No description available.

No description available.

Analysis of complex protein samples is demanding due to the wide dynamic range of expression levels and the limited detection range of technology. Proteomics relies heavily on the development of new fractionation strategies to help reduce complexity, and overcome the technological and biological challenges associated with proteome analysis. Here, the development of a prototype instrument named ??Microflow MF10?? was explored to enrich for particular classes of proteins. The MF10 was found to have a number of advantages over commercially available fractionation systems. Due to the reduced separation electrode distance, fractionation was rapid, occuring within ~0.125 kVH over 2-6 fractions under native conditions but longer under denaturing conditions. As low as 2 ng peptide could be fractionated with recovery for downstream analysis achievable. The ability to alter protein charge by changing the pH (acidic (pI 3.6) to basic environments (pI 10.4)) allows selection of proteins based on charge/mobility, size, shape, buffer ionic strength, pH and field strength. Proteins <10 kDa are also not routinely analysed because current technology is unable to cater for this region of the proteome. Peptide enrichment using the MF10 was achieved using a 7-protein/peptide standard mix (1-25 kDa), to the 1-5 kDa fraction with simultaneous fractionation of the higher mass protein standards. Plasma was also used to enrich for the peptidome (< 5 kDa) in the presence of the proteome. Enrichment of 73 proteins inclusive of 22 proteins in the 1-25 kDa fraction was achieved compared to a total equivalent of 42 proteins from unfractionated plasma. Rare samples (≤ 106 cells) from stem cell populations or derived clinically are challenging due to the absolute limits in protein copy number and abundance. CD34+ haematopoietic stem cells and CD4+/CD8+ T-cells were used to develop fractionation methods and elucidate the cell differentiation process. MF10 fractionation and analysis by SDS-PAGE and LC-MS/MS revealed 24 differentially expressed proteins between the 3 cell populations, which may be involved in cell differentiation. To quantify these expression differences, iTRAQ with 2-D LC-MS/MS was applied. This study has highlighted the challenges associated with samples of limited quantity. It has been successful in understanding the effects of various conditions on the electrophoretic mobility of proteins, which in proteomics, has remained largely unexplored.

Purpose: Fractionation in radiotherapy is the scheduled break up of a total treatment dose into individual doses. The goal of this thesis is to seek a mathematically optimal dose schedule, in the context of a biological tissue dose-response model, the linear-quadratic function. Methods: We examined the mathematical properties of the fractionation problem in the context of an arbitrary number of sensitive-structure constraints and determined the properties of the optima. We also implemented a numerical search technique to solve the problem. Results: On the theoretical side, we confirmed and extended the results in the literature. We showed the optima always occur at the intersection of two or more constraints or at the equal dose per fraction point (or at any arbitrary feasible point on the boundary, which includes the two points just mentioned). On the numerical side, we successfully implemented a simulated annealing algorithm to our problem.

Stable carbon isotope fractionation during methanogenesis is affected by the availability of substrates. The effects of different substrates on methanogen biomass, total lipid extract, biomarkers and methane under both abundant and limiting substrate conditions were studied. Methanosarcina barkeri was grown with methanol, acetate, trimethylamine (TMA) and H2/CO2, and carbon isotope fractionation in methane production was greatest with methanol, followed by H2/CO2, TMA and acetate. In contrast, biomass was isotopically lightest in M.barkeri grown on methanol, followed by TMA, H2/CO2 and acetate. Generally, fractionation was greater in cultures grown with abundant substrate availability as compared to those supplied with limiting substrate. During autotrophic growth, fractionation was greatest during slower growth for both methane and biomass production. The results of these fractionation studies under controlled laboratory conditions can be applied to the interpretation of isotopic signatures for methane and methanogen biomarkers, and ecological processes, in marine environments. Several hypersaline springs off the western shore of Lake Winnipegosis, MB support unique microbial mat communities. These low temperature springs contain water with a mean salinity as high as 6.1%. Studies were undertaken to contrast the anaerobic microbial communities of these springs, specifically the methanogens and sulphate-reducing bacteria (SRB), and their contributions to biogeochemical cycling in these mats. Comparisons of lipid profiles revealed changes in the proportions of the dominant fatty acids related to the amount of mat growth. Cultures of SRB and methanogens were established with six different substrates. Methanogenic cultures grew best on TMA and methanol, but could use formate, H2/CO2 and glycine betaine as well. In contrast, H2/CO2 was the preferred substrate of the SRB enrichment cultures, which were also able to use formate, but not TMA, the breakdown product of the compatible solute glycine betaine. Maximum methane production occurred at 5% salinity. The lipid composition of the mats, including methanogen biomarkers, and the results of the enrichments on different substrates and at different salinities, suggest that methanogenesis in these springs is supported by compatible solutes whereas sulphate reduction is linked to availability of hydrogen and formate. / February 2005

Stable carbon isotope fractionation during methanogenesis is affected by the availability of substrates. The effects of different substrates on methanogen biomass, total lipid extract, biomarkers and methane under both abundant and limiting substrate conditions were studied. Methanosarcina barkeri was grown with methanol, acetate, trimethylamine (TMA) and H2/CO2, and carbon isotope fractionation in methane production was greatest with methanol, followed by H2/CO2, TMA and acetate. In contrast, biomass was isotopically lightest in M.barkeri grown on methanol, followed by TMA, H2/CO2 and acetate. Generally, fractionation was greater in cultures grown with abundant substrate availability as compared to those supplied with limiting substrate. During autotrophic growth, fractionation was greatest during slower growth for both methane and biomass production. The results of these fractionation studies under controlled laboratory conditions can be applied to the interpretation of isotopic signatures for methane and methanogen biomarkers, and ecological processes, in marine environments. Several hypersaline springs off the western shore of Lake Winnipegosis, MB support unique microbial mat communities. These low temperature springs contain water with a mean salinity as high as 6.1%. Studies were undertaken to contrast the anaerobic microbial communities of these springs, specifically the methanogens and sulphate-reducing bacteria (SRB), and their contributions to biogeochemical cycling in these mats. Comparisons of lipid profiles revealed changes in the proportions of the dominant fatty acids related to the amount of mat growth. Cultures of SRB and methanogens were established with six different substrates. Methanogenic cultures grew best on TMA and methanol, but could use formate, H2/CO2 and glycine betaine as well. In contrast, H2/CO2 was the preferred substrate of the SRB enrichment cultures, which were also able to use formate, but not TMA, the breakdown product of the compatible solute glycine betaine. Maximum methane production occurred at 5% salinity. The lipid composition of the mats, including methanogen biomarkers, and the results of the enrichments on different substrates and at different salinities, suggest that methanogenesis in these springs is supported by compatible solutes whereas sulphate reduction is linked to availability of hydrogen and formate.

No description available.

Purpose: Fractionation in radiotherapy is the scheduled break up of a total treatment dose into individual doses. The goal of this thesis is to seek a mathematically optimal dose schedule, in the context of a biological tissue dose-response model, the linear-quadratic function. Methods: We examined the mathematical properties of the fractionation problem in the context of an arbitrary number of sensitive-structure constraints and determined the properties of the optima. We also implemented a numerical search technique to solve the problem. Results: On the theoretical side, we confirmed and extended the results in the literature. We showed the optima always occur at the intersection of two or more constraints or at the equal dose per fraction point (or at any arbitrary feasible point on the boundary, which includes the two points just mentioned). On the numerical side, we successfully implemented a simulated annealing algorithm to our problem.

Stable carbon isotope fractionation during methanogenesis is affected by the availability of substrates. The effects of different substrates on methanogen biomass, total lipid extract, biomarkers and methane under both abundant and limiting substrate conditions were studied. Methanosarcina barkeri was grown with methanol, acetate, trimethylamine (TMA) and H2/CO2, and carbon isotope fractionation in methane production was greatest with methanol, followed by H2/CO2, TMA and acetate. In contrast, biomass was isotopically lightest in M.barkeri grown on methanol, followed by TMA, H2/CO2 and acetate. Generally, fractionation was greater in cultures grown with abundant substrate availability as compared to those supplied with limiting substrate. During autotrophic growth, fractionation was greatest during slower growth for both methane and biomass production. The results of these fractionation studies under controlled laboratory conditions can be applied to the interpretation of isotopic signatures for methane and methanogen biomarkers, and ecological processes, in marine environments. Several hypersaline springs off the western shore of Lake Winnipegosis, MB support unique microbial mat communities. These low temperature springs contain water with a mean salinity as high as 6.1%. Studies were undertaken to contrast the anaerobic microbial communities of these springs, specifically the methanogens and sulphate-reducing bacteria (SRB), and their contributions to biogeochemical cycling in these mats. Comparisons of lipid profiles revealed changes in the proportions of the dominant fatty acids related to the amount of mat growth. Cultures of SRB and methanogens were established with six different substrates. Methanogenic cultures grew best on TMA and methanol, but could use formate, H2/CO2 and glycine betaine as well. In contrast, H2/CO2 was the preferred substrate of the SRB enrichment cultures, which were also able to use formate, but not TMA, the breakdown product of the compatible solute glycine betaine. Maximum methane production occurred at 5% salinity. The lipid composition of the mats, including methanogen biomarkers, and the results of the enrichments on different substrates and at different salinities, suggest that methanogenesis in these springs is supported by compatible solutes whereas sulphate reduction is linked to availability of hydrogen and formate.

Analysis of complex protein samples is demanding due to the wide dynamic range of expression levels and the limited detection range of technology. Proteomics relies heavily on the development of new fractionation strategies to help reduce complexity, and overcome the technological and biological challenges associated with proteome analysis. Here, the development of a prototype instrument named ??Microflow MF10?? was explored to enrich for particular classes of proteins. The MF10 was found to have a number of advantages over commercially available fractionation systems. Due to the reduced separation electrode distance, fractionation was rapid, occuring within ~0.125 kVH over 2-6 fractions under native conditions but longer under denaturing conditions. As low as 2 ng peptide could be fractionated with recovery for downstream analysis achievable. The ability to alter protein charge by changing the pH (acidic (pI 3.6) to basic environments (pI 10.4)) allows selection of proteins based on charge/mobility, size, shape, buffer ionic strength, pH and field strength. Proteins <10 kDa are also not routinely analysed because current technology is unable to cater for this region of the proteome. Peptide enrichment using the MF10 was achieved using a 7-protein/peptide standard mix (1-25 kDa), to the 1-5 kDa fraction with simultaneous fractionation of the higher mass protein standards. Plasma was also used to enrich for the peptidome (< 5 kDa) in the presence of the proteome. Enrichment of 73 proteins inclusive of 22 proteins in the 1-25 kDa fraction was achieved compared to a total equivalent of 42 proteins from unfractionated plasma. Rare samples (≤ 106 cells) from stem cell populations or derived clinically are challenging due to the absolute limits in protein copy number and abundance. CD34+ haematopoietic stem cells and CD4+/CD8+ T-cells were used to develop fractionation methods and elucidate the cell differentiation process. MF10 fractionation and analysis by SDS-PAGE and LC-MS/MS revealed 24 differentially expressed proteins between the 3 cell populations, which may be involved in cell differentiation. To quantify these expression differences, iTRAQ with 2-D LC-MS/MS was applied. This study has highlighted the challenges associated with samples of limited quantity. It has been successful in understanding the effects of various conditions on the electrophoretic mobility of proteins, which in proteomics, has remained largely unexplored.