- 4 - Abstract in English This diploma thesis is focused on the evaluation and comparison of the enantioselective potential of two columns CHIRAL ART Amylose-SA and CDShell-RSP using a set of 29 chiral drugs in high performance liquid chromatography. The separations of enantiomers were performed in three modes: reversed-phase, normal-phase, and polar- organic mode. The CHIRAL ART Amylose-SA column was tested in normal-phase mode, the CDShell-RSP column in reversed-phase and polar-organic modes. The CHIRAL ART Amylose-SA column contains amylose tris(3,5- dimethylphenylcarbamate) immobilized on 3 µm porous silica gel particles. The CDShell-RSP column contains a chiral selector hydroxypropyl-β-cyclodextrin, which is covalently bonded on 2.7 µm superficially porous particles. In the normal-phase mode, mobile phases composed of hexane and propane-2-ol were used. Furthermore, the effect of various additives (triethylamine, diethylamine, trifluoroacetic acid, and the mixture of diethylamine and trifluoroacetic acid) in the mobile phase on the enantioseparation of chiral drugs was tested. The most universal additive was a mixture of diethylamine and trifluoroacetic acid. A total of 22 chiral drugs were enantioseparated on the CHIRAL ART Amylose-SA column, 10 of them were baseline separated. Mobile phases for...

For the first time, germania-based hybrid organic-inorganic sol-gel materials were developed for analytical sample preparation and chromatographic separation. Being an isostructural analog of silica (SiO2), germania (GeO2) is compatible with silica network. This structural similarity, which is reflected by the relative positions of germanium and silicon in the periodic table, stimulated our investigation on the development of germania-based sol-gel hybrid organic-inorganic coatings for analytical applications. Sol-gel sorbents and stationary phases reported to date are predominantly silica-based. Poor pH stability of silica-based materials is a major drawback. In this work, this problem was addressed through development of germania-based organic-inorganic hybrid sol-gel materials. For this, tetramethoxygermane (TMOG) and tetraethyoxygermane (TEOG) were used as precursors to create a sol-gel network via hydrolytic polycondensation reactions to provide the inorganic component (germania) of the organic-inorganic hybrid coating. The growing sol-gel germania network was simultaneously reacted with an organic ligand that contained sol-gel-active sites in its chemical structure. Hydroxy-terminated polydimethylsiloxane (PDMS) and 3-cyanopropyltriethoxysilane (CPTES) served as sources of nonpolar and polar organic components, respectively. The sol-gel reactions were performed within fused silica capillaries. The prepared sol-gel germania coatings were found to provide excellent pH and thermal stability. Their extraction characteristics remained practically unchanged after continuous rinsing of the sol-gel germania-PDMS capillary for 24 hours with highly basic (pH=13) and/or acidic (pH = 1.3) solution. They were very efficient in extracting non-polar and moderately polar analytes such as polycyclic aromatic hydrocarbons, aldehydes, ketones. Possessing the polar cyanopropyl moiety, sol-gel germania cyanopropyl-PDMS capillaries were found to effectively extract polar analytes such as alcohols, fatty acids, and phenols. Besides, they also showed superior thermal stability compared with commercial cyano-PDMS coatings thanks to the covalent attachment of the coating to capillary surface achieved through sol-gel technology. Their extraction characteristics remained unchanged up to 330°C which is significantly higher than the maximum operation temperature ( < 280 ºC) for commercial cyano-PDMS coatings. Low ng/L detection limits were achieved for both non-polar and polar test solutes. Our preliminary results also indicated that sol-gel hybrid germania coatings have the potential to offer great analytical performance as GC stationary phases.

The impact parameter (IP) approximation is a semiclassical model in quantum scattering theory wherein N large masses interact with one small mass. We study this model in one spatial dimension using the tools of time-dependent scattering theory, considering a system of two large-mass particles and one small-mass particle. We demonstrate that the model's predictive power becomes arbitrarily good as the masses of the two heavy particles are made larger by studying the S-matrix for a particular scattering channel. We also show that the IP wave functions can be made arbitrarily close to the full three-body solution, uniformly in time, provided one of the large masses is fixed in place, and that such a result probably will not hold if we allow all the masses to move. / Ph. D.

The work in this thesis has demonstrated the chiral recognition through the adaptation of chromatographically derived chiral recognition systems by electrospray ionization mass spectrometry (ESI-MS). Mass-labeled, pseudoenantiomeric chiral selectors (where each pseudoenantiomer had the opposite stereochemistry, but was slightly different in mass due to labeling of one enantiomer) were prepared as soluble analogues of Pirkle type chiral stationary phases. When mixed with a chiral analyte, solutions containing these pseudoenantiomeric selectors afforded selector-analyte complexes in the ESI-MS, and the relative peak intensities of the complexes could be related back to the enantiomeric composition of the analyte. In each case of this study, the complex intensity fraction for either of the selector-analyte complexes in the ESI-MS varies linearly with the enantiomeric composition of the analyte. This linear relationship provides a measure of the extent of enantioselectivity and allows quantitative analysis of the enantiomeric composition of analyte.

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<p>The sigma S subunit (RpoS) of RNA polymerase acts as the master regulator of stress in <em>Escherichia coli</em>, allowing adaptation and survival under unfavourable conditions such as nutrient deprivation. RpoS regulates and integrates the signals of hundreds of genes (about 10% of the genome) organized into complex networks and modules that govern the response to stress and entry of the cell into stationary phase. Although microarray studies have been performed on starvation models in <em>E. coli</em>, the expression of the RpoS regulon has not been studied in long-term cultures due to the difficulty of isolating RNA from starving cells. In this study, the development of a protocol for isolating RNA from stationary phase cells, employing hot acid phenol without the use of DNase, is described. In addition, the expression of several genes during different phases of growth is analyzed by RT-qPCR in order to validate preliminary microarray data obtained from 24 and 48 hour-old cultures. Although the results obtained by RT-qPCR agree well with the literature, they do not corroborate preliminary microarray data at the 24 and 48 hour timepoint.</p> / Master of Science (MSc)

<p><em>Escherichia coli </em>cells undergo many morphological and physiological changes to survive in late stationary phase due, in part, to the exhaustion of nutrients and accumulation of inhibitory metabolites. Previous microarray data in our lab provided the general profile of gene expression from exponential phase to 48 h of incubation. The goal of this study is to use qPCR to validate the preferentially expressed genes in late stationary phase determined by the microarray data. The expression of three genes (<em>hha, tomB </em>and <em>emrK</em>) with increased expression levels from 24 h to 48 h of incubation and another two RpoS-dependent genes (<em>bolA</em> and <em>osmY</em>) with decreased expression from 24 h to 48 h of incubation were chosen as the target genes. RNA was first extracted from exponential phase, early stationary phase, 24 h and 48 h bacterial cultures. Hot phenol-chloroform and a commercial kit were used to isolate RNA. All these methods recovered low RNA in late stationary phase. qPCR partly confirmed the previous microarray data. The expression of <em>hha</em>, <em>tomB</em> and <em>emrK</em> was validated to increase from 24 h to 48 h of incubation. The discordant results between qPCR and microarray data may be due to the low transcript abundance of target genes and genomic DNA contamination in RNA extracted from 48 h bacterial cultures.</p> / Master of Science (MSc)

Escherichia coli survives in both nutrient rich nutrient-limited environments. As such, understanding the gene and protein level activity that occurs during stationary phase is considered an important aspect of bacterial survival. Escherichia coli has been studied for decades providing substantial insight into gene expression profiles in exponential phase and recently, during adaptation to stationary phase. This led to the discovery of RpoS as a growth phase-dependent sigma factor. Further studies indicated that there are many genes that are expressed in an RpoS-independent but stationary phase-specific manner. However, proteins represent the functional molecules of the cell. Additionally, protein expression does not always correlate with the corresponding gene expression patterns. Therefore, to obtain an in depth understanding of the proteins that play a role in long-term growth in E. coli, TMT- (Tandem Mass Tags) based quantitative proteomic analysis was performed to identify proteins that are preferentially expressed during prolonged starvation. We identified proteins that were both positively and negatively regulated by RpoS during stationary phase, such as GadA and TnaA, respectively. RpoS levels peaked during early stationary phase and declined thereafter. However, proteins that were RpoS-dependent continued to increase during prolonged stationary phase. Additionally, we identified proteins that were expressed in an RpoS-independent manner during stationary phase. This suggests that protein expression during early stationary phase is distinct from prolonged stationary phase. Furthermore, RpoS-independent proteins may also play an important role during long-term growth. / Thesis / Master of Science (MSc) / Escherichia coli adapts to shifts in nutrient availability using the alternative sigma factor RpoS which controls morphological and physiological changes. Although gene expression during growth has been extensively studied, comparable information regarding changes in protein abundance during prolonged incubation is not available. We employed a quantitative proteomics approach to identify proteins that are preferentially expressed during stationary phase in E. coli. We identified classes of proteins that are upregulated and downregulated by RpoS in addition to proteins regulated independently of RpoS. Global analysis of protein expression during growth can aid in understanding the adaptation of E. coli under starvation conditions.