Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xvi, 176 p.; also includes graphics (some col.). Includes abstract and vita. Advisor:, Dept. of Biochemistry. Includes bibliographical references (p. 168-176).

Thesis (Ph. D.)--University of Texas at Austin, 2003. / Vita. Includes bibliographical references. Available also from UMI Company.

Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 173-189). Also available on the World Wide Web.

Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xxii, 172 p. : ill., some col. Includes abstract and vita. Advisor: Michael K. Chan, Dept. of Chemistry. Includes bibliographical references (p. 165-172).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (p. 146-162).

Preliminary studies have shown that manuka honey affects the cell cycle of MRSA by impeding cell division, but mode of action was unknown. Cell division depends on the formation of septa and cleavage of peptidoglycan at cytokinesis. This study investigated how manuka honey might alter the cell cycle of EMRSA-15. Physiological and chemical changes in the bacteria exposed to manuka honey were determined using time to kill studies, confocal and electron microscopy. Data indicated that honey had a bactericidal effect on MRSA, inhibiting the cell cycle cytokinesis. Increased septum formation was noted in honey treated cells by transmission electron microscopy. Cell division components including FtsZ and Endo-B-N-Acetylglucosaminidase were investigated using cell wall turbidity assays, zymography, immunofluorescence and immuno gold labelling. Manuka honey treated MRSA cells showed a marked reduction in hydrolase activity after 12 hours compared to untreated cells. The immunofluorescence indicated an initial increase in FtsZ production followed by a significant decrease by 24 hours. PCR of FtsZ showed a 10% increase in production after 1 and 4 hours. Localization by gold labelling gave inconclusive results. Immunofluorescence of Endo-B-N-Acetylglucosaminidase showed a decrease in the amount of enzyme over 24 hours and localization by gold labelling indicated altered distribution of this enzyme. PCR showed no significant difference in expression. 2-D electrophoresis showed a differing proteomic profile between control cells and those treated with honey, with a potential target protein being identified. Methylglyoxal (an antibacterial component of manuka honey) was investigated after a report named this as potentially the active component of manuka honey. Results showed it has an effect but is not wholly responsible for the effects induced by manuka honey. It was concluded that increased numbers of cells with septa were formed and alteration in production of proteins and enzymes resulted in MRSA cells exposed to bactericidal concentrations of manuka honey. The work was also carried out with artificial honey controls, indicating that effects seen were not due to sugar content within honey or methylglyoxal content.

published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

Malaria is a global health concern accounting for approximately 219 million cases and an estimated 660 000 deaths in 2010. The most fatal strain of malarial parasite, Plasmodium falciparum is found to contain 3 Adenylate Kinases (PfAK1, PfAK2 and PfGAK). Adenylate Kinases are important enzymes that essentially catalyze and regulate energy metabolism processes. PfAK1 and PfAK2 catalyze the reversible MG2+ reaction ATP + AMP ←→ 2ADP whereas, the PfGAK catalyzes the Mg2+ dependent reaction GTP+AMP ←→ ADP+GDP. Of all malarial strains, only the Plasmodium falciparum Adenylate Kinase 2 (PfAK2) was found to contain a N-myristoylation sequence and subsequently formed a stable heterodimer with Plasmodium falciparum N-myristoyl transferase (PfNMT). The myristoylation of PfAK2 by PfNMT is believed to help transport PfAK2 to the parasitophorous vacuole membrane (PVM) so that the enzyme can perform its essential functions. With these enzymes being key components in the parasite’s survival, the structural study of these enzymes would provide a lot of insight into targeting these proteins for drug design that would effectively kill the parasite without affecting the human host. In this study, PfAK1 was able to be expressed, purified and crystallized with a dataset collected at 4.3Å. PfGAK was expressed and purified. A GTP analogue called GP5A was used to soak the purified PfGAKand the PfGAK bound to GP5A was crystallized and diffracted. Moreover, PfAK2 and PfNMT was successfully expressed and co-purified. The purified PfAK2-PfNMT heterodimer are undergoing crystal screening for possible crystallization conditions. / published_or_final_version / Physiology / Master / Master of Philosophy

Not available / text

The focus of this dissertation is the study of two enzymes, DXR and HppE. DXR catalyzes the first committed step in the MEP pathway, which is the pathway most eubacteria, archeabacteria, algae, and the plastids of plants use for the biosynthesis of isoprenoid. Since mammals utilize the mevalonate pathway and isoprenoids are essential for survival, all enzymes in the MEP pathway are excellent antibiotic targets. One antibiotic that has promise in the fight against malaria is the natural product fosmidomycin, whose antibiotic activity is due to its ability to bind and inhibit DXR. With a deeper understanding of DXR's catalyzed reaction, it will be possible to design a more sophisticated and potent antibiotic. To probe the mechanism of DXR, two fluorinated substrate analogues, 3F-DXP and 4F-DXP, and a fluorinated product analogue, FCH₂-MEP were designed and analyzed as possible substrates or inhibitors. To further analyze the mechanism of DXR, a 2° [²H]-KIE study was conducted using the equilibrium perturbation method. The second enzyme this dissertation examines is HppE, which catalyzes the final step in the biosynthesis of the antibiotic, fosfomycin. Fosfomycin is a clinically useful antibiotic for the treatment of limb-threatening diabetic foot infections and urinary tract infections. Chemically speaking, HppE is unique for two reasons. First, HppE's epoxidation differs from Nature's standard method of epoxide formation by alkene oxidation, where the epoxide oxygen is derived from molecular oxygen. For HppE, the epoxide is formed through the dehydrogenation of a secondary alcohol; thus the epoxide oxygen is derived from the substrate. Second, HppE is a unique member of the mononuclear non-heme iron-dependent family of enzymes. HppE differs from all other mononuclear non-heme iron-dependent enzymes by requiring NADH and an external electron mediator for turnover but not requiring [alpha]-KG, pterin, ascorbate, or an internal iron-sulfur cluster. After a study was published on the activity of zinc-reconstituted HppE from Streptomyces wedmorensis, the proposed iron and NADH dependent mechanism of HppE was reevaluated and was reconfirmed. The HppE from Pseudomonas syringae (Ps-HppE) was also purified and was characterized biochemically and spectroscopically. The results of [²H] and [¹⁸O]-KIE studies on Ps-HppE are also reported. / text