Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Lippia alba (Mill.) N. E. Brown is a native medicinal plant of South America. Known popularly as false-melissa , its leaves and roots are used in the treatment of some illnesses, including some of infective etiology. The presence of essential oils, among other metabolites with recognized antimicrobial activity, can explain its medicinal use. This paper describes the chemical analyses and antimicrobial activity of the essential oils from Lippia alba at different seasons of the year, as well as the bio-guided fractionation, the isolation and the structure elucidation of its main constituents. Lippia alba was cultivated in São Luiz Gonzaga, RS, Brazil, and its leaves were collected in 2005 and 2006 in the middle period (January, April, July and October) of each season. A voucher specimen (SMDB n° 10.050) was deposited in the herbarium of the Department of Biology, UFSM. The extraction of the essential oils was performed by the hydrodistillation method of fresh leaves. The oils were analyzed by GC-MS and their constituents were identified by the comparison of Kovat s retention indexes and mass spectra with literature data. The essential oil was fractionated by CC over silica gel for the isolation of germacrene D-4-ol and linalool, both identified by GC-MS, 13C and 1H NMR, DEPT-135, COSY and HETCOR. The antimicrobial activity of the essential oils, fractions and isolated compounds were determined by the broth microdilution method. The essential oil showed qualitative and semi-quantitative variability in relation to the seasons of the year. The yield ranged from 0.3 to 0.6%, being lower in winter and higher in summer, autumn and spring. The essential oil was composed of monoterpenoids (69 - 84%) and sesquiterpenoids (6 - 24%), being most of them oxygenated and hidrocarbonated, respectively. Moreover, the major compounds (> 15%) were not the same between the years of the analysis. The seasonal variability influenced the antimicrobial activity of the essential oils. With the exception of P. aeruginosa, all other microorganisms were susceptible to the essential oil. The most sensitive ones were S. cerevisiae (MIC and MFC of 800 μg/mL for the oil obtained in January), S. aureus (MIC of 800 μg/mL and MBC of 1600 μg/mL for the oils obtained in April and July) and K. pneumoniae (MIC and MFC of 1600 μg/mL for the oil extracted in July). In view of the antimicrobial activity, winter and summer are the best seasons for the collection and extraction of essential oils. The bio-guided fractionation of the essential oil showed that the fractions containing greater amounts of oxygenates are more active than those containing hydrocarbon compounds. Germacrene D-4-ol presented activity against S. cerevisiae (MIC and MFC of 200 μg/mL), P. zopfii (MIC and MFC of 400 μg/mL) and S. aureus (MIC and MBC of 800 μg/mL), while linalool was inactive against the tested microorganisms up to 3200 mg/mL. / Lippia alba (Mill.) N. E. Brown é uma planta medicinal nativa da América do Sul. Conhecida popularmente como falsa-melissa , suas folhas e raízes são empregadas no tratamento de várias doenças, incluindo algumas de etiologia infecciosa. A presença dos óleos essenciais, entre outros metabólitos com reconhecida atividade antimicrobiana, pode explicar o seu uso como medicinal. Este trabalho descreve a analise química e a atividade antimicrobiana dos óleos essenciais de Lippia alba nas diferentes estações do ano, bem como o fracionamento bio-guiado, o isolamento e a elucidação estrutural dos seus principais constituintes. Lippia alba foi cultivada no município de São Luiz Gonzaga, RS, Brasil, e suas partes aéreas foram coletadas no período médio de cada estação (janeiro, abril, julho e outubro) durante o ano de 2005 e 2006. Material testemunha (SMDB n° 10.050) encontra-se depositado no Herbário do Departamento de Biologia da UFSM. Para a realização dos experimentos, foram utilizadas as folhas frescas. A extração dos óleos essenciais foi realizada pelo método da hidrodestilação. Os óleos foram analisados por CG-EM e seus constituintes identificados pela comparação dos índices de retenção de Kovats e espectros de massas com dados da literatura. O óleo essencial também foi fracionado por CC sobre gel de sílica até o isolamento do germacreno D-4- ol e do linalol, ambos identificados por CG-EM, RMN 13C e 1H, DEPT-135, COSY e HETCOR. A atividade antimicrobiana dos óleos essenciais, das frações e das substâncias isoladas foi determinada pelo método de microdiluição em caldo. O óleo essencial apresentou variabilidade qualitativa e semiquantitativa em relação às estações do ano. O rendimento variou na ordem de 0,3 a 0,6%, sendo menor no inverno, e maior no verão, outono e primavera. O óleo essencial foi composto de monoterpenóides (69 - 84%) e sesquiterpenóides (6 24%), sendo a maioria deles oxigenados e hidrocarbonados, respectivamente. Os compostos majoritários (>15%) não foram os mesmos entre os diferentes anos de análise. A variabilidade sazonal, por sua vez, influenciou a atividade antimicrobiana dos óleos essenciais. Com exceção de P. aeruginosa, todos os outros microorganismos foram sensíveis ao óleo essencial de pelo menos uma estação. Os microorganismos com menor CIM foram S. cerevisiae (CIM e CFM de 800 μg/mL para o óleo obtido em janeiro), S. aureus (CIM de 800 μg/mL e CBM 1600 μg/mL para os óleos obtidos em abril e julho) e K. pneumoniae (CIM e CBM de 1600 μg/mL para o óleo extraído em julho). Tendo em vista a atividade antimicrobiana, o inverno e o verão constituem as melhores épocas para a coleta e extração dos óleos essenciais. O fracionamento bio-guiado do óleo essencial mostrou que frações contendo maiores quantidades de compostos oxigenados são mais ativas do que aquelas contendo compostos hidrocarbonados. Germacreno D-4-ol apresentou atividade contra S. cerevisiae (CIM e CFM de 200 μg/mL), P. zopfii (CIM e CFM de 400 μg/mL) e S. aureus (CIM e CBM de 800 μg/mL), ao passo que linalol foi inativo contra os microorganismos testados até a concentração de 3200 mg/mL.

La composition minérale du sol influence la physiologie de la vigne, le potentiel qualitatif des baies et la qualité du vin. Parmi les éléments que la vigne prélève du sol, l'azote impacte son développement, la composition des baies ainsi que la synthèse de certains métabolites secondaires. Son effet peut être direct sur la voie métabolique, ou indirecte par exemple à travers une modification de la vigueur de la vigne. Le présent travail s’intéresse à un possible effet direct de l’azote sur la synthèse des thiols volatils, en particulier le 3SH (3-sulfanylhexan-1-ol) à l’odeur de pamplemousse et ses précurseurs (Glut-3SH et Cys-3SH), ainsi que l'IBMP (3-isobutyl-2-méthoxypyrazine) à l’odeur de poivron vert, en absence d’une contrainte hydrique et d’une modification de la vigueur des ceps. La vigne a été étudiée en faisant varier le statut azoté, en conditions contrôlées et en plein champ. Cependant l’état hydrique et la vigueur ont été similaires entre les modalités. Une augmentation de la teneur en Glut-3SH dans les baies et les moûts ainsi que du contenu en 3SH dans le vin a été observée en réponse à une augmentation de l’alimentation en azote. En revanche, aucun effet n’a été perçu sur la teneur en Cys-3SH, ni sur l'expression de VviGST3, VviGST4 et VviGGT de sa voie de synthèse. Cette observation semble montrer qu’en réponse à l’azote, le 3SH est synthétisé à partir du Glut-3SH, indépendamment du Cys-3SH et des trois gènes mentionnés. De nouveaux gènes candidats de la voie de biosynthèse des précurseurs du 3SH ont été identifiés par séquençage d’ARN. Le statut azoté de la vigne n’a pas influencé la teneur en IBMP, ni l'expression de VviOMT3 et VviOMT4, gènes clés de sa voie de biosynthèse. L’effet de N sur l’IBMP décrit précédemment est probablement indirect et dû à une modification du microclimat dans la zone de la grappe, induite par une vigueur plus forte des vignes ayant un statut azoté élevé. / Soil is a major component of the viticultural terroir. Its mineral composition influences vine physiology, berry quality potential and wine quality. Among the elements that vines pick up from the soil, nitrogen (N) has the strongest impact on vine physiology, berry composition and the synthesis of compounds that have an important sensory impact. This study has investigated a possible direct effect of N on the synthesis of volatile thiols i.e. 3SH (3-sulfanylhexan-1-ol) responsible for grapefruit aroma and its S-conjugates precursors (Glut-3SH and Cys-3SH), as well as IBMP (3-isobutyl-2-methoxypyrazine) responsible for green pepper aroma, without interference with water status and vigor variations. Vines were grown under variable N status in controlled conditions and in field trials. Water status was controlled during the season and vine vigor was similar among treatments. N supply increased Glut-3SH level in berries and in must and 3SH content in wines. In contrast, no effect of N was perceived on Cys-3SH concentration and on the expression of VviGST3, VviGST4 and VviGGT genes, which play a major role in its biosynthetic pathway. This observation indicates that, in response to N supply, 3SH could be synthetized from Glut-3SH, independently from Cys-3SH and the three mentioned genes. New candidate genes from the 3SH precursors pathway were also identified by whole transcriptome shotgun sequencing. There was no significant effect of vine N status on IBMP levels in berries, musts and wines, neither on the expression of VviOMT3 and VviOMT4, which are key genes in the biosynthetic pathway of IBMP. This result indicates that when an effect of N on IBMP was found in previous studies, it was likely mediated through the modification of bunch-zone microclimate, induced by the increased vigor of high N-status vines.

The grape variety Sauvignon Blanc (SB) is the flagship of New Zealand’s wine industry and accounted for over 75 % of the value of total wine exports in 2008. Two volatile thiols, 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl-acetate (3MHA), reminiscent of grapefruit and passion fruit respectively, are critical for the main varietal characters in New Zealand SB. These aromatic thiols are not present in the grape juice, but are synthesized and released by the yeast during alcoholic fermentation from non-aromatic precursors. The aim of this work was to elucidate the underlying genetics of volatile thiol synthesis in yeast (Saccharomyces cerevisiae) during alcoholic fermentation of grape juice. A gene-deletion strategy was chosen for the investigation of putative genes influencing 3MH and 3MHA release. The first part of this thesis optimized fermentation conditions in grape-juice-based media, which enabled auxotrophic laboratory strains, derived from S288C, to ferment grape juice to completion with high efficiency. Key steps to achieving this goal were the supplementation of the grape juice with higher than recommended amounts of amino acids, which increased the fermentation rate of auxotrophic yeast strains. Lysine auxotrophic strains especially benefited from this measure. In combination with the dilution of SB grape juice by 25 % with synthetic grape juice without sugars, the auxotrophic laboratory yeast BY4743 was able to metabolize all sugars in the grape juice-based media in a time frame similar to that of a commercial wine yeast. The key properties of the resulting wine were comparable to wine made with a commercial wine yeast under the same conditions. In the second part, these newly developed fermentation conditions were employed to screen 69 single-gene deletion strains in the laboratory yeast background BY4743. The list of the 69 candidate genes was compiled by combining existing knowledge about thiol production in yeast with the mining of several biological databases. Screening of the single-gene deletions revealed 17 genes which caused biologically relevant increases or decreases in volatile thiol production, but none abolished it. The majority of the 17 genes were related to the sulfur and nitrogen metabolism in yeast. A subset of these thiol-influencing genes were also deleted in a wine yeast, and were overexpressed in both wine yeast and laboratory yeast, to gain more insight in their regulatory effects. The findings confirmed that sulfur and nitrogen metabolism in yeast were important in regulating 3MH and 3MHA synthesis. Different sulfur and nitrogen sources were added to the grape must prior to fermentation and their effect on thiol release was studied. It was found that nitrogen sources urea and DAP, as well as, the sulfur compound S-ethyl-L-cysteine (SEC) increased 3MH and 3MHA concentrations in the resulting wines. The addition of cysteine to grape juice fermented with wine yeast deleted in genes CYS3 and CYS4 more than doubled total thiol production. Mapping approaches to investigate thiol production in yeast were employed in the final part of this thesis. Genetically mapped F2 progeny of a cross between a low thiol-producing yeast strain and a high-thiol producer were screened for their thiol phenotype. The 3MH and 3MHA phenotypes across 48 screened F2 progeny resembled normal distributions, indicating a quantitative trait. Subsequent mapping identified a locus on chromosome 14 with a small effect on the 3MHA phenotype, but no obvious candidate genes were evident in the region. Another approach to investigate the evolution of volatile thiols in yeast included the use of SEC, a thiol compound resembling the cysteinylated precursor of 3MH, as a sole nitrogen source in a yeast growth assay. It was found that most wine yeast, European yeast isolates and laboratory yeasts could utilize SEC as a nitrogen source, whereas various other S. cerevisiae isolates could not. Crosses between three pairs of Sec- and Sec+ yeast strains strongly indicated that this trait was monogenically inherited. However, no direct correlation between the SEC phenotype and volatile release could be observed. Genetic mapping experiments in one SEC-segregating yeast population linked this SEC phenotype to the leu2-D0 deletion in a cross between a Leu+ and Leu- yeast strain. It was shown that leucine auxotrophy most likely caused the Sec- phenotype. In a second F2 population of a cross between prototrophic Sec+ and Sec- strains, strong linkage was established to a region on chromosome 6 containing two candidate genes, DUG1 and IRC7. DUG1 was proved not to be the cause of the SEC phenotype, whereas IRC7 remains a strong candidate gene.

The grape variety Sauvignon Blanc (SB) is the flagship of New Zealand’s wine industry and accounted for over 75 % of the value of total wine exports in 2008. Two volatile thiols, 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl-acetate (3MHA), reminiscent of grapefruit and passion fruit respectively, are critical for the main varietal characters in New Zealand SB. These aromatic thiols are not present in the grape juice, but are synthesized and released by the yeast during alcoholic fermentation from non-aromatic precursors. The aim of this work was to elucidate the underlying genetics of volatile thiol synthesis in yeast (Saccharomyces cerevisiae) during alcoholic fermentation of grape juice. A gene-deletion strategy was chosen for the investigation of putative genes influencing 3MH and 3MHA release. The first part of this thesis optimized fermentation conditions in grape-juice-based media, which enabled auxotrophic laboratory strains, derived from S288C, to ferment grape juice to completion with high efficiency. Key steps to achieving this goal were the supplementation of the grape juice with higher than recommended amounts of amino acids, which increased the fermentation rate of auxotrophic yeast strains. Lysine auxotrophic strains especially benefited from this measure. In combination with the dilution of SB grape juice by 25 % with synthetic grape juice without sugars, the auxotrophic laboratory yeast BY4743 was able to metabolize all sugars in the grape juice-based media in a time frame similar to that of a commercial wine yeast. The key properties of the resulting wine were comparable to wine made with a commercial wine yeast under the same conditions. In the second part, these newly developed fermentation conditions were employed to screen 69 single-gene deletion strains in the laboratory yeast background BY4743. The list of the 69 candidate genes was compiled by combining existing knowledge about thiol production in yeast with the mining of several biological databases. Screening of the single-gene deletions revealed 17 genes which caused biologically relevant increases or decreases in volatile thiol production, but none abolished it. The majority of the 17 genes were related to the sulfur and nitrogen metabolism in yeast. A subset of these thiol-influencing genes were also deleted in a wine yeast, and were overexpressed in both wine yeast and laboratory yeast, to gain more insight in their regulatory effects. The findings confirmed that sulfur and nitrogen metabolism in yeast were important in regulating 3MH and 3MHA synthesis. Different sulfur and nitrogen sources were added to the grape must prior to fermentation and their effect on thiol release was studied. It was found that nitrogen sources urea and DAP, as well as, the sulfur compound S-ethyl-L-cysteine (SEC) increased 3MH and 3MHA concentrations in the resulting wines. The addition of cysteine to grape juice fermented with wine yeast deleted in genes CYS3 and CYS4 more than doubled total thiol production. Mapping approaches to investigate thiol production in yeast were employed in the final part of this thesis. Genetically mapped F2 progeny of a cross between a low thiol-producing yeast strain and a high-thiol producer were screened for their thiol phenotype. The 3MH and 3MHA phenotypes across 48 screened F2 progeny resembled normal distributions, indicating a quantitative trait. Subsequent mapping identified a locus on chromosome 14 with a small effect on the 3MHA phenotype, but no obvious candidate genes were evident in the region. Another approach to investigate the evolution of volatile thiols in yeast included the use of SEC, a thiol compound resembling the cysteinylated precursor of 3MH, as a sole nitrogen source in a yeast growth assay. It was found that most wine yeast, European yeast isolates and laboratory yeasts could utilize SEC as a nitrogen source, whereas various other S. cerevisiae isolates could not. Crosses between three pairs of Sec- and Sec+ yeast strains strongly indicated that this trait was monogenically inherited. However, no direct correlation between the SEC phenotype and volatile release could be observed. Genetic mapping experiments in one SEC-segregating yeast population linked this SEC phenotype to the leu2-D0 deletion in a cross between a Leu+ and Leu- yeast strain. It was shown that leucine auxotrophy most likely caused the Sec- phenotype. In a second F2 population of a cross between prototrophic Sec+ and Sec- strains, strong linkage was established to a region on chromosome 6 containing two candidate genes, DUG1 and IRC7. DUG1 was proved not to be the cause of the SEC phenotype, whereas IRC7 remains a strong candidate gene.

The grape variety Sauvignon Blanc (SB) is the flagship of New Zealand’s wine industry and accounted for over 75 % of the value of total wine exports in 2008. Two volatile thiols, 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl-acetate (3MHA), reminiscent of grapefruit and passion fruit respectively, are critical for the main varietal characters in New Zealand SB. These aromatic thiols are not present in the grape juice, but are synthesized and released by the yeast during alcoholic fermentation from non-aromatic precursors. The aim of this work was to elucidate the underlying genetics of volatile thiol synthesis in yeast (Saccharomyces cerevisiae) during alcoholic fermentation of grape juice. A gene-deletion strategy was chosen for the investigation of putative genes influencing 3MH and 3MHA release. The first part of this thesis optimized fermentation conditions in grape-juice-based media, which enabled auxotrophic laboratory strains, derived from S288C, to ferment grape juice to completion with high efficiency. Key steps to achieving this goal were the supplementation of the grape juice with higher than recommended amounts of amino acids, which increased the fermentation rate of auxotrophic yeast strains. Lysine auxotrophic strains especially benefited from this measure. In combination with the dilution of SB grape juice by 25 % with synthetic grape juice without sugars, the auxotrophic laboratory yeast BY4743 was able to metabolize all sugars in the grape juice-based media in a time frame similar to that of a commercial wine yeast. The key properties of the resulting wine were comparable to wine made with a commercial wine yeast under the same conditions. In the second part, these newly developed fermentation conditions were employed to screen 69 single-gene deletion strains in the laboratory yeast background BY4743. The list of the 69 candidate genes was compiled by combining existing knowledge about thiol production in yeast with the mining of several biological databases. Screening of the single-gene deletions revealed 17 genes which caused biologically relevant increases or decreases in volatile thiol production, but none abolished it. The majority of the 17 genes were related to the sulfur and nitrogen metabolism in yeast. A subset of these thiol-influencing genes were also deleted in a wine yeast, and were overexpressed in both wine yeast and laboratory yeast, to gain more insight in their regulatory effects. The findings confirmed that sulfur and nitrogen metabolism in yeast were important in regulating 3MH and 3MHA synthesis. Different sulfur and nitrogen sources were added to the grape must prior to fermentation and their effect on thiol release was studied. It was found that nitrogen sources urea and DAP, as well as, the sulfur compound S-ethyl-L-cysteine (SEC) increased 3MH and 3MHA concentrations in the resulting wines. The addition of cysteine to grape juice fermented with wine yeast deleted in genes CYS3 and CYS4 more than doubled total thiol production. Mapping approaches to investigate thiol production in yeast were employed in the final part of this thesis. Genetically mapped F2 progeny of a cross between a low thiol-producing yeast strain and a high-thiol producer were screened for their thiol phenotype. The 3MH and 3MHA phenotypes across 48 screened F2 progeny resembled normal distributions, indicating a quantitative trait. Subsequent mapping identified a locus on chromosome 14 with a small effect on the 3MHA phenotype, but no obvious candidate genes were evident in the region. Another approach to investigate the evolution of volatile thiols in yeast included the use of SEC, a thiol compound resembling the cysteinylated precursor of 3MH, as a sole nitrogen source in a yeast growth assay. It was found that most wine yeast, European yeast isolates and laboratory yeasts could utilize SEC as a nitrogen source, whereas various other S. cerevisiae isolates could not. Crosses between three pairs of Sec- and Sec+ yeast strains strongly indicated that this trait was monogenically inherited. However, no direct correlation between the SEC phenotype and volatile release could be observed. Genetic mapping experiments in one SEC-segregating yeast population linked this SEC phenotype to the leu2-D0 deletion in a cross between a Leu+ and Leu- yeast strain. It was shown that leucine auxotrophy most likely caused the Sec- phenotype. In a second F2 population of a cross between prototrophic Sec+ and Sec- strains, strong linkage was established to a region on chromosome 6 containing two candidate genes, DUG1 and IRC7. DUG1 was proved not to be the cause of the SEC phenotype, whereas IRC7 remains a strong candidate gene.

The grape variety Sauvignon Blanc (SB) is the flagship of New Zealand’s wine industry and accounted for over 75 % of the value of total wine exports in 2008. Two volatile thiols, 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl-acetate (3MHA), reminiscent of grapefruit and passion fruit respectively, are critical for the main varietal characters in New Zealand SB. These aromatic thiols are not present in the grape juice, but are synthesized and released by the yeast during alcoholic fermentation from non-aromatic precursors. The aim of this work was to elucidate the underlying genetics of volatile thiol synthesis in yeast (Saccharomyces cerevisiae) during alcoholic fermentation of grape juice. A gene-deletion strategy was chosen for the investigation of putative genes influencing 3MH and 3MHA release. The first part of this thesis optimized fermentation conditions in grape-juice-based media, which enabled auxotrophic laboratory strains, derived from S288C, to ferment grape juice to completion with high efficiency. Key steps to achieving this goal were the supplementation of the grape juice with higher than recommended amounts of amino acids, which increased the fermentation rate of auxotrophic yeast strains. Lysine auxotrophic strains especially benefited from this measure. In combination with the dilution of SB grape juice by 25 % with synthetic grape juice without sugars, the auxotrophic laboratory yeast BY4743 was able to metabolize all sugars in the grape juice-based media in a time frame similar to that of a commercial wine yeast. The key properties of the resulting wine were comparable to wine made with a commercial wine yeast under the same conditions. In the second part, these newly developed fermentation conditions were employed to screen 69 single-gene deletion strains in the laboratory yeast background BY4743. The list of the 69 candidate genes was compiled by combining existing knowledge about thiol production in yeast with the mining of several biological databases. Screening of the single-gene deletions revealed 17 genes which caused biologically relevant increases or decreases in volatile thiol production, but none abolished it. The majority of the 17 genes were related to the sulfur and nitrogen metabolism in yeast. A subset of these thiol-influencing genes were also deleted in a wine yeast, and were overexpressed in both wine yeast and laboratory yeast, to gain more insight in their regulatory effects. The findings confirmed that sulfur and nitrogen metabolism in yeast were important in regulating 3MH and 3MHA synthesis. Different sulfur and nitrogen sources were added to the grape must prior to fermentation and their effect on thiol release was studied. It was found that nitrogen sources urea and DAP, as well as, the sulfur compound S-ethyl-L-cysteine (SEC) increased 3MH and 3MHA concentrations in the resulting wines. The addition of cysteine to grape juice fermented with wine yeast deleted in genes CYS3 and CYS4 more than doubled total thiol production. Mapping approaches to investigate thiol production in yeast were employed in the final part of this thesis. Genetically mapped F2 progeny of a cross between a low thiol-producing yeast strain and a high-thiol producer were screened for their thiol phenotype. The 3MH and 3MHA phenotypes across 48 screened F2 progeny resembled normal distributions, indicating a quantitative trait. Subsequent mapping identified a locus on chromosome 14 with a small effect on the 3MHA phenotype, but no obvious candidate genes were evident in the region. Another approach to investigate the evolution of volatile thiols in yeast included the use of SEC, a thiol compound resembling the cysteinylated precursor of 3MH, as a sole nitrogen source in a yeast growth assay. It was found that most wine yeast, European yeast isolates and laboratory yeasts could utilize SEC as a nitrogen source, whereas various other S. cerevisiae isolates could not. Crosses between three pairs of Sec- and Sec+ yeast strains strongly indicated that this trait was monogenically inherited. However, no direct correlation between the SEC phenotype and volatile release could be observed. Genetic mapping experiments in one SEC-segregating yeast population linked this SEC phenotype to the leu2-D0 deletion in a cross between a Leu+ and Leu- yeast strain. It was shown that leucine auxotrophy most likely caused the Sec- phenotype. In a second F2 population of a cross between prototrophic Sec+ and Sec- strains, strong linkage was established to a region on chromosome 6 containing two candidate genes, DUG1 and IRC7. DUG1 was proved not to be the cause of the SEC phenotype, whereas IRC7 remains a strong candidate gene.

The grape variety Sauvignon Blanc (SB) is the flagship of New Zealand’s wine industry and accounted for over 75 % of the value of total wine exports in 2008. Two volatile thiols, 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl-acetate (3MHA), reminiscent of grapefruit and passion fruit respectively, are critical for the main varietal characters in New Zealand SB. These aromatic thiols are not present in the grape juice, but are synthesized and released by the yeast during alcoholic fermentation from non-aromatic precursors. The aim of this work was to elucidate the underlying genetics of volatile thiol synthesis in yeast (Saccharomyces cerevisiae) during alcoholic fermentation of grape juice. A gene-deletion strategy was chosen for the investigation of putative genes influencing 3MH and 3MHA release. The first part of this thesis optimized fermentation conditions in grape-juice-based media, which enabled auxotrophic laboratory strains, derived from S288C, to ferment grape juice to completion with high efficiency. Key steps to achieving this goal were the supplementation of the grape juice with higher than recommended amounts of amino acids, which increased the fermentation rate of auxotrophic yeast strains. Lysine auxotrophic strains especially benefited from this measure. In combination with the dilution of SB grape juice by 25 % with synthetic grape juice without sugars, the auxotrophic laboratory yeast BY4743 was able to metabolize all sugars in the grape juice-based media in a time frame similar to that of a commercial wine yeast. The key properties of the resulting wine were comparable to wine made with a commercial wine yeast under the same conditions. In the second part, these newly developed fermentation conditions were employed to screen 69 single-gene deletion strains in the laboratory yeast background BY4743. The list of the 69 candidate genes was compiled by combining existing knowledge about thiol production in yeast with the mining of several biological databases. Screening of the single-gene deletions revealed 17 genes which caused biologically relevant increases or decreases in volatile thiol production, but none abolished it. The majority of the 17 genes were related to the sulfur and nitrogen metabolism in yeast. A subset of these thiol-influencing genes were also deleted in a wine yeast, and were overexpressed in both wine yeast and laboratory yeast, to gain more insight in their regulatory effects. The findings confirmed that sulfur and nitrogen metabolism in yeast were important in regulating 3MH and 3MHA synthesis. Different sulfur and nitrogen sources were added to the grape must prior to fermentation and their effect on thiol release was studied. It was found that nitrogen sources urea and DAP, as well as, the sulfur compound S-ethyl-L-cysteine (SEC) increased 3MH and 3MHA concentrations in the resulting wines. The addition of cysteine to grape juice fermented with wine yeast deleted in genes CYS3 and CYS4 more than doubled total thiol production. Mapping approaches to investigate thiol production in yeast were employed in the final part of this thesis. Genetically mapped F2 progeny of a cross between a low thiol-producing yeast strain and a high-thiol producer were screened for their thiol phenotype. The 3MH and 3MHA phenotypes across 48 screened F2 progeny resembled normal distributions, indicating a quantitative trait. Subsequent mapping identified a locus on chromosome 14 with a small effect on the 3MHA phenotype, but no obvious candidate genes were evident in the region. Another approach to investigate the evolution of volatile thiols in yeast included the use of SEC, a thiol compound resembling the cysteinylated precursor of 3MH, as a sole nitrogen source in a yeast growth assay. It was found that most wine yeast, European yeast isolates and laboratory yeasts could utilize SEC as a nitrogen source, whereas various other S. cerevisiae isolates could not. Crosses between three pairs of Sec- and Sec+ yeast strains strongly indicated that this trait was monogenically inherited. However, no direct correlation between the SEC phenotype and volatile release could be observed. Genetic mapping experiments in one SEC-segregating yeast population linked this SEC phenotype to the leu2-D0 deletion in a cross between a Leu+ and Leu- yeast strain. It was shown that leucine auxotrophy most likely caused the Sec- phenotype. In a second F2 population of a cross between prototrophic Sec+ and Sec- strains, strong linkage was established to a region on chromosome 6 containing two candidate genes, DUG1 and IRC7. DUG1 was proved not to be the cause of the SEC phenotype, whereas IRC7 remains a strong candidate gene.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-08-29T19:32:36Z No. of bitstreams: 1 MayaraCristinaMendesMaia_DISSERT.pdf: 3094006 bytes, checksum: 6eb9d0d3601a7e6c6d840f4eb7fcdeb9 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-09-02T23:34:57Z (GMT) No. of bitstreams: 1 MayaraCristinaMendesMaia_DISSERT.pdf: 3094006 bytes, checksum: 6eb9d0d3601a7e6c6d840f4eb7fcdeb9 (MD5) / Made available in DSpace on 2016-09-02T23:34:57Z (GMT). No. of bitstreams: 1 MayaraCristinaMendesMaia_DISSERT.pdf: 3094006 bytes, checksum: 6eb9d0d3601a7e6c6d840f4eb7fcdeb9 (MD5) Previous issue date: 2016-04-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Acreditamos que as recorrentes mudan?as do cinema cl?ssico, abrindo espa?o no cinema para o pensamento feminista, tem chegado ao Brasil de diferentes formas e com diversas tem?ticas fortemente dentro do cinema independente. Neste sentido, nossa pesquisa surge da necessidade de compreender as transforma??es sociais e na produ??o audiovisual sobre a mulher atleta e suas poss?veis rela??es com a Teoria Feminista do Cinema. Deste modo, nosso objetivo de trabalho foi analisar a obra cinematogr?fica brasileira ?Mulheres Ol?mpicas?, estabelecendo suas poss?veis rela??es com a teoria feminista do cinema. Realizamos uma an?lise contextual da obra atrav?s dos estudos de Gumbrecht. Nossas fichas t?cnicas de an?lise foram organizadas em dois momentos. O primeiro momento ? composto pelos objetos e pelas condi??es da experi?ncia est?tica do filme debatidos a partir dos elementos do texto f?lmico. O segundo momento, regido pelos conte?dos e efeitos da experi?ncia est?tica, est? dividido nas categorias que surgem partir da an?lise de conte?do de Bardin, a saber: desigualdade de sexo; desvaloriza??o da atleta; lutas pol?ticas e conquistas de espa?o; diferentes mulheres brasileiras; objetos e marcas simb?licos e; desconhecimento da hist?ria do esporte ol?mpico brasileiro. Ao estudarmos as categorias, conclu?mos que o document?rio n?o s? apresenta rela??es com a Teoria Feminista do Cinema, como retrata imagens de mulheres brasileiras atletas que h? muito tempo foram ofuscadas em sua visibilidade midi?tica e que, tribut?ria a emerg?ncia dessa teoria, ganham olhares mais generosos ? problematiza??o dos espa?os sociais ocupados pela mulher que predominam na obra. / Our research arises from the need to understand the social, and audiovisual production on the female athletes and their possible relationship with the Feminist Theory of Cinema. After conducting a survey of Brazilian works that address the theme of Brazilian female athlete by access Brazilian film libraries and the Internet, defined as a general goal of our work is to analyze the Brazilian cinematographic work "Mulheres Ol?mpicas", establishing their possible relationships with feminist theory cinema. We build a contextual analysis of the work through Gumbrecht studies. Our analysis of data sheets were organized in two stages. The first time was composed of objects and the conditions of the aesthetic experience of the film discussed from the filmic text elements. The second time, governed by the content and effects of the aesthetic experience, was divided into the categories that emerge from the Bardin content analysis, namely: sex inequality; devaluation of the athlete; political struggles and space conquests; different Brazilian women; objects and symbolic brands and; ignorance of the history of Brazilian Olympic sport. In studying the categories, we conclude that the documentary not only has relations with the Theory Feminist Cinema, as portrayed by images of women Brazilian athletes who have long been overshadowed by its media visibility and that tax the emergence of this theory, make more generous looks to problematize the social spaces occupied by women predominate in the work.

Made available in DSpace on 2015-04-01T12:09:01Z (GMT). No. of bitstreams: 1 parte1.pdf: 3938300 bytes, checksum: d776c692585937cc573fe392dbb20e02 (MD5) Previous issue date: 2012-02-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Terpinen-4-ol (4TRP) is a monoterpe alcohol and component of the essential oils of several aromatic plants. Similarly to 4TRP, other monoterpenoid alcohols have showed anticonvulsant activity in convulsion animal models. The present report aimed to investigate the psychopharmacological effects of terpinen-4-ol, specifically the anticonvulsant activity. Mice (Mus musculus) swiss male and and rats (Rattus norvegicus) wistar male were used. In experiments in vivo, the 4TRP was administered at doses of 25 to 200 mg/kg, ip, and concentrations of 10, 20 and 40 ng/2μL, icv. For in vitro experiments, concentrations were 0.1 mM and 1.0 mM. General tests as pharmacological behavioral screening, spontaneous movement, potentiation of sleeping time induced by pentobarbital and rota rod test were initially performed to investigate the profile of action of this monoterpene in the CNS. In all these methods, the results suggest that this drug has psycholeptics profile and does not cause significant changes in motor coordination of animals. Next, more specific experiments were performed to evaluate the psicodepressor profile: the elevated plus maze (EPM) test and the perforated plate have been selected to investigate possibleanxiolytic effects. In these experiments, and the doses tested, were not observed consistently indicative of anxiolytic activity related to 4TRP. Later, tests were performed to evaluate the possibility of this substance present a profile of anticonvulsant activity. According to the behavioral parameters evaluated, 4TRP (via ip) was able to inhibit seizures induced by both Pentylenetetrazole (PTZ) and Maximal electroshock (MES), presenting a potential anticonvulsant effect. In a subsequent step, following the animals by electroencephalographic recordings, we observed that animals treated with 4TRP (via icv) were protected against PTZ-induced seizures, corroborating the results of the previous step. In the search for a characterization of the possible mechanisms by which this substance exerts this action, we investigate more clearly the involvement of the GABAergic system employing the methodologies convulsions induced by picrotoxin (PIC) and seizures induced by 3-mercapto-propionic acid (3-MP). In accordance with the results, it is possible to infer that the action exerted by monoterpenoid is related to the GABAergic system and that the presence of flumazenil, a selective antagonist of the benzodiazepine site of GABA A receptors, was unable to reverse the anticonvulsant effect of 4TRP, demonstrating that this is not the same binding site of benzodiazepines. In a subsequent step, using the technique of "Whole-Cell Patch Clamp , demonstrated that 4TRP was able to decrease the sodium current in sodium channels of voltage-dependent in isolated cells of neurons in dorsal root ganglia (DRG), and possibly its effect is related to changes in neuronal excitability in consequence of modulation of these channels. It was concluded that 4TRP has psychopharmacological effects, with an anticonvulsant profile. Its mechanism of action appears to be mediated by interference with the GABAergic system and does not involve activation, at least directly, the benzodiazepine site of GABAA receptors and that involves the blockade of sodiumchannels voltage-dependent. / O 4-terpineol (4TRP) é um monoterpeno álcool monocíclico, que pode ser encontrado em óleos essenciais de plantas aromáticas diversas. Vários estudos têm relatado os efeitos apresentados por monoterpenos estruturalmente análogos ao 4TRP sobre o Sistema Nervoso Central (SNC). Justificando-se pela lacuna existente na farmacoterapia das epilepsias, a realização deste trabalho teve como objetivo avaliar os efeitos psicofarmacológicos do 4TRP. Camundongos (Mus musculus) Swiss, machos e ratos (Rattus novergicus) Wistar, machos foram utilizados. Nos experimentos in vivo, o 4TRP foi administrado em doses variando entre 25 a 200 mg/kg, i.p., e nas concentrações de 10, 20 e 40 ng/2µL, i.c.v. Para os experimentos in vitro, as concentrações utilizadas foram 0,1 mM e 1,0mM. Como triagem farmacológica, para investigar o perfil de ação deste monoterpeno no SNC, foram realizados inicialmente testes gerais comportamentais como o teste da movimentação espontânea, potencialização do tempo de sono induzido por pentobarbital e o teste da barra giratória para investigar o perfil de ação deste monoterpeno no SNC. Em todas essas metodologias, os resultados obtidos sugerem que este apresenta perfil de droga psicoléptica, sem alterar, no entanto, a coordenação motora dos animais de forma significativa. Posteriormente, para avaliar possíveis efeitos ansiolíticos, foram realizados os testes do labirinto em cruz elevado (LCE) e o teste da placa perfurada. Nesses experimentos, nas doses testadas, o 4TRP não apresentou indicativos consistentes de atividade ansiolítica. Em seguida, foram realizados testes com o objetivo de avaliar a possibilidade de esta substância apresentar um perfil de atividade anticonvulsivante. De acordo com os parâmetros comportamentais avaliados, 4TRP (via i.p.), foi capaz de inibir convulsões induzidas tanto pelo pentilenotetrazol (PTZ) como pelo eletrochoque máximo (ECM), apresentando um potencial efeito anticonvulsivante. Em uma etapa posterior, acompanhando os animais através de registros eletroencefalográficos, percebe-se que animais tratados com 4TRP (via i.c.v.), foram protegidos contra convulsões induzidas por PTZ, corroborando com os resultados da etapa anterior. Para uma caracterização dos possíveis mecanismos pelos quais esta substância exerce sua ação, buscou-se evidenciar a participação do sistema GABAérgico empregando as metodologias: convulsões induzidas pela picrotoxina (PIC) e convulsões induzidas pelo ácido 3-mercapto-propiônico (3-MP). Em conformidade com os resultados, pode-se afirmar que a ação deste monoterpenóide está relacionada ao sistema GABAérgico e ainda que a presença do flumazenil, um antagonista seletivo do sítio benzodiazepínico dos receptores GABAA, não foi capaz de reverter o efeito anticonvulsivante de 4TRP, demonstrando que este não atua no mesmo sítio de ligação dos benzodiazepínicos. Em uma etapa subsequente, utilizando a técnica de Patch Clamp-Whole Cell , demonstrou-se que 4TRP foi capaz de inibir significaticamente a corrente de canais de sódio dependentes de voltagem em células isoladas de neurônios de gânglios da raiz dorsal (GRD), estando o seu efeito, possivemente relacionado à mudanças na excitabilidade neuronal em conseqüência da modulação desses canais. Conclui-se que 4TRP apresenta efeitos psicofarmacológicos, com perfil de fármaco anticonvulsivante, que seu mecanismo de ação parece ser mediado pela interferência com o sistema GABAérgico e não envolve a ativação, pelo menos de forma direta, do sítio benzodiazepínico dos receptores GABAA e que envolve o bloqueio dos canais para sódio dependentes de voltagem.

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