Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 78-98).

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 92-104). Also available in print.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 97-108). Also available in electronic version. Access restricted to campus users.

Thesis--M. Phil., University of Hong Kong. / Typewritten.

In order to reduce malaria prevalence worldwide, a better understanding of parasite transmission and the effect of drug resistance is needed. The effect of drug resistance on malaria transmission has been examined for some drugs, but not for mitochondrial inhibitors such as atovaquone and the current basis of malaria therapy, artemisinin. Therefore, the goal of this study was to produce gametocytes, the life cycle stage that transmits from mosquito to human, in several different drug resistant patient isolates as well as to determine the effect of drug resistance on gametocyte development and transmission. Previous studies have shown that the mutation that confers resistance to atovaquone, a common antimalarial, occurs de novo after treatment and transmission of this resistance is not seen in the field. Therefore, to determine whether or not the resistance mutation can be transmitted, mosquito-feeding experiments were conducted using atovaquone resistant parasites and resulting oocyst DNA was analyzed. In addition to these atovaquone studies, artemisinin resistant gametocytes were also grown in vitro and drug pressure was added to determine if resistance mechanisms affect gametocyte development. This study is the first examine gametocyte development in these resistant strains and the first to report that transmission of the atovaquone resistant mutation may be possible. However, data is currently inconclusive on the effect of artemisinin resistance on gametocyte development.

We have engineered a mutant of HIV Reverse Transcriptase that can be fluorescently labeled by covalent attachment of the environmentally sensitive fluorophore 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC). The result is a polymerase that is kinetically indistinguishable from the wild-type enzyme, but provides a signal to monitor changes in enzyme structure that result from conformational changes induced by substrate binding. Using this system, we have expanded the kinetic model governing nucleotide binding to include an enzymatic isomerization following initial nucleotide binding. In doing so, we define the role of induced-fit in nucleotide specificity and mismatch discrimination. Additionally, we have characterized the kinetics governing the specificity and discrimination of several widely administered Nucleotide Reverse Transcriptase Inhibitors (NRTI’s) used to combat HIV infection including 3TC (Lamivudine), FTC (Emtricitabine), and AZT (Zidovudine) for the wild-type polymerase and mutants with clinical resistance to these compounds. Our findings resolve the apparent tighter binding of these inhibitor compounds compared to the correct nucleotide by showing that the affinity for the correct nucleotide is stronger than the inhibitors. The apparent weaker binding of the correct nucleotide is a result of a incomplete interpretation of binding data that fails to account for the importance of the reverse rate of the conformational change. The apparent Kd (Kd,app) measurements for correct nucleotide estimates Km rather than Kd because nucleotide binding does not reach equilibrium. The conformationally sensitive enzyme has also been used to characterize the kinetics governing DNA association. We show that DNA binding is governed by a two-step process where a fast initial association is followed by a second, slow isomerization that is off the pathway for nucleotide binding and incorporation. Finally, we have implemented single molecule techniques using fluorophore labeled nucleotides to study the effects of AZT incorporation on the DNA translocation dynamics of the polymerase. We find that primer termination with AZT results in DNA that fails to translocate, therefore occluding the next nucleotide from binding. This shift in translocation equilibrium exposes the newly formed phosphodiester bond to ATP- or pyrophosphate-mediated AZT excision; thereby rescuing productive polymerization. This finding represents the first kinetic measurement of DNA translocation by a polymerase. / text

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Glioblastoma multiforme (GBM), a grade IV malignant astrocytic tumor according to WHO classification, is one of the most common and malignant brain tumor. Temozolomide (TMZ) is the current standard treatment for GBM. Nevertheless, resistance to chemotherapy in GBM is common and therefore a major obstacle to successful treatment. Adducin 3 (ADD3), a cytoskeletal protein, has been found to be associated with chemoresistance in osteosarcoma, but its potential role in glioblastoma is unclear. A TMZ-resistant model was established by chronically exposing the glioma cells (D54 cell line) to an increasing dose of TMZ. A resistant subclone (D54-R) was successfully generated. ADD3 expression level was found to be upregulated in the D54-R when compared to the parental D54 cells (D54-C). CD133 is a putative cancer stem cell marker. Its expression level was found also to be higher in D54-R when compared to D54-C cells. Among the D54-R cells, a subgroup of cells was found to express ADD3 intensely. The proportion of these spherical cells was higher in D54-R than D54-C. Moreover, these cells were spherical in morphology and expressed putative cancer stem cell markers: CD133, NANOG and OCT-3/-4. Therefore, ADD3 is associated with cancer stem cells in human glioma. The upregulation of ADD3 expression is associated with TMZ-resistance in GBM. / published_or_final_version / Surgery / Master / Master of Research in Medicine

The global emergence of drug resistance is posing increasing difficulties in the public health control and treatment of tuberculosis (TB). Fluoroquinolones (FQs) are regarded as having a pivotal role among the antimicrobial agents in multidrug regimens against multidrug-resistant tuberculosis (MDR-TB). Thus, early diagnosis of fluoroquinolone-resistant (FQr) MDR-TB and extensively drug-resistant tuberculosis (XDR-TB) by molecular tests has predictive value for the guidance of TB therapy. The pharmacokinetic (PK) and pharmacodynamic (PD) indices are valuable parameters to evaluate the activity and efficacy of fluoroquinolones (FQs) based upon the bactericidal effect and prevention of the emergence of resistance. In the first part of this study, the potencies of ofloxacin (OFX) and moxifloxacin (MXF) against clinical isolates of MDR-TB in terms of their PK/PD indices (Cmax/MIC90, AUC/MIC90, Cmax/MPC90 and AUC/MPC90) were investigated and compared. The results revealed that MXF displays higher ratios of PK/PD in vitro and could serve as a promising agent for the treatment of MDR-TB. Molecular tests on resistance genes are reliable and rapid technology for diagnosis of drug-resistant TB which facilitates timely patient management and public health control of TB. In the second part of the study, the feasibility of a PCRsequencing assay for the examination of mutations in the quinolone-resistance-determining- region (QRDR) of the gyrase A (gyrA) gene in FQ-resistant (FQr) Mycobacterium tuberculosis in direct clinical specimens was evaluated. As determined by gyrA QRDR DNA sequencing analysis, complete concordance of phenotypic and genotypic outcomes was demonstrated. The results indicate that the molecular assay is an accurate and effective method for the diagnosis of FQr TB and allows identification of mixed resistant variants in the same patient. GyrA mutations that associated with FQr in clinical isolates of M. tuberculosis were clustered in hotspot codons 88, 90, 91 and 94, corroborating other reports. We also detected a novel gyrA Ala74Ser mutation in M. tuberculosis directly from the respiratory specimens by using the PCR-DNA sequencing assay. In the third part of this study, the functional effect of the Ala74Ser mutant was verified through study of the DNA supercoiling inhibitory activities of OFX and MXF against the recombinant DNA gyrase. Fifty percent inhibitory concentrations (IC50) of FQs against the DNA supercoiling activities of the recombinant DNA gyrase complex reconstituted with gyrA Ala74Ser were eight-fold and 14-fold greater than the wild-type H37Rv reference strain, and results correlated well with their phenotypic drug susceptibilities. Besides, a combination of gyrA mutations (Glu21Gln, Ser95Thr and Gly668Asp) was also characterized to be non-functional polymorphisms. The impact of the gyrA Ala74Ser mutation on drug binding affinity was elucidated through a crystal structure model of the gyrA-MXF-DNA cleavage complex. Alanine at position 74 of gyrA in M. tuberculosis, which corresponds to the alanine at position 67 of gyrA in Escherichia coli, is an amino acid lying in the α3 helix domain which forms a hydrophobic interface between the gyrA-gyrA dimer. Perturbation of the gyrA-gyrA dimer interface caused by the Ala74Ser mutation probably disturbs the putative drug binding pocket, and leads to the reduction of the binding affinity of FQ due to the distance effect. This is the first report verifying that gyrA Ala74Ser mutation alone is responsible for FQr in M. tuberculosis. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Drug efflux represents an important protection mechanism against antibiotics and environmental toxic compounds in bacteria. Efflux genes constitute from 6% to 18% of all transporters in bacterial genomes, yet their regulation, natural substrates, and physiological functions are poorly understood. Among the 20 chromosomally encoded efflux genes in Escherichia coli K-12, only the AcrAB-TolC efflux system is constitutively expressed under the ordinary laboratory growth of E. coli. To explore conditions and circumstances that trigger the expression of additional efflux genes as well as their physiological functions, I examined the expression of all 20 efflux genes under a physiologically relevant circumstance for E. coli, which is anaerobic condition in this study. I found that expression of an RND type efflux pump MdtEF is up-regulated more than 20 fold when E. coli is cultured under anaerobic conditions. Mutagenesis studies revealed that the anaerobically induced expression of mdtEF is subject to the regulation of the anaerobic global transcription factor ArcA. Direct drug efflux and tolerance assay showed that anaerobically grown E. coli cells display an increased efflux activity and enhanced drug tolerance in an MdtEF dependent manner, confirming the functional up-regulation of the efflux pump MdtEF in the anaerobic physiology of E. coli. Since the up-regulation of mdtEF by anaerobic growth occurs in the absence of antibiotics and drugs, I speculate that MdtEF has physiological functions under the anaerobic growth of E. coli. To explore this, I first compared the viability of ΔmdtEF and WT MG1655 strains and found that ΔmdtEF caused a decreased cell survival during prolonged anaerobic growth of E. coli. Interestingly, this defect became more pronounced when cells grow in the presence of 10 mM nitrate, but no defect was observed in ΔmdtEF strain when cells grow in the presence of 40 mM fumarate under the same anaerobic conditions, suggesting that MdtEF has physiological roles relevant to the anaerobic respiration of nitrate. I further found that E. coli cells harboring the deletion of mdtEF are susceptible to indole nitrosative derivatives, a class of toxic by-products formed and accumulated within E. coli when the bacterium respires nitrate under anaerobic conditions, and deletion of the genes responsible for the biosynthesis of indole, tnaAB, restores the growth defect of the ΔmdtEF strain during anaerobic respiration of nitrate. Taken together, I conclude that the multidrug efflux pump MdtEF expels the nitrosated indole derivatives out of E. coli cells under anaerobic conditions. Since the production and accumulation of nitrosyl indole derivatives is ascribed to the reactive nitrogen species elicited when E. coli consumes nitrate, I propose that the up-regulated multidrug efflux pump MdtEF functions to protect E. coli from nitrosative damage in its anaerobic ecological niches. / published_or_final_version / Biological Sciences / Master / Master of Philosophy