Master of Science (MS), Bowling Green State University, 2021, Biological Sciences

An oomycete pathogen, Globisporangium ultimum (also known as Pythium ultimum), causes damping-off on a wide range of hosts. This disease is one of the major constraints on soybean production. Although fungicide seed treatments are often used to combat the disease, significant losses occur in cool and moist conditions. In addition, the emergence of fungicide-resistant isolates, the lack of resistant cultivars, and the ineffectiveness of crop rotations pose further challenges in managing the disease. Hence, new molecular targets are needed to control G. ultimum. In this study, G. ultimum and G. sylvaticum were isolated from the soybean fields (Bowling Green, Ohio). Pathogenicity assays were evaluated on two soybean cultivars: William and William 82. The seed-and seedling rot assays determined that both the isolates were pathogenic to both the seeds and seedlings of soybeans. Globisporangium ultimum showed a 100% disease severity index (DSI) on both cultivars, while G. sylvaticum had a DSI of 73.1% and 93% on William and William 82, respectively. The seedling root rot assay showed a similar rate of infection in both cultivars, based on the root surface area compared to the control (healthy plant). Kazal-type serine protease inhibitors (KPIs) are produced and secreted by many pathogens, including G. ultimum. They neutralize plant defense proteins in the protease family, allowing pathogens to colonize the hostile apoplast. In silico analysis via FungiDB showed that G. ultimum genome encodes 13 putative secreted KPIs. Four genes: PYU1_1G000142, PYU1_G009682, PYU1_G013310, and PYU1_G002778 were selected for gene expression analysis during infection using qPCR. Results showed that PYU1_1G000142 and PYU1_G009682 showed the highest expression at 48 hours post inoculation (hpi), indicating that they were associated with late infection in both soybean cultivars. In contrast, PYU1_G013310 showed highest expression at 6 hpi in William and at 24 hpi in William 82, indicating th (open full item for complete abstract)

Committee: Vipaporn Phuntumart Ph.D. (Advisor); Raymond Larsen Ph.D. (Committee Member); Paul Morris Ph.D. (Committee Member) Subjects: Agriculture; Bioinformatics; Biology; Molecular Biology

Doctor of Philosophy, Case Western Reserve University, 2007, Chemistry

The ATP-dependent serine protease Lon is responsible for degrading damaged and certain regulatory proteins in vivo. The importance of Lon activity in bacterial pathogenicity has led to its emergence as a target in the development of novel antibiotics however no potent or specific inhibitors had been reported. This study focused on identifying a lead compound(s) for the development of potent inhibitors of the proteolytic activity of Lon. Steady-state kinetic characterization of the ATP and peptide hydrolysis activities of human and Salmonella enterica serovar Typhimurium (S. Typhimurium) Lon revealed no kinetic differences in ATP hydrolysis, but marked differences in substrate specificity. This suggests a peptide-based inhibitor may be developed which exploits these differences to target an inhibitor to a single homolog, minimizing cross-reactivity. Screening of commercially available peptide-based inhibitors highlight the utility of transition state analogs in inhibiting peptide hydrolysis. The peptidyl boronate, MG262, was the most potent inhibitor tested and was effective against both human and S. Typhimurium homologs (IC50 = 160 ± 10 nM and 122 ± 9 nM, respectively). Peptidyl boronates inhibit peptide hydrolysis through a two-step time-dependent mechanism with an overall Ki of ~ 20 nM. The first step is rapid and involves binding of the inhibitor and formation of a covalent adduct with the active site serine. A second slow step occurs in which the protease undergoes a conformational change or isomerization to enhance the interaction of the inhibitor with the proteolytic active site. Although inhibition of serine and threonine proteases by peptidyl boronates has been detected previously, Lon is the first protease which requires the binding of ATP to observe inhibition. Finally, the purification of the human homolog of the steroidogenic acute regulatory protein (StAR) is described. It is shown to be a substrate of human Lon and provides a starting point for the dev (open full item for complete abstract)

Committee: Irene Lee (Advisor) Subjects: Chemistry, Biochemistry

Doctor of Philosophy, Miami University, 2011, Chemistry and Biochemistry

Enzyme-substrate interactions are a concept that spans various topics in biochemistry and molecular biology (e.g., kinetics, metabolism, and translation), and there are a vast amount of representations used to teach this concept that lack common conventions. Deficiencies in understanding of enzyme-substrate interactions or lack thereof could hinder students' understandings of later concepts in biochemistry. However, prior to this dissertation, there was no way to efficiently measure students' understandings of enzyme-substrate interactions. Therefore, this dissertation set out to (1) investigate biochemistry students' understandings of enzyme-substrate interactions and how multiple representations of the phenomena influence the understanding and (2) use misconceptions uncovered to create the Enzyme-Substrate Interactions Concept Inventory (ESICI) to allow for efficient measurement of students' understandings. Multiple theoretical frameworks guided the development, collection, and analysis of the data in this dissertation. A sequential mixed methods design was used to address the aims of this dissertation. This design consisted of student interviews using multiple representations to elicit students' understandings of enzyme-substrate interactions, followed by the development of the ESICI based on the findings from the interviews. The ESICI was subsequently administered to 707 students at 16 institutions from across the United States. Students were found to have a range of prior knowledge that they used to interpret the representations. The use of multiple representations provided evidence of cognitive dissonance, representational competence, and misconceptions. The ESICI further provided evidence of misconceptions, the most significant being students' sole focus on electronic complementarity and students' lack of energetic understanding of enzyme-substrate interactions. The findings from this dissertation could be used in the classroom to measure students' unders (open full item for complete abstract)

Committee: Dr. Stacey Lowery Bretz (Advisor); Dr. Ellen J. Yezierski (Committee Chair); Dr. Marcy H. Towns (Committee Member); Dr. Michael W. Crowder (Committee Member); Dr. Kathryn B. McGrew (Committee Member) Subjects: Biochemistry; Biophysics; Chemistry; Educational Tests and Measurements; Educational Theory; Molecular Biology; Science Education; Teaching