Theses and Dissertations from UMD
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Item Spatiotemporal proteomic approaches for investigating patterning during embryonic development(2024) Pade, Leena Rajendra; Nemes, Peter; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Characterization of molecular events as embryonic cells give rise to tissues and organs raises a potential to better understand normal development and design remedies for diseases. In this work, I integrated bioanalytical chemistry with neurodevelopmental biology to uncover mechanisms underlying tissue induction in a developing embryo. Specifically, I developed ultrasensitive proteomic approaches to study the remodeling of the proteome as embryonic cells differentiate in space and time to induce tissue formation. This dissertation discusses the design and development of proteomic strategies to deepen proteomic coverage from limited embryonic tissues. A novel sample preparation workflow and detection strategy was developed to address the challenge of interference from abundant proteins such as yolk in Xenopus tissues which in turn boosts the sensitivity of detecting low abundant proteins from complex limited amounts of tissues. The refined analytical workflow was implemented to study the development of critical signaling centers and stem cell populations and the tissues they induce to form in developing embryos.Item DIFFERENTIATION AND REGULATION OF BOVINE TH2 CELLS(2024) Kandel, Anmol; Xiao, Zhengguo Zhengguo; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Memory CD4+ T cells, specifically type-2 (Th2) cells, are pivotal in defending against infections caused by extracellular pathogens, including several economically important parasites. However, whether interleukin-4 (IL4) expression is a signature feature of bovine Th2 cells likewise in mice and humans is unclear. Pasture-raised cattle, routinely exposed to extracellular parasites such as Ostertagia ostertagi (OO), are likely to develop a typical Th2 memory response. Therefore, using cytokine induction assay, we evaluated the circulatory memory bovine T cell profile of these cattle and also analyzed if the expression of presumptuous memory marker, CD45RO, is reliable in identifying memory bovine T cells. Surprisingly, the majority of the memory CD4+ T cells dominantly produced interferon-gamma (IFNγ), with only a small fraction co-expressing IL4, and memory bovine T cell identification did not correlate with CD45RO expression. Results suggested that cattle naturally exposed to extracellular parasites do not develop typically IL4 dominant Th2 response. To further investigate these results, resting CD4+ T cells isolated from healthy cattle blood were cultured under simple in vitro Th2 culture. Analysis of differentiated cells through flow cytometry revealed limited IL4 protein detection, which was in line with the lack of upregulation of IL4 and its master regulator GATA3 transcripts shown by the quantitative polymerase chain reaction (qPCR) assay. To validate whether differentiated cells were actually Th2, unbiased proteomic analysis was conducted. Based on differentially expressed 397 proteins between differentiated cells and naïve phenotype, bovine Th2 differentiation was validated; nonetheless, the process was not found to be associated with IL4 induction. Moreover, despite using published strategies from mice and humans, such as reducing T cell receptor (TCR) stimulation strength and adding exogenous recombinant bovine IL4, the expression of IL4 could not be significantly enhanced. Interestingly, differentiated bovine Th2 cells proliferated in the presence of OO antigens, suggesting that extracellular parasites could influence bovine Th2 differentiation, at least in vitro. To validate the results from pathogen-infected tissues and in vitro culture, a panel of anti-parasitic CD4+ single T cell clones was established from five pasture-raised cattle that were infected with OO. Evaluation of memory responses exhibited by the anti-parasitic CD4+ single T cell clones strongly supported IFNγ dominant memory response, and only 20% of them co-expressed IL4 through a small subset of IFN γ + cells. All the data pointed out that bovine CD4+ T cell differentiation is partially distinct from those in mice and humans, and IL4 expression is not a hallmark feature of the bovine Th2 cells.Item Development of single-neuron proteomics by mass spectrometry for the mammalian brain.(2021) Choi, Bok Dong; Nemes, Peter; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Single-neuron proteomics holds the potential to advance our understanding of important biological processes during neuron maturation and development. However, to characterize proteins from single neurons, further technological advances are still required. This dissertation discusses the development and application of single-cell mass spectrometry (MS) technologies to investigate proteins and its role in different neurons. The work presented herein demonstrates the strategies to develop and advance single-neuron analysis using capillary electrophoresis (CE)-MS. In addition, this work features several contributions to our understanding of neuron-to-neuron heterogeneity, providing new information to advance cell biology and neuroscience.Item Towards the synthesis of PNAG crosslinkers to identify protein binding partners(2019) Mrugalski, Kevin R; Poulin, Myles; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Bacterial biofilms are an area of major concern in the medical field due to natural drug resistance. Many pathogenetic species of bacteria that infect humans including Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Vibrio cholera form biofilms and their associated infections are becoming harder to treat. Poly β-(1→6)-N-acetyl-D-glucosamine (PNAG) is a major component of biofilms across multiple species and has been found to play a key role in the early stages of the biofilm life-cycle. However, little information is known about what proteins interact with this important polysaccharide. Our goal is to create small PNAG analogues to covalently capture and identify PNAG binding partners in E. coli, an important model organism. PNAG analogues will contain photoaffinity groups, that when activated, covalently link associated proteins to the probe. Then, using a proteomics-mass spectrometry-based approach, we will identify PNAG binding partners. Here, we describe the efforts and challenges encountered synthesizing the final PNAG probes. New synthetic routes are proposed based on literature precedent that will enable synthesis of the desired compounds.Item INTERROGATING PROTEIN CARGOES OF MDSC-DERIVED EXOSOMES ON THE BASIS OF POST-TRANSLATIONAL MODIFICATIONS(2017) Chauhan, Sitara; Fenselau, Catherine; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Myeloid-derived suppressor cells (MDSC) are immature myeloid cells which accumulate in cancer patients and tumor-bearing mice. Their function in the tumor microenvironment is to inactivate the immune response to cancer by suppressing both the adaptive and innate immune system. Therefore, MDSC are a major obstacle in immunotherapeutic approaches designed to cure cancer. MDSC-derived from tumor bearing mice have been found to shed exosomes. Exosomes are nano-sized vesicles that carry biologically active molecules and play a role in intercellular communication. MDSC-derived exosomes have been reported to mediate the immunosuppressive functions of the parental cells by stimulating the accumulation of MDSC and also by converting macrophages to a tumor-promoting phenotype. Recent developments in government policy have launched a goal of curing cancer using immune-based therapies (Cancer MoonShot 2020). The understanding of the mechanisms and functions of MDSC immune suppression will be crucial in the success of these therapeutic endeavors. Our current study focuses on interrogating the protein cargo carried by MDSC-derived exosomes based on differential post-translational modifications (PTMs). Post-translational modifications have important roles in functions, signaling, location and interactions of proteins. Selecting proteins based on a specific post-translational modification can aid in the identification of low-abundance proteins which may not be identified in a shotgun proteomics approach. The first aim of this work was to successfully modify an existing surface chemistry method to use on exosomes. We then used a proteomic strategy to identify glycoproteins on the surface of MDSC-derived exosomes, and then test if selected glycoproteins contribute to exosome-mediated chemotaxis and migration of MDSCs. Furthermore, we also aimed at examining the ubiquitome of the MDSC-derived exosomes, using top-down and bottom-up proteomics. Since inflammation has been reported to enhance the tumor promoting activity of the MDSC, the bottom-up analysis focused on the effects of increased inflammation on the ubiquitination of the protein cargo of MDSC-derived exosomes. Spectral counting was used to estimate differences in abundance of proteins found with ubiquitinated proteoforms in high and basal levels of inflammation. The top-down analysis aimed at characterizing the length and topology of ubiquitin linkages on substrate proteins in MDSC-exosomes.Item CHARACTERIZATION OF POLYUBIQUITIN CHAINS BY MASS SPECTROMETRY(2016) Lee, Amanda Elizabeth; Fenselau, Catherine; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The work outlined in this dissertation will allow biochemists and cellular biologists to characterize polyubiquitin chains involved in their cellular environment by following a facile mass spectrometric based workflow. The characterization of polyubiquitin chains has been of interest since their discovery in 1984. The profound effects of ubiquitination on the movement and processing of cellular proteins depend exclusively on the structures of mono and polyubiquitin modifications anchored or unanchored on the protein within the cellular environment. However, structure-function studies have been hindered by the difficulty in identifying complex chain structures due to limited instrument capabilities of the past. Genetic mutations or reiterative immunoprecipitations have been used previously to characterize the polyubiquitin chains, but their tedium makes it difficult to study a broad ubiquitinome. Top-down and middle-out mass spectral based proteomic studies have been reported for polyubiquitin and have had success in characterizing parts of the chain, but no method to date has been successful at differentiating all theoretical ubiquitin chain isomers (ubiquitin chain lengths from dimer to tetramer alone have 1340 possible isomers). The workflow presented here can identify chain length, topology and linkages present using a chromatographic-time-scale compatible, LC-MS/MS based workflow. To accomplish this feat, the strategy had to exploit the most recent advances in top-down mass spectrometry. This included the most advanced electron transfer dissociation (ETD) activation and sensitivity for large masses from the orbitrap Fusion Lumos. The spectral interpretation had to be done manually with the aid of a graphical interface to assign mass shifts because of a lack of software capable to interpret fragmentation across isopeptide linkages. However, the method outlined can be applied to any mass spectral based system granted it results in extensive fragmentation across the polyubiquitin chain; making this method adaptable to future advances in the field.Item PROTEOMIC CHARACTERIZATION OF EXOSOMES SHED BY MYELOID-DERIVED SUPPRESSOR CELLS(2015) Burke, Meghan Catherine; Fenselau, Catherine; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Exosomes are a class of extracellular vesicles that have been shown to contribute to metastasis when derived from tumor cells. Myeloid-derived suppressor cells (MDSC) are an immature population of myeloid cells that accumulate in the tumor microenvironment and inhibit anti-tumor immunity. Given the role of the source cells, it is our hypothesis that MDSC-derived exosomes may contribute to or mediate the effects of MDSC in the tumor microenvironment. The goal of this work is to use mass-spectrometry based proteomics to characterize exosomes produced by MDSC that are induced by 4T1 mammary carcinoma. The protein content of the exosomes will be analyzed to determine if the exosomal proteome is representative of the parental cells or if it reflects active protein sorting. Increased inflammation in the tumor microenvironment is associated with an increased population of MDSC, which further increases the level of immune suppression. Here, the relative change in abundance of exosomal proteins under a heightened level of inflammation in the tumor microenvironment will be performed using the spectral count method. While it is known that exosomes first form through invagination at the plasma membrane, the mechanism(s) through which the protein cargo is sorted into exosomes remains poorly understood. Given the role of ubiquitination in protein localization and trafficking, immunoaffinity enrichment coupled to mass spectrometry has been employed to identify exosomal proteins that carry this modification. Identification of the substrate proteins in MDSC-derived exosomes may provide insight into exosome formation and/or function.Item Biochemical characterizations of extracellular vesicles shed by vegetative and sporulating Bacillus subtilis(2015) Kim, Yeji; Fenselau, Catherine; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Sporulation of Bacilli is a developmental process that provides long-term viability in unfavorable environments. Recently, biogenesis of extracellular vesicles (EVs) from Bacilli has also been reported to participate in various physiological and pathogenic phenomena. In this study, EVs were isolated from vegetative and sporulating Bacillus subtilis cells and characterized using mass spectrometry (MS)-based proteomics, microscopy, and fluorescence spectrophotometry. The microscopic approach demonstrated that both vegetative and sporulating cells produce EVs. In the proteomic analysis, 156 proteins were identified with statistical significance in EVs collected at the vegetative phase and 185 proteins in EVs shed during sporulation. The two EV cargos showed qualitatively and quantitatively different proteome patterns. Sporulation-associated proteins had greater abundances in EVs at the sporulation stage. Additionally, a fusion-like event of EVs with B. subtilis cells was observed by a fluorescence de-quenching assay. Based on these observations, B. subtilis EVs are proposed to support intercellular communication and sporulation.Item A Proteomics Approach to the Examination of Proteins in Marine Systems(2014) Faux, Jessica Felicia; Harvey, Henry R; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The response of global carbon and nitrogen cycles to future climate change is uncertain. In order to understand the impacts that future changes to climate will have on these cycles, a more detailed understanding of them is essential. This dissertation utilizes a combined approach of molecular biomarkers and proteomic investigations to elucidate historic source material contributions and microbial protein production to contribute to a more thorough understanding of the marine carbon and nitrogen cycles. The examination of molecular organic biomarkers throughout an Arctic sediment core showed the dominant input in the area was from marine sources with lower but steady contributions from terrestrial sources during the Holocene. Attempts to recover proteins from deeper sediments to correlate with lipid biomarkers were unsuccessful but led to the optimization of an extraction protocol for an added protein standard, bovine serum albumin, from sediments. An investigation into the expressed proteome of the heterotrophic marine bacterium, Ruegeria pomeroyi, under environmentally realistic carbon supply conditions during exponential and stationary growth phases identified over 2000 proteins. The most abundant proteins identified were responsible for porins, transport, binding, translation, and protein refolding and could represent potential biomarkers of bacterial processes and/or activity. A parallel study of R. pomeroyi, in which 13C-labeled leucine was added to the culture during exponential growth phase, showed labeled incorporation ranging from 16 to 21% of the total proteins produced depending on growth phase. The widespread distribution of the label among the growth phases indicates active recycling by the bacteria. This study demonstrates a method through which bacterial protein synthesis can be tracked. A study of the marine diatom Thalassiosira pseudonana acclimated to iron replete or iron-limited conditions showed iron-limited organisms increased proteins involved in pathways associated with intracellular protein recycling, the pentose phosphate pathway, lower photosynthetic energy production, enhancement of photorespiration, and increased polysaccharide production. This application of proteomics to the examination of proteins in marine sediments, a marine diatom, and a heterotrophic marine bacterium shows the potential for these techniques to help elucidate the fate of proteins in marine environments and could be used in conjunction with well-established molecular organic marker studies.Item Identifying Molecular Functions of Heliotropic Motor Tissue Through Proteomic Analysis of Soybean Pulvini(2013) Lee, Hakme; Sullivan, Joseph; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heliotropic and nyctinastic leaf movement are mediated in soybean through turgor changes in the motor cells of the pulvinus, located at the base of the leaves. While some elements of the signaling pathways have been studied, a broad-scale protein identification has not yet been reported. In my research pulvini proteins were extracted in light- and dark-harvested soybean using the TCA/acetone method and identified by LC-MS/MS. Gene ontology analysis revealed proteins involved in proton transport were enriched in the soybean pulvinus proteome compared to a background soybean proteome. Proteins more highly expressed in the light were mostly stress response proteins, while under-expressed proteins were categorized as energy proteins. Further investigations using more sensitive extraction protocols and a multitude of sample times will build on these initial results to provide a thorough examination of heliotropic mechanisms at the molecular level.