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 UTILIZATION OF FUNCTIONALIZED CATANIONIC SURFACTANT VESICLES TO INVESTIGATE PROTEIN-GLYCAN INTERACTIONS(2014) Mahle, Amanda Caroline; Beckett, Dorothy; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Despite the estimate that more than half of the human proteome is glycosylated, the field of glycomics lags substantially behind proteomics and genomics. The ubiquity of carbohydrates in and on the cell is directly related to the extensive roles they play in the folding and stabilization of protein structure, and in cellular recognition processes. The slow progress in deciphering glycan interactions at a molecular level is in large part due to the absence of a functional system to express, on a large scale, carbohydrates of known structure, in the context of a biologically relevant assay system. The goals of my research are to prepare and characterize glycan-functionalized catanionic surfactant vesicles as a platform for glycan synthesis, and to demonstrate that the resulting glycan-functionalized vesicles serve as a scaffold for the interrogation of protein-glycan interactions. To accomplish this goal, a high-yield method for the purification of N. gonorrhoeae lipooligosaccharide (LOS) glycosyltransferase LgtE, an enzyme that catalyzes the addition of galactose onto a terminal glucose found on LOS, was first optimized. Preliminary evidence is presented, suggesting that catanionic vesicles solubilize macromolecules found in inclusion bodies, indicating that surfactant vesicles facilitate the purification of insoluble species. A novel method exploiting differential lectin binding, measured by flow cytometry, was developed to demonstrate LgtE activity on whole cell bacteria and on catanionic vesicles functionalized with LOS or a synthetic glycolipid acceptor substrate. The data from these studies confirm the vesicles' robustness in the highly sensitive assay, and demonstrates LgtE mediated oligosaccharide biosynthesis on vesicles, regardless of acceptor origin. Enzyme activity was then characterized on whole cells and LOS functionalized vesicles. Unexpectedly, LgtE is observed to have approximately the same affinity for both terminal glucose and galactose as acceptor substrates for galactosyl transfer. Finally, enzymatic synthesis and retention of the vesicles by hydrophobically modified chitosan coated electrodes is demonstrated by differential antibody binding. This dissertation presents proof-of-concept that glycan-functionalized catanionic vesicles can be used to create a high-specificity and high-throughput glycan array. This array will allow for the investigation of a variety of protein-glycan interactions, and will undoubtedly have applications in many fields of glycomics.Item What Makes a Pathogen? Genetic and Structural Heterogeneity of Neisserial Lipooligosaccharide(2006-01-24) O'Connor, Ellen Therese; Stein, Daniel C; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The genus Neisseria consists of both pathogenic and commensal species that colonize mucosal niches. Specific structures of neisserial lipooligosaccharide (LOS) expressed by the gonococcus and meningococcus have been shown to play a role in pathogenesis. Commensal Neisseria also produce LOS, but can express additional structures not found in the pathogenic strains. N. sicca 4320 differs from other commensal Neisseria because it caused disease in a seemingly healthy individual. Therefore, this strain was used to research the extent to which a non-pathogen differs from a pathogen. A bioinformatic examination of the nucleotide sequence of the 4320 chromosome revealed strong homologies with N. meningitidis MC58. These findings suggest that 4320 is a member of the Neisseria and that commensal and pathogenic Neisseria have similar genomic content. Through MALDI-TOF, exoglycosidase digestion, and MSn analyses, 4320 was shown to express both LOS and LPS. The 4320 LOS resembles that of Haemophilus sp. in that it contained 3 heptoses. The LPS consists of a N-acetylglucosamine and rhamnose disaccharide repeat that is not attached to a lipid A, a novel molecule. PEA transfer onto pathogenic Neisseria LOS was examined, as strains expressing PEA at 3-HepII as opposed to 6-HepII are more serum resistant. Southern hybridization was used to identify the presence of phosphoethanolamine (PEA) transfer genes in N. sicca strains. 4320 showed differences in the presence of Lpt3 transfer genes compared to other strains within the species. Lpt3 was isolated and biochemically shown to mediate the addition of PEA to 3-HepII of LOS that was constructed to lack the 3-HepII decoration. lpt3 nucleotide sequence analysis was done on a diverse group of gonococcal isolates, and phylogenetic analysis showed a high degree of sequence divergence. These data support the idea that the presence of PEA at 3-HepII of LOS increases the pathogenic ability of the Neisseria, as lpt3 was only found in N. sicca strains isolated from fatal endocarditis cases.