Single-cell genomics assays, particularly single-cell RNA sequencing that enables genome-wide profiling of gene expression, have been driven forward by a combination of technological and computational advances. While producing extraordinary large amounts of data for biological discovery, methods for mining results currently rely heavily on heuristics and lack of modeling has resulted in limited mechanistic biological insight. This thesis presents two models for normalization and trajectory inference in single-cell RNA sequencing analysis to demonstrate how biophysical modeling, when combined with principled statistical inference, can yield interpretable insights grounded in rigorous theoretical frameworks.
\r\n\r\nWe begin by explaining the two cultures in single-cell RNA sequencing analysis. Next, we present the chemical master equation, which forms the theoretical foundation for biophysically informed stochastic models of gene expression, and explore an existing gap in developing uniform approximations over time under the large-volume limit. Returning to single-cell RNA sequencing data analysis, we introduce two mechanistic models for normalization and trajectory inference, which are essential components of single-cell RNA sequencing analysis.
", "doi": "10.7907/asek-t904", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:16412", "collection": "thesis", "collection_id": "16412", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05222024-204650454", "primary_object_url": { "basename": "laubscher_emily_2024.pdf", "content": "final", "filesize": 38141307, "license": "other", "mime_type": "application/pdf", "url": "/16412/1/laubscher_emily_2024.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Deep Learning-Enabled Integrated Measurements of Immune Signaling in Primary Human Macrophages", "author": [ { "family_name": "Laubscher", "given_name": "Emily Chiu", "orcid": "0009-0008-0242-0507", "clpid": "Laubscher-Emily-Chiu" } ], "thesis_advisor": [ { "family_name": "Van Valen", "given_name": "David A.", "orcid": "0000-0001-7534-7621", "clpid": "Van-Valen-D" } ], "thesis_committee": [ { "family_name": "Wei", "given_name": "Lu", "orcid": "0000-0001-9170-2283", "clpid": "Wei-Lu" }, { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Van Valen", "given_name": "David A.", "orcid": "0000-0001-7534-7621", "clpid": "Van-Valen-D" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Examination of biological systems at the single-cell level reveals heterogeneity in both time and space. Single-cell temporal and spatial heterogeneity allow communities of cells to process noisy stimuli and perform complex tasks. We leveraged state-of-the-art imaging technologies to characterize cell-to-cell heterogeneity in responses to environmental stimuli to reveal mechanisms of information transmission. Fluorescent live-cell reporters enable real-time visualization of the activity state of cell signaling proteins. Signaling dynamics allow cells to translate information about environmental stimuli into cellular behaviors. Chapter 2 explores the variety of live-cell reporters designed to characterize the dynamic patterns of activity of key signaling pathways, and covers the development of two live-cell reporters. Spatial transcriptomics assays, on the other hand, excel at capturing heterogeneity in spatial gene expression patterns, which is often required to enable a tissue to perform complex functions. Chapter 3 details the development of Polaris, a deep learning-enabled analysis method for spatial transcriptomics data. Polaris is an assay-agnostic, turnkey solution for analyzing images from spatial transcriptomics experiments, minimizing the time and expertise require to extract biological insights. In chapter 4, we pair dynamic measurements of live-cell reporters with a spatial transcriptomics measurement in an integrated imaging assay in primary human macrophages. This imaging assay revealed transcriptional sub-populations of cells with differing distributions of dynamic immune signaling responses and morphological states.
\r\n\r\nThis work contributes a number of methodological developments, including live- cell reporter expression in primary human macrophages and deep learning-enabled spatial transcriptomics image analysis. Expression of live-cell reporters in primary macrophages will enable the investigation of environmental cues shape macrophages\u2019 cell state, which is highly plastic and shaped by external stimuli. Polaris expedites the analysis of this multi-modal imaging data set, extracting single-cell gene expression values without manual parameter tuning. However, Polaris\u2019 impact extends beyond the scope of this work to the broader spatial biology field as its spot detection and gene decoding capabilities generalize to data sets from a variety of sample types and imaging modalities. Finally, our paired dynamics-spatial transcriptomics imaging assay can be generally applied to characterize information transmission from environmental stimuli through signaling dynamics to the expression of downstream genes for a wide variety of signaling pathways in primary and immortalized cell types.
", "doi": "10.7907/hayp-kx45", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16147", "collection": "thesis", "collection_id": "16147", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07272023-175309910", "primary_object_url": { "basename": "Goronzy_CalTechThesis_Final2.pdf", "content": "final", "filesize": 5387002, "license": "other", "mime_type": "application/pdf", "url": "/16147/2/Goronzy_CalTechThesis_Final2.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Higher-Order Chromatin States and Nuclear Structures Regulating Gene Expression", "author": [ { "family_name": "Goronzy", "given_name": "Isabel Nadine", "orcid": "0000-0002-6713-9192", "clpid": "Goronzy-Isabel-Nadine" } ], "thesis_advisor": [ { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" }, { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Pachter", "given_name": "Lior S.", "orcid": "0000-0002-9164-6231", "clpid": "Pachter-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Although the same genome is present in every cell, each cell type orchestrates a distinct gene expression program, which can be rapidly adapted in response to stimuli. Accordingly, gene regulation is a highly complex, context-specific process that involves the dynamic interplay between numerous regulatory factors. Most methods to study these regulatory factors only measure pairwise interactions between molecules and are limited to mapping one regulatory protein at a time. Consequently, the combinatorial complexity of gene regulation at individual genomic loci and the functional consequence of many regulatory factors remain underexplored. To address this, we have developed new sequencing-based approaches and computational analyses to comprehensively profile, at unprecedented scale, the diverse gene regulatory landscape and directly establish the link between regulatory factors and transcriptional outcomes. In Chapter 2, we present Chromatin Immunoprecipitation Done-In-Parallel (ChIP-DIP), a highly multiplexed method for mapping hundreds of proteins to DNA within a single sample. ChIP-DIP increases the throughput of existing methods by > 100-fold and enables the production of consortium-scale, cell type-specific data within a single lab. Capitalizing on the scale and diversity provided by ChIP-DIP, we uncover unique quantitative combinations of histone modifications that define distinctive classes of regulatory elements. Specifically, we find features distinguishing classes of promoters that correspond to different polymerase activity, transcriptional levels, and gene types and find acetylation patterns distinguishing classes of enhancers that exhibit distinct activity states, induction potential, and regulatory potential. Next, in Chapter 3, we apply RNA-DNA SPRITE (RD-SPRITE), a method for simultaneous measurement of RNA and DNA organization, to investigate the functional relationship between genome structure and transcription. We demonstrate that RD-SPRITE precisely detects individual, nascent pre-mRNAs at their transcriptional locus and, as a result, can be used to assess the 3D genome structure present during active transcription. We find that RNA polymerase II transcription occurs within genomic structures previously thought to be inactive, such as the B compartment and DNA regions near the nucleolus. This suggests that active transcription can occur throughout the nucleus and argues against structural domains that preclude transcription. Overall, our findings highlight the ability of RD-SPRITE to establish a structure-function link. Finally, in Chapter 4, we apply RD-SPRITE to study the transcriptional dependence of nuclear organization. We demonstrate that transcriptional inhibition leads to the loss of high-order structure around multiple RNA-processing bodies \u2014 the nucleolus, the scaRNA hub and the histone locus body \u2014 that are responsible for essential nuclear functions such as RNA processing and gene regulation. These findings suggest a role for RNA and nascent transcription in the formation and maintenance of long-range 3D contacts and critical nuclear compartments. In summary, we have developed new approaches to explore epigenomic and organizational complexity within the mammalian nucleus and have uncovered genome-wide principles of gene regulation.
", "doi": "10.7907/8gm2-jn84", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16358", "collection": "thesis", "collection_id": "16358", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04182024-125718373", "primary_object_url": { "basename": "Strehle_Mackenzie_Thesis.pdf", "content": "final", "filesize": 28785152, "license": "other", "mime_type": "application/pdf", "url": "/16358/1/Strehle_Mackenzie_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Mechanisms of Xist-Mediated Gene Silencing During the Initiation and Maintenance of X Chromosome Inactivation", "author": [ { "family_name": "Strehle", "given_name": "Mackenzie", "orcid": "0000-0003-1410-8701", "clpid": "Strehle-Mackenzie" } ], "thesis_advisor": [ { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Zernicka-Goetz", "given_name": "Magdalena", "orcid": "0000-0002-7004-2471", "clpid": "Zernicka-Goetz-M" }, { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "X chromosome inactivation (XCI) is a critical development process during which one of the two X chromosomes in female mammals is silenced to balance gene expression with males. XCI is initiated by upregulation of the long noncoding RNA (lncRNA) Xist from the future inactive X chromosome (Xi), which recruits a variety of proteins in cis to mediate transcriptional repression that is maintained throughout the lifetime of the organism. Recent studies have demonstrated that silencing following Xist expression is dependent on direct recruitment of the transcriptional silencing protein SHARP (also known as SPEN); however, the mechanism underlying formation of the Xi silencing compartment has remained poorly defined. Similarly, it has long been thought that maintenance of XCI occurs independently of Xist and depends on differential DNA methylation enrichment on the Xi, but the evidence in support of these views is lacking. Here, we show how low copy numbers of Xist can recruit SHARP in super-stoichiometric excess to initiate gene silencing on the X and mediate formation of the silent Xi compartment. We also provide preliminary evidence suggesting that maintenance of XCI is Xist independent, but dependent on DNA methylation and histone deacetylation. Together, these results offer a more holistic view of the molecular mechanisms underlying both initiation and maintenance XCI, as well as provide a framework for further investigation into lncRNA biology and epigenetic regulation more broadly.", "doi": "10.7907/nxq5-hj97", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16366", "collection": "thesis", "collection_id": "16366", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05012024-121056794", "primary_object_url": { "basename": "JGuo_Thesis.pdf", "content": "final", "filesize": 20442455, "license": "other", "mime_type": "application/pdf", "url": "/16366/1/JGuo_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Defining the Universe of Functional RNA-Protein Interactions", "author": [ { "family_name": "Guo", "given_name": "Jimmy Kang", "orcid": "0000-0002-7211-4117", "clpid": "Guo-Jimmy-Kang" } ], "thesis_advisor": [ { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" } ], "thesis_committee": [ { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Flynn", "given_name": "Ryan A.", "orcid": "0000-0001-5013-0442", "clpid": "Flynn-Ryan-A" }, { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" }, { "family_name": "Voorhees", "given_name": "Rebecca M.", "orcid": "0000-0003-1640-2293", "clpid": "Voorhees-R-M" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "RNA has been proposed to mediate many central mechanisms of cell biology, including protein recruitment to chromatin, genome structure organization, and gene expression. In most cases, these critical functions have been widely attributed to the proteins to which RNAs bind. One paradigm example of this is the Xist long non-coding RNA, which complexes with many distinct proteins to orchestrate X-chromosome inactivation. Beyond Xist, there are many critical non-coding RNAs (ncRNAs) that are not yet functionally characterized because we lack information on what proteins they bind to. In this thesis, Chapter 1 discusses the growing gap between the vast potential of ncRNA functions and what has been demonstrated to be functionally meaningful. We highlight critical discrepancies between biochemical evidence supporting specific RNA-protein interactions and genetic evidence demonstrating the same interactions are often dispensable for function. Chapter 2 explores previously reported RNA-protein interactions for many chromatin proteins (i.e., PRC2, CTCF, etc.), demonstrating that they do not represent bona fide interactions in cells. We present Covalent Linkage Affinity Purification (CLAP), a method that employs denaturing purification of RNA-protein complexes, showing that CLAP accurately removes false signals that do not occur in vivo, while retaining known RNA-protein interactions. Chapter 3 details a highly multiplexed method of mapping RBPs and their in vivo binding sites across dozens to hundreds of targets within a single experiment. We present Split and Pool Identification of RBP targets (SPIDR), which enables the rapid, de novo discovery of RNA-protein interactions at an unprecedented scale and separates bona fide RBPs from non-RBPs. Using SPIDR, we uncover a previously unknown LARP1 binding site on the 18S ribosomal RNA that is directly adjacent to the mRNA entry channel, which may explain how LARP1 achieves translational control of sequence-specific mRNAs. Finally, Chapter 4 proposes new experimental and analytical approaches to evaluate the potentially wide universe of ncRNA-protein functions at scale. Together, these results provide a comprehensive framework for evaluating RNA-protein interactions and underscore the growing importance of RNA-mediated functions in cell biology.", "doi": "10.7907/wbvq-bz46", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16062", "collection": "thesis", "collection_id": "16062", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022023-194724728", "type": "thesis", "title": "Stochastic Foundations for Single-Cell RNA Sequencing", "author": [ { "family_name": "Gorin", "given_name": "Gennady", "orcid": "0000-0001-6097-2029", "clpid": "Gorin-Gennady" } ], "thesis_advisor": [ { "family_name": "Pachter", "given_name": "Lior S.", "orcid": "0000-0002-9164-6231", "clpid": "Pachter-L" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" }, { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Pachter", "given_name": "Lior S.", "orcid": "0000-0002-9164-6231", "clpid": "Pachter-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Single-cell RNA sequencing, which quantifies cell transcriptomes, has seen widespread adoption, accompanied by proliferation of analysis methods. However, there has been relatively little systematic investigation of its best practices and their underlying assumptions, leading to challenges and discrepancies in interpretation. I present a set of generic, principled strategies for modeling the biological and technical components of sequencing experiments and use case studies to motivate their application to sequencing data.
", "doi": "10.7907/jn6n-x368", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" } ]