Microrganismos geneticamente modificados (OGMs), obtidos através das técnicas de biologia molecular são amplamente utilizados para as mais diversas finalidades, atingindo o ambiente, solo, águas, animais e até mesmo o Homem. Contudo, o comportamento e o destino destes microrganismos no ambiente, depois que sua função tenha sido completada, e onde estes não podem ser controlados, tem sido objeto de preocupação. Para diminuir os riscos potenciais associados à liberação (acidental ou intencional) dos OGMs no ambiente pesquisadores desenvolveram sistemas de contenção de microrganismos geneticamente engenheirados, baseados na clonagem de genes que codificam proteínas tóxicas, sob regulação de promotores indutíveis (para revisão ver Molin et aI., 1993). Estes sistemas permitem o controle da morte celular em tempo pré-determinado, com a indução do promotor que regula o gene que codifica a proteína tóxica. Contudo, embora as leveduras sejam tão importantes e úteis quanto as bactérias, não existem sistemas suicidas usados para contenção destes microrganismos. No presente trabalho, construímos um sistema suicida para a levedura Saccharomyces cerevisiae o qual apresenta o gene da nuclease de Serratia marcescens clonado sob controle do promotor ADH2GAPDH, que é reprimido por glicose. Desde que a nuclease é capaz de destruir DNA e RNA, tal sistema não permitirá que o material genético do OGM em questão, seja transferido para outros microrganismos, após a morte celular. A atividade da nuclease foi detectada in vivo, através da diminuição da viabilidade dos transformantes de levedura, após o consumo da glicose. Quando leveduras mutantes rad52 foram usadas como células hospedeiras, o efeito letal do plasmídeo foi dramaticamente aumentado. Por outro lado, a atividade da nuclease produzida nos tranformantes de levedura foi analisada in vitro, através da incubação de extratos celulares com DNA plasmidiano mostrando a completa degradação deste em gel de agarose. Além disso, em ensaio de microcosmos, a viabilidade das leveduras portadoras do plasmídeo suicida no ambiente diminuiu drasticamente quando comparada com células controles. Estes resultados mostram que a nuclease de Serratía marcescens é expressa de forma ativa em S. cerevísíae sendo capaz de evitar a permanência dos transformantes de leveduras no ambiente, em laboratório, ou em condições simulando o ambiente, sem a necessidade de um indutor adicional. / Genetic Engineered Microorganisms (GEMs), obtained by molecular biology techniques, are becoming of widespread use for different purposes thus reaching the environment, including soil, water, plants, animais and Man. However, the behavior and fate of these microorganisms after their function has been completed in environments where they cannot be physically contained is a matter of concern. To minimize the potential risks associated with the release (deliberate or accidental) of GEMs in the environment, researchers have developed biological containment systems for bacteria which are based on the cloning of genes coding for toxic proteins, under the regulation of regulatable promoters (for review, see Molin et al., 1993). Such suicide systems allow the predetermined control of cell death from the moment the promoter of the killer gene is switched on. However, even if yeasts are as useful and important in biotechnological processes as bacteria, there is no suicide system described so far for the containment of these microorganisms. In the present work, we constructed a suicide system for the yeast Saccharomyces cerevisiae, consisting of the Serratia marcescens nuclease gene, cloned under the control of the yeast AOH2GAPOH promoter, which is repressed by glucose. Since this nuclease is able to destroy DNA and RNA, such suicide system does not allow the genetic material of the GEM to be transferred to other microorganisms upon cell death. The nuclease activity in S. cerevisiae was detected in vivo, through the decrease in cell viability of the yeast transformants, upon glucose consumption. When yeast rad52 mutants were used as the host cells, the lethal effect of the suicide plasmid was dramatically increased. On the other hand, the activity of the nuclease produced in yeast transformants was analyzed in vitro, by incubation of cell-free extracts with plasmid DNA, showing complete degradation on agarose gels. Furthermore, in microcosmos assays, the viability of the yeast cells carrying the suicide plasmid decreased drastically when compared to control cells. These results showed that the S. marcescens nuclease is expressed in an active form in S. cerevisiae, being able to avoid the maintenance of the yeast transformants, either in the laboratory, or in conditions simulating the environment, without the need of adding any inducer.

O Experimento foi realizado com o objetivo de avaliar a substituição parcial e total de plasma bovino por levedura hidrolisada na dieta de leitões desmamados no período de 21 a 47 dias de idade. Foram utilizados 1600 leitões da linhagem PIC, distribuídos em blocos ao acaso, com quatro tratamentos. As dietas foram divididas em quatro fases (pré-inicial I 22 a 28 dias; pré-inicial II 29 a 35 dias; inicial I - 36 a 47 dias e inicial II 48 a 63 dias). A inclusão percentual, Plasma: Levedura, nas dietas foi: T1 (6:0; 4:0; 2:0 e 0:0); T2 (3: 4; 2: 3; 1: 2 e 0: 0); T3 (1,5: 6; 1: 4,5; 0,5: 3 e 0: 0) e T4 (0: 8; 0: 6; 0: 4 e 0: 0). Cada tratamento teve 10 repetições (cinco de machos e cinco de fêmeas) totalizando 40 unidades experimentais com 40 animais cada. As variáveis zootécnicas avaliadas durante o período experimental foram: consumo de ração, ganho de peso, e conversão alimentar. Foram tomadas amostras de sangue de 5 animais por tratamento no dia de início do experimento, e de 10 animais por tratamento 25 dias depois. Foram quantificadas a IgA e a IgG. Os dados obtidos foram submetidos a análise de variância ANOVA, utilizando-se o teste Tukey para comparação das médias ao nível de significância de 5% utilizando o pacote estatístico SAS. Não houve diferença estatística nas quantidades de Ig A e Ig G circulante, nem na variação destas imunoglobulinas no tempo, entre os tratamentos. Considerando a análise dos dados conclui-se que nas condições estudadas, a utilização da relação percentual de (1,5: 6; 1: 4,5; 0,5: 3 e 0: 0) de plasma: levedura hidrolisada resultou um maior consumo de ração e ganho de peso dos animais, em comparação às outras proporções, e consequentemente podendo trazer um maior lucro para o produtor / The experiment was performed with the objective of evaluating the partial and complete substitution of bovine plasma for hydrolyzed yeast in the diet of weaned piglets from 21 to 47 days old. 1600 piglets of PIC lineage were used and randomly distributed in blocks where they received four treatments. Their diets were divided into four phases (pre-initial I: 22 to 28 days; pre-initial II- 29 to 35 days; initial I- 36 to 47 days and initial II-48 to 63 days). The percentage inclusion Plasma:Yeast in the diets were: T1 (6:0; 4:0; 2:0 and 0:0); T2 (3: 4; 2: 3; 1: 2 and 0: 0); T3 (1.5: 6; 1: 4.5; 0.5: 3 and 0: 0) and T4 (0: 8; 0: 6; 0: 4 and 0: 0). Each treatment had 10 repetitions (five times with the males and five times with the females), totaling 40 experimental units with 40 animals each. Husbandry variables evaluated during the trial period were: feed intake, body weight gain and feed conversion. Blood samples of 5 animals per treatment were taken at the beginning of the experiment day, and 10 animals per treatment 25 days later. IgA and IgG were quantified. The data obtained were analyzed by ANOVA analysis of variance, using the Tukey test to compare means at the 5% significance level, using the statistical package SAS. There was no statistical difference in the quantities of Ig A and Ig G circulating, or the variation of these immunoglobulins in the time, between treatments. Considering the analysis of the data it was concluded that the conditions studied, the percentage utilization ratio of (1.5: 6; 1: 4.5: 0.5: 3 and 0: 0) plasma: hydrolyzed yeast resulted in a higher feed intake and weight gain of animals compared to other proportions, and consequently can bring higher profits to the producer

Não consta resumo na publicação. / The biological value of the proteins of Saccharomyces cerevisiae grown in sugar cane molasses was studied in rats during 3 generations. The animals were fed two levels of yeast protein either 10% or 25% and compared with casein fed controls. In the first generation the PER and body weight were significantly lower for the animals receiving the yeast in their diet. In the 2rd and 3rd generation the growth was retarded even more in relation to what had been observed for the first one, specially at 10% protein level. A decreased male fertility was also observed at litter born from rats fed either 10% or 25% protein. The digestibility for casein was 90% and 80% for single cell protein.

Orientador: José Augusto Rosário Rodrigues / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-22T20:09:11Z (GMT). No. of bitstreams: 1 Chaves_MichelRicardodeBarros_M.pdf: 6595019 bytes, checksum: 834b04f04c23dc9c48e6103fda01f3ae (MD5) Previous issue date: 2013 / Resumo: A redução microbiológica de a-metileno-b-cetoésteres é capaz de fornecer produtos com redução quimio, enantio e diasterosseletiva da ligação C=C, da ligação C=O, ou mesmo de ambas, fazendo uso das enoato redutases e álcool desidrogenases presentes nos microrganismos. Sendo assim, os substratos em estudo foram sintetizados empregando diferentes protocolos, entretanto, o que mostrou ser mais eficiente consistiu do preparo a-metileno-b-hidroxi-ésteres, conhecidos como adutos de Morita-Baylis-Hillman, seguido de oxidação com ácido 2-iodoxibenzóico. Os substratos 2-(benzoil)-prop-2-enoato de metila, 2-[(4-clorofenil)carbonil]-prop-2-enoato de metila e 2-[(4-bromofenil)carbonil]-prop-2-enoato de metila foram biorreduzidos com Saccharomyces cerevisiae tipo II (Sigma-Aldrich®), Pichia stiptis CCT 2617, Rhodotorula glutinis CCT 2182 e Pichia kluyveri CCT 3365 como biocatalisadores. Os a-metil-b-hidróxi-ésteres foram formados tendo Saccharomyces cerevisiae tipo II como melhor biocatalisador, com rendimentos de 70 a 79% além de apresentarem razões syn/anti de até 9:1 com excessos enantioméricos de 99% para ambos diastereoisômeros e predominância do diastereoisômero syn (2S,3S) sobre o anti (2R,3S). Foram avaliados como suporte para o substrato a resina polimérica XAD7HP, que, apesar de fornecer incrementos na diastereosseletividade, apresentou resultados insatisfatórios para a conversão dos a-metil-b-hidroxi-ésteres. A alternativa empregada para contornar os baixos rendimentos apresentados foi o uso de papéis de filtro como suporte para o substrato, realizando o fornecimento do substrato ao meio reacional. O produto 3-(p-bromofenil)-3-hidroxi-2-metil-propanoato de metila teve sua configuração absoluta determinada por RMN de H empregando o método de Mosher, obtendo a configuracao (2S,3S). / Abstract: The microbial reduction of a-methylene-b-ketoesters can furnish products with chemio, enantio and diasteroselective reduction of C=C, C=O or both bonds, making use of the enoate reductases and alcohol dehydrogenases present on microorganisms. Thus, the substrates studied here were prepared using different protocols, but, the one that showed better results consisted of the preparing of Morita-Baylis-Hillman aducts followed by their oxidation with 2-iodoxybenxoic acid. Methyl 2-benzoylprop-2-enoate, methyl 2-[(4-chlorophenyl)carbonyl]prop-2-enoate and methyl 2-[(4-bromophenyl)carbonyl]prop-2-enoate were bioreduced by type II Saccharomyces cerevisiae (Sigma-Aldrich®), Pichia stiptis CCT 2617, Rhodotorula glutinis CCT 2182 and Pichia kluyveri CCT 3365 as biocatalysts and the first one showed better activity. The a-methyl-b-hydroxy-esters were obtained up to 79% yield, showing syn/anti ratio up to 9:1 and e.e. of 99% for both diastereoisomers, also was observed that the syn (2S, 3S) prevailed over its anti diastereoisomer (2R,3S). The use of the polymeric resin XAD7HP as substrate reservoir was evaluated, obtaining good improvement on the diastereoselectivity, although the conversion was unsatisfactory. The chosen alternative to achieve the presented yields was the use of filter paper as substrate reservoir, performing the substrate feeding to the medium. Assignment of the absolute configuration of methyl 3-(p-bromophenyl)-3-hydroxy-2-methyl propanoate was determined by Mosher Ls method, obtaining the configuration (2S,3S). / Mestrado / Quimica Organica / Mestre em Química

Temperature is arguably the leading factor that drives adaptation of organisms and ecosystems. Remarkably, many sister species share the same habitat because of their different temporal or micro-spatial thermal adaptation. In this PhD, the underlying molecular mechanisms of the adaptation of closely related species to different temperatures are sought. A thermodynamic analysis was applied to a genome-scale metabolic model of S. cerevisiae at warm and cold temperatures to identify thermo-dependent reactions. Gene Ontology (GO) analysis of predicted cold-dependent reactions found that redox reactions were significantly enriched. A complementary large scale experimental approach was taken by competing 6,000 mutant strains at 16°C to identify genes that were responsible for the fitness at low temperatures. The experiment was carried out in three different nutritional conditions to test the plasticity of temperature dependency. A list of strains whose copy number significantly increased or decreased in all media conditions was constructed and analysed using Gene Ontology. Vitamin biosynthesis, lipid/fatty acid processes and oxido-reduction reactions were all found to be significantly affected by the cold condition. Combining the data from the two studies a list of candidate genes affected by temperature changes were generated. In particular, two genes, GUT2 and ADH3, were identified as potential cold favouring genes and studied in more detailed. Mutants for these two genes were created in a pair of natural sympatric cryotolerant and thermotolerant Saccharomyces yeasts, namely S. kudriavzevii CA111 and S. cerevisiae 96.2, representing an excellent ecological experimental model for differential temperature adaption. My results showed that when compared to the parental strains, both mutants showed lower fitness at cold temperatures as predicted, and in S. kudriavzevii CA111 these mutations significantly improve growth at warm temperatures. Results from all aspects of this work indicate that oxidation reduction reactions are important for cold acclimation. It is known that heat stress causes redox imbalances which are compensated by increasing glycerol production or cytosolic acetaldehyde. Since GUT2 and ADH3 are involved in these processes, mutations in these genes may not be able to compensate for temperature changes. My data also shows that vitamins may also play an important role in cold acclimation which would be an interesting line of investigation for future work. Overall this PhD thesis has incorporated in silico and in vivo work to identify potential processes and genes involved in the temperature adaptation of sister Saccharomyces yeast species. The approach and results provided in this study support the use of a systems biology framework to studying species adaptation to environmental changes, and show that such models can yield testable predictions that may lead to new biological discoveries.

O exossomo é um complexo constituído por até 11 subunidades (Rrp4p, Rrp6p, Rrp40p, Rrp41p, Rrp42p, Rrp43p, Rrp44p, Rrp45p, Rrp46p, Csl4p, Mtr3p) que possui atividade exorribonucleolítica 3`→5` e está envolvido no processamento e degradação de vários tipos de RNAs na célula eucariótica. O complexo tem sido estudado em diversos organismos, como leveduras, insetos, plantas, humanos e também em várias espécies de archaea. Apesar da conservação da estrutura do exossomo ao longo da evolução e de oito subunidades do exossomo eucariótico apresentarem domínios de RNase, apenas duas proteínas, Rrp6p e Rrp44p têm atividade catalítica. A despeito da importância do exossomo para a célula, ainda não está claro o papel de cada subunidade na atividade do complexo. Neste trabalho foram utilizados mutantes da subunidade Rrp43p a fim de avaliar como mutações pontuais nesta subunidade afetam a montagem e estabilização do complexo do exossomo de Saccharomyces cerevisiae. Ensaios de purificação do exossomo com TAP-Rrp43p revelaram que os mutantes co-purificam Mtr3p e Rrp44p menos eficientemente. Além disso, os mutantes também apresentam atividade exorribonucleolítica 3`→5` reduzida, indicando que o defeito na montagem do complexo pode afetar a sua atividade enzimática. / The exosome is a protein complex comprised of up to eleven subunits (Rrp4p, Rrp6p, Rrp40p, Rrp41p, Rrp42p, Rrp43p, Rrp44p, Rrp45p, Rrp46p, Csl4p and Mtr3p) that has 3`→5` exoribonucleolytic activity and is involved in degradation and processing pathways of several kinds of RNA in eukaryotes. This complex has also been identified in several organisms, such as yeast, insects, plants, humans and also many species of archaea. Despite the overall structure conservation of the complex throughout evolution and eight of the eukaryotic exosome subunits displaying RNase domains, only two proteins, Rrp6p and Rrp44p have catalytic activity. Although the exosome has been shown to be involved in many different aspects of RNA metabolism, the role that each subunit plays in the activity of the complex has not yet been determined. In this work we used of TAP-purified exosome complexes to study the effect of Rrp43p mutations on the assembly and stabilization of the complex in Saccharomyces cerevisiae. Co-immunoprecipitation assays revealed that Rrp43p mutants co-purify Mtr3p and Rrp44p subunits less efficiently. Besides, Rrp43p mutants also present decreased activity, indicating that an assembly defect may affect its enzymatic activity

Saccharomyces cerevisiae est l’acteur principal de la fermentation du moût de raisin, mais l’influence de facteurs sur sa distribution dans les vignobles est peu connue. La région bordelaise, par son histoire et ses appellations, est une région d’intérêt pour étudier la diversité de S.cerevisiae. Au total, 2422 isolats de S.cerevisiae provenant de prélèvements de raisins et de cuves en fermentation spontanées sur deux années consécutives ont été analysés par 15 à 17 marqueurs microsatellites. Une très grande diversité génétique est mise en évidence, supérieure en mode de conduite conventionnel par rapport au mode biologique. Le mode de conduite influence faiblement la structure de la population de S.cerevisiae au vignoble. L’appellation et le domaine impactent significativement la structure de population, sans que des gradients de diversité n‘apparaissent, mais nos analyses révèlent des connections importantes dans le sens Pessac-Léognan vers les autres appellations du Bordelais, en particulier le Médoc. Des flux importants bidirectionnels sont mis en évidence entre les compartiments vigne et chai, illustrés par la présence de 25% de souches apparentées à des levures commerciales au vignoble, retour des souches du chai au vignoble jusqu’alors sous-estimé, alors qu’un flux d’importance similaire est observé entre le vignoble et le chai. La présence de populations ancestrales communes dans des prélèvements anciens (plus de 20 ans) et récents révèle la stabilité des populations sur le long terme à l’échelle d’une appellation. Une succession temporelle des populations du chai pourrait être favorisée par la mise en œuvre de pied de cuve avec repiquages successifs. / Saccharomyces cerevisiae is the main actor of wine fermentation but still little is known about the factors impacting its distribution in the vineyards. Bordeaux region, by its history and its appellations, is one of the regions of interest to study S. cerevisiae diversity. A total of 2422 isolates of S.cerevisiae sampled from grapes and spontaneous fermentation tanks during two consecutive years were analyzed by 15 to 17 microsatellite markers. A very high genetic diversity is demonstrated, greater in conventional farming system than in organic one. The type of farming system weakly influences the population structure of S.cerevisiae in the vineyard. The appellation and the wine estate significantly impact the population structure, without appearance of diversity gradients, but our analyses reveal important connections from the Pessac-Léognan to other Bordeaux appellations, especially to the Medoc. Bidirectional strong flows are highlight between the vineyard and the cellar compartments as illustrated by the presence of 25% of commercial related strains in the vineyard, due to the unexpected return of strains through cellar to the vineyard, while a flow of similar magnitude is observed between the vineyard and the cellar. The presence of common ancestral populations in old (over 20 years) and recent samples showed population stability over the long-term at an appellation scale. A temporal succession of cellar populations was highlighted that could be link with the implementation of the Pied de Cuve method through successive inoculations.

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