Thesis (M. Sc.)--University of Canterbury, 2009. / Typescript (photocopy). "June 2009." Includes bibliographical references. Also available via the World Wide Web.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 175-187). Also available on the Internet.

Thesis (M.Sc.(Chemistry))--University of Pretoria, 2006. / Includes abstract in English. Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 175-187). Also available on the Internet.

Thesis (Ph. D.)--University of California, San Diego, 2001. / Vita. Includes bibliographical references.

Thesis (M.S.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains vii, 88 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / "December 2002." Typescript. Vita. Includes bibliographical references (leaves 202-211). Also available on the Internet.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is a primary constituent of chronic, polymicrobial infections in the lungs of individuals with cystic fibrosis (CF). A significant consequence of CF is production of thick mucus along epithelial surfaces. In the lungs, this mucus collects and serves as an excellent growth substrate for a range of bacteria including. CF lung fluids (sputum) also enhance the virulence of P. aeruginosa, as production of a signaling molecule critical for virulence, the Pseudomonas quinolone signal (PQS), is enhanced in the presence of phenylalanine and tyrosine in CF sputum. The goal of this dissertation is to better understand how phenylalanine and tyrosine affect PQS production and ultimately P. aeruginosa virulence. To address this, I use transcriptome profiling to determine that genes for phenylalanine and tyrosine catabolism, PQS biosynthesis, and a transcriptional regulator called PhhR are up-regulated in the presence of phenylalanine and tyrosine. I determine that PhhR regulates genes for aromatic amino acid catabolism but not genes for PQS biosynthesis. The PhhR regulon is further characterized by mapping of PhhR-regulated promoters with primer extension, and evidence for direct regulation is presented. To explain enhanced production of PQS in CF sputum, I favor a model in which flux of a shared metabolic precursor, chorismate, toward PQS biosynthesis is enhanced when phenylalanine and tyrosine are present. I investigate this model by examining the first step in PQS biosynthesis, conversion of chorismate to anthranilate by an anthranilate synthase (AS). P. aeruginosa possesses two AS enzymes encoded by the trpEG and phnAB genes, with the former generating anthranilate specifically for tryptophan biosynthesis while the latter generates anthranilate for PQS biosynthesis. I investigate the evolutionary origins of these two enzymes and generate unmarked deletion mutants to dissect their roles in tryptophan and PQS biosynthesis. The ability of PhnAB to compensate for loss of TrpEG at high cell densities is documented, and a model explaining anthranilate sequestering is developed. Knowledge gained from these studies will be useful in developing novel therapeutic strategies. / text

Unnatural amino acids (UAAs) have been used in bacteria and yeast to pinpoint protein binding sites, identify binding partners, PEGylate proteins site-specifically (vs. randomly), and attach small molecule fluorophores to proteins. The process of UAA incorporation involves the manipulation of the genetic code, which is established by the proper function of aminoacyl tRNA synthetases (RSs) and their cognate transfer RNAs (tRNAs). It has been discovered that certain regions of RS proteins can either block or enable cross-species reactivity of RSs. In essence, a bacterial RS can function with a human tRNA by transferring the human CP1 region to the bacterial RS, and vice versa. This knowledge has been used to engineer a tRNA capable of recognizing a stop codon (tRNA*), rather than an amino acid codon, and a cognate RS capable of recognizing only tRNA* and no endogenous tRNAs. We have previously described the use of this methodology to engineer a UAA incorporation system capable of amber stop codon suppression in HEK293T cells. Since UAAs are so useful, and their use has now been enabled in mammalian systems, we applied UAA incorporation to pluripotent cells. Stem and pluripotent cells have been the focus of cutting edge research for years, but much of the work done on these cell lines is done in the ignorance of basic biological processes underlying differentiation, dedifferentiation, and tumorigenesis. In order to facilitate the study of these basic biological processes and enable more adept manipulation of differentiation, dedifferentiation, and tumorigenesis, the development and use of two separate UAA incorporation systems is described herein. The overarching goal of this project is to facilitate the study of protein-protein interactions in stem and pluripotent cells. Since we have also previously described the development of a mammalian two-hybrid system, the use of that system in pluripotent cells is also described. / text

The electrochemical detector provides a promising detection mode for capillary electrophoresis (CE) due to its excellent sensitivity, good portability, high selectivity, easy miniaturization, low capital and running cost. To widen its scope for determining trace analytes in complex samples, three dual-electrode detectors were fabricated to enable the determination of electro-inactive analytes, to assess co-eluted peaks and to give a large enhancement of the detection sensitivity by modifying electrode surface using multi-walled carbon nanotubes (MWNTs). To determine trace non-electroactive amino acids present in human tears, a serial dual-electrode detector was developed using an upstream on-capillary Pt film electrode to oxidize bromide to bromine at +1.0 V and a downstream Pt disk electrode to detect the residual bromine at +0.2 V after their reaction with amino acids eluted out from the separation capillary. The bromide reagent was introduced after CE separation by a newly designed coaxial post-column reactor fabricated onto the PMMA chip. Using optimized CE buffer containing 20 mM borate, 20 mM SDS at pH 9.8, L-glutamine, L-alanine and taurine were baseline separated with detection limits ranging from 0.56-0.65 μM and a working range of 2-200 μM for L-glutamine and of 2-300 μM for both L-alanine and taurine. Method reliability was established by close to 100% recoveries for spiked amino acids and good agreement between the measured and the literature reported amino acid concentrations in tears. For the determination of polyphenols in wine, a microchip-CE device was fabricated with a dual-opposite carbon fiber microelectrode operated in a parallel mode to assess peak purity. Under optimized conditions, (+)-catechin, trans-resveratrol, quercetin, (-)-epicatechin and gallic acid were baseline separated within 16 min with detection limits ranging from 0.031- 0.21 mg/L and repeatability of 2.0-3.3 % (n=5). The use of an opposite dual-electrode enables the simultaneous determination of peaks and measurement of their current ratios at +0.8 V and +1.0 V vs Ag/AgCl. The capability of using current ratio to identify the presence of co-migrating impurities was demonstrated in a mixed standard solution with overlapping (+)-catechin and (-)-epicatechin peaks and in a commercial red wine with interfering impurities. Matching of both the migration time and the current ratio reduce false positive and validate polyphenol quantitation in red wine. Lastly, a dual-opposite MWNTs modified carbon fiber microelectrode (CFME) was developed to determine the biomarkers (4-nitrophenol, 4-nitrophenyl-glucuronide and 4-nitrophenyl-sulfate) needed to assess exposure to methyl parathion. Use of the MWNTs modified CFME showed a much higher sensitivity than bare CFME, with a detection limit of 0.46 μM for 4-nitrophenol. Baseline separation of all three biomarkers was obtained within 31 min by a 45 cm long capillary under 12 kV in a 20 mM phosphate buffer at pH 7.0. The method developed was successfully utilized to determine low levels of biomarkers in human urine without using complex pretreatment steps and delivered recoveries ranging from 95.3 - 97.3% and RSDs within 5.8% (n=3). Using a parallel dual-electrode detector was shown to deliver reliable results with matching current ratios and comparable migration time to those obtained from biomarker standards. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy