Mastitis is the most prevalent and major cause of economic losses in dairy farms. Bovine mastitis caused by strains of <i>S. aureus</i> is a major economically important disease affecting the dairy industry worldwide. <i>S. aureus</i> is one of the most common udder pathogens that cause either clinical or sub-clinical mammary gland infections. Different treatment regimes have failed to cure <i>S. aureus</i> intramammary infections. Most mastitis vaccination strategies have focused on the enhancement of systemic humoral immunity rather than strengthening local intramammary immunity. Vaccines aimed at enhancing intramammary immunity of dairy cows against <i>S. aureus</i> mastitis have had limited success. Commercially available vaccines show various degrees of success and work in research laboratories with experimental vaccines suggest that in part, the failure of these vaccines lies in the limited antigenic repertoire contained in the vaccine formulations. Moreover, not only does variation in the antigenic composition but also presence of capsular polysaccharide in most pathogenic strains and decreased activity of immune effectors in milk affect the success of vaccines. In addition to these, the ability of <i>S. aureus</i> to attach and internalize into mammary epithelial cells, enables bacteria to escape from the effect of immunity and antibiotics by being hidden in the intracellular niche and thereby causing chronic recurrent intramammary infection. <i>S. aureus</i> also has the ability to become electron-transport-defective and to form slow-growing small colonies that are non haemolytic and less virulent. These small colony variants might hide from the immune surveillance in the intracellular area and revert to the parental strain causing chronic recurrent infections. If immunization targets antigenic molecules that are conserved throughout all pathogenic strains, even the small colony variants can be controlled since the immune system will clear the parental strain which causes lethal infection. Thus, immunization trials should focus on conserved immunogenic antigen molecules among pathogenic strains formulated with an adjuvant and delivered by a route of immunization to induce maximum stimulation of the immune system. Moreover, immunization should focus on inducing Th1 responses, which is protective against <i>S. aureus</i> mastitis. It has been reported that proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity might be used as such antigens to induce protection against parasitic and microbial infections. Previous study in our laboratory on mastitis-causing streptococci indicates that GapC proteins of <i>S. uberis</i> and <i>S. dysgalactiae</i> have potential as vaccine antigens to protect dairy cows against mastitis caused by environmental streptococci. Two conserved cell wall associated proteins with iii glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, GapB and GapC have been identified from <i>S. aureus</i> isolates from bovine intramammary infections. The overall goal of this study was to improve our understanding on intramammary immunity using the GapC and GapB proteins of <i>S. aureus</i> as model antigens for mastitis and to determine the regulation of expression of <i>gapB</i> and <i>gapC</i> genes and their roles in the pathogenesis of bovine <i>S. aureus</i> mastitis. We hypothesized that strengthening local intramammary immunity using GapB and GapC proteins of <i>S. aureus</i> as antigens will protect against bovine <i>S. aureus</i> mastitis. To test this hypothesis we took the approach of using the <i>gapB</i> and <i>gapC</i> genes and constructed plasmids encoding GapB, GapC and GapB::GapC (GapC/B) chimeric proteins. We set six objectives to test our hypothesis using these proteins to enhance the intramammary immunity. In aim 1 we constructed plasmids encoding the GapB, GapC proteins and also constructed a chimeric gene encoding the GapC and GapB proteins as a single entity (GapC/B chimera) as the basis for a multivalent vaccine. In this objective the humoral and cellular immune responses to GapC/B were compared to the responses to the individual proteins alone or in combination in C57 BL/6 mice. Our results showed that the GapC/B protein elicited strong humoral and cellular immune responses as judged by the levels of total IgG, IgG1, IgG2a, IL-4 and IFN-ã secretion and lymphocyte proliferation. These results strongly suggest the potential of this chimeric protein as a target for vaccine production to control mastitis caused by <i>S. aureus</i>. In aim 2 we continued our studies on GapC/B by testing the effects of DNA vaccination with plasmids encoding the individual gapB and gapC genes as well as the gapC/B protein gene with or without a boost with the recombinant proteins. The results showed that DNA vaccination alone was unable to elicit a significant humoral response and barely able to elicit a detectable cell-mediated response to the recombinant antigens but subsequent immunization with the proteins elicited an excellent response. In addition, we found that DNA vaccination using a plasmid encoding the GapC/B chimera followed by a boost with the same protein, although successful, is less effective than priming with plasmids encoding GapB or GapC followed by a boost with the individual antigens. In aim 3 we optimized immune responses in cows by comparing route of vaccination (subcutaneous versus intradermal), site of vaccination (locally at the area drained by the supramammary lymph node versus distantly at area drained by parotid lymph node. Our results showed that both subcutaneous and intradermal immunizations with the GapC/B protein at the area drained by the supramammary and parotid lymph nodes resulted in significantly increased serum and milk titers of total IgG, IgG1, IgG2, iv and IgA in all vaccinated groups as compared to placebo. The anti-GapC/B IgG1 serum and milk titers were significantly higher in all vaccinated group as compared to the placebo group. These results indicated that vaccination at the area drained by the supramammary lymph node resulted in better immune responses. In aim 4 we tested different formulations of the GapC/B antigen with adjuvants such as PCPP, CpG, PCPP + CpG and VSA-3. We found that the VSA-3 formulation induced the best immune responses in cows. In this objective we also monitored immune responses longitudinally over one lactation cycle to determine the duration of immune responses by measuring IgG, IgG1, IgG2, and IgA on monthly blood and milk samples. We found that the duration of immune responses was about four months. In aim 5 we tested the role of GapC in the virulence of <i>S. aureus</i> mastitis using the <i>S. aureus</i> wild type strain RN6390 and its isogenic GapC mutant strain H330. Our results from both in vitro adhesion and invasion assays on MAC- T cells and in vivo infection of ovine mammary glands showed that GapC is an important virulence factor in <i>S. aureus</i> mastitis. In aim 6 we examined the role of sar and agr loci on the expression of <i>gapC</i> and <i>gapB</i> genes by qRT- PCR using <i>S. aureus</i> RN6390 and its isogenic mutants defective in agrA, sarA and sar/agr (double mutant) at exponential and stationary phases of growth. Our results showed that both <i>gapB</i> and <i>gapC</i> expression were down regulated in the mutant strains, indicating that the expression of the <i>gapB</i> and <i>gapC</i> genes is controlled by the universal virulence gene regulators, agr and sar. We also checked the role of environmental factors such as pH, growth media, and oxygen tension on the expression of <i>gapB</i> and <i>gapC</i> using q-RT-PCR. Our results showed that the expression of <i>gapB</i> and <i>gapC</i> genes in different strains of <i>S. aureus</i> was not consistent under the above-mentioned environmental conditions.

Mastitis is the most prevalent and major cause of economic losses in dairy farms. Bovine mastitis caused by strains of <i>S. aureus</i> is a major economically important disease affecting the dairy industry worldwide. <i>S. aureus</i> is one of the most common udder pathogens that cause either clinical or sub-clinical mammary gland infections. Different treatment regimes have failed to cure <i>S. aureus</i> intramammary infections. Most mastitis vaccination strategies have focused on the enhancement of systemic humoral immunity rather than strengthening local intramammary immunity. Vaccines aimed at enhancing intramammary immunity of dairy cows against <i>S. aureus</i> mastitis have had limited success. Commercially available vaccines show various degrees of success and work in research laboratories with experimental vaccines suggest that in part, the failure of these vaccines lies in the limited antigenic repertoire contained in the vaccine formulations. Moreover, not only does variation in the antigenic composition but also presence of capsular polysaccharide in most pathogenic strains and decreased activity of immune effectors in milk affect the success of vaccines. In addition to these, the ability of <i>S. aureus</i> to attach and internalize into mammary epithelial cells, enables bacteria to escape from the effect of immunity and antibiotics by being hidden in the intracellular niche and thereby causing chronic recurrent intramammary infection. <i>S. aureus</i> also has the ability to become electron-transport-defective and to form slow-growing small colonies that are non haemolytic and less virulent. These small colony variants might hide from the immune surveillance in the intracellular area and revert to the parental strain causing chronic recurrent infections. If immunization targets antigenic molecules that are conserved throughout all pathogenic strains, even the small colony variants can be controlled since the immune system will clear the parental strain which causes lethal infection. Thus, immunization trials should focus on conserved immunogenic antigen molecules among pathogenic strains formulated with an adjuvant and delivered by a route of immunization to induce maximum stimulation of the immune system. Moreover, immunization should focus on inducing Th1 responses, which is protective against <i>S. aureus</i> mastitis. It has been reported that proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity might be used as such antigens to induce protection against parasitic and microbial infections. Previous study in our laboratory on mastitis-causing streptococci indicates that GapC proteins of <i>S. uberis</i> and <i>S. dysgalactiae</i> have potential as vaccine antigens to protect dairy cows against mastitis caused by environmental streptococci. Two conserved cell wall associated proteins with iii glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, GapB and GapC have been identified from <i>S. aureus</i> isolates from bovine intramammary infections. The overall goal of this study was to improve our understanding on intramammary immunity using the GapC and GapB proteins of <i>S. aureus</i> as model antigens for mastitis and to determine the regulation of expression of <i>gapB</i> and <i>gapC</i> genes and their roles in the pathogenesis of bovine <i>S. aureus</i> mastitis. We hypothesized that strengthening local intramammary immunity using GapB and GapC proteins of <i>S. aureus</i> as antigens will protect against bovine <i>S. aureus</i> mastitis. To test this hypothesis we took the approach of using the <i>gapB</i> and <i>gapC</i> genes and constructed plasmids encoding GapB, GapC and GapB::GapC (GapC/B) chimeric proteins. We set six objectives to test our hypothesis using these proteins to enhance the intramammary immunity. In aim 1 we constructed plasmids encoding the GapB, GapC proteins and also constructed a chimeric gene encoding the GapC and GapB proteins as a single entity (GapC/B chimera) as the basis for a multivalent vaccine. In this objective the humoral and cellular immune responses to GapC/B were compared to the responses to the individual proteins alone or in combination in C57 BL/6 mice. Our results showed that the GapC/B protein elicited strong humoral and cellular immune responses as judged by the levels of total IgG, IgG1, IgG2a, IL-4 and IFN-ã secretion and lymphocyte proliferation. These results strongly suggest the potential of this chimeric protein as a target for vaccine production to control mastitis caused by <i>S. aureus</i>. In aim 2 we continued our studies on GapC/B by testing the effects of DNA vaccination with plasmids encoding the individual gapB and gapC genes as well as the gapC/B protein gene with or without a boost with the recombinant proteins. The results showed that DNA vaccination alone was unable to elicit a significant humoral response and barely able to elicit a detectable cell-mediated response to the recombinant antigens but subsequent immunization with the proteins elicited an excellent response. In addition, we found that DNA vaccination using a plasmid encoding the GapC/B chimera followed by a boost with the same protein, although successful, is less effective than priming with plasmids encoding GapB or GapC followed by a boost with the individual antigens. In aim 3 we optimized immune responses in cows by comparing route of vaccination (subcutaneous versus intradermal), site of vaccination (locally at the area drained by the supramammary lymph node versus distantly at area drained by parotid lymph node. Our results showed that both subcutaneous and intradermal immunizations with the GapC/B protein at the area drained by the supramammary and parotid lymph nodes resulted in significantly increased serum and milk titers of total IgG, IgG1, IgG2, iv and IgA in all vaccinated groups as compared to placebo. The anti-GapC/B IgG1 serum and milk titers were significantly higher in all vaccinated group as compared to the placebo group. These results indicated that vaccination at the area drained by the supramammary lymph node resulted in better immune responses. In aim 4 we tested different formulations of the GapC/B antigen with adjuvants such as PCPP, CpG, PCPP + CpG and VSA-3. We found that the VSA-3 formulation induced the best immune responses in cows. In this objective we also monitored immune responses longitudinally over one lactation cycle to determine the duration of immune responses by measuring IgG, IgG1, IgG2, and IgA on monthly blood and milk samples. We found that the duration of immune responses was about four months. In aim 5 we tested the role of GapC in the virulence of <i>S. aureus</i> mastitis using the <i>S. aureus</i> wild type strain RN6390 and its isogenic GapC mutant strain H330. Our results from both in vitro adhesion and invasion assays on MAC- T cells and in vivo infection of ovine mammary glands showed that GapC is an important virulence factor in <i>S. aureus</i> mastitis. In aim 6 we examined the role of sar and agr loci on the expression of <i>gapC</i> and <i>gapB</i> genes by qRT- PCR using <i>S. aureus</i> RN6390 and its isogenic mutants defective in agrA, sarA and sar/agr (double mutant) at exponential and stationary phases of growth. Our results showed that both <i>gapB</i> and <i>gapC</i> expression were down regulated in the mutant strains, indicating that the expression of the <i>gapB</i> and <i>gapC</i> genes is controlled by the universal virulence gene regulators, agr and sar. We also checked the role of environmental factors such as pH, growth media, and oxygen tension on the expression of <i>gapB</i> and <i>gapC</i> using q-RT-PCR. Our results showed that the expression of <i>gapB</i> and <i>gapC</i> genes in different strains of <i>S. aureus</i> was not consistent under the above-mentioned environmental conditions.

Baker's yeast (Saccharomyces cerevisiae) undergoes major biochemical and structural rearrangements in order to survive cycles of desiccation and rehydration, yet a firm understanding of the response is lacking. The purpose of this study was to examine the response of S. cerevisiae to desiccation and rehydration at both the physiological and molecular levels. Transmission electron microscopy was used to show that loss of vacuolar structure, enlarged nuclear boundaries, as well as cell wall thickening were all associated with the desiccation response. Molecular analysis focused on glyceraldehyde-3-phosphate dehydrogenase (GAPDH, E.C. 1.2.1.13), a multifunctional protein that is involved in several cellular processes other than glycolysis, including nuclear translocation under stress and intracellular sensing of oxidative stress during apoptosis. Here, GAPDH was studied primarily to determine its potential role in mediating the changes in cell wall physiology identified through our structural studies. GAPDH appears to be shuttled between the cell wall and the cytoplasm during the desiccation/rehydration process. Western analyses in combination with the use of inhibitors of translation (cycloheximide) suggest that the shuttling process does not require de novo protein synthesis. Western analyses also identified an immuno-reactive peptide in the cell wall and cytoplasmic fractions of lower molecular mass than native GAPDH (27 KDa vs. 37 KDa). This lower molecular weight peptide exhibited the translocation process similar to that of the full length GAPDH. Studies with GAPDH deletion strains suggested that the 27 kDa fragment is encoded by tdh3. The importance of this lower molecular weight form is yet to be determined. / Master of Science in Life Sciences

Chong, King Wai Isaac. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 99-104). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Table of Contents --- p.vi / Lists of Figures --- p.ix / List of Tables --- p.xi / List of Abbreviations --- p.xii / Chapter Chapter One: --- Introduction --- p.1 / Chapter 1.1 --- Overview of Glyceraldehyde 3-phosphate Dehydrogenases --- p.1 / Chapter 1.2 --- Properties And Molecular Structures of GAPDH --- p.3 / Chapter 1.3 --- Action Mechanism of GAPDH --- p.6 / Chapter 1.4 --- Novel Functions of GAPDH Unrelated to Carbohydrate Metabolism --- p.8 / Chapter 1.5 --- Effects of Heavy Metal on Enzyme Activity And Gene Expression of GAPDH --- p.10 / Chapter 1.6 --- Metal Binding Properties And Metal Binding Sites of GAPDH --- p.12 / Chapter 1.7 --- Isolation And Purification of GAPDH from Different Organisms --- p.13 / Chapter 1.8 --- Development of New Purification Method of GAPDH Using Immobilized Metal Affinity Chromatography --- p.15 / Chapter 1.9 --- Study of GAPDH from Chironomidae Larvae --- p.16 / Chapter 1.10 --- Aims of Study --- p.18 / Chapter Chapter Two: --- Methods And Materials --- p.19 / Chapter 2.1 --- Isolation of Native Chironomidae GAPDH --- p.19 / Chapter 2.1.1 --- Chemicals And Reagents --- p.19 / Chapter 2.1.2 --- Reagents --- p.19 / Chapter 2.1.3 --- Preparation of Crude Protein Extract from Chironomidae Larvae --- p.24 / Chapter 2.1.4 --- Immobilized Metal Affinity Chromatography --- p.24 / Chapter 2.1.5 --- Large Scale Preparation of Crude Protein Extract --- p.25 / Chapter 2.1.6 --- Ammonium Sulfate Fractionation --- p.25 / Chapter 2.1.7 --- Copper Affinity Column Chromatography --- p.26 / Chapter 2.1.8 --- Dye Affinity Column Chromatography --- p.26 / Chapter 2.2 --- Identification of Chironomidae GAPDH --- p.27 / Chapter 2.2.1 --- Chemicals And Reagents --- p.27 / Chapter 2.2.2 --- Reagents --- p.28 / Chapter 2.2.3 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.32 / Chapter 2.2.4 --- Non-Denaturing Polyacrylamide Gel Electrophoresis --- p.33 / Chapter 2.2.5 --- Protein Extraction from Coosmassie Blue Stained Polyacrylamide Gel --- p.33 / Chapter 2.2.6 --- N-terminal Amino Acid Analysis --- p.33 / Chapter 2.2.7 --- Sequence Analysis --- p.34 / Chapter 2.3 --- Kinetic Analysis of Chironomidae GAPDH --- p.34 / Chapter 2.3.1 --- Chemcials And Reagents --- p.34 / Chapter 2.3.2 --- Reagents --- p.34 / Chapter 2.3.3 --- Determination of Enzyme Concentration And GAPDH Activity --- p.35 / Chapter 2.4 --- Molecular Cloning of Chironomidae GAPDH --- p.36 / Chapter 2.4.1 --- Chemicals And Reagents --- p.36 / Chapter 2.4.2 --- Reagents --- p.37 / Chapter 2.4.3 --- RNA Extraction from Chironomidae Larvae --- p.41 / Chapter 2.4.4 --- DNase I Removal of Genomic DNA Contamination --- p.42 / Chapter 2.4.5 --- RNA Concentration Determination And RNA Agarose Electrophoresis --- p.42 / Chapter 2.4.6 --- First Strand cDNA Synthesis --- p.43 / Chapter 2.4.7 --- pRSet A B C Vectors --- p.43 / Chapter 2.4.8 --- Cloning Primer Design --- p.45 / Chapter 2.4.9 --- Polymerase Chain Reaction --- p.45 / Chapter 2.4.10 --- DNA Agarose Electrophoresis --- p.46 / Chapter 2.4.11 --- Restriction Enzyme Digestion of Insert And Plasmid --- p.46 / Chapter 2.4.12 --- Ligation of Plasmid And Insert DNA --- p.46 / Chapter 2.4.13 --- Preparation of Chemically Competent E. coli --- p.47 / Chapter 2.4.14 --- Transformation of Plasmid by Heat Shock --- p.47 / Chapter 2.4.15 --- Colony PCR --- p.48 / Chapter 2.5 --- Recombinant Protein Expression And Purification --- p.48 / Chapter 2.5.1 --- Chemicals And Reagents --- p.48 / Chapter 2.5.2 --- Reagents --- p.49 / Chapter 2.5.3 --- Protein expression by IPTG --- p.51 / Chapter 2.5.4 --- Protein purification by Nickel Affinity Column Chromatography --- p.52 / Chapter 2.5.5 --- EnterokinaseMax ´ёØ Removal of Polyhistidine Fusion Tag --- p.52 / Chapter 2.5.6 --- Western Blotting of Protein --- p.53 / Chapter Chapter Three: --- Results --- p.54 / Chapter 3.1 --- Two Affinity Chromatography Methods for GAPDH Purification --- p.54 / Chapter 3.2 --- Isolation And Purification of Native Chironomidae GAPDH --- p.54 / Chapter 3.3 --- Identification of Chironomidae GAPDH --- p.62 / Chapter 3.3.1 --- N-terminal amino acid analysis --- p.62 / Chapter 3.3.2 --- Sequence Analysis --- p.62 / Chapter 3.4 --- Molecular Cloning of Chironomidae GAPDH --- p.63 / Chapter 3.5 --- Isolation And Purification of recombinant Chironomidae GAPDH --- p.70 / Chapter 3.6 --- Protein Gel Electrophoresis Analysis of GAPDHs --- p.74 / Chapter 3.7 --- "Effects of Heavy Metals, pH And Temperature on GAPDHs" --- p.76 / Chapter 3.7.1 --- Heavy Metal Effect --- p.76 / Chapter 3.7.2 --- pH Effect --- p.76 / Chapter 3.7.3 --- Temperature --- p.77 / Chapter 3.8 --- Kinetic Analysis of GAPDHs --- p.84 / Chapter Chapter Four: --- Discussion --- p.89 / Chapter 4.1 --- New Method for The Isolation and Purification of Chironomidae GAPDH --- p.89 / Chapter 4.2 --- "Effects of Heavy Metals, pH And Temperature on GAPDHs" --- p.91 / Chapter 4.3 --- Kinetic Analysis of GAPDHs --- p.91 / Chapter 4.4 --- Zinc Activation of Chironomidae GAPDH --- p.92 / Chapter 4.5 --- Future Study --- p.93 / Chapter 4.5.1 --- Sequence Analysis Using Prediction Programmes --- p.94 / Chapter 4.5.2 --- Protein Crystallization --- p.95 / Chapter 4.5.3 --- Site-Directed Mutagenesis --- p.95 / Chapter 4.5.4 --- Biacore Surface Plasmon Resonance --- p.95 / Chapter Chapter Five: --- Conclusion --- p.98 / Chapter Chapter Six: --- References --- p.99

A oxidação fotocatalítica do glicerol (OFG) é uma possível alternativa para aproveitamento do excedente de glicerol proveniente do processo de produção do biodiesel, porém são poucos os registros desta reação na literatura. Neste trabalho, primeiramente foram testadas amostras comerciais de ZnO e TiO2, com o objetivo de identificar e quantificar os principais produtos de oxidação para cada semicondutor. Também foram realizados testes para determinação da influência dos parâmetros reacionais sobre a conversão e seletividade para gliceraldeído (GAD) e dihidroxiacetona (DHA), variando-se as condições experimentais tanto isoladamente quanto simultaneamente, através planejamento de experimentos. Por fim, foram sintetizadas amostras de ZnO visando-se obter uma amostra com melhores resultados em termos de conversão e seletividade para GAD e DHA que a amostra comercial. Os testes fotocatalíticos foram conduzidos em reator batelada (slurry) sob radiação ultravioleta, usando soluções aquosas de glicerol. Os produtos de oxidação foram analisados por cromatografia líquida de alta eficiência (CLAE). Os catalisadores sintetizados foram caracterizados por MEV, área BET, DRX, espalhamento de raios X a baixo ângulo e medidas de Potencial Zeta. Os resultados mostraram que ZnO e TiO2 diferem significativamente quanto à seletividade. O ZnO apresentou maior seletividade para produtos de maior valor agregado, como o gliceraldeído (GAD) e a dihidroxiacetona (DHA), indicando maior contribuição da fotocatálise indireta. O TiO2 apresentou seletividade maior para produtos resultantes da quebra da molécula de glicerol, como o formaldeído e o glicolaldeído, o que indica maior contribuição da fotocatálise direta. Nos testes da influência dos parâmetros reacionais para o ZnO, determinou-se que a conversão é afetada principalmente pelo pH inicial e concentração de catalisador e que a seletividade é afetada principalmente pela temperatura de reação e pH inicial. Em relação às amostras de ZnO sintetizadas em laboratório, nenhuma foi mais fotoativa que a amostra comercial, porém a amostra ZnO-B foi a mais fotoativa dentre as sintetizadas e foi mais seletiva ao GAD do que o ZnO comercial. Pôde-se determinar que o potencial zeta foi a característica mais influente na fotoatividade destes catalisadores, sendo que quanto mais positivo este valor, mais fotoativo o catalisador. Finalmente, em relação às amostras de ZnO com diferentes proporções de planos polares, a amostra com baixa proporção foi cerca de 2 vezes mais fotoativa que a amostra com alta proporção destes planos, o que se opõe aos resultados encontrados na literatura sobre fotoatividade relacionada a planos cristalinos. / The photocatalytic oxidation of glycerol emerges as a potential alternative to contribute to the utilization of glycerol surplus from biodiesel production. However, there are few reports about this reaction in the literature. In this work, as a first approach, commercial samples of ZnO and TiO2 were tested in order to identify the main products for each semiconductor. Next, the influence of the reaction parameters on the conversion and selectivity to glyceraldehyde (GAD) and dihydroxyacetone (DHA) was studied through a design of experiments, using ZnO as catalyst. Finally, additional ZnO samples were synthesized as an attempt to obtain a catalyst with conversion and/or selectivity higher than the commercial sample. The photocatalytic tests were carried out in a batch reactor (slurry) under ultraviolet radiation, using aqueous solution of glycerol. The oxidation products were analysed by liquid chromatography (HPLC). The synthesized catalysts were characterized by SEM, BET surface area, XRD, SAXS and Zeta Potential measurements. The results showed that ZnO and TiO2 differ significantly in selectivity. ZnO was more selective to products with higher commercial value (GAD and DHA), indicating greater contribution from indirect photocatalysis. On the other hand, TiO2 showed higher selectivity to products from the cleavage of the glycerol molecule, such as formaldehyde and glycolaldehyde, which indicates greater contribution from direct photocatalysis. In reference to the tests about the influence of the reaction conditions using ZnO, it was found that conversion was affected mainly by the initial pH and catalyst concentration, whereas selectivity was affected mainly by the temperature and initial pH. Regarding the synthesized ZnO catalysts, none of them reached conversion higher than the commercial sample, yet the sample ZnO-B showed the highest selectivity to GAD, even higher than the commercial sample. It was also determined that the zeta potential was the most influential characteristic on the catalyst activity. Finally, regarding the ZnO catalysts with different proportions of polar planes, the sample with low proportion of polar planes was about 2 times more active than the sample with high proportion of polar planes, which contradicts the records found in the literature about this specific topic.

A oxidação fotocatalítica do glicerol (OFG) é uma possível alternativa para aproveitamento do excedente de glicerol proveniente do processo de produção do biodiesel, porém são poucos os registros desta reação na literatura. Neste trabalho, primeiramente foram testadas amostras comerciais de ZnO e TiO2, com o objetivo de identificar e quantificar os principais produtos de oxidação para cada semicondutor. Também foram realizados testes para determinação da influência dos parâmetros reacionais sobre a conversão e seletividade para gliceraldeído (GAD) e dihidroxiacetona (DHA), variando-se as condições experimentais tanto isoladamente quanto simultaneamente, através planejamento de experimentos. Por fim, foram sintetizadas amostras de ZnO visando-se obter uma amostra com melhores resultados em termos de conversão e seletividade para GAD e DHA que a amostra comercial. Os testes fotocatalíticos foram conduzidos em reator batelada (slurry) sob radiação ultravioleta, usando soluções aquosas de glicerol. Os produtos de oxidação foram analisados por cromatografia líquida de alta eficiência (CLAE). Os catalisadores sintetizados foram caracterizados por MEV, área BET, DRX, espalhamento de raios X a baixo ângulo e medidas de Potencial Zeta. Os resultados mostraram que ZnO e TiO2 diferem significativamente quanto à seletividade. O ZnO apresentou maior seletividade para produtos de maior valor agregado, como o gliceraldeído (GAD) e a dihidroxiacetona (DHA), indicando maior contribuição da fotocatálise indireta. O TiO2 apresentou seletividade maior para produtos resultantes da quebra da molécula de glicerol, como o formaldeído e o glicolaldeído, o que indica maior contribuição da fotocatálise direta. Nos testes da influência dos parâmetros reacionais para o ZnO, determinou-se que a conversão é afetada principalmente pelo pH inicial e concentração de catalisador e que a seletividade é afetada principalmente pela temperatura de reação e pH inicial. Em relação às amostras de ZnO sintetizadas em laboratório, nenhuma foi mais fotoativa que a amostra comercial, porém a amostra ZnO-B foi a mais fotoativa dentre as sintetizadas e foi mais seletiva ao GAD do que o ZnO comercial. Pôde-se determinar que o potencial zeta foi a característica mais influente na fotoatividade destes catalisadores, sendo que quanto mais positivo este valor, mais fotoativo o catalisador. Finalmente, em relação às amostras de ZnO com diferentes proporções de planos polares, a amostra com baixa proporção foi cerca de 2 vezes mais fotoativa que a amostra com alta proporção destes planos, o que se opõe aos resultados encontrados na literatura sobre fotoatividade relacionada a planos cristalinos. / The photocatalytic oxidation of glycerol emerges as a potential alternative to contribute to the utilization of glycerol surplus from biodiesel production. However, there are few reports about this reaction in the literature. In this work, as a first approach, commercial samples of ZnO and TiO2 were tested in order to identify the main products for each semiconductor. Next, the influence of the reaction parameters on the conversion and selectivity to glyceraldehyde (GAD) and dihydroxyacetone (DHA) was studied through a design of experiments, using ZnO as catalyst. Finally, additional ZnO samples were synthesized as an attempt to obtain a catalyst with conversion and/or selectivity higher than the commercial sample. The photocatalytic tests were carried out in a batch reactor (slurry) under ultraviolet radiation, using aqueous solution of glycerol. The oxidation products were analysed by liquid chromatography (HPLC). The synthesized catalysts were characterized by SEM, BET surface area, XRD, SAXS and Zeta Potential measurements. The results showed that ZnO and TiO2 differ significantly in selectivity. ZnO was more selective to products with higher commercial value (GAD and DHA), indicating greater contribution from indirect photocatalysis. On the other hand, TiO2 showed higher selectivity to products from the cleavage of the glycerol molecule, such as formaldehyde and glycolaldehyde, which indicates greater contribution from direct photocatalysis. In reference to the tests about the influence of the reaction conditions using ZnO, it was found that conversion was affected mainly by the initial pH and catalyst concentration, whereas selectivity was affected mainly by the temperature and initial pH. Regarding the synthesized ZnO catalysts, none of them reached conversion higher than the commercial sample, yet the sample ZnO-B showed the highest selectivity to GAD, even higher than the commercial sample. It was also determined that the zeta potential was the most influential characteristic on the catalyst activity. Finally, regarding the ZnO catalysts with different proportions of polar planes, the sample with low proportion of polar planes was about 2 times more active than the sample with high proportion of polar planes, which contradicts the records found in the literature about this specific topic.

A oxidação fotocatalítica do glicerol (OFG) é uma possível alternativa para aproveitamento do excedente de glicerol proveniente do processo de produção do biodiesel, porém são poucos os registros desta reação na literatura. Neste trabalho, primeiramente foram testadas amostras comerciais de ZnO e TiO2, com o objetivo de identificar e quantificar os principais produtos de oxidação para cada semicondutor. Também foram realizados testes para determinação da influência dos parâmetros reacionais sobre a conversão e seletividade para gliceraldeído (GAD) e dihidroxiacetona (DHA), variando-se as condições experimentais tanto isoladamente quanto simultaneamente, através planejamento de experimentos. Por fim, foram sintetizadas amostras de ZnO visando-se obter uma amostra com melhores resultados em termos de conversão e seletividade para GAD e DHA que a amostra comercial. Os testes fotocatalíticos foram conduzidos em reator batelada (slurry) sob radiação ultravioleta, usando soluções aquosas de glicerol. Os produtos de oxidação foram analisados por cromatografia líquida de alta eficiência (CLAE). Os catalisadores sintetizados foram caracterizados por MEV, área BET, DRX, espalhamento de raios X a baixo ângulo e medidas de Potencial Zeta. Os resultados mostraram que ZnO e TiO2 diferem significativamente quanto à seletividade. O ZnO apresentou maior seletividade para produtos de maior valor agregado, como o gliceraldeído (GAD) e a dihidroxiacetona (DHA), indicando maior contribuição da fotocatálise indireta. O TiO2 apresentou seletividade maior para produtos resultantes da quebra da molécula de glicerol, como o formaldeído e o glicolaldeído, o que indica maior contribuição da fotocatálise direta. Nos testes da influência dos parâmetros reacionais para o ZnO, determinou-se que a conversão é afetada principalmente pelo pH inicial e concentração de catalisador e que a seletividade é afetada principalmente pela temperatura de reação e pH inicial. Em relação às amostras de ZnO sintetizadas em laboratório, nenhuma foi mais fotoativa que a amostra comercial, porém a amostra ZnO-B foi a mais fotoativa dentre as sintetizadas e foi mais seletiva ao GAD do que o ZnO comercial. Pôde-se determinar que o potencial zeta foi a característica mais influente na fotoatividade destes catalisadores, sendo que quanto mais positivo este valor, mais fotoativo o catalisador. Finalmente, em relação às amostras de ZnO com diferentes proporções de planos polares, a amostra com baixa proporção foi cerca de 2 vezes mais fotoativa que a amostra com alta proporção destes planos, o que se opõe aos resultados encontrados na literatura sobre fotoatividade relacionada a planos cristalinos. / The photocatalytic oxidation of glycerol emerges as a potential alternative to contribute to the utilization of glycerol surplus from biodiesel production. However, there are few reports about this reaction in the literature. In this work, as a first approach, commercial samples of ZnO and TiO2 were tested in order to identify the main products for each semiconductor. Next, the influence of the reaction parameters on the conversion and selectivity to glyceraldehyde (GAD) and dihydroxyacetone (DHA) was studied through a design of experiments, using ZnO as catalyst. Finally, additional ZnO samples were synthesized as an attempt to obtain a catalyst with conversion and/or selectivity higher than the commercial sample. The photocatalytic tests were carried out in a batch reactor (slurry) under ultraviolet radiation, using aqueous solution of glycerol. The oxidation products were analysed by liquid chromatography (HPLC). The synthesized catalysts were characterized by SEM, BET surface area, XRD, SAXS and Zeta Potential measurements. The results showed that ZnO and TiO2 differ significantly in selectivity. ZnO was more selective to products with higher commercial value (GAD and DHA), indicating greater contribution from indirect photocatalysis. On the other hand, TiO2 showed higher selectivity to products from the cleavage of the glycerol molecule, such as formaldehyde and glycolaldehyde, which indicates greater contribution from direct photocatalysis. In reference to the tests about the influence of the reaction conditions using ZnO, it was found that conversion was affected mainly by the initial pH and catalyst concentration, whereas selectivity was affected mainly by the temperature and initial pH. Regarding the synthesized ZnO catalysts, none of them reached conversion higher than the commercial sample, yet the sample ZnO-B showed the highest selectivity to GAD, even higher than the commercial sample. It was also determined that the zeta potential was the most influential characteristic on the catalyst activity. Finally, regarding the ZnO catalysts with different proportions of polar planes, the sample with low proportion of polar planes was about 2 times more active than the sample with high proportion of polar planes, which contradicts the records found in the literature about this specific topic.

Blastocystis is an important parasite that infects humans and a wide range of animals like rats, birds, reptiles, etc. infecting a sum of 60% of world population. It belongs to the Stramenopiles, a Heterologous group that includes for example the Phythophthora infestans the responsible for the Irish potato famine. Previous work had reported the presence of an unusual fusion protein that is composed of two of the main glycolytic enzymes; Triosephosphate isomerase-glyceraldehyde-3-phosphate dehydrogenase (TPI-GAPDH). Little is known about this protein. Blastocystis TPI-GAPDH and Blastocystis enolase were both characterized biochemically and biophysically in this project. The phylogenetic relationships of those two proteins among other members of either Stramenopiles, or other members of the kingdom of life were examined and found to be grouping within the chromalveolates. Our studies revealed that those two proteins, Blastocystis enolase and Blastocystis TPI-GAPDH, had a peptide signal targeting them to the mitochondria. This was an unusual finding knowing that text books always referred to the glycolytic pathway as a canonical cytoplasmic pathway. Structural studies had also been conducted to unravel the unknown structure of the fusion protein Blastocystis TPI-GAPDH. X-ray crystallography had been conducted to solve the protein structure and the protein was found to be a tetrameric protein composed of a central tetrameric GAPDH protein flanked with two dimmers of TPI protein. Solving its structure would be the starting point towards reviling the role that TPI-GAPDH might play in Blastocystis and other organisms that it was found in as well. Although a fusion protein, the individual components of the fusion were found to contain all features deemed essential for function for TPI and GAPDH and contain all expected protein motifs for these enzymes.

>Magister Scientiae - MSc / Knowledge of haematological 'normdata', of experimental animals, and the biological variables that affect it is essential in order to recognise variations from the normal. In addition, the haemopoietic system may be regarded in principle as good material for studies of the cellular events associated with ageing. These considerations, together with the well documented effects of age on various physiological processes, prompted an investigation into the effects of donor age on several blood parameters. Review of the literature revealed that age-related changes in blood parameters have been reported for several species, but the documentation thereof is incomplete, inconsistent and inconclusive in many respects. Blood samples from male Wistar rats of nine different biological ages, ranging from birth to 96 weeks of age, were analysed for haematological and biochemical parameters. These included the blood cell counts, erythrocytic indices, haemoglobin concentration, haematocrit, erythrocytic 2,3-diphosphoglycerate and adenosine triphosphate levels, and erythrocytic glucose 6-phosphate dehydrogenase and pyruvate kinase activities. Data was obtained which demonstrates that all blood parameters measured underwent significant, although not al~ays regular, age-related changes. These changes were found to be more marked during the first month of life than at any other period. Evidence is also presented to show that the depressed haemoglobin concentration during the early postnatal life may not imply a condition of 'physiologic anaemia' as was previously thought. Since the blood profile exhibits only slight changes from about 24 weeks of age, it does not seem that the haemopoietic system of the old rat deteriorates significantly as to constitute a limiting factor for the animal's life. However, the importance of taking an animal's age into account when blood parameters constitute experimental results is emphasised. The second phase of this study involved a detailed investigation of the effect of the animal's age on erythrocytes in particular. These cells have limited life-spans, and are often used as models in studies of cellular ageing. Special emphasis was therefore placed on comparing the relative effects of host and cellular ageing on the properties of these cells. Erythrocytes from rats between one and 48 weeks of age were separated into two populations by a modification of the conventional density gradient centrifugation technique. The two populations were assumed to differ in mean cell age and were analysed for erythrocytic indices, phosphate ester concentrations and the activities of glucose 6-phosphate dehydrogenase and pyruvate kinase. Evidence is presented to show that ageing rat erythrocytes exhibit a decrease in volume, phosphate ester content and enzyme activities while the cellular haemoglobin concentration increases. Differences in the mean cell age however, does not seem to account for the donor-age-related effects observed in the whole blood parameters. Rather, the significant differences found in the characteristics of similarly aged red cells, between variously aged donors, demonstrate that the biological age of the organism influences the red cells and probably the ageing thereof in vivo. The contribution of the changing status of the erythrocyte's environment of progressively older animals, to alterations which take place in the ageing red cell is discussed.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24109号 / 農博第2514号 / 新制||農||1093(附属図書館) / 学位論文||R4||N5400(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 佐藤 健司, 教授 菅原 達也, 准教授 木下 政人 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM