_advisor": [ { "family_name": "Kornfield", "given_name": "Julia A.", "clpid": "Kornfield-J-A" } ], "thesis_committee": [ { "family_name": "Kornfield", "given_name": "Julia A.", "clpid": "Kornfield-J-A" }, { "family_name": "Guo", "given_name": "Chin-Lin", "clpid": "Guo-Chin-Lin" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Proper vision depends on the physical properties of the eye tissues. Diseases that alter the chemical and mechanical states of the tissue can result in a loss of functionality. Degenerative myopia and keratoconus are diseases that exhibit such changes in the sclera and cornea, respectively. These diseases may be treatable by engineering the mechanical properties of the sclera and cornea.
\r\n\r\nWe have developed an intact globe expansion method for mechanical characterization of eyes in vitro, which provides reliable measurements, with relatively few samples, and mimics the type and distribution of stresses inherent in the natural boundary conditions of the eye. Furthermore, application of high intraocular pressures provides a way to study shape changes of the sclera and cornea which are similar to those exhibited in myopia and keratoconus. Potential treatments that show an ability to prevent ocular distension in this method have a chance of preventing the deformations that occur in vivo in the diseases.
\r\n\r\nOur studies in vitro indicate that crosslinking can improve tissue mechanical stability and resistance to deformation. Light activated crosslinking provides spatial and temporal control of treatments, and photoinitiator systems such as Eosin Y (EY) and triethanolamine (TEOA) allow this control with safe doses of visible light.
\r\n\r\nFor myopia treatment, we demonstrate in vitro stabilization of eyes using the intact globe method after drug is delivered to the sclera in vitro or in vivo on 2\u20133 week old rabbits. Biocompatibility studies of the scleral treatments indicate excellent tolerance to the light and drug in both rabbits and guinea pigs. Further, we have developed treatment protocols for use in a guinea pig form-deprivation model of myopia. In normally growing guinea pig eyes, treatments can create substantial changes to eye shape. These changes are manifested in shifts in the refractive error and ocular length that persist for the duration over which the animals are monitored.
\r\n\r\nFor keratoconus, treatment in vitro on rabbit eyes using EY/TEOA demonstrates similar capabilities of corneal stabilization as a treatment currently in clinical trials. The EY/TEOA treatment shows advantages of reduced treatment time and the possibility of treatment without removal of the epithelium.
", "doi": "10.7907/DXGR-GV57", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:5209", "collection": "thesis", "collection_id": "5209", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05282008-103138", "primary_object_url": { "basename": "Wonhee_Thesis_Final.pdf", "content": "final", "filesize": 4633312, "license": "other", "mime_type": "application/pdf", "url": "/5209/1/Wonhee_Thesis_Final.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Microfluidic Chip Calorimeters for Biological Applications", "author": [ { "family_name": "Lee", "given_name": "Wonhee", "orcid": "0000-0003-0119-4372", "clpid": "Lee-Wonhee" } ], "thesis_advisor": [ { "family_name": "Roukes", "given_name": "Michael Lee", "orcid": "0000-0002-2916-6026", "clpid": "Roukes-M-L" } ], "thesis_committee": [ { "family_name": "Roukes", "given_name": "Michael Lee", "orcid": "0000-0002-2916-6026", "clpid": "Roukes-M-L" }, { "family_name": "Guo", "given_name": "Chin-Lin", "clpid": "Guo-Chin-Lin" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "The usage of calorimeters is limited due to its long measurement time and large sample consumption, despite its many advantages including universal applicability and simple sample preparation. Miniaturization of calorimeters not only resolves these problems, it also enables high-throughput measurements with array operations. We have developed microfluidic chip calorimeters with high sensitivity and reliable microfluidics-based sample handling. Immense sensitivity improvements are attained through reduction of the thermal conductance via on-chip vacuum insulation. This is enabled by Parylene thin-film microfluidic systems. Polydimethylsiloxane microfluidic systems, combined with the Parylene microfluidic system, gives easy and accurate control of picoliter-scale sample volume in a manner that is easily scalable to large, complex systems. Two device classes have been realized.
\r\n\r\nHeat conduction calorimeters for biochemical reactions with 3.5 nL sample volume were built and validated by measurements of the heat of mixing and of enzyme activity. The thermal conductance of these devices was 15.5 \u00b5W/K and their power sensitivity was 4.2 nW. These devices can be built as calorimetric arrays to enable high-throughput heat of reaction measurements upon libraries of biomolecular interactions.
\r\n\r\nFlow calorimeters were designed for sensor applications and measurements of cellular metabolism. The thermal conductance of these devices was 4.7 \u00b5W/K and their power sensitivity was 1.5 nW. Further reduction of thermal conductance and optimal thermocouple materials will deliver sensitivity of order ~1 pW, which will enable real-time measurement of single cell metabolism.
", "doi": "10.7907/W8E0-4W22", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:5209", "collection": "thesis", "collection_id": "5209", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05282008-103138", "primary_object_url": { "basename": "Wonhee_Thesis_Final.pdf", "content": "final", "filesize": 4633312, "license": "other", "mime_type": "application/pdf", "url": "/5209/1/Wonhee_Thesis_Final.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Microfluidic Chip Calorimeters for Biological Applications", "author": [ { "family_name": "Lee", "given_name": "Wonhee", "orcid": "0000-0003-0119-4372", "clpid": "Lee-Wonhee" } ], "thesis_advisor": [ { "family_name": "Roukes", "given_name": "Michael Lee", "orcid": "0000-0002-2916-6026", "clpid": "Roukes-M-L" } ], "thesis_committee": [ { "family_name": "Roukes", "given_name": "Michael Lee", "orcid": "0000-0002-2916-6026", "clpid": "Roukes-M-L" }, { "family_name": "Guo", "given_name": "Chin-Lin", "clpid": "Guo-Chin-Lin" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "The usage of calorimeters is limited due to its long measurement time and large sample consumption, despite its many advantages including universal applicability and simple sample preparation. Miniaturization of calorimeters not only resolves these problems, it also enables high-throughput measurements with array operations. We have developed microfluidic chip calorimeters with high sensitivity and reliable microfluidics-based sample handling. Immense sensitivity improvements are attained through reduction of the thermal conductance via on-chip vacuum insulation. This is enabled by Parylene thin-film microfluidic systems. Polydimethylsiloxane microfluidic systems, combined with the Parylene microfluidic system, gives easy and accurate control of picoliter-scale sample volume in a manner that is easily scalable to large, complex systems. Two device classes have been realized.
\r\n\r\nHeat conduction calorimeters for biochemical reactions with 3.5 nL sample volume were built and validated by measurements of the heat of mixing and of enzyme activity. The thermal conductance of these devices was 15.5 \u00b5W/K and their power sensitivity was 4.2 nW. These devices can be built as calorimetric arrays to enable high-throughput heat of reaction measurements upon libraries of biomolecular interactions.
\r\n\r\nFlow calorimeters were designed for sensor applications and measurements of cellular metabolism. The thermal conductance of these devices was 4.7 \u00b5W/K and their power sensitivity was 1.5 nW. Further reduction of thermal conductance and optimal thermocouple materials will deliver sensitivity of order ~1 pW, which will enable real-time measurement of single cell metabolism.
", "doi": "10.7907/W8E0-4W22", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:2547", "collection": "thesis", "collection_id": "2547", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06112007-102627", "primary_object_url": { "basename": "BalagaddeThesis.pdf", "content": "final", "filesize": 4579992, "license": "other", "mime_type": "application/pdf", "url": "/2547/1/BalagaddeThesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Microfluidic Technologies for Continuous Culture and Genetic Circuit Characterization", "author": [ { "family_name": "Balagadd\u00e9", "given_name": "Frederick Kiguli", "orcid": "0000-0003-2796-8874", "clpid": "Balagadd\u00e9-Frederick-Kiguli" } ], "thesis_advisor": [ { "family_name": "Quake", "given_name": "Stephen R.", "orcid": "0000-0002-1613-0809", "clpid": "Quake-S-R" } ], "thesis_committee": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Scherer", "given_name": "Axel", "orcid": "0000-0002-2160-9064", "clpid": "Scherer-A" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Quake", "given_name": "Stephen R.", "orcid": "0000-0002-1613-0809", "clpid": "Quake-S-R" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "In this thesis, I have used microfluidics--the science and technology of systems that manipulate small amounts of fluids (10-9 to 10-18 liters) in microsized channels--to invent and implement a miniaturized continuous culture device or microchemostat. It relies on a novel in silicone sterilization approach to circumventing biofilm formation. The microchemostat system has inbuilt automation, which allows it to run, unattended, indefinitely (for up to months at a time). With a working volume of ~10 nL, the microchemostat is capable of culturing extremely small populations of bacteria (100 to ~104 cells vs ~109 in macroscale cultures). The microsized population reduces the number of cell-division events per unit time and hence slows down microbial evolution. This aspect facilitates long-term monitoring of the behavior of genetically engineered microbes while preserving their genetic homogeneity. Unlike its conventional continuous-culture counterparts, the microchemostat allows simultaneous operation of fourteen (or more) independent microreactors which enjoy ultralow consumption of medium and biological reagents, allowing high-throughput research at low cost. It also facilitates automated, noninvasive monitoring of bacterial behavior in terms of bacterial count, cell morphology as well as single-cell resolved gene-expression dynamics reported by fluorescence or luminescence. The unprecedented temporal and single cell resolution readings allow the microchemostat to capture dynamics such as delicate oscillations that have eluded detection in conventional settings.
\r\n\r\nThanks to its unique capability for long-term culturing and suppression of microbial evolution, the microchemostat promises to become integrated as an ingredient of a multicomponent monolithic entity in future applications. The microchemostat would mainly be responsible for in silicone production and supply of genetically homogeneous bacteria for use in various capacities.
", "doi": "10.7907/NJ5Z-XV43", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:1921", "collection": "thesis", "collection_id": "1921", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05212007-162731", "primary_object_url": { "basename": "GEMurphy_EntireThesis.pdf", "content": "final", "filesize": 17586889, "license": "other", "mime_type": "application/pdf", "url": "/1921/8/GEMurphy_EntireThesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Cryoelectron Tomography of Bacteria and their Macromolecular Machines", "author": [ { "family_name": "Murphy", "given_name": "Gavin Erick", "clpid": "Murphy-Gavin-Erick" } ], "thesis_advisor": [ { "family_name": "Jensen", "given_name": "Grant J.", "clpid": "Jensen-G-J" } ], "thesis_committee": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "clpid": "Bjorkman-P-J" }, { "family_name": "Jensen", "given_name": "Grant J.", "clpid": "Jensen-G-J" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "clpid": "Leadbetter-J-R" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" } ], "local_group": [ { "literal": "div_biol" } ], "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.", "doi": "10.7907/517B-0Z68", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:1281", "collection": "thesis", "collection_id": "1281", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-04062007-163445", "primary_object_url": { "basename": "bao-thesis-double-sided-printing.pdf", "content": "final", "filesize": 12330960, "license": "other", "mime_type": "application/pdf", "url": "/1281/1/bao-thesis-double-sided-printing.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Lost in a Crowd: Observations of Single DNA Knots and Single Mammalian Cells", "author": [ { "family_name": "Bao", "given_name": "Xiaoyan Robert", "orcid": "0000-0001-7931-2944", "clpid": "Bao-Xiaoyan-Robert" } ], "thesis_advisor": [ { "family_name": "Quake", "given_name": "Stephen R.", "orcid": "0000-0002-1613-0809", "clpid": "Quake-S-R" }, { "family_name": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Quake", "given_name": "Stephen R.", "orcid": "0000-0002-1613-0809", "clpid": "Quake-S-R" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "The overarching theme for the two main experiments presented here is that standard biochemical and cell biological techniques, which need fairly large samples and hence average over large numbers of things, be they molecules or cells, tend to smear out interesting phenomena that occur to a small fraction of those things. Averaging also collapses the entire population distribution into one single value, and furthermore prevents tracking properties of individuals over the lifetime of the experiment. The first project described here involves mechanically tying knots into linear DNA molecules stretched out between optically trapped beads. Without mechanical intervention, the molecules we used contained knots only rarely, and those knots are expected to involve small portions of the DNA contour and hence give only small perturbations to the overall polymer dynamics. By tying and observing them singly, we were able to show that, while knots collapse and tighten under tension, even the most complex ones we tied retained mobility to quite a surprising degree. The observed knot sizes and diffusivities correlated well with theoretical predictions for knots in ideal ropes of finite thickness, indicating that even under high tension the different parts of the molecular knots are kept away from each other because of electrostatic repulsion. The differences between knots of different topologies, both in size and speed, open up the possibility that, with further refinement, this approach may allow us to observe the stepwise actions of single topoisomerases in chemically undoing complex knots. The second project was to develop a microfluidic system to perform many signaling experiments on cells simultaneously within a single field-of-view of a microscope. Single cell sensitivity has been pivotal both in verifying data quality and in understanding cell-to-cell variabilities in signaling strengths. In the course of these two projects I also had a few side ideas which, sadly, I wasn\u2019t able to develop to the degree that I would have liked. I've included them here as minor digressions, in the hopes that someone will see them and find them useful.", "doi": "10.7907/F2XV-MC44", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:5044", "collection": "thesis", "collection_id": "5044", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-12182006-115817", "primary_object_url": { "basename": "thesis.pdf", "content": "final", "filesize": 18696961, "license": "other", "mime_type": "application/pdf", "url": "/5044/1/thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "In Vitro Synthetic Transcriptional Networks", "author": [ { "family_name": "Kim", "given_name": "Jongmin", "orcid": "0000-0002-2713-1006", "clpid": "Kim-Jongmin" } ], "thesis_advisor": [ { "family_name": "Winfree", "given_name": "Erik", "clpid": "Winfree-E" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" }, { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Winfree", "given_name": "Erik", "clpid": "Winfree-E" } ], "local_group": [ { "literal": "div_biol" } ], "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.", "doi": "10.7907/Q9YA-N192", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:2404", "collection": "thesis", "collection_id": "2404", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06022006-154329", "primary_object_url": { "basename": "drummond-thesis.pdf", "content": "final", "filesize": 2942072, "license": "other", "mime_type": "application/pdf", "url": "/2404/1/drummond-thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Misfolding Dominates Protein Evolution", "author": [ { "family_name": "Drummond", "given_name": "David Allan", "orcid": "0000-0001-7018-7059", "clpid": "Drummond-David-Allan" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Adami", "given_name": "Christoph Carl", "orcid": "0000-0002-2915-9504", "clpid": "Adami-C-C" }, { "family_name": "Winfree", "given_name": "Erik", "orcid": "0000-0002-5899-7523", "clpid": "Winfree-E" }, { "family_name": "Bruck", "given_name": "Jehoshua", "orcid": "0000-0001-8474-0812", "clpid": "Bruck-J" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "The diverse array of protein functions depends upon these molecules' reliable ability to fold into the native structures determined by their amino-acid sequences. Because mutations that alter a protein's sequence frequently disrupt its folding, protein evolution explores protein sequence space conservatively, either by point mutations or recombination between related sequences. Attempts to engineer proteins by co-opting the evolutionary algorithm have also largely proceeded by the stepwise accumulation of beneficial mutations. Other strategies for directed evolution have focused on introducing many mutations at once as a way to increase the likelihood of finding improved variants, attempting to balance higher mutational diversity with lower retention of folding. Using simple models, I explore this tradeoff and find that protein misfolding dominates whether increasing mutation levels increase the number of improved variants. I analyze results of a popular mutagenesis protocol, error-prone PCR, for evidence that coupling between mutations might favor higher mutation levels, as claimed by several groups. A comparison of high-mutation-rate mutagenesis to protein recombination between distantly related proteins reveals qualitative differences in protein tolerance for sequence changes introduced by each method. Mutational tolerance may also be reflected in the rate at which proteins accumulate sequence changes over evolutionary time; why proteins evolve at different rates remains a major open question in biology. An analysis of rate determinants suggests that one major variable, linked to how highly expressed the encoding gene is, dominates the rate of yeast protein evolution. To explain this trend, I hypothesize that proteins are selected to fold properly despite mistranslation, a property I call translational robustness, and test it using genomic data. To examine protein evolution at a higher level of detail, a large-scale simulation is constructed in which simulated organisms, with genomes containing genes expressing computationally foldable proteins at different levels, evolve over millions of generations with protein misfolding imposing the only fitness cost. The results suggest that protein misfolding suffices to explain many significant trends in genome evolution observed across taxa, predict a novel genomic trend which is then identified in yeast, and create insight into the causes of evolutionary rate variation in proteins.", "doi": "10.7907/DH8E-2N10", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:5252", "collection": "thesis", "collection_id": "5252", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-09302006-140602", "primary_object_url": { "basename": "thesis-c.lacenere.pdf", "content": "final", "filesize": 14263807, "license": "other", "mime_type": "application/pdf", "url": "/5252/1/thesis-c.lacenere.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Advances in Single Molecule Nucleic Acid Sequencing", "author": [ { "family_name": "Lacenere", "given_name": "Christopher J.", "clpid": "Lacenere-Christopher-J" } ], "thesis_advisor": [ { "family_name": "Quake", "given_name": "Stephen R.", "clpid": "Quake-S-R" } ], "thesis_committee": [ { "family_name": "Deshaies", "given_name": "Raymond Joseph", "clpid": "Deshaies-R-J" }, { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Quake", "given_name": "Stephen R.", "clpid": "Quake-S-R" } ], "local_group": [ { "literal": "div_biol" } ], "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.", "doi": "10.7907/CA4B-S389", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:923", "collection": "thesis", "collection_id": "923", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-03112006-214258", "primary_object_url": { "basename": "Rajan_Kulkarni_Thesis.pdf", "content": "final", "filesize": 1931379, "license": "other", "mime_type": "application/pdf", "url": "/923/1/Rajan_Kulkarni_Thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Mechanics of the Cytoskeleton: Examining the Dynamics of Cytoplasmic Transport through Fluorescence Microscopy", "author": [ { "family_name": "Kulkarni", "given_name": "Rajan P.", "clpid": "Kulkarni-Rajan-P" } ], "thesis_advisor": [ { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" } ], "thesis_committee": [ { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" } ], "local_group": [ { "literal": "div_biol" } ], "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.
", "doi": "10.7907/85ea-ed40", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:5146", "collection": "thesis", "collection_id": "5146", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-12272005-160649", "primary_object_url": { "basename": "CHC_Thesis.pdf", "content": "final", "filesize": 9441271, "license": "other", "mime_type": "application/pdf", "url": "/5146/1/CHC_Thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Directed Evolution of the Transcriptional Activator LuxR", "author": [ { "family_name": "Collins", "given_name": "Cynthia Hollie", "orcid": "0000-0003-1647-860X", "clpid": "Collins-Cynthia-Hollie" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Parker", "given_name": "Carl Stevens", "orcid": "0000-0001-9795-4211", "clpid": "Parker-C-S" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "LuxR-type proteins are responsible for density-dependent transcriptional regulation in quorum-sensing systems that employ acyl-homoserine lactones (acyl-HSLs) as signal molecules. The Vibrio fischeri lux operon, which includes LuxR, has been well studied and shown to function in E. coli. The components of the lux operon have been used to engineer de novo genetic circuits because they provide a versatile intercellular communications system. We have used directed evolution to engineer LuxR to generate new components for engineering genetic circuits with a wide range of communications functions and to explore the evolution of LuxR specificity and response.
\r\n\r\nWe developed and validated a screening system to identify mutants of LuxR that activate gene expression with non-cognate acyl-HSLs. We screened libraries of luxR mutants for variants exhibiting increased gene activation in response to octanoyl-HSL (C8HSL). Eight LuxR variants were identified that showed a 100-fold increase in sensitivity to C8HSL. These variants displayed increased sensitivities to a broadened range of acyl-HSLs while maintaining a wild-type or greater response to LuxR\u2019s cognate signal, 3-oxo-hexanoyl-homoserine lactone (3OC6HSL).
\r\n\r\nTo generate a LuxR with a new signaling specificity, we used a dual selection system to identify LuxR variants that either activate gene expression (ON) or do not (OFF) under desired sets of conditions. The dual selection system was evaluated prior to its use, and a 490-fold enrichment in functional clones per round of ON/OFF selection was observed. We used the dual selection system to identify a LuxR variant that responds to straight-chain acyl-HSLs, but no longer responds to 3OC6HSL. A single mutation in the N terminal signal-binding domain reduces the response to acyl-HSLs having a carbonyl substituent at the third carbon of the acyl chain.
\r\n\r\nWe used the dual selection system to identify LuxR variants that activate transcription upon binding to a promoter containing a mutated operator site. An amino acid position critical for modulating the DNA-binding specificity of LuxR for the new DNA target was identified. The dual selection system provides a rapid and reliable method for identifying LuxR variants that have the desired response, or lack thereof, to a given set of acyl-HSL signals or DNA targets.
", "doi": "10.7907/61qd-8j79", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:2064", "collection": "thesis", "collection_id": "2064", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05252006-223101", "primary_object_url": { "basename": "02_sv_thesis.pdf", "content": "final", "filesize": 2213812, "license": "other", "mime_type": "application/pdf", "url": "/2064/2/02_sv_thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Manipulating Fluids: Advances in Micro-Fluidics, Opto-Fluidics and Fluidic Self-Assembly", "author": [ { "family_name": "Vyawahare", "given_name": "Saurabh", "clpid": "Vyawahare-Saurabh" } ], "thesis_advisor": [ { "family_name": "Scherer", "given_name": "Axel", "clpid": "Scherer-A" } ], "thesis_committee": [ { "family_name": "Scherer", "given_name": "Axel", "clpid": "Scherer-A" }, { "family_name": "Bockrath", "given_name": "Marc William", "clpid": "Bockrath-M-W" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "This dissertation describes work in three inter-related areas \u2013 micro-fluidics, opto-fluidics and fluidic self-assembly. Micro-fluidics has gotten a boost in recent years with the development of multilayered elastomeric devices made of poly (dimethylsiloxane) (PDMS), allowing active elements like valves and pumps. However, while PDMS has many advantages, it is not resistant to organic solvents. New materials and/or new designs are needed for solvent resistance. I describe how novel fluorinated elastomers can replace PDMS when combined with three dimensional (3-D) solid printing. I also show how another 3-D fabrication method, multilayer photo-lithography, allows for fabrication of devices integrating filters. In general, 3-D fabrications allow new kinds of micro-fluidic devices to be made that would be impossible to emulate with two dimensional chips.
\r\n\r\nIn opto-fluidics, I describe a number of experiments with quantum dots both inside and outside chips. Inside chips, I manipulate quantum dots using hydrodynamic focusing to pattern fine lines, like a barcode. Outside chips, I describe our attempts to create quantum dot composites with micro-spheres. I also show how evaporated gold films and chemical passivation can then be used to enhance the emission of quantum dots.
\r\n\r\nFinally, within fluids, self-assembly is an attractive way to manipulate materials, and I provide two examples: first, a DNA-based energy transfer molecule that relies on quantum mechanics and self-assembles inside fluids. This kind of molecular photonics mimics parts of the photosynthetic apparatus of plants and bacteria. The second example of self-assembly in fluids describes a new phenomena - the surface tension mediated self assembly of particles like quantum dots and micro-spheres into fine lines. This self assembly by capillary flows can be combined with photo-lithography, and is expected to find use in future nano- and micro-fabrication schemes.
\r\n\r\nIn conclusion, advances in fluidics, integrating materials like quantum dots and solvent resistant elastomers along with 3-D fabrication and methods of self assembly, provide a new set of tools that significantly expand our control over fluids.
", "doi": "10.7907/04XY-C430", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:1797", "collection": "thesis", "collection_id": "1797", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05142004-100932", "primary_object_url": { "basename": "thesis_final_version.pdf", "content": "final", "filesize": 1432148, "license": "other", "mime_type": "application/pdf", "url": "/1797/1/thesis_final_version.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Single-Molecule Detection and DNA Sequencing-by-Synthesis", "author": [ { "family_name": "Kartalov", "given_name": "Emil Paskalev", "orcid": "0000-0003-0521-9194", "clpid": "Kartalov-Emil-Paskalev" } ], "thesis_advisor": [ { "family_name": "Quake", "given_name": "Stephen R.", "clpid": "Quake-S-R" } ], "thesis_committee": [ { "family_name": "Quake", "given_name": "Stephen R.", "clpid": "Quake-S-R" }, { "family_name": "Bockrath", "given_name": "Marc William", "clpid": "Bockrath-M-W" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Painter", "given_name": "Oskar J.", "clpid": "Painter-O" }, { "family_name": "Anderson", "given_name": "W. French", "clpid": "Anderson-W-F" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Fluorescence detection has established itself as one of the main techniques of interrogation of biological systems. Extending those techniques to decrease the sample size to single molecules provides an absolute standard for bulk sample calibrations, as well as better insights since individual behavior is observed instead of population averages. We observed a number of fluorophores, including GFP, at the single-molecule level at room temperature. Calibrations gave a correct estimate of bulk surface densities over four orders of magnitude, through an optical, non-invasive, non-destructive means. Novel surface chemistry enabled visualization of single tagged nucleotide incorporations inside DNA immobilized on a glass surface at the single-molecule level. This technology was later extended to successful single-molecule DNA sequencing.\r\n\r\nAt the same time, PDMS microfluidics was developed to provide the plumbing control, speed, and economy of scale for a broad range of applications. Novel surface chemistry anchored DNA to the PDMS microchannels, which allowed sequencing-by-synthesis to be conducted in the microfluidic environment using optical techniques. Materials, device, and architecture problems were also solved. Finally, all technology was put together and successful microfluidic bulk-fluorescence DNA sequencing was demonstrated. The same technology is applicable to any DNA studies in microfluidic environments and can eventually be extended to close the circle to single-molecule detection.", "doi": "10.7907/50YQ-0S81", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:2350", "collection": "thesis", "collection_id": "2350", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06012004-144201", "primary_object_url": { "basename": "HansenPC.pdf", "content": "final", "filesize": 13507896, "license": "other", "mime_type": "application/pdf", "url": "/2350/1/HansenPC.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Microfluidic Technologies for Structural Biology", "author": [ { "family_name": "Hansen", "given_name": "Carl Lars Genghis", "clpid": "Hansen-Carl-Lars-Genghis" } ], "thesis_advisor": [ { "family_name": "Quake", "given_name": "Stephen R.", "orcid": "0000-0002-1613-0809", "clpid": "Quake-S-R" } ], "thesis_committee": [ { "family_name": "Quake", "given_name": "Stephen R.", "orcid": "0000-0002-1613-0809", "clpid": "Quake-S-R" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Painter", "given_name": "Oskar J.", "orcid": "0000-0002-1581-9209", "clpid": "Painter-O" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "In the post-genomic era, X-ray crystallography has emerged as the workhorse of large-scale structural biology initiatives that seek to understand protein function and interaction at the atomic scale. Despite impressive technological advances in X-ray sources, phasing techniques, and computing power, the determination of protein structure has been severely hampered by the difficulties in obtaining high-quality protein crystals. Emergent technologies utilizing microfluidics now have the potential to solve these problems on several levels, both by allowing researchers to conduct efficient assays in nanoliter reaction volumes, and by exploiting the properties of mass-transport at the micron scale to improve the crystallization process. The technique of Multilayer Soft Lithography (MSL) has been used to developed a set of microfluidic tools suitable for all stages of protein crystallogenesis, including protein solubility phase-space mapping, crystallization screening, harvesting, and in silicone diffraction studies. These tools represent the state of the art in on-chip fluid handling functionality and have been demonstrated to dramatically improve protein crystallization.\r\n", "doi": "10.7907/N9T3-7114", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" } ]