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

More information is available at Theses and Dissertations at University of Maryland Libraries.

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    METABOLIC VIRULENCE DETERMINANTS AND RAPID MOLECULAR DIAGNOSTICS OF PATHOGENIC SPIROCHETES
    (2016) Backstedt, Brian; Pal, Utpal; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Borrelia burgdorferi and Leptospira interrogans are pathogenic spirochetes that elicit serious health threats, termed as Lyme disease and leptospirosis. Key areas of spirochete research involve a better understanding of their intriguing biology and infection, including identification of novel virulence factors and improvements in diagnostic and preventive methods. Notably, certain bacterial metabolic enzymes are surface-exposed, having evolved to acquire additional functions referred to as protein moonlighting that contributes in microbial virulence. Comparative genome analysis revealed that certain components of sugar metabolism pathways are either absent or seemingly inactive in pathogenic spirochetes, which were studied herein for their potential roles as metabolic virulence factors. Of nine borrelial enzymes investigated, only phosphomannose isomerase (PMI) was found to be surface-exposed and remained enzymatically active in the spirochete outer membrane. PMI is critical for mannose metabolism and facilitates the interconversion of fructose 6-phosphate and mannose-6-phosphate, although its occurrence in borrelial surface remains enigmatic. PMI may provide a critical function for B. burgdorferi viability as it is constitutively expressed and all attempts to create genetic mutants remained unsuccessful. Active immunization studies using recombinant PMI did not influence the outcome of infection within tick or murine hosts, although a significant reduction in bacterial levels within the joints of mice was recorded, suggesting its involvement in spirochete persistence in a tissue-specific manner. Despite substantial advancement, the development of more effective diagnostics for leptospirosis and Lyme disease still remains a critical need since human vaccines are unavailable. Antibiotic treatment can resolve these infections but is most effective when administered early during infection, prior to pathogen dissemination to distant organs. As diagnostic methods for spirochete infection still depends on ineffective and antiquated technologies, we sought to develop novel RNA-based assays for better detection of early spirochete infection. Results indicated that targeting specific regions of 16S and 23S ribosomal RNA targets provided the highest possible sensitivity and specificity of detection, which was far superior to current serological, microbiological or molecular methods used to detect presence of invading pathogens.
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    Ribosome integrity and translational fidelity require accurate modification and processing of rRNA in the yeast Saccharomyces cerevisiae
    (2006-11-21) Roshek, Jennifer Lynn Baxter; Dinman, Jonathan D; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Translating mRNA sequences into functional proteins is a fundamental process necessary for the viability of organisms throughout all kingdoms of life. The ribosome carries out this process with a delicate balance between speed and accuracy. Although kinetic and biochemical studies along with high resolution crystal structures have provided much information about the ribosome, many of the underlying mechanisms of ribosome function are still poorly understood. This work seeks to understand how ribosome structure and function are affected by changes in rRNA as caused by two very different mechanisms. mof6-1, originally isolated as a recessive mutation which promoted increased efficiencies of programmed -1 ribosomal frameshifting, was found to be an allele of RPD3 which encodes a histone deacetylase that is involved in transcriptional activation and silencing. This mutant demonstrated a delay in ribosomal RNA (rRNA) processing leading to changes in reading frame maintenance and ribosomal A-site specific defects. To understand the role of cis-acting changes to rRNA, yeast strains deficient in rRNA modifications in the peptidyl transferase center of the ribosome were monitored for changes in ribosome structure and translational fidelity. Analyses revealed mutant phenotypes including sensitivity to translational inhibitors; changes in reading frame maintenance, nonsense suppression and aa-tRNA selection; and increased rates of A-site tRNA binding to the mutant ribosome. One mutant in particular, spb1DA/snr52Δ, promoted increased rates of programmed -1 ribosomal frameshifting, increased rates of near cognate tRNA selection and A-site tRNA binding. Structural analysis of spb1DA/snr52Δ revealed changes consistent with a more accessible ribosomal A-site. These results suggest that rRNA nucleotide modifications produce small but distinct changes in ribosome structure and function contributing to overall translational fidelity. Taken together, these data suggest that rRNA, a main component of the ribosome, contributes directly to translational fidelity. Defects in rRNA caused by changes in both its processing and modification can cause changes in reading frame maintenance, nonsense suppression, aa-tRNA selection and binding as well as ribosome structure.