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:17314", "collection": "thesis", "collection_id": "17314", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05302025-191432965", "primary_object_url": { "basename": "caltech_thesis_Yujing.pdf", "content": "final", "filesize": 28670824, "license": "other", "mime_type": "application/pdf", "url": "/17314/1/caltech_thesis_Yujing.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Exploring Cell Diversity in Complex Tissues through Spatial Genomics and Spatial Transcriptomics", "author": [ { "family_name": "Yang", "given_name": "Yujing", "orcid": "0000-0002-2338-6263", "clpid": "Yang-Yujing" } ], "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": "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" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The study of cellular diversity is a fundamental requirement for understanding how multicellular organisms function. During the development of multicellular organisms, cells differentiate into various cell types with different molecular compositions, exhibit different phenotypes, and show distinct morphologies. Each single cell occupies a specific spatial location within different tissues and organs and performs a unique function. A holistic understanding of cells requires the integration of multiple \u201comics\u201d modalities, including genomics, epigenomics, transcriptomics, and proteomics. Current well-established single-cell sequencing methods have been used to build enormous single-cell transcriptomic atlases. While single-cell sequencing methods are now capable of multi-omic profiling, they all require cell dissociation, during which important spatial context information is lost. To study cellular diversity within its native spatial context, our lab has developed innovative spatial genomics and transcriptomics tools that enable multi-omics profiling at single-cell resolution while preserving intact tissue organization. This thesis presents two projects that leverage these tools to investigate cellular diversity in complex tissues across different biological scales, from subnuclear to tissue-level organization. In Chapter 2, we applied spatial multi-omics to the mouse cerebellum, achieving single-cell resolution profiling of 100,049 genomic loci, 17,856 nascent transcripts, 60 mature mRNAs, and 28 immunofluorescently labeled subnuclear structures. To achieve this, we developed innovative two-layer barcodes for DNA sequential fluorescence in situ hybridization (seqFISH). Combining cell-type information from nascent and mature transcriptomes, we captured the three-dimensional genomic architecture and its interactions with subnuclear compartments in a cell-type-specific manner. Our findings show that repressive chromatin compartments have greater cell-type specificity than active chromatin compartments in the mouse cerebellum. In Chapter 3, we integrated single-cell multiome sequencing, which profiles single-nucleus RNA and chromatin accessibility (ATAC) from the same cells, with seqFISH spatial transcriptomics. This approach was applied to the 17- to 18-week-old human fetal kidney, targeting 224 marker genes. By combining sequencing and spatial profiling data, we constructed a comprehensive developmental atlas of human kidney organogenesis, providing new insights into the tissue organization and gene expression patterns during kidney development.", "doi": "10.7907/r85x-qs80", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:16525", "collection": "thesis", "collection_id": "16525", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06152024-132652470", "primary_object_url": { "basename": "Wang_Zitong_2025.pdf", "content": "final", "filesize": 14747054, "license": "other", "mime_type": "application/pdf", "url": "/16525/2/Wang_Zitong_2025.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Theoretical and Computational Analysis of Cell Migration in Complex Tissue Environments", "author": [ { "family_name": "Wang", "given_name": "Zitong (Jerry)", "orcid": "0000-0001-8008-7318", "clpid": "Wang-Zitong-Jerry" } ], "thesis_advisor": [ { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" } ], "thesis_committee": [ { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Eberhardt", "given_name": "Frederick", "clpid": "Eberhardt-Frederick" }, { "family_name": "Merchant", "given_name": "Akil Abid", "orcid": "0000-0001-7472-822X", "clpid": "Merchant-Akil-Abid" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Cells sense and respond in spatially structured environments, including soils and tissue. My Ph.D. projects centered on developing new theoretical models and computational methods to understand how cells migrate in complex environments.
\r\n \r\nThe first project is more theoretical in nature, leveraging information theory to study how the spatial organization of cell signaling pathways are adapted to the cell's natural environment. In tissue and soil, cells must localize to their targets by navigating distributions of extracellular ligands that are spatially discontinuous, consisting of local concentration peaks, due to binding a non-uniform network of ECM fibers. It is unclear how cells navigate patchy environments while not getting trapped in local concentration peaks. To answer this question, we framed navigation as a problem of maximizing mutual information in space and developed a computational algorithm for computing signaling pathway architectures that maximize mutual information in simulated natural environments. We found that for cells in tissues and soils, dynamic localization of membrane receptors dramatically boosts sensing precision and enables cells to navigate to chemical sources 30 times faster, but this receptor localization strategy is relatively inconsequential for cells in purely diffusive environments. Further, we found that anisotropic receptor dynamics previously observed in immune cells and growth cones are nearly optimal as predicted by our model.
\r\n\r\nThe second project is more computational in nature, leveraging multiplexed tissue imaging to understand T-cell migration in tumor microenvironments. Immunotherapies can halt or slow down cancer progression by activating either endogenous or engineered T-cells to detect and kill cancer cells. T-cells must infiltrate the tumor core for immunotherapies to be effective. However, many solid tumors resist T-cell infiltration, challenging the efficacy of current therapies. In collaboration with clinician scientists at Cedars-Sinai Medical Center, we developed an integrated deep learning framework, Morpheus, that takes large-scale spatial omics profiles of patient tumors, and combines a formulation of T-cell infiltration prediction as a self-supervised machine learning problem with a counterfactual optimization strategy to generate minimal tumor perturbations predicted to boost T-cell infiltration. We applied Morpheus to 368 metastatic melanoma and colorectal cancer samples assayed using 40-plex imaging mass cytometry, discovering cohort-dependent, combinatorial perturbations, involving CXCL9, CXCL10, CCL22 and CCL18 for melanoma and CXCR4, PD-1, PD-L1 and CYR61 for colorectal cancer, predicted to support T-cell infiltration across large patient cohorts. Using only raw image data, Morpheus also identified distinct therapeutic strategies for different patient strata such as cancer stage or fatty liver presence. Our work presents a paradigm for counterfactual-based prediction and design of cancer therapeutics using spatial omics data.
", "doi": "10.7907/mj08-b258", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:16533", "collection": "thesis", "collection_id": "16533", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07052024-170119371", "primary_object_url": { "basename": "Thesis.pdf", "content": "final", "filesize": 43525786, "license": "other", "mime_type": "application/pdf", "url": "/16533/1/Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Active Acquisition Methods for Single Cell Genomics", "author": [ { "family_name": "Chen", "given_name": "Xiaoqiao", "orcid": "0000-0003-4685-3466", "clpid": "Chen-Xiaoqiao" } ], "thesis_advisor": [ { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" } ], "thesis_committee": [ { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Yue", "given_name": "Yisong", "orcid": "0000-0001-9127-1989", "clpid": "Yue-Yisong" }, { "family_name": "Bouman", "given_name": "Katherine L.", "orcid": "0000-0003-0077-4367", "clpid": "Bouman-K-L" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "We introduce two novel computational methodologies, ActiveSVM and Active Cell Inference, aimed at reducing the costs and enhancing the efficiency of single-cell mRNA sequencing and spatial transcriptomics, respectively. ActiveSVM employs an active learning approach to identify minimal yet highly informative gene sets for cell-type classification, physiological state identification, and genetic perturbation responses in single-cell datasets. By focusing on misclassified cells through an iterative process, ActiveSVM efficiently scales to analyze over a million cells, demonstrating around 90% accuracy across various datasets, including cell atlas and disease characterization studies.
\r\n\r\nActive Cell Inference complements this by utilizing ordered gene sets, developed through ActiveSVM, to streamline spatial genomics measurements. This end-to-end pipeline significantly reduces measurement time and costs by up to 100-fold in scientific and clinical settings. It optimizes the gene probing process by identifying well-classified cells early, allowing for targeted gene application based on cell classification certainty. This method's efficacy is further enhanced by a temporal scaling calibration scheme, improving calibration accuracy throughout its iterative process.
\r\n\r\nBoth methodologies were rigorously tested on the expansive Human Cell Atlas dataset, using the advanced computational tool, CellxGene-Census, involving over 60 million cells. This integration facilitated the creation of precise gene sets for various human tissues, dramatically improving the efficiency and reliability of these cutting-edge genomic techniques. Together, ActiveSVM and Active Cell Inference represent significant advancements in the application of genomics to clinical diagnostics, therapeutic discovery, and genetic screens, promising substantial reductions in the operational complexities and costs associated with next-generation sequencing technologies.
", "doi": "10.7907/nsn8-nd79", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17117", "collection": "thesis", "collection_id": "17117", "cite_using_url": "https://resolver.caltech.edu/CaltechThesis:03312025-203601435", "primary_object_url": { "basename": "KatsuyaColon_Thesis_Final.pdf", "content": "final", "filesize": 13949697, "license": "other", "mime_type": "application/pdf", "url": "/17117/1/KatsuyaColon_Thesis_Final.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "In Situ Signal Amplification for Spatial Transcriptomics Using Programmable DNA Assemblies", "author": [ { "family_name": "Col\u00f3n", "given_name": "Katsuya Lex", "orcid": "0000-0002-7347-6128", "clpid": "Col\u00f3n-Katsuya-Lex" } ], "thesis_advisor": [ { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "thesis_committee": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "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_chem" } ], "abstract": "Sequential Fluorescent In Situ Hybridization (seqFISH) has been an invaluable tool in imaging-based spatial transcriptomics, aiding researchers in elucidating spatially-resolved, gene expression patterns in intact tissues and cell culture models. However, methods that rely on smFISH, such as seqFISH, suffer from poor signal-to-noise ratio in certain tissue types or target RNA, require many fluorescently labeled RNA targeting probes which prohibits imaging of small RNA species, and exhibit poor sample throughput due to the need of high magnification objective or long exposure times. Herein, we develop solutions to these limitations by developing and utilizing a robust signal amplification strategy. While various amplification technologies exist, their limitations often hinder broad applicability. Moreover, we desire an amplification platform that is amenable to the denaturing wash conditions used in seqFISH. We will begin Chapter I by discussing the background, technical challenges, and utility of various in situ signal amplification technologies. Chapter II details the exploration and technical limitations of rolling circle amplification (RCA) and branched DNA (bDNA) assembly utilizing ssDNA padlock amplifier strands. Chapter III discusses the design and development of a novel amplification strategy called Signal amPlicAtion by Recursive Crosslinking (SPARC), which builds upon the knowledge gained from Chapter II. We highlight SPARC as a unique photochemical signal amplification method that iteratively deposits amplifier strands near the primary probe target for linear signal amplification. Then, the deposited amplifier strands act as a scaffold for branched DNA assembly, leading to an exponential signal amplification. Through each deposition and assembly step, amplifier strands are photo-crosslinked to the extracellular matrix, forming highly stable DNA nanostructures that can withstand harsh denaturing wash conditions. We demonstrate the utility of SPARC in amplifying signal of both single-molecule transcripts and proteins.", "doi": "10.7907/pp5f-pk64", "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:16431", "collection": "thesis", "collection_id": "16431", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05282024-221603734", "primary_object_url": { "basename": "MorganSchwartz_Thesis_20240601.pdf", "content": "final", "filesize": 21199702, "license": "other", "mime_type": "application/pdf", "url": "/16431/2/MorganSchwartz_Thesis_20240601.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Accelerating Biological Discovery with Deep Learning and Spatial Optical Barcodes", "author": [ { "family_name": "Schwartz", "given_name": "Morgan Sarah", "orcid": "0000-0001-8131-9125", "clpid": "Schwartz-Morgan-Sarah" } ], "thesis_advisor": [ { "family_name": "Van Valen", "given_name": "David A.", "orcid": "0000-0001-7534-7621", "clpid": "Van-Valen-D" } ], "thesis_committee": [ { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "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" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Van Valen", "given_name": "David A.", "orcid": "0000-0001-7534-7621", "clpid": "Van-Valen-D" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Methodological advances in biology have given us a powerful suite of tools for measuring the state of the cell. Among these methods, next-generation sequencing, including single-cell methods, enables comprehensive measurement of gene expression; however, sequencing-based methods often preclude the collection of other visible phenotypic information. In contrast, light microscopy supports many different measurements that can be acquired in sequential rounds of labeling and imaging because light microscopy does not destroy the sample. Furthermore, light microscopy supports live cell imaging, including the use of fluorescent reporters to observe signaling dynamics in real time. In order to fully understand cellular function, multimodal data collection is needed that encompasses live cell response, end-point phenotypes, and finally perturbations to test the components of relevant signaling networks. In this thesis, I present key advances to create a unified experimental platform for interrogating the cell state. This platform uses light microscopy to collect multimodal measurements of cell state while supporting high-throughput perturbation screening. This platform is supported by a suite of deep learning analysis tools to enable quantitative analysis of these high-dimensional datasets. In Chapter 2, I introduce Caliban, our deep learning method for nuclear segmentation and tracking. In Chapter 3, I present a new method of optical barcodes to enable microscopy-based pooled perturbation screens. Finally, in Chapter 4, I describe preliminary work that leverages the previously described cell tracking and barcoding methodologies to explore the interdependencies of signaling pathway dynamics.", "doi": "10.7907/55c7-8142", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:15163", "collection": "thesis", "collection_id": "15163", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05112023-130637882", "primary_object_url": { "basename": "Liaw_Eric_2023_Thesis.pdf", "content": "final", "filesize": 10234218, "license": "other", "mime_type": "application/pdf", "url": "/15163/2/Liaw_Eric_2023_Thesis.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "A Novel, Rapid Phenotypic Assay for a Beta-Lactam Antibiotic Susceptibility and an Analysis of its Theoretical Limits", "author": [ { "family_name": "Liaw", "given_name": "Eric Jer-Jiun", "orcid": "0000-0003-2244-8335", "clpid": "Liaw-Eric-Jer-Jiun" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Current management of bacterial infections is limited by the slow turnaround time of culture-based antibiotic susceptibility testing (AST). Culture-free phenotypic AST methods, though faster, are limited not only by analytical sensitivity but also by the low number, density, and purity of live pathogens present in clinical specimens before culturing. Separating and concentrating pathogens from clinical specimen matrices and improving the analytic sensitivity of phenotypic measurement technologies remain active areas of research. However, to date, the literature lacks consensus over what is a reasonable goal for the minimum number of pathogens in a clinical specimen needed to accurately perform phenotypic AST.
\r\n\r\nI describe \"bulk filtration AST\" and \"digital filtration AST,\" two new filtration-based AST methods that improve an AST method previously published by others and myself. These methods use nucleic acid quantification to assess the activity of antibiotic classes (and only those classes) targeting peptidoglycan turnover, specifically the beta-lactams, which are the most frequently prescribed class of antibiotics. I use filtration AST to quantify the in vitro pharmacodynamics of beta-lactam antibiotics over time scales shorter than two hours, and I simultaneously validate the methods' accuracies on clinical isolates of Enterobacteriaceae. To analyze filtration AST results, either for fitting parameter values or for predicting susceptibility, I derive probabilistic models for the outcomes of each of the two filtration AST methods, then perform Bayesian parameter inference from my data.
\r\n\r\nI then propose a general mathematical framework for defining the concepts of the phenotypic assay and the ideal phenotypic assay. Within this framework, I calculate the ideal filtration AST performance as a function of the number of cells assayed, my fitted pharmacodynamic parameters, and other variables. Interestingly, the observed performance of my implementation of digital filtration AST is consistent with the implementation's approaching the ideal performance. I hope my demonstration of these new methods and my theoretical framework will help guide future research into rapid phenotypic AST.
", "doi": "10.7907/qhvg-7q92", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:14382", "collection": "thesis", "collection_id": "14382", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10012021-223453672", "primary_object_url": { "basename": "20211001_PhD_Thesis_MA_Proofread_Final.pdf", "content": "final", "filesize": 2995353, "license": "other", "mime_type": "application/pdf", "url": "/14382/1/20211001_PhD_Thesis_MA_Proofread_Final.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Development of Single-Cell SPRITE: a Tool for Measuring Heterogeneity of 3D DNA Organization", "author": [ { "family_name": "Arrastia", "given_name": "Mary Villanueva", "orcid": "0000-0002-0723-3574", "clpid": "Arrastia-Mary-Villanueva" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Across eukaryotic cells, DNA from each nucleus is organized in three dimensions in order to help regulate transcriptional activity. Decades of chromosome capture technologies have revealed fundamental chromatin structures, providing information about how DNA is assembled genome-wide. The majority of these methods utilize direct physical ligation of DNA molecules to generate pairwise interactions, which have provided information about short-range interactions and intra-chromosomal structures. Recent technologies have moved toward identifying multiple DNA interactions simultaneously without physical ligation of DNA molecules, revealing information about long-range interactions and inter-chromosomal structures. One of the biggest limitations of these methods is that they only study DNA organization in bulk, which misses the heterogeneity of chromosomal structures at the single-cell level. As a result, single-cell chromosome capture methods have been developed to begin probing into the cell-to-cell variability of DNA organization and answer long-standing questions regarding single-cell structure. However, single-cell methods are currently limited to identifying low-resolution, intra-chromosomal DNA interactions with few numbers of cells. This creates a need for an improved, high-throughput single-cell method that can capture high-resolution structures and simultaneous mapping of both intra- and inter-chromosomal interactions to better elucidate single-cell DNA organization. In this thesis, we describe the development of 'single-cell split-pool recognition of interactions by tag extension' (scSPRITE), a single-cell chromosome capture method that allows for mapping of high-resolution, intra- and inter-chromosomal structures across thousands of cells. Through scSPRITE, we were not only able to reveal fundamental information about single-cell DNA organizations, but we can also quantitatively measure the variability of DNA interactions from cell to cell.
", "doi": "10.7907/w70x-2294", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14409", "collection": "thesis", "collection_id": "14409", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10282021-191743624", "type": "thesis", "title": "Quantitative Sequencing and its Application to Studies of the Human Small-Intestine Microbiota", "author": [ { "family_name": "Barlow", "given_name": "Jacob T.", "orcid": "0000-0002-1842-4835", "clpid": "Barlow-Jacob-T" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "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" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Our understanding of the interplay between microbial species and the hosts they live on and in is continually expanding. New insights have focused not only microorganisms that drive specific disease states but also those that help maintain human health. As research drives towards mechanistic understanding of host-microbe relationships new quantitative tools are needed to help interrogate these complex interactions. Chapter I of this thesis discusses formulation of a method for rapid detection of antibiotic resistance in Neisseria gonorrhoeae. Our approach identified RNA signatures from transcriptional profiling of Neisseria gonorrhoeae after 10-minute antibiotic exposure. Utilization of these RNA markers allowed for rapid identification of antibiotic susceptibility or resistance to the antibiotic ciprofloxacin. Chapter II shifts focus to the development of a quantitative sequencing technique for the measurement of absolute taxon abundances in complex microbial communities. Combining the precision of digital PCR with the high-throughput nature of 16S rRNA gene amplicon sequencing allowed for simultaneous quantitative profiling of all bacterial taxa in host-associated microbial communities. We extensively characterized our quantitative sequencing methodology in the presence of high host nucleic acid levels and low microbial loads to understand the limits of quantification and detection in complex sample types. Last, Chapter III applies the quantitative sequencing technology from Chapter II to investigate the microbial community of the human small intestine, specifically the duodenum. Data from the duodenum of 250 individuals revealed a wide range of total microbial loads and a distinct subset of microbes, termed disruptor taxa, that were associated with small intestinal bacterial overgrowth (SIBO) and GI symptom severity.
", "doi": "10.7907/ca28-fk21", "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:14204", "collection": "thesis", "collection_id": "14204", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05302021-051953086", "primary_object_url": { "basename": "CheeHuat(Linus)Eng_thesis.pdf", "content": "final", "filesize": 6063063, "license": "other", "mime_type": "application/pdf", "url": "/14204/1/CheeHuat(Linus)Eng_thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Plus Ultra: Genome-Wide Spatial Transcriptomics with RNA seqFISH+", "author": [ { "family_name": "Eng", "given_name": "Chee Huat (Linus)", "orcid": "0000-0002-2521-9696", "clpid": "Eng-Chee-Huat-Linus" } ], "thesis_advisor": [ { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "thesis_committee": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Visualizing single cells and their organization in intact tissue is crucial to understanding their governing biological function. Even though single cell RNA sequencing has provided many insights into the heterogeneity and gene expression profiles across many tissue types, the dissociation process which loses the spatial information is hindering our deeper understanding of how these transcriptional distinct cell types are organized and interacting in their native tissue environment.
\r\n\r\nThe thesis begins by giving a background on how single cell RNA sequencing has transformed biology and the emergence of spatial technology such as sequential fluorescence in situ hybridization (seqFISH). While spatial methods are useful for mapping the cell types identified from single cell RNA sequencing, the need for turning spatial technology such as seqFISH, which has high detection efficiency of the transcriptome with spatial information, into an in situ discovery tool is discussed as the scientific community\u2019s goal heads towards building spatial atlases for every human tissues and organs such as the brain.
\r\n \r\nWhile seqFISH has high detection efficiency, it is still limited in the number of genes capable of profiling at once. The major obstacle is the optical crowding problems when more RNA species are targeted and imaged using a fluorescence microscope. In Chapter 2, we first investigated, if the RNA molecules are instead captured on a coverslip and profiled with sequential barcoding strategy, the FISH-based method will reliably characterize the transcriptome when molecular crowding is not an issue.
\r\n \r\nFinally, in Chapter 3, we demonstrate the barcoding strategy to break through the molecular crowding limit of multiplexed FISH. From being able to profile hundreds to a thousand genes by various multiplexed FISH methods at that time in the field, we succeeded in profiling 10,000 genes by RNA seqFISH+, an evolved version of seqFISH, in various intact tissue sections, turning seqFISH+ into a spatial discovery technology with its genome-wide coverage and high detection efficiency. The work described in this part of the thesis is highlighted in Nature Method\u2019s Method of The Year 2020- Spatially-resolved Transcriptomic article.
", "doi": "10.7907/nvfe-5j74", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:14062", "collection": "thesis", "collection_id": "14062", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01292021-130102777", "type": "thesis", "title": "Single-Cell Analysis of Normal and Perturbed Early T-Cell Developmental Processes", "author": [ { "family_name": "Zhou", "given_name": "Wen", "orcid": "0000-0003-0357-2744", "clpid": "Zhou-Wen" } ], "thesis_advisor": [ { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" } ], "thesis_committee": [ { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Bronner", "given_name": "Marianne E.", "orcid": "0000-0003-4274-1862", "clpid": "Bronner-M-E" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Early T-cell development converts multipotent precursors to committed pro-T cells, silencing progenitor genes while inducing T-cell genes. However, both the underlying steps of developmental progression and the regulations involved have remained obscure. Although some of the expressions of important regulators in early T-cell development have been studied in bulk populations, the nature of heterogeneity in this constantly refreshed developmental continuum makes it difficult to understand the developmental trajectories that the cells have undergone using bulk analysis, both in natural conditions and under gene perturbations.
\r\n\r\nCombining droplet-based single cell RNA sequencing (scRNA-seq), deep-sequenced whole-transcript scRNA-seq, and seqFISH for key regulatory genes, we established regulatory phenotypes of sequential ETP subsets; confirmed initial co-expression of progenitor- with T-cell specification genes; defined stage-specific relationships between cell-cycle and differentiation; and generated a pseudotime model from ETP to T-lineage commitment, supported by RNA velocity and transcription factor perturbations. This model was validated by developmental kinetics of ETP subsets at population and clonal levels. The results imply that multilineage priming is integral to T-cell specification in natural developing pro-T cells in the thymus.
\r\n\r\nMoreover, we examined the functional implications of some of the transcription factors (TFs) through bone marrow (BM) derived ex-vivo differentiation systems. Using scRNA-seq, Cell Hashing, and a pool-based CRISPR/Cas9 perturbation system, we established the normal and perturbed developmental trajectories before and after the T-lineage commitment stages. Our analysis revealed that, without the essential lineage commitment TF, Bcl11b, the developing early T cells immediately realized the lack of the essential regulator around the proliferating late DN2a stage. But instead of pushing the developmental path backwards to resemble the earlier stage of uncommitted cells, cells lacking Bcl11b underwent a diverging route of accumulation of 'non-T' genes that are not naturally expressed in earlier stages, potentially leading to the eventual loss of Notch responses. Our results also revealed the complex regulations by TFs that set up the earliest T-lineage progression and commitment conditions. The SCENIC analysis suggested that Gata3 and Tcf7, despite both being important regulatory factors for T-lineage progression, have very different regulatory roles in controlling proliferation and suppressing myeloid lineages. Furthermore, pseudotime analysis also showed that some of the stem and progenitor genes and 'multilineage' associated genes expressed by early pro-T cells potentially hold back the T-lineage differentiation speed. In summary, our study leveraged both in vivo thymic pro-T cells' developmental trajectory obtained through single-cell analysis and ex-vivo derived T cells for internal-controlled perturbations, and revealed some profound roles of TFs in regulating early T-cell differentiation processes.
", "doi": "10.7907/733t-mg82", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "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:11718", "collection": "thesis", "collection_id": "11718", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06072019-153337463", "primary_object_url": { "basename": "Thesis-Yandong Zhang-Final.pdf", "content": "final", "filesize": 2182617, "license": "other", "mime_type": "application/pdf", "url": "/11718/1/Thesis-Yandong Zhang-Final.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Highly Multiplexed Imaging of E. Coli Chromosome and Sensitive Detection of Single-Cell Protein", "author": [ { "family_name": "Zhang", "given_name": "Yandong", "orcid": "0000-0003-3291-9209", "clpid": "Zhang-Yandong" } ], "thesis_advisor": [ { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" } ], "thesis_committee": [ { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "clpid": "Ismagilov-R-F" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The driving force for biology research is the development of new techniques which allow high-sensitivity, high-throughput measurement in various contexts. Over the past decade, the emerging of a variety of single-cell techniques have greatly transformed our understanding of biological system. My thesis work was therefore focused on development of new single- cell techniques and use the techniques to generate new insights into biological system. Specifically, in the first part of my thesis work, we developed DNA seqFISH, a technique that allows us to image more than 100 different loci on the chromosome in single cells. We applied this technique to image E. coli chromosome with 50kb genomic resolution and 50nm spatial precision. Our data allows us to parse the E. coli chromosome structure according to their different spatial conformations and different cell-cycle stages. We identified two chromosome conformations with distinct domain structures, which is obscured from previous population-average research. We further characterized the domain structure dynamics during daughter chromosome segregation. Therefore, our data provides a high- resolution, dynamic view of E. coli chromosome structure.
\r\n\r\nIn the second part, we developed a novel method for sensitive detection of targeted protein and its post-translational modification (PTM) isoform in single cells. Instead of depending on antibodies to distinguish targeted protein and its PTM isoform, we developed an efficient covalent barcoding strategy to barcode targeted protein inside the cells. Thereafter, targeted protein and its PTM isoform are separated by conventional gel electrophoresis, while their single-cell identity is preserved in the covalently attached oligo. By counting the attached DNA oligos using next-generation sequencing, targeted protein, and its PTM isoform can be accurately measured. We demonstrated the utility of the technology by quantification of histone protein, H2B and its mono-ubiquitination isoform, H2Bub at single-cell level. Our method revealed the single-cell heterogeneities of H2Bub/H2B ratio and its cell-cycle dynamics. Our method therefore provides an antibody-free method for sensitive detection of proteins and its isoforms in single cells.
", "doi": "10.7907/CDSX-MR28", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:10382", "collection": "thesis", "collection_id": "10382", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08182017-161833257", "primary_object_url": { "basename": "MDavis_THESIS_ASSEMBLED_FINAL.pdf", "content": "final", "filesize": 7374405, "license": "other", "mime_type": "application/pdf", "url": "/10382/1/MDavis_THESIS_ASSEMBLED_FINAL.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Computational Studies of Noncovalent Interactions in Ligand-Gated Ion Channels \u2013 and - Synthesis and Characterization of Red and Near Infrared Cyanine Dyes", "author": [ { "family_name": "Davis", "given_name": "Matthew Robert", "orcid": "0000-0002-6374-4555", "clpid": "Davis-Matthew-Robert" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This thesis is presented in two parts. The first part, Chapter 2, 3, and 4, offers a series of studies on noncovalent interactions in Ligand Gation Ion Channels (LGICs). The second part describes a series of studies involving the synthesis and characterization of cyanine dyes. The common thread in this work is the use of Density Functional Theory (DFT) to study chemical-scale phenomenon. Chapter 1 offers a brief introduction to DFT and a comparison with other traditional computational chemistry methodology; Hartree-Fock (HF). Summaries of the use of DFT to study both noncovalent interations and electronically excited states are also presented. In addition, the author comments on the correct application of DFT.
\r\n\r\nChapter 2 details a computational study of the cation-\u03c0 interaction of complex cations to substituted benzenes and indoles. The cation-\u03c0 interaction is the electrostatic interaction between a cation and the negative electrostatic potential on an aromatic ring originating from its large permanent quadrupole moment. This chapter, in addition to establishing the correct computational parameters, establishes a large set of substituent effects with which to study cation-\u03c0 interactions in vivo. These binding energy values are compared to previous applications of cation-\u03c0 binding energies from our lab, and it was found that the derived binding energies are sufficiently accurate.
\r\n\r\nChapter 3 applies the foundational knowledge from the previous chapter to study cation-\u03c0 interactions of cationic ligands to multiple aromatics. This is a common motif in vivo known as the aromatic box. Using this methodology, it is established that cation binding in this form is cooperative. Further, many aromatic boxes from crystal structures were evaluated energetically.
\r\n\r\nChapter 4 describes work to develop a new amino acid to study hydrogen bonds in Xenopus laevis oocytes. These fluorinated aliphatic amino acids inductively attenuate the hydrogen bond accepting ability of the carbonyl. This new strategy was used to probe for a hydrogen bond between the indole NH \u03b14 TrpB and a backbone carbonyl associated with L119 on the \u03b22 subunit of the \u03b14\u03b22 nicotinic acetylcholine receptor (nAChR). The fluorinated amino acids were validated computationally and with NMR studies. This new strategy showed that the \u03b14-\u03b22 interfacial hydrogen prediction was false.
\r\n\r\nChapter 5 describes the synthesis and characterization of a series of meso-aromatic-acetylene cyanine dyes which feature a very large Stokes shift. Synthesis of the dyes features a key Sonagashira reaction. These dyes are investigated photophysically and computationally using time dependent DFT (TDDFT). The mechanism for this Stokes shift is an excitation to the S2 state, relaxation to the S1 state, and normal cyanine fluorescence.
\r\n\r\nChapter 6 describes three separate strategies to construct a cyanine-based photocage to release drugs in vivo using an ortho-quinone methide strategy. One strategy utilized an acetylene-aromatic cyanine dye much like those described in Chapter 5, the second utilized an ethynyl-trimethylphenyl cation dye, and the third a photoinduced electron transfer cyanine dye. None of these strategies produced a usable photocage. The failure of these strategies are ascribed to both the short excited state lifetime of cyanine dyes and the direction of the transition dipole moment.
\r\n\r\nFinally, three appendices are presented. Appendix A describes early work to synthesize and characterize a meso-hydroxy substituted Cy5 dye. Appendix B offers many of the same computations as Chapters 2 and 3 using HF instead of DFT. Appendix C describes orbital mixing of cyanine dyes from Chapter 5 using HF instead of DFT.
", "doi": "10.7907/Z9XK8CQF", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10966", "collection": "thesis", "collection_id": "10966", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292018-192944407", "type": "thesis", "title": "Noncommutative Biology: Sequential Regulation of Complex Networks and Connected Matter", "author": [ { "family_name": "Letsou", "given_name": "William Peter", "orcid": "0000-0002-4969-2330", "clpid": "Letsou-William-Peter" } ], "thesis_advisor": [ { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" } ], "thesis_committee": [ { "family_name": "Weitekamp", "given_name": "Daniel P.", "clpid": "Weitekamp-D-P" }, { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Murray", "given_name": "Richard M.", "clpid": "Murray-R-M" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "During animal development from zygote to adult, a limited set of regulatory molecules are autonomously deployed in the service of tissue-specific gene expression (reviewed in chapter 1). Inherent in the process is the tension that single cells sample heterogeneous expression states while robustly maintaining a collective final outcome. This thesis addresses theoretical issues that help resolve the paradox that one cell simultaneously contains the fate information of many.
\r\n\r\nPrevious models of development have likened cell fate to minima on a smooth potential energy surface. Such static pictures can be misleading because they suggest the egg knows the path it will take to the adult before it divides even once. Recognition that the potential analogy is an oversimplification has led others to propose that the surface is actually nonsmooth. Chapter 2 reviews the theoretical basis for smooth potentials and resolves these problems by appealing to the tangent space of gene expression. It is then shown that if the potential difference is sufficient to characterize the difference between egg and adult, then the tangent space controls on gene expression are one-dimensional. Furthermore, a shortcoming of models ignoring the connectivity and common origin of dividing cells is that they erect artificial barriers between alternative fates. A fundamentally different picture is sketched wherein the difference between egg and adult is schematized as the shape of the locus of equipotential fates accessible at the same point in time. The conjugacy of space and time is invoked to explain how the requirement that each fate be on a line of equipotential is the same as requiring that each alternative fate move the same distance down the surface at each step. The developmental trajectory is deterministic but not known in advance because it needs to be ascertained at each step which way cells \"turn\" in order to maintain their equipotential relationship. Chapters 3 and 4 refine this sequential model of collective development with specific examples.
\r\n\r\nA simple solution to the problem of cell-type specific gene expression is combinatorial binding of transcription factors at promoters. It is shown in chapter 3 that such models result in substantial information bottlenecks, because all cell fate information is concentrated at the start. We explore a novel, noncommutative model of gene regulation—known as sequential logic—that spreads the information out over time. It is shown using time sequences of noncommutative controllers that targets which otherwise would have been activated together can be regulated independently. We derive scaling laws for two noncommutative models of regulation, motivated by phosphorylation/neural networks and chromosome folding, respectively, and show that they scale super-exponentially in the number of regulators. It is also shown that specificity in control is robust to loss of a regulator. Consequently, sequential logic overcomes the information bottleneck in complex problems and enables novel solutions through roundabout strategies. The theoretical results are connected to real biological networks demonstrating specificity in the context of promiscuity.
\r\n\r\nNoncommutative sequential logic has improved storage capacity, but it does not specify who or what supplies the sequences of input that determine cell fate. Chapter 4 offers a solution by way of the seemingly unrelated problem of looping in twisted strings. Cells and strings obey a set of common space-time constraints, ultimately due to the conservation of energy. It is argued that the most parsimonious allocation of energy from the straight to strained string is the one in which each segment sees the same share of the total. Planar looping is shown to be a consequence of the parsimony principle and the Euler-Poincaré equations for rotational motion in the presence an applied torque. We then solve the problem for the looping of a twisted string; with two strains, the Euler-Poincaré predict a different answer than the classical Frenet-Serret equations. Using the results of chapter 2, it is concluded that the Frenet-Serret curvatures assigned ahead of time are not guaranteed to generate space curves that conserve energy: the predicted string has localized strains the Euler-Poincaré solution lacks. Rotational dynamics of strings are connected to developing organisms by postulating conserved RNA polymerase as an analog of angular momentum, and transcriptional activity as energy. Alternative fates along a one-dimensional \"string\" of dividing cells are possible by finding the RNAP distribution that conserves transcriptional activity along a curve of constant developmental potential. Consequently, each alternative fate samples a different sequence of changes to the distribution as it follows a local gradient downhill from high to low developmental potential over time.
\r\n\r\nIn conclusion, regulation in the tangent space of gene expression resolves the paradox that development has a unique solution specified in the DNA of the egg which cannot be determined with certainty until completion of the adult. Noncommutative sequential logic generates complexity that cannot be realized at the start, while interdependent cells (and strings) require time to ensure that each fate is at the same potential difference from a common ancestor. This fundamental reimagining of the Waddington framework can be tested using new multiplexed mRNA imaging technologies that preserve the spatial context of cells in developing tissue.
", "doi": "10.7907/9B5E-F105", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10349", "collection": "thesis", "collection_id": "10349", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07222017-155020423", "primary_object_url": { "basename": "Stone_Thesis.pdf", "content": "final", "filesize": 28125680, "license": "other", "mime_type": "application/pdf", "url": "/10349/13/Stone_Thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Cell-Selective Chemoproteomics for Biological Discovery", "author": [ { "family_name": "Stone", "given_name": "Shannon Elizabeth", "orcid": "0000-0002-6617-3874", "clpid": "Stone-Shannon-Elizabeth" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Cellular protein synthesis changes rapidly in response to internal and external cues in ways that vary from cell to cell. Global proteomic analyses of microbial communities, tissues, and organisms have provided important insights into the behavior of such systems, but can obscure the diversity of responses characteristic of different cellular subpopulations. Recent advances in cell-specific proteomics\u2014fueled in part by the development of bioorthogonal chemistries, more sensitive mass spectrometers and more advanced mining algorithms\u2014have yielded unprecedented glimpses into how proteins are expressed in space and time. Whereas previous cell-specific proteomic analyses were confined to abundant cells in relatively simple systems, recent advances in chemoproteomics allow researchers to map the protein expression patterns of even rare cells in complex tissues and whole organisms.
\r\n\r\nChapter 1 highlights recently developed strategies for cell-selective proteomics, including metabolic labeling strategies such as bioorthogonal noncanonical amino acid tagging (BONCAT). Bioorthogonal noncanonical amino acid tagging (BONCAT) is a chemoproteomic technique that enables temporal labeling of proteins. In cell-selective BONCAT, expressing a mutant aminoacyl-tRNA synthetase under the control of cell-specific genetic elements affords cellular resolution; only cells of interest can selectively incorporate a noncanonical amino acid into proteins for subsequent detection and identification. Chapter 2 details protocols to set up a cell-selective BONCAT system.
\r\n\r\nWhile BONCAT had previously been applied to studies of microbial pathogenesis in tissue culture-based models of infection, we sought to further develop the method to identify the proteome of methicillin-resistant Staphylococcus aureus (MRSA) within a mouse model of infection, as detailed in Chapter 3. We used this technique to enrich for staphylococcal proteins made within the host and in addition to finding many factors known to be important for infection, we also found many that had not previously been associated with infection. Screening several of these previously unknown factors in vivo led to the discovery of a novel protein important for MRSA infection. This unbiased approach to cell-selectively label pathogenic proteins during infection could be used as a global discovery tool for novel anti-infective strategies.
\r\n\r\nIn Chapter 4, we combine this cell-selective BONCAT strategy with microbial identification after passive clarity technique (MiPACT) to visualize both staphylococcal protein synthesis and ribosomal RNA within whole skin abscesses during infection. In Chapter 5, we continue developing cell-selective BONCAT to study microbial protein synthesis in the context of a living mouse by extending the system to Bacteroides fragilis, a common human gut commensal.
\r\n\r\nFinally, cell-selective BONCAT is wholly dependent on the bioorthogonal nature of the azide and its detection reagents. Fishing out an azide-tagged molecule from the rest of the cellular milieu requires optimization of enrichment-based strategies. In Chapter 6, we describe the development of a peptide to quantitate the gain of our enrichments.
\r\n\r\nWhile innovations in mass spectrometry and computational algorithms have facilitated the identification and quantification of thousands of proteins simultaneously from complex samples, this abundance of data does not necessarily lead to biological insight. Cell-specific proteomic techniques will play a key role in the identification of the mechanisms that govern cell specialization and that allow organisms to respond to changing environments. Overall, this work demonstrates the power of cell-selective chemoproteomics to ascertain biological insights in complex systems.
", "doi": "10.7907/Z9V122ZF", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "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:10226", "collection": "thesis", "collection_id": "10226", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05302017-222932176", "primary_object_url": { "basename": "Chan-Ken-Thesis-05-31-2017.pdf", "content": "final", "filesize": 6002407, "license": "other", "mime_type": "application/pdf", "url": "/10226/23/Chan-Ken-Thesis-05-31-2017.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Engineered Viral Vectors and Developed Tissue Clearing Methods for Single-cell Phenotyping in Whole Organs", "author": [ { "family_name": "Chan", "given_name": "Ken Yee", "orcid": "0000-0002-8853-5186", "clpid": "Chan-Ken-Yee" } ], "thesis_advisor": [ { "family_name": "Gradinaru", "given_name": "Viviana", "clpid": "Gradinaru-V" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" }, { "family_name": "Allman", "given_name": "John Morgan", "clpid": "Allman-J-M" }, { "family_name": "Gradinaru", "given_name": "Viviana", "clpid": "Gradinaru-V" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "A central question in biology is how different cell types interact with each other and their native environment to form complex functional systems and networks. Although our ability to investigate this question has considerably expanded from the development of genetically encoded tools, some limitations still persist. For instance, we are limited in our ability to visualize the native three dimensional environments of whole organs. Additionally, it is challenging to efficiently deliver transgenes into difficult-to-target areas through direct-injections, such as the cardiac ganglia, or broadly distributed networks, such as the myenteric nervous system, which limits our ability to extensively study these areas. Therefore, tools and methods that overcome these limitations are needed. Towards this end, my thesis work has been focused on developing tools for single-cell resolution phenotyping in whole organs. I have been developing tissue clearing technologies to render whole organs transparent for optical interrogation and characterizing viral capsids and engineering viral vectors for noninvasive widespread gene delivery to the central and peripheral nervous system.
\r\n\r\nTissue clearing techniques for three dimensional optical interrogation were invented over a century ago. However, these earlier methods used harsh organic chemicals and failed to retain the tissue\u2019s native fluorescence or epitopes. These earlier methods eventually became incompatible to the hundreds of newly generated transgenic mouse lines that allowed for cell type-specific expression of fluorescent transgenes or to fluorescent labeling techniques, such as immunohistochemistry (IHC). The first part of my dissertation is aimed at addressing these limitations by further developing and standardizing a tissue clearing method that utilizes the vasculature to perfuse clearing reagents. This technique, called perfusion assisted agent release in situ (PARS) enables (i) whole organ clearing of soft tissue, (ii) preservation of native fluorescence, and (iii) preservation of epitopes compatible with IHC.
\r\n\r\nAlthough PARS allows us to optically investigate whole soft tissue organs, it is unsuitable for clearing bone tissue. The clearing of bone is important as it may provide optical access to delicate environments, such as the lymphatic vessels lining the dural sinuses beneath the skull that would otherwise be damaged through traditional methods. However, clearing bone tissue is challenging since it is composed of both soft (bone marrow) and hard (mineral) tissue. To overcome this challenge, I developed a clearing method that rendered intact bone tissue transparent by using EDTA to decalcify bones and by constructing a convective flow chamber to efficiently clear bones. This method, called Bone CLARITY, is able to preserve native fluorescence and epitopes. In order to demonstrate the utility of Bone CLARITY, I collaborated with colleagues to quantitatively access a rare and non-uniformly distributed population of osteoprogenitor cells in their native three dimensional environment. Bone CLARITY in conjunction with light-sheet microscope enabled the early detection of an increase to this osteoprogenitor population after administration of a novel anabolic drug, which may have been undetected with traditional techniques.
\r\n\r\nTowards my second goal, I have been working on characterizing adeno-associated viruses (AAVs) for non-invasive widespread gene delivery across the central or peripheral nervous system. Through systemic delivery, these novel AAVs are able to efficiently deliver transgenes to (i) difficult-to-target areas, such as the dorsal root ganglia; (ii) cellular populations that are widely distributed across the mouse body, such as neurons in the myenteric nervous system, and (iii) through highly selective barriers, such as the blood-brain barrier. These viruses enable rapid expression of transgenes for perturbing and monitoring cellular circuits, or for potentially treating neurological diseases. In addition, I worked on engineering or validating several different gene regulatory elements to achieve cell type restricted expression in transgenic and non-transgenic animals with AAVs. These viral vectors may prove useful in rapidly testing newly developed genetic tools. Finally, I developed and characterized two different two-component viral vector systems to control the density of labeling when systemically delivering genes with our novel engineered viruses. I utilized this two-component system to perform single-cell morphology studies in the CNS and PNS. Collectively, these capsids and vectors expand the AAV toolbox and enable efficient and versatile gene delivery into the CNS and PNS of transgenic and non-transgenic animals.
\r\n", "doi": "10.7907/Z9NC5Z7H", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:9898", "collection": "thesis", "collection_id": "9898", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07292016-195927111", "primary_object_url": { "basename": "David Selck Thesis.pdf", "content": "final", "filesize": 11243250, "license": "other", "mime_type": "application/pdf", "url": "/9898/73/David Selck Thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Microfluidics for Molecular Measurements and Quantitative Distributable Diagnostics", "author": [ { "family_name": "Selck", "given_name": "David Anthony", "orcid": "0000-0002-0591-4165", "clpid": "Selck-David-Anthony" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" }, { "family_name": "Guttman", "given_name": "Mitchell", "clpid": "Guttman-M" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "clpid": "Ismagilov-R-F" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A major challenge in global health care is a lack of portable and affordable quantitative diagnostic devices. This is because classic quantification of biomolecules is typically performed using kinetic assays that require strict control only found in controlled laboratory environments. By using the power of microfluidics, quantitative assays can be performed robustly in a \"digital\" format that is decoupled from precise kinetics through highly parallelized qualitative reactions. The benefits of performing quantitative assays in a digital format extend beyond just assay robustness to reduction of instrumental complexity, increase in quantitative precision, and an increase in the amount of information that can be gained from a single experiment. These microfluidic architectures, however, are not limited to usage in scenarios of quantification of biomolecules. These architectures can also potentially be extended to answering complex biological questions in single cells, such as determining the 3-dimensional organization of nuclear DNA and RNA.", "doi": "10.7907/Z9ZC80XT", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:9941", "collection": "thesis", "collection_id": "9941", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10172016-103350209", "type": "thesis", "title": "Experiments on Gas Phase Chemistry with High Sensitivity Laser Spectroscopy", "author": [ { "family_name": "Mertens", "given_name": "Laura Anna", "clpid": "Mertens-Laura-Anna" } ], "thesis_advisor": [ { "family_name": "Okumura", "given_name": "Mitchio", "clpid": "Okumura-M" } ], "thesis_committee": [ { "family_name": "Marcus", "given_name": "Rudolph A.", "clpid": "Marcus-R-A" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" }, { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" }, { "family_name": "Wennberg", "given_name": "Paul O.", "clpid": "Wennberg-P-O" }, { "family_name": "Okumura", "given_name": "Mitchio", "clpid": "Okumura-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Gas phase chemistry is important to many environments on Earth and beyond. The Earth\u2019s atmosphere is dominated by free radical reactions that, when perturbed by pollution, can lead to serious environmental problems like stratospheric ozone depletion and urban smog. Outside Earth, many other planetary atmospheres are affected by gas phase, radical chemistry, including the atmosphere of Saturn\u2019s Moon Titan. Gas phase chemistry in interstellar clouds can synthesize the molecular building blocks of our universe. Studying gas phase chemistry has also led to basic chemical knowledge of how chemical reactions proceed and how intermolecular forces work.
\r\n\r\nThis work is dedicated to studying gas phase chemical reactions with high-sensitivity laser spectroscopy. Laser spectroscopy can be a sensitive and selective way to detect gas phase species. Since laser pulses can both create reactants and detect the products, laser techniques allow the study of chemical kinetics in real time. Consequently, many different laser techniques have been developed to study gas phase chemistry. This thesis is divided into two sections: a longer first section on my work at the California Institute of Technology in Pasadena, CA and a smaller second section based on my work at the Universit\u00e9 de Rennes 1 in Rennes, France. These two sections, while on different topics \u2013 atmospheric chemical reactions and collisional rotational energy transfer at ultra-low temperatures \u2013 are united by their study of gas phase with laser spectroscopy, which shows the breadth of this experimental approach. This thesis will both look at kinetics (the rate of chemical reactions) and product yield of chemical reactions, both key pieces of information to modeling gas phase reactions.
\r\n\r\nThe first part of this work outlines my work at the California Institute of Technology, studying atmospheric radical chemistry with cavity-ringdown spectroscopy (CRDS). Chapter 1 put this work in a broader picture of current scientific work on Earth\u2019s atmosphere. Chapter 2 provides a detailed description of our cavity-ringdown spectrometer and temperature-controlled flow cell. Next, I discuss work on three important atmospheric reactions: the isomerization of simple alkoxy radicals (Chapter 3), the reaction of HO\u2082 with NO (Chapter 4), and the reaction of OH with NO2 (Chapter 5).
\r\n\r\nThe second, and smaller, part of this work, contains one chapter \u2013 chapter 6 \u2013 on work done at the Universit\u00e9 de Rennes 1, which describes work on the rotational energy transfer in collisions between CO and Ar at temperatures from 293 to 30 K with infrared-vacuum ultraviolet double resonance CRESU experiments.
", "doi": "10.7907/Z95X26XD", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:10179", "collection": "thesis", "collection_id": "10179", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05172017-103505376", "primary_object_url": { "basename": "Zhou_Edward Haojiang_2017.pdf", "content": "final", "filesize": 54748561, "license": "other", "mime_type": "application/pdf", "url": "/10179/44/Zhou_Edward Haojiang_2017.pdf", "version": "v13.0.0" }, "type": "thesis", "title": "Optical Focusing and Imaging through Scattering Media", "author": [ { "family_name": "Zhou", "given_name": "Edward Haojiang", "orcid": "0000-0001-7020-9502", "clpid": "Zhou-Edward-Haojiang" } ], "thesis_advisor": [ { "family_name": "Yang", "given_name": "Changhuei", "clpid": "Yang-Changhuei" } ], "thesis_committee": [ { "family_name": "Yang", "given_name": "Changhuei", "clpid": "Yang-Changhuei" }, { "family_name": "Judkewitz", "given_name": "Benjamin", "clpid": "Judkewitz-Benjamin" }, { "family_name": "Wang", "given_name": "Lihong", "clpid": "Wang-Lihong" }, { "family_name": "Vaidyanathan", "given_name": "P. P.", "clpid": "Vaidyanathan-P-P" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Optical techniques, which have been widely used in various fields including bio-medicine, remote sensing, astronomy, and industrial production, play an important role in modern life. Optical focusing and imaging, which correspond to the basic methods of utilizing light, are key to the implementation of optical techniques. In free space or a nearly transparent medium, optical imaging and focusing can be easily realized by using conventional optical elements, such as lenses and mirrors, due to the ballistic propagation of light in these media. However, in scattering media like biological tissue and fog, refractive index inhomogeneities cause diffusive propagation of light that increases with depth, which restricts the use of optical methods in thick, scattering media. Generally speaking, scattering media poses three challenges to optical focusing and imaging: wavefront aberrations, glare, and decorrelation. Wavefront aberrations can randomize light traveling through a scattering medium, disrupt the formation of focus, and break the conjugate relation in imaging. Glare caused by backscattering will largely impair the visibility of imaging, and decorrelation in dynamic media requires systems that counter the effect of scattering to operate faster than the decorrelation time. In this thesis, we explored solutions to the problem of scattering from different aspects. We presented Time Reversal by Analysis of Changing wavefronts from Kinetic targets (TRACK) technique to realize noninvasive optical focusing through a scattering medium. We showed that by taking the difference between time-varying scattering fields caused by a moving object and applying optical phase conjugation, light can be focused back to the location previously occupied by the object. To tackle the decorrelation of living tissue, we built up a fast digital optical phase conjugation (DOPC) system based on FPGA and DMD, which has a response time of 5.3 ms and was the fastest DOPC system in the world before 2017. We demonstrated that the system is fast enough to focus light through 2.3mm-thick living mouse skin. As for glare, inspired by noise canceling headphones, we invented an optical analogue termed coherence gated negation (CGN) technique. CGN can optically cancel out the glare in an active illumination imaging scenario to realize imaging through scattering media, like fog. In the experiment, we suppressed the glare by an order of magnitude and allowed improved imaging of a weak target. Finally, we demonstrated a method to image a moving target through scattering media noninvasively. Its principle roots are in the speckle-correlation-based imaging (SCI) invented by Ori Katz. We improved the technique and extended its application to bright field imaging of a moving target.
", "doi": "10.7907/Z9TX3CD1", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:9762", "collection": "thesis", "collection_id": "9762", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05262016-050606547", "type": "thesis", "title": "Structural and Biochemical Characterization of Ligand Bound States of the FeMo-Cofactor of Nitrogenase", "author": [ { "family_name": "Perez", "given_name": "Kathryn A.", "clpid": "Perez-Kathryn-A" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" }, { "family_name": "Okumura", "given_name": "Mitchio", "clpid": "Okumura-M" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Nitrogenase is the only known enzyme capable of nitrogen fixation, the reduction of dinitrogen to ammonia, a metabolically available form of nitrogen. Developing an understanding of the complex mechanism required for biological nitrogen fixation requires that the enzyme be characterized in catalytically relevant states, such as those involving ligand binding and reduction. Nitrogenase catalyzes this reaction through the cyclic interaction of two metalloproteins, the Fe-protein and the MoFe-protein which contain three distinct metalloclusters, in an ATP-hydrolysis dependent electron transfer reaction. The binding and subsequent reduction of substrates requires multiple electrons donated from the Fe-protein to the MoFe-protein, in which the active site is located. In this study, we have structurally characterized the binding of two inhibitors to the FeMo-cofactor, CO and the Se of SeCN-. Both interactions involve the displacement of a single S, and the Se was used as a label to follow the interchange of three S sites within the FeMo-cofactor during catalysis. These finding change any future approaches to characterize the mechanism of biological nitrogen fixation, requiring that structural changes be considered for substrate binding and reduction.", "doi": "10.7907/Z9M043DX", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9688", "collection": "thesis", "collection_id": "9688", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04282016-051723211", "primary_object_url": { "basename": "Xiaoze_Thesis_Caltech_04282016.pdf", "content": "final", "filesize": 13112405, "license": "other", "mime_type": "application/pdf", "url": "/9688/1/Xiaoze_Thesis_Caltech_04282016.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Computational Microscopy: Breaking the Limit of Conventional Optics", "author": [ { "family_name": "Ou", "given_name": "Xiaoze", "orcid": "0000-0001-9918-0221", "clpid": "Ou-Xiaoze" } ], "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": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Computational imaging is flourishing thanks to the recent advancement in array photodetectors and image processing algorithms. This thesis presents Fourier ptychography, which is a computational imaging technique implemented in microscopy to break the limit of conventional optics. With the implementation of Fourier ptychography, the resolution of the imaging system can surpass the diffraction limit of the objective lens's numerical aperture; the quantitative phase information of a sample can be reconstructed from intensity-only measurements; and the aberration of a microscope system can be characterized and computationally corrected. This computational microscopy technique enhances the performance of conventional optical systems and expands the scope of their applications.", "doi": "10.7907/Z9M32SRZ", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9585", "collection": "thesis", "collection_id": "9585", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02252016-123239637", "primary_object_url": { "basename": "CIT Thesis - Naeem S. Husain.pdf", "content": "final", "filesize": 58468950, "license": "other", "mime_type": "application/pdf", "url": "/9585/1/CIT Thesis - Naeem S. Husain.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "Mapping mRNA and Protein Expression with High Signal-to-Background in Diverse Organisms", "author": [ { "family_name": "Husain", "given_name": "Naeem Shahab", "clpid": "Husain-Naeem-Shahab" } ], "thesis_advisor": [ { "family_name": "Pierce", "given_name": "Niles A.", "clpid": "Pierce-N-A" } ], "thesis_committee": [ { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" }, { "family_name": "Pierce", "given_name": "Niles A.", "clpid": "Pierce-N-A" }, { "family_name": "Prober", "given_name": "David A.", "clpid": "Prober-D-A" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "In situ hybridization (ISH) techniques allow for the study of the nucleic acid expression within whole biological samples. The quality of probes for ISH dictates how accurate and bright the signal is for the experiment; however, there is currently not a systematic way to determine what the best probe set would be. In response to this, we have developed a framework to optimize an ISH probe set to achieve the greatest signal-to-background ratio. As methods like ISH help obtain more information about biological processes, there is a growing desire to simultaneously analyze various targets within the same sample to examine these complex genetic interactions. To facilitate this, a novel amplification technique called hybridization chain reaction (HCR) has allowed for the in situ detection of multiple target mRNAs concurrently in zebrafish embryos. We have now expanded this technology further by adapting HCR amplification for ISH to other model organisms, particularly, whole mount Drosophila melanogaster embryos and formalin-fixed parafin-embedded human tissue sections. Beyond looking at mRNA, immunohistochemistry (IHC) provides another tool to understand biological systems by analyzing protein expression patterns. The ability to easily look at both mRNAs and proteins in the same sample offers significant advantages as each provides unique information, but current methods are technically difficult and labor intensive. In response, we have engineered a scheme to use HCR to amplify signal for IHC. We then used this advancement to develop a straightforward protocol using HCR amplification for simultaneous detection of multiple proteins and mRNAs with a high signal-to-background ratio.", "doi": "10.7907/Z9DF6P73", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "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:9753", "collection": "thesis", "collection_id": "9753", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05252016-131059745", "primary_object_url": { "basename": "0_HSegal_Thesis_Final_Post Proof Reader.pdf", "content": "final", "filesize": 5688249, "license": "other", "mime_type": "application/pdf", "url": "/9753/4/0_HSegal_Thesis_Final_Post Proof Reader.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "Electrochemical Methods to Study Iron-Sulfur Cluster Proteins", "author": [ { "family_name": "Segal", "given_name": "Helen Muriel", "clpid": "Segal-Helen-Muriel" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Electron transfer between proteins is an important mechanism in multiple biological processes. In this thesis, methods were developed to study electron transfer in two biological contexts: 1) DNA-mediated signaling between DNA binding proteins with 4Fe-4S clusters and 2) nitrogenase.
\r\n\r\nThe first portion of this thesis focuses on the spectroscopic and electrochemical characterization of the iron-sulfur cluster in Dna2. Dna2 is a helicase-nuclease that is involved in Okazaki fragment maturation, double strand break repair, mitochondrial genome maintenance, and telomere maintenance. Dna2 is one of multiple DNA repair and replication proteins that contain a 4Fe-4S cluster, a cofactor that generally participates in electron transfer processes. It has been proposed that these enzymes may use their 4Fe-4S clusters to signal one another over large molecular distances to coordinate their activity on biological time scales through DNA-mediated redox chemistry. A combination of EPR and UV-visible absorption spectroscopy along with electrochemistry studies on DNA-modified gold electrodes was performed to provide insight into the chemical characteristics of the 4Fe-4S cluster in Dna2. These studies also provide a foundation for how DNA charge transport might coordinate the action of eukaryotic DNA repair and replication proteins with 4Fe-4S clusters.
\r\n\r\nThe second portion of this thesis describes the development of electrochemical methods to study nitrogenase, the enzyme that catalyzes the reduction of atmospheric dinitrogen to bioavailable ammonia. First, flavodoxin II, the biological reductant of the Fe-protein of nitrogenase, was characterized using a combination of electrochemical and structural methods to determine the molecular interactions that facilitate reduction of the nitrogenase iron protein. Second, two electrochemical methods, edge-plane pyrolytic graphite electrodes and single crystal gold electrodes modified with \u03c9-functionalized alkane-thiols, were adapted to study the redox chemistry at the iron-sulfur cluster of the Fe-protein. These studies provided insight into both the fundamental characteristics of electron transfer reactions involving nitrogenase, as well as insight into how to better study this enzyme using electrochemical methods.
\r\n\r\n", "doi": "10.7907/Z92Z13HT", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:8978", "collection": "thesis", "collection_id": "8978", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06032015-151007228", "primary_object_url": { "basename": "Thesis _ Thinh Bui.pdf", "content": "final", "filesize": 12279597, "license": "other", "mime_type": "application/pdf", "url": "/8978/1/Thesis _ Thinh Bui.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Cavity Enhanced Spectroscopies for Applications of Remote Sensing, Chemical Kinetics and Detection of Radical Species", "author": [ { "family_name": "Bui", "given_name": "Thinh Quoc", "clpid": "Bui-Thinh-Quoc" } ], "thesis_advisor": [ { "family_name": "Okumura", "given_name": "Mitchio", "clpid": "Okumura-M" } ], "thesis_committee": [ { "family_name": "Marcus", "given_name": "Rudolph A.", "clpid": "Marcus-R-A" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" }, { "family_name": "Libbrecht", "given_name": "Kenneth George", "clpid": "Libbrecht-K-G" }, { "family_name": "Blake", "given_name": "Geoffrey A.", "clpid": "Blake-G-A" }, { "family_name": "Okumura", "given_name": "Mitchio", "clpid": "Okumura-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This thesis describes applications of cavity enhanced spectroscopy towards applications of remote sensing, chemical kinetics and detection of transient radical molecular species. Both direct absorption spectroscopy and cavity ring-down spectroscopy are used in this work. Frequency-stabilized cavity ring-down spectroscopy (FS-CRDS) was utilized for measurements of spectral lineshapes of O2 and CO2 for obtaining laboratory reference data in support of NASA\u2019s OCO-2 mission. FS-CRDS is highly sensitive (> 10 km absorption path length) and precise (> 10000:1 SNR), making it ideal to study subtle non-Voigt lineshape effects. In addition, these advantages of FS-CRDS were further extended for measuring kinetic isotope effects: A dual-wavelength variation of FS-CRDS was used for measuring precise D/H and 13C/12C methane isotope ratios (sigma>0.026%) for the purpose of measuring the temperature dependent kinetic isotope effects of methane oxidation with O(1D) and OH radicals. Finally, direct absorption spectroscopic detection of the trans-DOCO radical via a frequency combs spectrometer was conducted in collaboration with professor Jun Ye at JILA/University of Colorado. ", "doi": "10.7907/Z96D5QXW", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8850", "collection": "thesis", "collection_id": "8850", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05072015-162723691", "primary_object_url": { "basename": "Yi-Ju-Chen-Thesis-2015.pdf", "content": "final", "filesize": 21452452, "license": "other", "mime_type": "application/pdf", "url": "/8850/1/Yi-Ju-Chen-Thesis-2015.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "The Mechanical Genome in Regulation and Infection", "author": [ { "family_name": "Chen", "given_name": "Yi-Ju", "clpid": "Chen-Yi-Ju" } ], "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": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_pma" } ], "abstract": "Biological information storage and retrieval is a dynamic process that requires the genome to undergo dramatic structural rearrangements. Recent advances in single-molecule techniques have allowed precise quantification of the nano-mechanical properties of DNA [1, 2], and direct in vivo observation of molecules in action [3]. In this work, we will examine elasticity in protein-mediated DNA looping, whose structural rearrangement is essential for transcriptional regulation in both prokaryotes and eukaryotes. We will look at hydrodynamics in the process of viral DNA ejection, which mediates information transfer and exchange and has prominent implications in evolution. As in the case of Kepler's laws of planetary motion leading to Newton's gravitational theory, and the allometric scaling laws in biology revealing the organizing principles of complex networks [4], experimental data collapse in these biological phenomena has guided much of our studies and urged us to find the underlying physical principles.", "doi": "10.7907/Z9PC308X", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "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:7921", "collection": "thesis", "collection_id": "7921", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08142013-092554646", "primary_object_url": { "basename": "MinorPaul2013thesis.pdf", "content": "final", "filesize": 11555022, "license": "other", "mime_type": "application/pdf", "url": "/7921/1/MinorPaul2013thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Wnt and FGF Signaling in C. elegans Vulval Cell Lineage Polarity", "author": [ { "family_name": "Minor", "given_name": "Paul Joseph", "clpid": "Minor-Paul-Joseph" } ], "thesis_advisor": [ { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "thesis_committee": [ { "family_name": "Bronner", "given_name": "Marianne E.", "clpid": "Bronner-M-E" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" }, { "family_name": "Hay", "given_name": "Bruce A.", "clpid": "Hay-B-A" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The interpretation of extracellular cues leading to the polarization of intracellular components and asymmetric cell divisions is a fundamental part of metazoan organogenesis. The C. elegans vulva, with its invariant cell lineage and interaction of multiple cell signaling pathways, provides an excellent model for the study of cell polarity within an organized epithelial tissue. Herein I discuss the interaction of Wnt and FGF signaling in controlling vulval cell lineage polarity with emphasis on the posterior-most cell that forms the vulva, P7.p.
\r\n\r\nThe mirror symmetry of the C. elegans vulva is achieved by the opposite division orientation of the vulval precursor cells (VPCs) flanking the axis of symmetry. Opposing Wnt signals control the division patterns of the VPCs by controlling the localization of SYS-1/ \u03b2-catenin toward the direction of the Wnt gradient. Multiple Wnt signals, expressed at the axis of symmetry, promote the wild-type, anterior-facing, P7.p orientation, whereas Wnts EGL-20 and CWN-1 from the tail and posterior body wall muscle, respectively, promote the daughter cells of P7.p to face the posterior. EGL-20 acts through a member of the LDL receptor superfamily, LRP-2, along with Ror/CAM-1 and Van Gogh/VANG-1. All three transmembrane proteins control orientation through the localization of the SYS-1.
\r\n\r\nThe Fibroblast Growth Factor (FGF) pathway acts in concert with LIN-17/Frizzled to regulate the localization of SYS-1. The source of the FGF ligand is the 1\u00b0 VPC, P6.p, which controls the polarity of the neighboring 2\u00b0 VPC, P7.p, by signaling through the sex myoblasts (SMs), activating the FGF pathway. The Wnt, cwn-1, is expressed in the posterior body wall muscle of the worm as well as the SMs, making it the only Wnt expressed on the posterior and anterior sides of P7.p at the time of the polarity decision. Both sources of cwn-1 act instructively to influence P7.p polarity in the direction of the Wnt gradient. The FGF pathway leads to the regulation of cwn-1 transcripts in the SMs. These results illustrate the first evidence of the interaction between FGF and Wnt in C. elegans development and vulval cell lineage polarity as well as highlight the promiscuous nature of Wnt signaling within C. elegans.
", "doi": "10.7907/99ZE-5Y87", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8504", "collection": "thesis", "collection_id": "8504", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06072014-140700155", "type": "thesis", "title": "Biophysics of V(D)J Recombination and Genome Packaging: In Singulo Studies on RAG, HMGB1, and TFAM", "author": [ { "family_name": "Lovely", "given_name": "Geoffrey A.", "clpid": "Lovely-Geoffrey-A" } ], "thesis_advisor": [ { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "thesis_committee": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Fraser", "given_name": "Scott E.", "orcid": "0000-0002-5377-0223", "clpid": "Fraser-S-E" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The recombination-activating gene products, RAG1 and RAG2, initiate V(D)J recombination during lymphocyte development by cleaving DNA adjacent to conserved recombination signal sequences (RSSs). The reaction involves DNA binding, synapsis, and cleavage at two RSSs located on the same DNA molecule and results in the assembly of antigen receptor genes. Since their discovery full-length, RAG1 and RAG2 have been difficult to purify, and core derivatives are shown to be most active when purified from adherent 293-T cells. However, the protein yield from adherent 293-T cells is limited. Here we develop a human suspension cell purification and change the expression vector to boost RAG production 6-fold. We use these purified RAG proteins to investigate V(D)J recombination on a mechanistic single molecule level. As a result, we are able to measure the binding statistics (dwell times and binding energies) of the initial RAG binding events with or without its co-factor high mobility group box protein 1 (HMGB1), and to characterize synapse formation at the single-molecule level yielding insights into the distribution of dwell times in the paired complex and the propensity for cleavage upon forming the synapse. We then go on to investigate HMGB1 further by measuring it compact single DNA molecules. We observed concentration dependent DNA compaction, differential DNA compaction depending on the divalent cation type, and found that at a particular HMGB1 concentration the percentage of DNA compacted is conserved across DNA lengths. Lastly, we investigate another HMGB protein called TFAM, which is essential for packaging the mitochondrial genome. We present crystal structures of TFAM bound to the heavy strand promoter 1 (HSP1) and to nonspecific DNA. We show TFAM dimerization is dispensable for DNA bending and transcriptional activation, but is required for mtDNA compaction. We propose that TFAM dimerization enhances mtDNA compaction by promoting looping of mtDNA.\r\n", "doi": "10.7907/Z9W9573H", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8218", "collection": "thesis", "collection_id": "8218", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05052014-142031176", "primary_object_url": { "basename": "Bing_Sun_2014_thesis_complete.pdf", "content": "final", "filesize": 3582945, "license": "other", "mime_type": "application/pdf", "url": "/8218/67/Bing_Sun_2014_thesis_complete.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Mechanistic Studies of Reactions at the Single-Molecule Level using Microfluidics with Applications in Molecular Diagnostics", "author": [ { "family_name": "Sun", "given_name": "Bing", "clpid": "Sun-Bing" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" }, { "family_name": "Pierce", "given_name": "Niles A.", "clpid": "Pierce-N-A" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "clpid": "Ismagilov-R-F" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Motivated by needs in molecular diagnostics and advances in microfabrication, researchers started to seek help from microfluidic technology, as it provides approaches to achieve high throughput, high sensitivity, and high resolution. One strategy applied in microfluidics to fulfill such requirements is to convert continuous analog signal into digitalized signal. One most commonly used example for this conversion is digital PCR, where by counting the number of reacted compartments (triggered by the presence of the target entity) out of the total number of compartments, one could use Poisson statistics to calculate the amount of input target.
\r\n\r\nHowever, there are still problems to be solved and assumptions to be validated before the technology is widely employed. In this dissertation, the digital quantification strategy has been examined from two angles: efficiency and robustness. The former is a critical factor for ensuring the accuracy of absolute quantification methods, and the latter is the premise for such technology to be practically implemented in diagnosis beyond the laboratory. The two angles are further framed into a \u201cfate\u201d and \u201crate\u201d determination scheme, where the influence of different parameters is attributed to fate determination step or rate determination step. In this discussion, microfluidic platforms have been used to understand reaction mechanism at single molecule level. Although the discussion raises more challenges for digital assay development, it brings the problem to the attention of the scientific community for the first time.
\r\n\r\nThis dissertation also contributes towards developing POC test in limited resource settings. On one hand, it adds ease of access to the tests by incorporating massively producible, low cost plastic material and by integrating new features that allow instant result acquisition and result feedback. On the other hand, it explores new isothermal chemistry and new strategies to address important global health concerns such as cyctatin C quantification, HIV/HCV detection and treatment monitoring as well as HCV genotyping.
\r\n", "doi": "10.7907/BT81-YX06", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:7865", "collection": "thesis", "collection_id": "7865", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06072013-111912342", "primary_object_url": { "basename": "Kiwook_Hwang_2013_thesis_final.pdf", "content": "final", "filesize": 24703251, "license": "other", "mime_type": "application/pdf", "url": "/7865/49/Kiwook_Hwang_2013_thesis_final.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Biotechnologies for Cancer Diagnostics: Cell Sorting, Protein Analysis and Imaging of Cellular Metabolism", "author": [ { "family_name": "Hwang", "given_name": "Kiwook", "clpid": "Hwang-Kiwook" } ], "thesis_advisor": [ { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" } ], "thesis_committee": [ { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" }, { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This thesis presents the development of chip-based technology for informative in vitro cancer diagnostics. In the first part of this thesis, I will present my contribution in the development of a technology called \u201cNucleic Acid Cell Sorting (NACS)\u201d, based on microarrays composed of nucleic acid encoded peptide major histocompatibility complexes (p/MHC), and the experimental and theoretical methods to detect and analyze secreted proteins from single or few cells.
\r\n \r\nSecondly, a novel portable platform for imaging of cellular metabolism with radio probes is presented. A microfluidic chip, so called \u201cRadiopharmaceutical Imaging Chip\u201d (RIMChip), combined with a beta-particle imaging camera, is developed to visualize the uptake of radio probes in a small number of cells. Due to its sophisticated design, RIMChip allows robust and user-friendly execution of sensitive and quantitative radio assays. The performance of this platform is validated with adherent and suspension cancer cell lines. This platform is then applied to study the metabolic response of cancer cells under the treatment of drugs. Both cases of mouse lymphoma and human glioblastoma cell lines, the metabolic responses to the drug exposures are observed within a short time (~ 1 hour), and are correlated with the arrest of cell-cycle, or with changes in receptor tyrosine kinase signaling.
\r\n \r\nThe last parts of this thesis present summaries of ongoing projects: development of a new agent as an in vivo imaging probe for c-MET, and quantitative monitoring of glycolytic metabolism of primary glioblastoma cells. To develop a new agent for c-MET imaging, the one-bead-one-compound combinatorial library method is used, coupled with iterative screening. The performance of the agent is quantitatively validated with cell-based fluorescent assays. In the case of monitoring the metabolism of primary glioblastoma cell, by RIMChip, cells were sorting according to their expression levels of oncoprotein, or were treated with different kinds of drugs to study the metabolic heterogeneity of cancer cells or metabolic response of glioblastoma cells to drug treatments, respectively.
\r\n", "doi": "10.7907/Z6DN-0483", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7858", "collection": "thesis", "collection_id": "7858", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06062013-235430660", "primary_object_url": { "basename": "thesis_anag.pdf", "content": "final", "filesize": 18690309, "license": "other", "mime_type": "application/pdf", "url": "/7858/91/thesis_anag.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Developing Peptide Based Capture Agents for Diagnostics and Therapeutics ", "author": [ { "family_name": "Nag", "given_name": "Arundhati", "clpid": "Nag-Arundhati" } ], "thesis_advisor": [ { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" }, { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Iterative in situ click chemistry (IISCC) is a robust general technology for development of high throughput, inexpensive protein detection agents. In IISCC, the target protein acts as a template and catalyst, and assembles its own ligand from modular blocks of peptides. This process of ligand discovery is iterated to add peptide arms to develop a multivalent ligand with increased affinity and selectivity. The peptide based protein capture agents (PCC) should ideally have the same degree of selectivity and specificity as a monoclonal antibody, along with improved chemical stability. We had previously reported developing a PCC agent against bovine carbonic anhydrase II (bCAII) that could replace a polyclonal antibody. To further enhance the affinity or specificity of the PCC agent, I explore branching the peptide arms to develop branched PCC agents against bCAII. The developed branched capture agents have two to three fold higher affinities for the target protein. In the second part of my thesis, I describe the epitope targeting strategy, a strategy for directing the development of a peptide ligand against specific region or fragment of the protein. The strategy is successfully demonstrated by developing PCC agents with low nanomolar binding affinities that target the C-terminal hydrophobic motif of Akt2 kinase. One of the developed triligands inhibits the kinase activity of Akt. This suggests that, if targeted against the right epitope, the PCC agents can also influence the functional properties of the protein. The exquisite control of the epitope targeting strategy is further demonstrated by developing a cyclic ligand against Akt2. The cyclic ligand acts as an inhibitor by itself, without any iteration of the ligand discovery process. The epitope targeting strategy is a cornerstone of the IISCC technology and opens up new opportunities, leading to the development of protein detection agents and of modulators of protein functions.", "doi": "10.7907/Y064-VD39", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" } ]