The marine bacterium Sphingopyxis alaskensis was isolated as one of the most numerically abundant bacteria from cold (4–10??C) nutrient depleted waters in the North Pacific Ocean. The objective of this study was to examine cold adaptation of S. alaskensis by using proteomics to examine changes in global protein levels caused by growth at low (10??C) and high (30??C) temperatures. Stable isotope labelling-based quantitative proteomics was used, and a rigorous post-experimental data processing workflow adapted from microarray-based methods was developed. The approach included metabolic labelling with 14N/15N and normalisation and statistical testing of quantitative proteomics data. Approximately 400,000 tandem mass spectra were generated resulting in the confident identification of 2,135 proteins (66% genome coverage) and the quantitation of 1,172 proteins (37% genome coverage). Normalisation approaches were evaluated using cultures grown at 30??C and labelled with 14N and 15N. For 10??C vs. 30??C experiments, protein quantities were normalised within each experiment using a multivariate lowess approach. Statistical significance was assessed by combining data from all experiments and applying a moderated t-test using the empirical Bayes method with the limma package in R. Proteins were ranked after calculating the B-statistic and the Storey-Tibshirani false discovery rate. 217 proteins (6% genome coverage) were determined to have significant quantitative differences. In achieving these outcomes a range of factors that impact on quantitative proteomics data quality were broadly assessed, resulting in the development of a robust approach that is generally applicable to quantitative proteomics of biological system. The significantly differentially abundant proteins from the proteomics data provided insight into molecular mechanisms of cold adaptation in S. alaskensis. Important aspects of cold adaptation included cell membrane restructuring, exopolysaccharide biosynthesis, lipid degradation, carbohydrate and amino acid metabolism, and increased capacity of transcriptional and translational processes. A number of cold adaptive responses in S. alaskensis were novel, including a specific cold-active protein folding pathway, a possible thermally-controlled stringent response, and biosynthesis of intracellular polyhydroxyalkanoate reserve material. The overall study provided important new insight into the evolution of growth strategies necessary for the effective competition of S. alaskensis in cold, oligotrophic environments.

Very little is known about the hydrophobic proteins of psychrophiles and their roles in cold adaptation. In light of this situation, methods were developed to analyse the hydrophobic proteome (HPP) of the model psychrophilic archaeon Methanococcoides burtonii. Central to this analysis was a novel differential solubility fractionation procedure, which resulted in a significant increase in the efficiency of resolving the HPP. Over 50% of the detected proteins were not identified in previous whole cell extract analyses, and these underwent an intensive manual annotation process producing high quality functional assignments. Utilising the functional assignments, biological context analysis of the HPP was performed, revealing novel and often unique biology. The analysis acted as a platform for differential proteomics of the organism???s response to both temperature and substrate using stable isotope labelling. The results of which revealed that low temperature growth was associated with an increase in the abundance of surface and secreted proteins, and translation apparatus. Conversely, growth at a higher temperature was associated with an increase in the abundance of general protein folding machinery and indications of an oxidative stress response, emphasising that the temperature for maximum growth rate is stressful. Through investigation of the response of M. burtonii to substrate it was found that growth on methanol was stressful, and its low energy yield resulted in an increase in the abundance of energy conserving systems. The extracellular polymeric substance (EPS) and morphology of M. burtonii was also investigated with respect to both temperature and substrate, using a number of techniques in microscopy. It was found that the EPS was comprised of proteins, sugars and RNA, and that growth at different temperatures resulted in the production of EPS that displayed significantly different properties on dehydration, thus indicating compositional variation. When cells were grown on methanol they took on highly irregular shapes and had electron transparent inclusions. The observations from the ultrastructural analysis were contemplated with respect to the proteomic findings, revealing novel avenues of research. This study has highlighted the roles of hydrophobic proteins in cold adaptation biology, and the value of comprehensive proteomics for the examination of adaptation in microorganisms

Thesis (Ph. D.)--Carleton University, 2002. / Includes bibliographical references (p. 166-178). Also available in electronic format on the Internet.

Thesis (M. Sc.)--Carleton University, 2004. / Includes bibliographical references (p. 147-165). Also available in electronic format on the Internet.

Thesis (M.Sc.) - Carleton University, 2005. / Includes bibliographical references (p. 134-145). Also available in electronic format on the Internet.

Thesis (Ph.D.) - Carleton University, 2006. / Includes bibliographical references (p. 118-138). Also available in electronic format on the Internet.

Thesis (M.Sc.) - Carleton University, 2006. / Includes bibliographical references (p. 151-170). Also available in electronic format on the Internet.

Although the relationship between the effects of cold and infection has been studied for a long time, the mechanisms contributing to this phenomenon have not yet been discovered. While short-term exposure to cold triggers a stress response and the production of proinflammatory cytokines, long-term cold exposure induces adaptation and anti- inflammatory shift. The role of cold adaptation in the development and the process of the organism's response to infection remains an unresolved issue. In this diploma thesis, we focused on determining the effect of cold stress or cold adaptation on the immune system of rats which was stimulated by ligands of Toll-like receptors (TLRs). The obtained results showed the importance of γδT lymphocytes and their cytokine production in the process of cold adaptation. To determine how cold adaptation affects the response to infectious agents, we studied changes in the proportion of different immune cell populations in rats treated with the TLR2/6 ligand Pam2CSK4. We determined significant changes in the activation of myeloid cells and B lymphocytes, but also in the population of γδT lymphocytes. Our obtained results suggested the importance of γδT lymphocytes and the protective effect of cold adaptation. Key words: immune system, cold stress, cold adaptation, infection,...

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