No description available.

Upon encountering stress conditions, cells must rapidly alter their gene expression and re-model their RNA complement to deal with the changing environment. As a consequence, both new RNA transcription as well as RNA degradation must take place. Accordingly, the RNA degradative machinery may adjust to the changes in RNA metabolism. Thus, a study of the response of the three major degradative exoribonucleases in Escherichia coli, polynucleotide phosphorylase, RNase II, and RNase R, to stress is of significant importance. RNase R, a processive 3' to 5' exoribonuclease, is unique among the known E. coli exoribonucleases in its ability to digest through RNAs containing extensive secondary structure without the aid of a helicase. In vivo, RNase R plays important roles in quality control of stable RNA, decay of mRNA with extensive repetitive extragenic palindromic (REP) sequences, cell-cycle regulated degradation of tmRNA in Caulobacter crescentus, as well as processing of rRNA under low temperature in P. syringae. In this dissertation, RNase R was shown to be unusual among the E. coli exoribonucleases in its dramatic response to a variety of stress conditions. Elevation of RNase R activity by as much as 10-fold was observed in response to entry into stationary phase, starvation and cold shock, and an ~3-fold increase was seen during growth in minimal medium compared to rich medium. The elevation in RNase R activity was associated with an increase in RNase R protein. Phenotypes of rnr mutants were also investigated, and RNase R was found to contribute to cell growth and viability. Further investigation of the regulation of RNase R during stress, primarily in stationary phase, revealed a novel regulation mechanism. Despite the large increase in RNase R protein and activity in stationary phase, rnr message actually decreased to only ~14% of its level in exponential phase. Further study revealed that RNase R is highly unstable in exponential phase and becomes stabilized during stationary phase, cold shock, and in minimal medium. Investigation of proteolysis on the unusual instability of RNase R indicated that both Lon and ClpXP play a role. In the absence of Lon, RNase R stability is increased ~10-fold. Based on these results, I propose that the increase in RNase R during stress is due to its enhanced stability under those conditions.

The main focus of this thesis is on hydrophilic interaction chromatography (HILIC) and the preparation of stationary phases for HILIC. The mechanism of HILIC is also discussed; a large part of the discussion has been adapted from a review written by me and professor Irgum for the Journal of Separation Science (ref 34). By reevaluating the literature we have revealed that the notion of HILIC as simply partitioning chromatography needed modification. However, our interest in the HILIC mechanism was mainly inspired by the need to understand how to construct the optimal HILIC stationary phase. The ultimate stationary phase for HILIC is still not found. My theory is that a non-charged stationary phase capable of retaining a full hydration layer even at extreme acetonitrile (> 85%) concentrations should give a HILIC stationary phase with a more pure partitioning retention behavior similar to that of a swollen C18 reversed phase. The preparation of a sorbitol methacrylate grafted silica stationary phase is one of our attempts at producing such a stationary phase. The preparation of such a grafted silica has been performed, but with huge difficulty and this work is still far from producing a column of commercial quality and reprodicibility. This thesis also discusses a new method for the initiation of atom transfer radical polymerization from chlorinated silica. This new grafting scheme theoretically results in a silica particle grafted with equally long polymer chains, anchored to the silica carrier by a hydrolytically stable silicon-carbon bond. The hydrolytic stability is especially important for HILIC stationary phases due to the high water concentration at the surface.

Thesis (Ph. D.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 124-133). Also available on the World Wide Web.

Thesis (Ph. D.)--Florida State University, 2003. / Advisor: Dr. John G. Dorsey, Florida State University, College of Arts and Sciences, Department of Chemistry and Biochemistry. Title and description from dissertation home page (viewed Aug. 24, 2004). Includes bibliographical references.

Streptococcus mutans is a member of the dental plaque and is the principal causative agent of dental caries. It can metabolize a wide array of sugars which results in the production of acid that causes demineralization of the tooth surface. S. mutans can also persist for extended periods of starvation, which may occur in different niches within the oral cavity. Previous studies have shown that mucin prolonged the survival of S. mutans in batch cultures and biofilms. Our laboratory has shown that the pyruvate dehydrogenase (pdh) operon is upregulated in stationary phase and is important for prolonged survival during nutrient starvation in chemically defined medium (CDM). In this study, I found that mucin enabled S. mutans to grow in a minimal chemically defined medium (MCDM) containing glucose and lacking all amino acids. A pdh mutant was found to be impaired in survival in CDM or MCDM supplemented with mucin. Additionally, I have shown that a galactose utilization mutant was impaired in survival in CDM with mucin and no exogenous glucose; galactose is the most abundant utilizable sugar residue in mucin. Mucin, supplemented in minimal medium with sucrose, also enabled S. mutans to form biofilms. Survival of the biofilms was pdh independent. Although biofilm formation was not seen in the absence of sucrose, cells did adhere to the glass coverslip; their survival was found to be pdh dependent. Using a previously constructed reporter strain, pdh expression was observed in a majority of cells in this condition. The expression of the pdh operon was also monitored in batch cultures. The starting inoculum size affected the percentage of fluorescent cells and the outcome of survival in media (CDM or MCDM) supplemented with mucin and no sugar. Greater than 50% of the stationary phase population expressed pdh in CDM with galactose. Less than 1% expressed pdh in CDM with glucose. However, galactose was unable to prolong survival of S. mutans in batch cultures in contrast to the effect of mucin. These results show that mucin may have a metabolic role in promoting long term survival of S. mutans. However it is possible that different components of mucin can be utilized in different conditions. / Microbiology and Immunology

The popularity of capillary electrophoresis (CE) as a separation technique has been established over the years. CE offers the advantages of high resolution, high separation efficiency, fast approaches of method development, a range of operational modes and low consumption of reagents. The strategy employed here for the development of chromatographic separations involved the utilization of experimental designs, multi-linear regression and response surface methodology to build empirical models that related the chromatographic quality to the factors influencing the separation. Separation of Nitrofuran antibiotics (NFAs) and their metabolites (NFMs) by using micellar electrokinetic capillary chromatography was successfully completed. The best conditions found to give optimum resolution from the optimization study was pH 9.0, 80 mM SDC concentration, 16 kV with running buffer consisting of 20 mM borate and 20 mM phosphate concentration using a 73 cm x 75 ƒÝm column, resulting in completely resolved NFAs and NFMs within 16 min. It is interesting that all the compounds can be reliably separated with the one mixture, and single CE condition. Whilst all antibiotics have shorter migration time than their respective derivatised metabolites, as a group apart from nitrofurantoin the antibiotics elute before the metabolites. The analytical figures of merit for CE analysis exhibited excellent reproducibility of absolute and relative migration times, and acceptable reproducibility of relative response areas. Successful separation of metabolite derivatives was achieved when the developed method was applied to a spiked prawn sample. The chiral separation of Triadimenol was successfully completed using micellar electrokinetic capillary chromatography. The best conditions found to give optimum resolution from the optimisation study were pH 6.0, 20% methanol, 50 mM SDS concentration, 18 kV with running buffer consisting of 20 mM borate and 20 mM phosphate concentration using a 64.5 cm x 50 ƒÝm column, resulted in baseline resolution of all Triadimenol isomers within 18 min. The optimised separation conditions were applied to a blank grape sample and to a spiked grape sample. No peaks were observed in the blank grape sample whereas the spiked grape sample had two diastereoismer peaks with poor detection sensitivity. Increase in detection sensitivity is necessary to determine the possibility of resolution of all the isomers of Triadimenol, in the spiked grape sample and the blank. Online preconcentration techniques were attempted to for Triadimenol isomer separation. When using online preconcentration technique of sweeping, a 30-fold increase in detection sensitivity of Triadimenol was observed compared to MEKC mode. However enantiomer separation was not possible with sulfated-£]-CD chiral selector. The best conditions were found to be pH 2.5, 50 mM SDS concentration, -20 kV with running buffer consisting of 20 mM phosphate concentration, using a 64.5 cm x 50 ƒÝm column, resulting in diastereoisomer separation within 8 min. Final stage of the project was to create stationary phase beds in capillaries and micro-channels that could be removed and re-created, thus providing a fresh stationary phase. The replaceable stationary phase (RSP) can be used as an operating mode of CE/CEC. Preparation of reversible stationary phase (RSP) inside the capillary column was successfully performed using low methoxy pectin (LMP). LMP renders a capability of reversible thermogelation. Electroosmotic flow (EOF) and sufficient hydrophobicity of LMP gel allow separation of analyates. The porosity of LMP RSP was adequate to support EOF. Successful separation with good reproducibility of areas and migration times was obtained for Caffeine, Aspartame, Benzoic acid, Saccharine (CABS) mixture and NFAs. After performing continuous analyses, the aging of RSP was observed. Temperature was the ¡¥switch¡¦, which applied to remove aged RSP. RSP was recreated for further analysis of analytes. RSP was UV transparent, capable of handling various analytes and diff erent buffer electrolytes including aqueous-organic solvents.

The Whelk-O1 chiral stationary phase is widely used in liquid and supercritical chromatography for the separation of enantiomers. The enantioselective mechanism of the Whelk-O1 chiral stationary phase is the main focus of this thesis. Semi-flexible models are developed based on ab initio calculations for the Whelk-O1 selector and a series of chiral analytes. Extensive molecular dynamics simulations are then applied to study the solvation, selectivity and in silico optimization of the chiral stationary phase. The solvation of the Whelk-O1 chiral stationary phase has been explored in a normal phase n-hexane/2-propanol solvent, a reversed phase water/methanol solvent, and a supercritical CO2/methanol solvent. We found that, in all three solvents, the Whelk-O1 selectors are open to the bulk, indicating readiness for docking of analyte. Significant solvent partitioning at the interfaces was noticed, which generates a polarity gradient between the stationary phase and the bulk, and may encourage a high analyte concentration at the interface. Hydrogen bonding activities on the amide hydrogen, amide oxygen, and nitro oxygen of the Whelk selector have also been analyzed. The selectivity of the Whelk selector was studied by molecular dynamics simulations of analyte docking on the chiral stationary phase. The elution orders and the separation factors for a series of analytes were predicted successfully. We found that hydrogen bonding and π-π stacking interactions are essential for the enantioselectivity as they are strong and specific, and they hold analytes to the cleft region of the Whelk selector. Other interactions, both stabilizing interactions such as the CH-π interaction and the edge-to-face π-π interaction, and destabilizing interactions such as steric hindrance and unfavorable conformational changes also contribute to the enantioselectivity. We identified a dominant docking arrangement for the most retained enantiomers. Other docking arrangements were found to be more frequent for the least retained enantiomers and these involve interactions with alternative selector sites. Based on the identified enantioselective mechanism obtained from the study, an optimization of the Whelk-O1 chiral stationary phase was undertaken and in silico evaluation of the modified chiral stationary phases was carried out. It was demonstrated that restriction of the alternative docking arrangements for the least retained enantiomers could possibly improve the enantioselectivity of the chiral stationary phase. / Thesis (Ph.D, Chemistry) -- Queen's University, 2008-10-08 11:54:20.249

No description available.

Submitted master thesis is by its character focused on development of polyaniline which would possess attributes typical for monolithic stationary phase. Developed polyaniline aims to be used in capillary liquid chromatography. Accompanying target of submitted master thesis is to provide solutions for variety of complications which follow the preparation of polyaniline in its desired form such as consistency in a form of solutions, gels and pastes, extremely fast polymerization reaction, limited solubility of cross-linking agents, solubility of monolithic polyaniline in polar mobile phases and insufficient bond of polyaniline to the inner wall of fused silica capillary tubing. Solutions for this complications are selection of ideal oxidation agent (ammonium persulfate), cross-linking agent (tris(4-aminophenyl)amine) and porogenic agent (methanol). Also, setting ideal ratio between oxidation agent, cross-linking agent and porogenic agent, heat treatment, choice of suitable mobile phase (tetrahydrofuran), adjustment of capillary wall using silanization agent (3-[3-(trimethoxysilyl)propyl]-aniline) and construction of equipment needed for quick filling of capillaries. Described development concluded in the preparation of solid polyaniline monolith which had a steady bond to the inner wall of fused...