UMD Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/3
New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a given thesis/dissertation in DRUM.
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Item CONSEQUENCES OF NUCLEAR CONFINEMENT IN CANCER METASTASIS(2021) Baird, Michelle; Waterman, Clare M; Stroka, Kimberly M; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Malignant melanoma is characterized by its mutational heterogeneity and aggressive metastatic spread. During metastasis, melanoma cells migrate through diverse microenvironments, including regions of dense tissue confinement to reach the vasculature. Microenvironmental confinement of tumor cells causes nuclear deformation, which can lead to loss of nuclear envelope (NE) integrity and DNA damage, improper repair of which leads to genomic aberrations and heterogeneity. We hypothesize that during metastatic progression, expression levels of NE genes are altered, facilitating nuclear deformability and NE fragility, mediating an increase in genetic heterogeneity within the population. In this dissertation, we show a novel bioinformatic analysis of orthogonal RNA-seq data sets from patient samples of metastatic melanoma and benign nevi, revealing several NE proteins upregulated in metastatic disease. Performing a targeted siRNA-based screen using a PDMS confinement device to assay for nuclear fragility, we found reduction of lamin B receptor (LBR) dramatically reduced NE fragility in melanoma cells, and ectopic overexpression of LBR was sufficient to increase NE fragility in benign melanocytes. Utilizing functional protein domain truncations and point mutations in LBR, we found the cholesterol synthase activity of LBR was specifically required for increased NE fragility, independent of LBRs additional roles tethering heterochromatin and lamin B to the NE. Additionally, we found that reduction of LBR in melanoma cells results in a reorganization of cholesterol in the NE. Thus, LBR generated cholesterol in the NE promotes NE fragility. To determine if LBR-mediated NE fragility was correlated with increased nuclear deformability, we assayed NE mechanics with atomic force microscopy. In melanoma cells, we find reduction of LBR results in an increase in nuclear stiffness and a decrease in deformability, while LBR overexpression in benign melanocytes results in an increase in nuclear deformability. These results show for the first time that upregulation of LBR in metastatic melanoma plays dual roles in reducing nuclear deformability and increasing NE rupture, specifically through alterations in cholesterol organization in the NE and open an exciting new direction to the role of cholesterol in NE integrity and mechanics.Item CHARACTERIZATION OF SURVIVAL ASSOCIATED GENE INTERACTIONS AND LYMPHOCYTE HETEROGENEITY IN CANCER(2019) Magen, Assaf; Hannenhalli, Sridhar; Computer Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cancer is the second leading cause of death globally. Tumors form intricate ecosystems in which malignant and immune cells interact to shape disease progression. Yet, the molecular underpinnings of tumorigenesis and immunological responses to tumors are poorly understood, limiting their manipulation to elicit favorable clinical outcomes. This thesis lays conceptual frameworks for investigating the molecular interactions taking place in tumors as well as the diversity of the immune response to cancer. In the molecular level of individual cancer cells, the phenotypic effect of perturbing a gene’s activity depends on the activity level of other genes, reflecting the notion that phenotypes are emergent properties of a network of functionally interacting genes. In the context of cancer, contemporary investigations have primarily focused on just one type of functional genetic interaction (GI) – synthetic lethality (SL). However, there may be additional types of GIs whose systematic identification would enrich the molecular and functional characterization of cancer. This thesis describes a novel data-driven approach called EnGIne, that applied to large-scale cancer data identifies 71,946 GIs spanning 12 distinct types, only a small minority of which are SLs. The detected GIs explain cancer driver genes’ tissue- specificity and differences in patients’ response to drugs, and stratify breast cancer tumors into refined subtypes. These results expand the scope of cancer GIs and lay a conceptual and computational basis for future studies of additional types of GIs and their translational applications. Furthermore, tumor growth is continuously shaped by the immune response. However, T cells typically adopt a dysfunctional phenotype may be reversed using immunotherapy strategies. Most current tumor immunotherapies leverage cytotoxic CD8+ T cells to elicit an effective anti-tumor response. Despite evidence for clinical potential of CD4+ tumor-infiltrating lymphocytes (TILs), their functional diversity has limited our ability to harness their anti-tumor activity. To address this issue, we have used single-cell mRNA sequencing (scRNAseq) to analyze the response of CD4+ T cells specific for a defined recombinant tumor antigen, both in the tumor microenvironment and draining lymph nodes (dLN). New computational approaches to characterize subpopulations identified TIL transcriptomic patterns strikingly distinct from those elicited by responses to infection, and dominated by diversity among T-bet-expressing T helper type 1 (Th1)-like cells. In contrast, the dLN response includes Follicular helper (Tfh)-like cells but lacks Th1 cells. We identify an interferon-driven signature in Th1-like TILs, and show that it is found in human liver cancer and melanoma, in which it is negatively associated with response to checkpoint therapy. Our study unveils unsuspected differences between tumor and virus CD4+ T cell responses, and provides a proof-of-concept methodology to characterize tumor- control CD4+ T cell effector programs. Targeting these programs should help improve immunotherapy strategies.