Thesis (Ph. D.)--University of Washington, 2005. / Includes bibliographical references (leaves 208-217).

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 132-138). Also available in electronic version.

Thesis (Ph.D.)--York University, 2005. Graduate Programme in Chemistry. / Typescript. Includes bibliographical references (leaves 140-150). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pNR11550

Chromosomal mediated antimicrobial resistance in Neisseria gonorrhoeae can develop as a result of three main processes including the alteration of target enzymes, changes in transmembrane transport channels and active efflux pump function. Single nucleotide polymorphisms (SNPs) of target genes such as DNA gyrase (gyrA) and topoisomerase (parC), together with mutations in the promoter regions of the efflux pumps norM and mtr can confer resistance to the macrolides, penicillins and fluoroquinolones. These SNPs were analyzed using the SNaPshot method to allow for rapid detection of resistant isolates. Oligonucleotides were developed in the 5’ to the 3’ direction, ending one nucleotide adjacent to the specific SNP of interest. Single base extension reactions were performed and were detected using capillary electrophoresis. The SNaPshot procedure from Applied Biosystems employed in this study adds a single fluorescently-labelled nucleotide complementary to this SNP at the 3’ end by a primer extension polymerase reaction. Then using capillary electrophoresis, the labelled nucleotide is detected, enabling differentiation between A, C, T, or G. SNP results obtained were verified using DNA sequencing and both single and multiplexed reactions were carried out to increase the efficiency of the procedure. Spiked urine samples were also observed to determine if SNPs could be detected clinically. Single reactions enabled the characterization of all confirmed and relevant SNPs. With multiplex primer extension, multiple peaks were observed, each corresponding to one of the SNPs in the gene. This technique was explored for its applicability to detect SNPs of gyrA and parC mutations. Observable SNP detection limits were seen in spiked urine samples at 108 cells/mL in as early as 4 hours. DNA sequencing results confirmed the SNPs identity in each case. Thus, capillary electrophoresis using the SNaPshot protocol is another way to rapidly identify clinically resistant strains of Neisseria gonorrhoeae. This technique has also been shown to reduce analysis time compared to DNA sequencing and produces the same results. / February 2005

Resumo: Clicar acesso eletrônico abaixo / Abstract: Click electronic access below / Orientador: Juliana Regina Peiró / Coorientador: Paulo César Ciarlini / Banca: Cáris Maroni Nunes / Banca: Carlos Augusto Araújo Valadão / Mestre

No description available.

Velocity-difference induced focusing (V-DIF) with a dynamic pH junction in capillary electrophoresis (CE) using a sample with a pH different from that of the background electrolyte (BGE) was developed in our group, but the mechanism was not well understood. In this work, the mechanism of this focusing technique was first studied using an appropriate dye to monitor the pH of the BGE and the sample during the focusing process. A mechanism was proposed based on the experimental results. This technique was then applied to serotonin to improve the detection limit when CE was used with a UV absorption detector. It was also applied to focus amino acids, peptides, and proteins to improve the concentration sensitivity. It is found that the pKa rather than the pI of the analytes is the key criterion for selecting the pH for the sample and for the BGE to obtain the optimum focusing for these molecules. Since UV detection only provides migration time information, more structure information is obtained by using a photodiode array (PDA) and mass spectrometer (MS) for peak identification. Comparisons were made between the PDA detection and MS detection for aromatic amino acids with V-DIF using a dynamic pH junction. This V-DIF technique was then applied to non-aromatic amino acids with MS detection. It was used at low pH with positive ESI-MS detection and at high pH with negative ESI-MS ionization. The results of the two methods were compared and discussed. Finally, the preparative operation of continuous flow counterbalanced CE (FCCE) was studied. The effects of larger sample volumes and multiple capillary systems on improving the purification yield were investigated. / Science, Faculty of / Chemistry, Department of / Graduate

No description available.

After standardizing a starch-gel electrophoretic technique, variations in the serum proteins of the genus Odocoileus due to the condition of the sample and the condition of the animal were studied. Significant changes in the serum sample were brought about by hemolysis, cloudiness, and decomposition. Cold storage for two years of adult deer serum, the addition of a bacteriostat to the sample, and the use of a muscle relaxant to procure samples from captive deer produced no significant changes in either the mobility or the percent composition of the protein fractions. A large individual variation was found in both the mobility and the percent composition of the protein fractions. The percent composition of the protein fractions was affected by sex, age, and season. The mobility of the protein fractions was affected by sex, but not by age or season. Captive deer at the University of British Columbia exhibited an additional negatively migrating protein fraction when compared to their wild counterparts. Comparisons of the mobilities in three groups of adult females of the genus Odocoileus indicate greater intra-subspecific differences than inter-specific differences. The technique of starch-gel electrophoresis, therefore, may be useful in individual and herd recognition but it is not useful in the recognition of subspecies or species of deer of the genus Odocoileus. / Science, Faculty of / Zoology, Department of / Graduate

Micellar electrokinetic chromatography is a widely used analytical separation technique. To achieve separation, it uses interaction of analytes with charged micelles present in the background electrolyte (BGE). Not only the mobilities of analytes but also mobilities of small inorganic ions, usually contained in the BGE, are influenced by the presence of micelles. Description of interactions between inorganic ions and micelles is needed for better understanding electrophoretic separations in BGEs containing micelles. Determination of physico-chemical constants (interaction constants, mobilities of complexes) by capillary electrophoresis is based on accurate determination of effective mobilities. For this reason, accurate determination of mobility of electroosmotic flow (EOF) is also necessary but complicated in BGEs containing charged micelles, because the neutral EOF marker can be mobilized by interaction with the micelles. In this work a new two-detector method for determination of effective mobility in interacting BGEs was proposed. In this method the analyte is placed in the BGE containing charged micelles, while the marker zone is in the BGE without micelles and so the possible interaction is avoided. Using this method, dependence of sodium cation mobility on concentration of lithium...