Orientador: Guilherme Targino Valente / Resumo: O bioetanol é o biocombustível mais utilizado no mundo, tendo como uma de suas principais vantagens o fato de contribuir para a redução da emissão de gases do efeito estufa. O principal organismo fermentador utilizado para a produção de bioetanol é a levedura Saccharomyces cerevisiae, que quando adicionada num meio com glicose ou outra fonte de carbono fermentável, utilizará a fermentação alcoólica como processo metabólico principal para a obtenção de energia e consequentemente o etanol. No presente trabalho testou-se 14 linhagens de S. cerevisiae, analisando o grau máximo de tolerância ao etanol bem como características celulares e moleculares. Para isso foi utilizado técnicas como citometria de fluxo, curvas de crescimento e analises de proteômica. O objetivo do trabalho foi analisar as proteínas mais abundantes, em um contexto comparativo para os experimentos de estresse máximo por etanol nas leveduras, identificando enzimas chaves presentes durante o tratamento e possivelmente responsável pela tolerância. Foram determinadas as concentrações máximas de etanol suportada por cada linhagem seguida por análises de viabilidade celular. Os resultados demonstraram que a capacidade de replicação e viabilidade celular não estão diretamente ligados ao fenômeno de tolerância. Além disso, a presença de proteínas relacionadas às vias fermentativas, vias oxidativas e manutenção da homeostase celular, parecem estar associadas a um perfil de maior tolerância ao etanol uma vez que estão ma... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Bioethanol is the most used biofuel in the world since its main advantage is the reduction of greenhouse gas emissions. The most used organism for bioethanol production is the yeast Saccharomyces cerevisiae, which uses glucose, or other fermentable carbon sources, for energy obtaining and ethanol production from the alcoholic fermentation pathway. In this work, 14 S. cerevisiae strains were evaluated concerning their highest ethanol tolerance degree and cellular/molecular characteristics as well. For this purpose, it was used techniques such as flow cytometry, growth curve analysis and proteomics. The objective of this work was to analyze the most abundant proteins, in a comparative context of maximum ethanol stress for yeasts, to identify keyenzymes present during cell treatments as well as their possible influence for the ethanol tolerance. Before that, it was determined the highest ethanol concentration each strain could support followed by cellular viabilities. The results demonstrated that replication capacity and cell viability are not closely related to the ethanol tolerance. Moreover, the presence of proteins related to fermentative pathways, oxidative pathways, and maintenance of cellular homeostasis, looks like to be linked to the highest ethanol tolerance profile, since those proteins are more present in higher ethanol tolerant strains. Furthermore, it was possible to understand that both tolerance and resistance features play different roles in the studied strains... (Complete abstract click electronic access below) / Mestre

L’étude de la réponse membranaire suite au stress éthanolique chez Saccharomyces cerevisiae vise à comprendre les mécanismes impliqués dans la survie des eucaryotes aux concentrations en éthanol élevées. La membrane cellulaire, par sa position entre l’environnement intra et extracellulaire, est la première cible des perturbations éthanoliques. Les expérimentations effectuées dans cette étude visent à caractériser le maintien de l’intégrité fonctionnelle en relation avec la composition en stérols membranaires chez la levure S. cerevisiae soumise à différents types de perturbations éthanoliques (augmentation de la concentrations en éthanol dans le milieu de croissance, chocs éthanoliques d’amplitude croissante et suivi au cours du temps ; choc à 20% pendant 15 minutes puis suivi au cours du temps après retour dans un milieu sans éthanol (« pulse » 20% d’éthanol)). Les résultats obtenus démontrent l’importance de l’ergostérol dans le maintien de l’intégrité membranaire et supportent également l’hypothèse du rôle « vecteur » de l’éthanol vis à vis d’une oxydation, dont l’efficacité serait dépendante de la nature des stérols présents au niveau membranaire. Les premiers résultats analysant la cinétique de transcription de gènes impliqués dans la réponse au stress oxydatif suggèrent une formation plus importante de formes réactives de l’oxygène (ROS), induite par le choc éthanol chez un mutant Δerg6, affecté dans la voie de biosynthèse de l’ergostérol. / The study of membrane response induced by ethanol stress in Saccharomyces cerevisiae aims to understand mechanisms involved in the survival of eukaryotic cells submitted to high ethanol concentrations. The cell membrane by its position between the intra- and extracellular environment is the first target of ethanolic perturbations. Experiments performed in this study aimed to characterize the maintain of the functional integrity of the membrane in relation to the sterol composition in the yeast S. cerevisiae submitted to different types of ethanolic disturbances: increasing concentrations of ethanol in the growth medium; ethanolic shocks of increasing magnitude; shock 20% ethanol for 15 minutes and then return in a medium without ethanol ("pulse" 20% ethanol)). Our results demonstrate the importance of ergosterol in maintaining membrane integrity and also support the hypothesis of the vector role of ethanol in cell oxidation, whose effectiveness is dependent on the nature of sterols at the membrane level. In addition, our results analyzing the kinetics of transcription of genes involved in oxidative stress response suggest an increased formation of reactive oxygen species (ROS) induced by ethanol in the Δerg6 mutant, affected in the biosynthetic pathway of ergosterol.

ROLE OF THE YXKO GENE OF BACILLUS SUBTILIS IN RESPONCE TO ENVIRONMENTAL STRESS Abstract Mutation of the yxkO gene, which encodes a putative ribokinase and belongs to the σB general stress response regulon, leads to reduced salt tolerance under potassium limitation in Bacillus subtilis. The biological function of the yxkO gene has not been determined yet, but it may be involved in the high affinity potassium uptake system, which has been described in Escherichia coli in contrast to Bacillus subtilis. Our goal was to describe another features of a mutant in the yxkO gene and to try to propose the role of this gene. Using the integration vector pMutin4, we prepared a Bacillus subtilis strain MP2 with a yxkO gene inactivation. The MP2 strain displays limited growth in a rich medium and it is a sensitive strain to tetracycline. Furthermore, this strain is unable to form endospores and the cells are longer, which indicates a septum formation defect. We accomplished a 2-D protein gel analysis to compare expression profiles of the MP2 strain and the 1A680 standard strain after salt and ethanol stress. The MP2 strain shows changes in productions of some energy metabolism enzymes and flagellin protein. We conclude that yxkO is a regulatory gene, whose product has a pleiotropic effect on many of cell functions.

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