Memoria para optar al Título Profesional de Médico Veterinario / Las enfermedades asociadas a circovirus porcino tipo 2 (PCV2) son consideradas el problema económico más grande de la industria porcina mundial. Además de mejorar la gestión y las prácticas de manejo (mejor higiene, menor hacinamiento y mejor ventilación), la disponibilidad de vacunas anti-PCV2 representa la opción inmunológica más eficaz para paliar el impacto de estas enfermedades. La proteína de la cápside de PCV2 (Cap) es un importante antígeno para el desarrollo de vacunas. Actualmente, la mayoría de las vacunas comerciales anti-PCV2 se producen como formulaciones inyectables de este antígeno. Aunque eficaces, estas vacunas tienen ciertas desventajas, incluyendo estrés animal e inmunosupresión concomitante, además de la implicancia de procedimientos laboriosos asociados y consumidores de tiempo. En este estudio, una cepa recombinante de la levadura Saccharomyces cerevisiae fue utilizada como vector y vehículo para entregar el antígeno de la cápside viral proveniente de un aislado nacional de PCV2. Este procedimiento se realizó con el fin de obtener datos dirigidos a estudiar el desarrollo de una vacuna oral contra el virus. De esta forma, el potencial inmunogénico de este sistema fue probado en ratones BALB/c, después de la administración de la levadura recombinante vía oral, bajo dos formulaciones distintas, como cepa viva y como extracto sonicado. Para esto, el gen cap químicamente sintetizado con la preferencia codogénica optimizada de la levadura (opt-cap) fue replicado en Escherichia coli y Saccharomyces cerevisiae a través del vector, pYES2. La expresión de la proteína Cap en la levadura fue confirmada a través de Western Blot con suero anti PCV2. Posteriormente, resultados de experimentación in vivo confirmaron que la administración oral en ratones de extractos sonicados de la levadura recombinante, estimulan la inducción de anticuerpos séricos y fecales específicos contra el antígeno Cap más eficientemente que la cepa viva. Estos resultados demostraron que es factible utilizar la levadura S. cerevisiae como un sistema seguro y simple de producir antígenos virales de PCV2, y de esta manera, su entrega vía oral podría ser una estratégica alternativa para inducir eficientemente anticuerpos anti-PCV2. Los resultados obtenidos en esta memoria en un modelo murino, están dirigidos a realizar investigación adicional en cerdos, el hospedero natural de PCV2 / Financiamiento: Proyecto Fondecyt de Iniciación en Investigación No. 1111013 y el Proyecto de Iniciación en Investigación de la Vicerrectoría de Investigación y Desarrollo de la Universidad de Chile, VID I 07/15-2

>Magister Scientiae - MSc / The yeast Saccharomyces cerevisiae is considered a good host used for heterologous protein production due to the organism’s microbial safety, rapid growth and eukaryotic post- translational processing. As a fermentative organism, S. cerevisiae is thus not only a useful platform for the production of biopharmaceuticals and industrial enzymes, but also a promising organism for second-generation biofuel production. Substantial effort has been focused on alleviating the many bottlenecks in recombinant gene expression, as well as in the secretory pathway to enhance heterologous protein titres. It was recently shown that highly active endocytosis could decrease the overall secreted protein titre in the supernatant. In this study, we aimed to block endocytotic and vacuolar complexes to ultimately disrupt, or impair, the endocytotic and vacuolar mechanisms of proteolysis and test the effect that this would have on secreted heterologous protein titres. This was accomplished by knocking out various genes involved in endocytosis and transforming the strains with genes encoding various hydrolases including β-glucosidase (Bgl), xylanase (Xyn2), endoglucanase (Eg2) and cellobiohydrylase (Cbh1). Our study demonstrated that genetic blocking of endocytotic mechanisms as well as vacuolar complexes could theoretically improve heterologous protein secretion in S. cerevisiae. Endoglucanase (Eg2) titres displayed improvement of 26% and 30% in strains which had the RVS161 and VRP1 genes deleted and xylanase titres displayed an improvement of 71% and 143% in strains with the END3 and SSA4 gene deletions. Several of the gene knockouts tested improved Xyn2 and Eg2 titres but the effect of the different gene targets varied widely. A double knock-out strain with deletions in CLC1 and RVS161 secreted 104% more Eg2 than its parental control strain on a per dry cell weight basis, a significant synergistic improvement. Other double knock-out strains displayed additive or similar activities when compared to their controls. Cbh1 secretion could not be improved through the gene deletions tested in our study and Bgl activity could not be measured in our transformants. These results demonstrate the different relationships of various heterologous proteins with various components of the secretion machinery and may also imply how endocytosis as well as vacuolar complexes affect the level of secreted protein.

Les potentialités liées aux cultures mixtes de micro-organismes sont immenses et ouvrent de vastes possibilités pour l’innovation dans les bioprocédés. Cependant, étant donné la complexité des phénomènes régulant la physiologie d’un seul micro-organisme, leur mise en oeuvre en culture mixte est d’autant plus difficile à maîtriser que des interactions entre ces micro-organismes viennent s’ajouter. Le travail présenté dans ce manuscrit est une étude d'un couple de levures oenologiques constitué de Saccharomyces cerevisiae et de Torulaspora delbrueckii. Son objectif est d'acquérir des informations expérimentales afin d’analyser et in fine modéliser l'évolution des cultures pures et des cultures mixtes. L'analyse des données expérimentales acquises lors des cultures pures de chaque levure a permis de quantifier l'effet favorable de la concentration initiale d'azote assimilable sur la croissance et la vitesse de fermentation des deux levures. Elle a aussi mis en évidence une prolongation de la phase de latence de T. delbrueckii induite par une augmentation des facteurs anaérobies. Les cultures mixtes ont été réalisées en présence et en absence de membrane de séparation permettant ainsi d'observer les effets du contact physique sur l'évolution des cocultures. Le contact physique influence la dynamique des populations. En culture mixte, S. cerevisiae domine T. delbrueckii dans un milieu synthétique classique. Une augmentation de la concentration initiale de facteurs anaérobies inverse complètement cette domination. L'analyse des résultats expérimentaux nous a orienté vers le développement d'un modèle «stoechio-cinétique» structuré dans lequel l'azote assimilé par la levure se répartit en deux compartiments: le compartiment constitutif et le compartiment de stockage. Ce modèle a permis une représentation fidèle des cinétiques observées en culture pure. La prise en compte des interactions s'est faite en intégrant la compétition pour les substrats, les interactions indirectes et les interactions directes. L'ensemble des hypothèses émises lors de ce travail de modélisation souligne la nécessité d'approfondir les connaissances scientifiques concernant le métabolisme deT. delbrueckii en anaérobiose stricte et l'effet des facteurs anaérobies sur les interactions microbiennes.

Orientador: Sandro Roberto Valentini / Coorientador: Cleslei Fernando Zanelli / Banca: Mário Henrique Bengtson / Banca: Carla Columbano de Oliveira / Resumo: O fator de tradução de eucariotos 5A (eIF5A) é altamente conservado em arqueas e eucariotos e sofre uma modificação pós-traducional única e essencial chamada hipusinação. Embora tenha sido sugerida inicialmente uma função para eIF5A no início da tradução, foi na etapa de elongação que sua função foi melhor demonstrada. Estudos prévios mostraram que eIF5A tem algum papel na translocação de proteínas pela via co-traducional e que mutantes desse fator possuem níveis aumentados de proteínas envolvidas com o estresse de retículo endoplasmático (RE). Desta forma, nesse trabalho, foi estudada a correlação entre eIF5A e a via de resposta ao estresse de RE (Unfolded Protein Response - UPR). Considerando que o fator de transcrição Hac1 é um elemento essencial da via UPR em Saccharomyces cerevisiae, foi inicialmente verificado se eIF5A afetava o splicing citoplasmático do mRNA de HAC1, o qual é dependente da ativação de Ire1, um sensor de proteínas desenoveladas no interior do RE. Para isto, foi realizada a quantificação dos níveis de mRNA maduro e imaturo de HAC1 por meio de ensaios de qPCR e RT-PCR semiquantitativa, análises estas que não revelaram qualquer diferença de comportamento do mutante hyp2-3 de eIF5A em relação ao selvagem. Por outro lado, diferentes mutantes de eIF5A mostraram sensibilidade a DTT e resistência a tunicamicina, um comportamento ainda não descrito na literatura e que difere de mutantes da via de SRP (via co-traducional de translocação de proteínas para o RE). ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The eukaryotic translation factor 5A (eIF5A) is highly conserved in archaea and eukaryotes and undergoes a unique and essential post-translational modification called hypusination. Although a function for eIF5A was initially suggested at the initiation of translation, it was in the elongation step that its function was best demonstrated. Previous studies have shown that eIF5A has a role in protein translocation by the co-translational pathway and that mutants of this factor have increased levels of proteins involved with endoplasmic reticulum (ER) stress. Therefore, in this work, the correlation of eIF5A with the stress response pathway of ER (the Unfolded Protein Response - UPR) was studied. Considering that the Hac1 transcription factor is an essential element of the UPR pathway in Saccharomyces cerevisiae, it was first verified whether eIF5A affects the cytoplasmic splicing of the HAC1 mRNA, which is dependent on the activation of Ire1, a sensor of unfolded proteins in the RE. To test it, we did the quantification of the mature and immature mRNA levels of HAC1 by means of qPCR and semi-quantitative RT-PCR assays, which did not reveal any difference in the behavior of the eIF5A mutant hyp2-3 in comparison to the wild-type. On the other hand, different mutants of eIF5A show sensitivity to DTT and resistance to tunicamycin, a behavior not yet described in the literature and that differs from mutants of the SRP pathway (co-translocation pathway for RE) and also this behavior of eIF5A mutants was not modified by overexpression of SRP. In addition, the eIF5A mutant phenotype in DTT and tunicamycin differs from that of knockout strains of ribosomal proteins, knockout of a ribosome biogenesis factor... (Complete abstract click electronic access below) / Mestre

O vinho é a bebida obtida por meio da fermentação alcoólica do mosto simples de uva sã, fresca e madura. Durante o processo fermentativo, realizado por leveduras Saccharomyces cerevisiae, ocorre uma sucessão de microrganismos. Espécies pertencentes aos gêneros Brettanomyces e Dekkera permanecem no produto final podendo influenciar de forma negativa ao produzirem compostos fenólicos voláteis. Estas leveduras apresentam como uma de suas características a lenta taxa de crescimento. Com isto, este trabalho teve como objetivo o desenvolvimento de meio de cultivo para isolamento e detecção destas leveduras e de metodologia molecular para detecção independente de cultivo. O meio de cultivo desenvolvido permitiu o isolamento e também a diferenciação de leveduras dos gêneros Brettanomyces e Dekkera das demais leveduras comumente encontradas em vinho. Uma metodologia molecular, baseada em PCR convencional, foi desenvolvida para a detecção dos gêneros em vinhos. Inicialmente foram construídos dois pares de oligonucleotídeos. Um par universal para leveduras e outro específico para D. bruxellensis. O primeiro par apresentou ampla aplicabilidade na avaliação da efetividade de diferentes processos de extração de DNA e na detecção de compostos inibidores presentes em amostras de vinho. A utilização de ambos os pares permitiu o desenvolvimento de um protocolo de extração e amplificação de DNA diretamente de amostras de vinho, facilitando a identificação de leveduras D. bruxellensis e permitindo a tomada de decisão em tempo hábil de evitar perdas econômicas. / The wine is a beverage obtained by alcoholic fermentation of simple must of healthy, fresh and mature grape. During the fermentation process, carried out by Saccharomyces cerevisiae yeasts, these is a succession of microorganisms. Species belonging to the genera Brettanomyces and Dekkera remain in the final product can influence negatively by producing volatile phenolic compounds. These yeasts present as one the features of the slow rate of growth. This work aimed at the development of culture media for isolation and detection of these strains and molecular methods for detection of independent culture. The medium developed also allowed the isolation and differentiation of yeasts of the genera Brettanomyces and Dekkera yeasts from other commonly found in wine. A molecular approach, based on conventional PCR, was developed for the detection of genera in wines. Initially we constructed two sets of primers. A universal pair for yeast and other specific for D. bruxellensis. The first pair showed a broad applicability in evaluating the effectiveness of various procedures for DNA extraction and detection of inhibitory compounds present in wine samples. The use of both pairs allowed development of a protocol for extracting and amplifying DNA directly from wine samples, facilitating the identification of yeasts D. bruxellensis and allowing the decision-making in a timely manner to avoid economic losses.

O Ácido Valproico (AV) é um fármaco antiepiléptico amplamente utilizado. Além disso, estudos recentes demonstram interessantes atividades antitumorais do AV contra diferentes tipos de tumores relacionado a sua atividade no remodelamento da cromatina. Entretanto, o uso do AV em pacientes possui limitações devido à sua reconhecida toxicidade sistêmica. Desta forma, é reforçada a necessidade do descobrimento de novos medicamentos com menor toxicidade e/ou maior efeito terapêutico. Para tanto, a estratégia mais sensata e econômica para se obter novos fármacos consiste em modificar quimicamente drogas já conhecidas. A síntese de complexos metálicos é uma abordagem recente para a obtenção de fármacos. Assim, neste trabalho foi analisado a citotoxicidade, mutagenicidade para o valproato de sódio (NaValp) e complexos de AV com Cu(II), Mg(II), 1,10-fenantrolina (Phen): [Cu2(Valp)4], [Cu(Valp)2Phen] e [Mg(Valp)2Phen] na levedura Saccharomyces cerevisiae proficientes e deficientes em vias de reparo de excisão de base – BER (apn1Δ, apn2Δ, ogg1Δ, rad27Δ, ntg1Δ, ntg2Δ mag1Δ), de excisão de nucleotídeos – NER (rad1Δ, rad4Δ, rad10Δ, rad14Δ), síntese de translesão – TLS (rev1Δ, rev3Δ, rad30Δ), reparação pós-replicação – PRR (rad6Δ, rad18Δ), recombinação homóloga – HR (rad52Δ) e junção final não homóloga – NHEJ (rad50Δ). Os resultados indicam que os complexos organometálicos têm maior citotoxicidade e são mais mutagênicos do que NaValp em fase de crescimento da levedura. Nos ensaios de mutagênese, não houve indução de mutação com NaValp, e os complexos organometálicos induziram substituições de pares de bases, identificadas pelo aumento da frequência de mutação nos loci his1 e lys1 na linhagem XV185-14C. As linhagens deficientes em proteínas da via BER foram sensíveis ao NaValp. Além disso, os danos no DNA causados pelo NaValp também podem ser tolerados pelas vias NER e TLS. As vias BER, NER, TLS, PRR e HR contribuíram com a tolerância ao dano induzido pelos complexos de cobre. O dano ao DNA induzido pelo complexo [Mg(Valp)2Phen] pode ser tolerado pelas vias NER, TLS, PRR, HR e NHEJ, enquanto a presença de endonucleases de BER resulta em morte celular. Desta forma, nosso estudo revela uma importante contribuição das vias de reparo do DNA sobre a sensibilidade ao NaValp e aos complexos organometálicos, indicando sua potencial aplicação como agentes citotóxicos. Os mecanismos de reparação diferiram para os compostos, sugerindo que induzem diferentes lesões no DNA. / Valproic Acid (VA) is an antiepileptic drug largely used. In addition, recent studies present interesting antitumor activities of VA against different types of tumors related to its activity in chromatin remodeling. However, the use of VA in patients has limitations due to its recognized systemic toxicity. Thus, it is necessary to discover new drugs with less toxicity and/or greater therapeutic effect. For that, the most sensible and economical strategy to obtain new drugs is to chemically modify known drugs. The synthesis of metal complexes is a recent approach to obtaining drugs. Therefore, the cytotoxicity and mutagenicity were analyzed for sodium valproate (NaValp) and VA complexes with Cu(II), Mg(II) and 1,10-phenantroline (Phen): [Cu2(Valp)4], [Cu(Valp)2Phen] and [Mg(Valp)2Phen] in Saccharomyces cerevisiae strains deficient in repair pathways, like base excision repair – BER (apn1Δ, apn2Δ, ogg1Δ, rad27Δ, ntg1Δ, ntg2Δ mag1Δ), nucleotide excision repair – NER (rad1Δ, rad4Δ, rad10Δ, rad14Δ), translesion synthesis – TLS (rev1Δ, rev3Δ, rad30Δ), post replicative repair – PRR (rad6Δ, rad18Δ), homologous recombination – HR (rad52Δ) e non-homologous end joining – NHEJ (rad50Δ). The results show that the organometallic complexes have higher cytotoxicity and are more mutagenic than NaValp in yeast growth phase. In the mutagenesis assay, there was not induction of mutation by the NaValp, whereas the organometallic complexes induced base pair substitutions, identified by the increase of mutation frequency in his1 and lys1 loci in the XV185-14C strain. The protein-deficient strains of the BER pathway were sensitive to NaValp. In addition, NER and TLS pathways can also tolerate DNA damage caused by NaValp. The BER, NER, TLS, PRR and HR pathways contributed to the damage tolerance induced by the copper complexes. DNA damage induced by [Mg(Valp)2Phen] complex can be tolerated by the NER, TLS, PRR, HR and NHEJ pathways, while the presence of BER endonucleases results in cell death. In this way, our study reveals an important contribution of the DNA repair pathways on sensitivity to NaValp and organometallic complexes, indicating the potential application as cytotoxic agents. The repair mechanisms are distinct for the compounds, suggesting that different lesions are induced in the DNA.

Both an intuitive observation and maybe the most mysterious process of biology, aging describes the progressive deterioration of cellular functions with time. Asymmetric cell divisions stand at the center of ability to reset age in offspring and for stem cells to self-renew. This requires the asymmetric segregation of age-determinants, many of which have been identified in the budding yeast Saccharomyces cerevisiae. We here use budding yeast to explore fundamental aspects underlying the asymmetric inheritance of mitochondria and the concurrent rejuvenation of daughter cells. We show that in addition to the preferential inheritance of high-functioning mitochondria to daughter cells, a distinct population of high-quality organelles must also be retained within the mother cell. We find that both physical retention and qualitative maintenance of a distinct mitochondrial population at the mother cell tip depends on Mitochondrial F-box protein (Mfb1p) and that MFB1-deletion leads to premature aging. Our findings outline a critical balance between the need for daughter cell rejuvenation and the requirement to conserve replicative potential within the mother cell. The particular mechanism by which Mfb1p functions further lead us to uncover a critical role of globally maintained cellular polarity in form of an axial budding pattern in lifespan regulation, the functional significance of which thus far remained essentially unexplored. We also find that the asymmetric localization of Mfb1p depends on potentially novel structures of the actin cytoskeleton and the loss of Mfb1p-polarization with age may accurately predict remaining cellular lifespan.

Life on Earth relies on a set of instructions encoded within an organism’s genome that is passed along from one generation to the next. Inherent to this mechanism of propagation is the need to copy the genetic material before passing it along to the progeny. Errors in this process coupled with stochastic damage will inevitably lead to changes in these instructions and may result in a reduction of fitness or even death of an individual. Yet, these same changes are also responsible for the adaptation mandated by our dynamic environment. Thus, there exists a delicate balance between maintenance and alteration of genetic material that is embodied to a large part at the various intersections of DNA replication, recombination and repair. Homologous recombination (HR) has been well studied and found to play vital roles in many cellular processes from the repair of the harrowing double-stranded break, the restart of a stalled or collapsed replication fork, as well as proper chromosome segregation during meiosis, all with the goal of striking this delicate balance. And yet, while HR is incumbent for the fitness of an organism, if left unchecked this same process can become detrimental by preventing better suited DNA repair pathways, permanently arresting cell cycle progression and creating some of the very problems it was meant to address such as aneuploidy or cancer. Despite a wealth of knowledge, the precise regulatory mechanisms remain an active area of research as they provide likely targets to combat these persistent diseases. Motor proteins that translocate along DNA have been particularly compelling and elusive due to their transitory nature, as well as the inevitability of collisions with bound protein(s) or nucleic acid structures that are likely regulated intermediates in the process. The yeast Srs2 helicase/translocase has long been regarded as the prototypical “anti-recombinase” as it has been shown to dismantle the Rad51 presynaptic filament, but also displays contradictory pro-recombinase functions. In vivo studies of Srs2 have been hampered by its involvement in multiple bioprocesses beyond recombination, while bulk in vitro approaches often produce conflicting results. Recent single molecule imaging of these players has shed light onto their involvement in the regulation of the various stages of the canonical pathway of HR. The Greene laboratory has developed ssDNA curtains to study the pre-synaptic filament and shown that Rad51-ssDNA filaments can create bonafide D-loop intermediates that would be incapable of repair and thus represent a toxic intermediate. These structures persist far longer than the entire process of DSBR in vivo and led us to hypothesize that motor proteins would be a key regulatory element to dismantle improperly paired intermediates for redistribution of the bound proteins and reengagement of the homology search process. Here I extend the use of ssDNA curtains to study Srs2 as it assembles into multimeric complexes to perform long-range disruption of various pre- and post-synaptic filament assemblies that include replication protein A (RPA), Rad51, Rad52, and D-loops. For the first time, direct observation of Srs2 translocating over RPA filaments is provided and shows these proteins are efficiently removed by Srs2. By including Rad52 on the RPA filament, I offer a refined model of the contradictory pro- and anti-recombinase activities of Srs2 through its antagonism of the single-strand annealing pathway in favor of HR. Additionally, Srs2 was found to initiate Rad51 disruption at breaks in the continuity of the filament marked by the persistence of replication protein A (RPA), Rad52, or the presence of an improper D-loop intermediate, the latter of which is efficiently disrupted before continuing translocation. In contrast to the prevailing model, we demonstrate that direct interaction between Srs2 and Rad51 is not necessary for long-range Rad51 clearance. These findings offer insights into the dynamic regulation of crucial HR intermediates by Srs2 and demonstrate that sub-nuclear concentrations of these proteins may be a likely driver for their activities.

RNA polymerase II (RNAP II) is responsible for transcribing messenger RNAs (mRNAs) as well as non-coding RNAs such as small nuclear RNAs (snRNAs) and microRNAs in eukaryotic cells. Rpb1, the largest catalytic subunit of this complex, possesses a unique C-Terminal Domain (CTD) that consists of tandem heptad repeats (the number varying from 26 to 52 by organism) with the consensus sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser (Y1S2P3T4S5P6S7). The CTD is extensively phosphorylated and dephosphorylated on non-proline residues during different steps of the transcription cycle, with roles for the threonine (Thr4) and tyrosine (Tyr1) attracting more attention. For example, in chicken cells, Thr4 functions in histone mRNA 3’ end formation, and Tyr1 phosphorylation is primarily associated with promoters and upstream antisense RNA formation, as well as preventing degradation of the polymerase, processes not found across all eukaryotes. A detailed introduction is described in Chapter 1. Taking advantage of the genetic tractability of yeast cells, we created a yeast (S. cerevisiae) strain with all CTD threonines substituted with valines (T4V) to study the role of CTD Thr4 in transcription in yeast, which prior to this study has been poorly characterized in S. cerevisiae. Using the T4V strain, we found that Thr4 was required for proper transcription of phosphate-regulated (PHO) and galactose-inducible (GAL) genes. We found genetic links between the T4V polymerase and genes encoding subunits of the Swr1 and Ino80 chromatin remodeling complexes, as well as the histone variant Htz1. We further provide evidence that CTD Thr4 is required for proper eviction of Htz1 by the Ino80 complex from genes requiring Thr4 for activation, presented in Chapter 2 of this thesis. Finally, Chapter 3 describes the functions of CTD Tyr1 in S. cerevisiae. Using a strategy similar to the T4V strain, I created a strain expressing an endogenous Rpb1 with all CTD tyrosine residues mutated to phenylalanine (Y1F). We found that this strain was viable, but with a severe slow-growth phenotype. We found genetic links between the Y1F polymerase and kinase/cyclin pair Srb10/Srb11, as well as an increase in occupancy on chromatin for the same. Further analysis indicated that RNA levels of genes associated with MAP Kinase associated stressors were dysregulated, and poly(A) site selection was biased towards distal poly(A) sites. Next, using an in vitro kinase assay, we showed Tyr1 phosphorylation on the CTD by MAP kinase Slt2, and in vivo CTD Tyr1 phosphorylation levels changed based on Slt2-associated stress response, as well as a decrease in in vivo Tyr1P-RNAP II from an Slt2 kinase-dead strain. Analysis of termination factors Nrd1 and Rtt103 showed transcription termination defects were likely the result of disruption of the interaction between the CTD interacting domains of these two proteins and the Y1F CTD. Extending this, we found additional disruptions in Slt2 recruitment to chromatin, increasing the depth of our knowledge of the interplay between induction of stress-associated genes, Slt2 function, and Nrd1-mediated termination.

Made available in DSpace on 2014-06-11T19:31:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-02-17Bitstream added on 2014-06-13T19:19:52Z : No. of bitstreams: 1 mirandajunior_m_dr_araiq.pdf: 884289 bytes, checksum: 19e1ba1c563a31b9cb660f1d7028b16c (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A principal meta deste trabalho foi a de realizar estudos com leveduras industriais brasileiras utilizadas em usinas, na tentativa de viabilizar o emprego da Tecnologia de Fermentação de Mostos com Altos Teores de Açúcares Fermentecíveis, na produção de etanol combustível. Inicialmente, foram realizados experimentos para obtenção de informações relativas as linhagens, na fermentação de sacarose, maltose e glicose, em meios contendo uma base nitrogenada e suplementado com fontes de nitrogênio com diferentes complexidades estruturais. Os estudos mostraram que as linhagens industriais apresentam diferentes perfis de crescimento nos meios suplementados com diferentes fontes de nitrogênio, que variaram de um simples sal de amônio, a um hidrolisado ácido de proteínas (casaminoácidos) e hidrolisado enzimático de proteínas (peptona). Entretanto, a maior capacidade de acúmulo de biomassa pode não ter um reflexo direto na capacidade de utilização de açúcares e a consequente produção de etanol pelas leveduras industriais. Além da natureza estrutural da fonte de nitrogênio e do tipo de açúcar, a presença do oxigênio, em maior quantidade nos cultivos agitados, interferiu sobremaneira no desempenho fermentativo das leveduras industriais, e até no acúmulo de trealose. No geral, foi a suplementação com peptona que propiciou maior produção de biomassa, preservação da viabilidade celular e consumo mais eficiente da fonte de carbono, em cultivos agitados e não agitados. Os estudos com alta densidade celular tiveram como objetivo definir condições experimentais para a condução da Fermentação de Mosto com Altos Teores de Açúcares Fermentescíveis, e que pudessem propiciar ao final do processo a completa utilização da sacarose, associada á preservação da viabilidade... / In this work studies with Brazilian yeast for ethanol production were conducted in order to verify the possibility of utilization of brazilian yeasts for ethanol production in an industrial process known as Very High Gravity Sucrose Fermentation Technology, to produce wine with high ethanol contends. This technology has the advantage to reduce production costs by increasing fermentation yields. Initially, studies were conducted with sucrose, maltose and glucose fermetation in medium containing YNB, supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Data suggest that yeast strains vary in their response to nitrogen source complex structure, kind of sugar and to oxygen availabity. In general, under peptone supplementation all strains, in shaking and static conditions, showed higher biomass accumulation, efficient sugar utilization and yeast viability was preserved. Sugar utilization by industrial strains not always was directly correlated with higher biomass accumulation. Trehalose accumulation was also influenced by the structural complexity of nitrogen sources, the kind of sugar and the presence of oxygen. Studies with high cell density were conducted to define experimental parameters for Very High Gravity Sucrose Fermentation, in order to induce at the end of process full sugar exhaustion together cell viability preservation. Complete sucrose utilization was detected only in media with 22 and 25% (w/v) sucrose. In the presence of higher sucrose contends (30% (w/v)), total sucrose exhaustion was obtained in a sugar cane based medium, supplemented with 2% (w/v) peptone. The results described in this thesis suggest that industrial yeasts show differing nitrogen demand, and the utilization of Very High Gravity Sucrose Fermentation technology could be carried only after finding the appropriated nutritional and fermentation conditions