Theses and Dissertations from UMD
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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 give thesis/dissertation in DRUM
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Item DEVELOPMENT OF A PROTEOMIC STRATEGY FOR ANALYSIS OF PLASMA MEMBRANE PROTEINS(2013) Choksawangkarn, Waeowalee; Fenselau, Catherine; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Plasma membrane (PM) proteins play crucial roles in cell signaling and communications, and they are the targets of more than two thirds of drugs currently under development. Studies on changes in protein content, quantity and modifications of the PM proteins indicate metabolic alteration of disease related cells; therefore, mass spectrometry-based proteomic studies may lead to improved understanding of the pathology, the characterization of novel biomarkers, and discovery of future drug targets. The main objectives of my research are to develop an effective enrichment strategy and to optimize the proteomic workflow for analysis of PM proteins from cells in suspension. Strategies were optimized with human multiple myeloma cells cultured in suspension, and optimized strategies were applied to study the PM proteome of myeloid-derived suppressor cells (MDSC) collected from an animal model. We focus on optimization of the cationic nanoparticle pellicle method for enrichment of PM proteins. The principle of this method is to attach cationic nanoparticles to the cell surface by electrostatic interaction between the positively charged nanoparticles and the negatively charged cell surface. Thus, the heavier coated-plasma membrane sheets can be separated more easily from cellular organelles by centrifugation after cell lysis. The isolated PM proteins were identified by LC-MS/MS analysis. We have also optimized the workflow for proteolysis to enhance identification of hydrophobic PM proteins. Our studies reveal that higher density nanoparticle pellicles provide higher enrichment efficiency of the PM proteins and that a procedure using digestion in the gel matrix enhances the analysis of highly hydrophobic proteins. The most effective enrichment technique and optimized proteomic procedures were applied to characterize the PM proteins from MDSC obtained from BALB/c mice carrying 4T1 mammary carcinomas. These cells are known to accumulate in individuals with cancer and suppress anti-tumor immunity. Their accumulation and activity are increased with heightened-levels of inflammation. Comparative studies of the PM proteins expressed in the cells derived from basal- and heightened- levels of inflammation were performed using the spectral counting method. This work reveals a set of protein candidates that have a high potential to be involved in the inflammation-driven immunosuppressive activity of the MDSC.Item Proteomic Analysis of Plasma Membrane Proteins from Drug Susceptible and Drug Resistant Breast Cancer Cell Lines(2004-11-08) Rahbar, Amir Mikel; Fenselau, Catherine C; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Drug discovery is an important field of research in the biotechnology and pharmaceutical industries. Plasma membranes are rich in drug targets and other proteins responsible for cell signaling, transport, signal transduction, and other cellular functions. Information obtained about these proteins, and the pathways they participate in, helps to facilitate the drug discovery process. Although these plasma membrane proteins play important roles in cellular function, they are usually expressed in very low abundance and are therefore hard to identify and analyze. Comparative proteomic analysis of plasma membranes in different types of cells or different disease states of the same cell or tissue type can help to design targeted therapies specific to particular cell or tissue types and can be used in the identification of biomarkers for early disease detection. In order to be able to identify proteins in the plasma membrane it is important to start out with a plasma membrane fraction that is free of contamination from other more abundant proteins from other portions of the cell. 2D gel electrophoresis is the primary protein separation tool for use with proteomics and drug discovery, however, the inability of membrane proteins to be separated using isoelectric focusing, which is the first step in the 2D gel protocol, excludes 2D gel electrophoresis as a viable technique for the separation of membrane proteins. This thesis develops and evaluates a method to identify proteins found in the plasma membranes of mammalian cells using a modified form of the cationic colloidal silica technique for plasma membrane isolation combined with analysis of these proteins using mass spectrometry. This method is then used in combination with metabolic stable isotope labeling to identify protein expression changes between the mitoxantrone drug susceptible and drug resistant MCF-7 breast cancer cell lines.