Orientador: Waldemar Gastoni Venturini Filho / Banca: Magali Leonel / Banca: Ricardo Figueira / Resumo: O objetivo deste trabalho foi avaliar a relação da concentração dos compostos fenólicos do mosto com a viabilidade celular, viabilidade de brotamento, brotamento celular da levedura alcoólica e a eficiência da fermentação por subprodutos durante a safra 2011/2012 na Usina São Manoel, São Manuel (SP). Esta unidade iniciou a safra com a levedura selecionada CAT-1. Durante a safra, as cepas nativas adentraram o processo fermentativo e também foram avaliadas quanto à sensibilidade a concentração dos compostos fenólicos. A pesquisa foi desenvolvida como um estudo de caso na planta industrial de fermentação contínua. As análises de concentração dos compostos fenólicos do mosto foram realizadas através do método FolinCiocalteu, enquanto que as análises de viabilidade celular, viabilidade de brotamento e brotamento da levedura foram feitas através de contagem em câmara de Neubauer utilizando corante azul de metileno. A eficiência da fermentação foi determinada por subprodutos gerados durante o processo fermentativo. A análise estatística foi avaliada pelo coeficiente de correlação de Pearson e sua significância através do teste t. Concluiu-se que, os compostos fenólicos contidos no mosto tiveram relação negativa sobre a viabilidade celularno período com leveduras 100% das nativas e no período geral da safra; sobre a viabilidade de brotamento somente o período com leveduras 100% nativas tiveram relação negativa. Os compostos fenólicosnão apresentaram relação com o brotamento celular da levedura e com a eficiência da fermentação em nenhum dos períodos estudados / Abstract: The aim of this project was to evaluate the relationship of the must phenolic compounds concentration with the cellular viability, budding viability, cellular budding of alcoholic yeast and the efficiency of the fermentation for by-products during the harvest 2011/2012 in São Manoel Sugar Mill, São Manuel (SP). This unit began the harvest with the selected yeast called CAT-1. During the harvest, the native stumps penetrated the fermentation process and they were also appraised as for the sensibility to the concentration of the phenolic compounds.The research was developed as a case study in the industrial plant of continuous fermentation. Analyses of concentration of the must phenolic compounds were accomplished through the FolinCiocalteu method, while the analyses of cellular viability, budding viability and budding yeast were made through counting in Neubauer chamber using a marker ( m-ethilen blue).The efficiency of the fermentation was determined using by-products generated during the fermentation process . Statistical analysis was evaluated by Pearson's correlation coefficient and its significance through the "t" test .It was concluded that the must phenolic compounds had negative relationship about the cellular viability in the periods with 100% of native yeasts along the general period of the harvest; about the budding viability only the period with 100% native yeasts had negative relationship. The phenolic compounds didn't present relationship with the budding cellular yeast and with the efficiency of the fermentation in none of the studied periods / Mestre

Orientador: Renato Wendhausen Júnior. / Dissertação (mestrado) - Universidade Regional de Blumenau, Centro de Ciências Exatas e Naturais, Programa de Pós-Graduação em Química.

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2015. / O aumento na demanda por energias sustentáveis impulsiona o desenvolvimento de estratégias biotecnológicas para a produção de biocombustíveis. Neste contexto, o aproveitamento eficiente da biomassa lignocelulósica como matéria prima é fundamental para a produção de etanol de segunda geração. A levedura Saccharomyces cerevisiae, organismo mais utilizado na produção industrial de bioetanol, é incapaz de utilizar pentoses, como a xilose, que é o segundo açúcar mais abundante em algumas biomassas. Neste trabalho, plasmídeos epissomais foram construídos para expressão de genes codificadores para xilose isomerase (XI) de Piromyces sp. e xiluloquinase (XK) de S. cerevisiae. As linhagens laboratoriais de S. cerevisiae CEN.PK 113.14A Δtrp1-289 (L2) e CEN.PK 113.3C Δtrp1-289, Δura-52 (L7) foram transformadas com os plasmídeos gerados. Desta forma, foram construídas linhagens recombinantes de S. cerevisiae expressando XI isoladamente (L2XI) ou em conjunto com XK (L7XIXK). Uma terceira linhagem, L7XIΦ, expressando XI e com o segundo plasmídeo vazio foi construída como controle. A linhagem L7XIXK apresentou melhores taxas fermentativas que as demais linhagens, confirmando o efeito positivo da expressão de XK. As linhagens L2XI, L7XIΦ e L7XIXK obtidas foram submetidas a um processo de condicionamento em meio seletivo contendo xilose como única fonte de carbono. Ao final do condicionamento, quando comparadas com as originais, apresentaram menor fase lag de crescimento e maior taxa de crescimento, maior consumo de xilose (entre 1,8 e 18,5 vezes) e rendimento de etanol (47% para L7XIXK), concomitante à diminuição do rendimento de xilitol (entre 87,6% e 91,8%). A linhagem L2XI, investigada anaerobicamente, também apresentou aumento no consumo específico de xilose (49,8%), rendimento de etanol (19%) e redução no rendimento de xilitol (75%). As linhagens condicionadas foram submetidas então a um processo de cura para remoção dos plasmídeos. Uma das linhagens obtidas, LC7, derivada de L7XIXK perdeu a capacidade de crescer em meio mínimo, indicando a perda dos plasmídeos. Entretanto o gene para XI ainda foi identificado na levedura. A retransformação desta levedura curada com os plasmídeos originais demonstrou que as melhorias da levedura condicionada podem estar associadas a mutações fora do plasmídeo. Esta linhagem curada tem grande potencial para desenvolvimento de uma linhagem de seleção para novas enzimas da via do catabolismo de xilose. / The increasing demand for sustainable energy drives the development of biotechnological strategies for the production of biofuels. In this context, efficient utilization of lignocellulosic biomass as feedstock is essential for the production of second generation biofuels. The yeast S. cerevisiae, main organism utilized in the industrial production of bioethanol, is unable to use pentoses, such as xylose, which is the second most abundant sugar in some biomasses. In this work, multi-copy plasmids were constructed for the expression of genes coding xylose isomerase (XI) from Piromyces sp. and xylulokinase (XK) from S. cerevisiae. The laboratory strains of S. cerevisiae CEN.PK 113.14A Δtrp1-289 (L2) and CEN.PK 113.3C Δtrp1-289, Δura-52 (L7) were transformed with the generated plasmids. Thus, recombinant strains of S. cerevisiae expressing solely XI (L2XI), or combined with XK (L7XIXK) were obtained. A third strain, L7XIΦ, expressing XI and with an empty second plasmid, was constructed as control. The L7XIXK strain presented better fermentative rates than the other strains, confirming the positive effect of XK expression. The obtained strains L2XI, L7XIΦ and L7XIXK underwent a conditioning process in selective medium with xylose as sole carbon source. At the end of the process, when compared to the original strains, conditioned strains presented shorter lag growth phase, and increased growth rate, increased xylose consumption (1,8 to 18,5 fold) and ethanol yield (47% for L7XIXK), along with reduction in xylitol production (between 87,6 and 91,8%). The strain L2XI was investigated under anaerobic conditions and also has presented improved xylose specific consumption (49,8%), ethanol yield (19%) and xylitol reduction (75%). The conditioned strains underwent a curing process, in order to remove the plasmids. One of the obtained strains, LC7, which derived from L7XIXK, has lost its ability to grow on minimal medium, a result consistent with the loss of plasmids. However the XI gene was still identified in the yeast. The retransformation of this curated yeast with the originally constructed plasmids showed that the improvements observed in conditioned strains may be associated with mutations outside of the plasmids. The curated strain has a great potential for the development of a screening strain for new enzymes of the xylose catabolic pathway.

Interstrand DNA crosslinks (ICLs) são importantes lesões genotóxicas que podem causar morte celular se não for devidamente reparadas. Diversas drogas antitumorais atuam através da indução de ICLs no DNA, e a resistência a essas drogas está muitas vezes relacionada à maior capacidade de células tumorais para reparar tais lesões. As ICLs são lesões complexas que requerem o envolvimento de diferentes vias de reparação, tais como BER NER, TLS e vias de reparação de DSBs (RH e NHEJ). Além disso, foi demonstrado que Pso2p desempenha um papel importante na reparação de ICLs. Muitos estudos também apontam para as alterações epigenéticas como essenciais para a sobrevivência da célula em resposta ao dano genotóxico, através da indução de checkpoints de ciclo celular e da modulação da eficiência (e, possivelmente, da escolha) das vias de reparação de DNA. Assim, a primeira parte desta Tese apresenta uma revisão dos mecanismos envolvidos na citotoxicidade dos ICLs induzidos pela furocumarina bifuncional 8-metoxipsoraleno (8-MOP) em Saccharomyces cerevisiae. A abordagem adotada discute a ação integrada de proteínas que atuam em vias de reparação de DNA envolvidas na remoção de ICLs, em combinação com fatores envolvidos no remodelamento da cromatina dependente de ATP. A fim de investigar o papel do remodelamento da cromatina em resposta a danos ao DNA induzidos pelo tratamento com furocumarinas mono e bifuncionais fotoativadas e ditelureto de difenila (DTDF), foram utilizadas linhagens de S. cerevisiae deficientes em diferentes proteínas relacionadas com a reparação do DNA e o remodelamento da cromatina. DTDF é um composto organotelurado proposto como um protótipo potencial para o desenvolvimento de novas drogas antitumorais. A sensibilidade do mutante pso2Δ ao DTDF, semelhante à observada para o 8-MOP, indica que este composto pode induzir lesões capazes de bloquear a replicação do DNA, tais como ligações cruzadas ou DSBs hairpin capped. Os resultados obtidos mostraram que os duplos mutantes pso2Δswr1Δ e rad52Δswr1Δ não são mais sensíveis aos agentes testados, DTDF, 3-CPS e 8-MOP, do que o mais sensível dos simples mutantes (pso2Δ e rad52Δ, respectivamente). Tendo em mente que Rad52p e Pso2p não têm interação epistática (aditiva ou sinergística) na reparação das lesões fotoinduzidas por 3-CPS e 8-MOP, sugere-se que Pso2p pode fornecer substrato para vias de reparação diferentes de HR, que poderiam ser NHEJ canônica e/ou MMEJ. O fato de que swr1Δ (deficiente em NHEJ) não aumenta a sensibilidade dos duplos mutantes, além da baixa sensibilidade observada para o mutante yku80Δ ao DTDF e à fotoadição de psoralenos, sugere que as lesões induzidas podem ser direcionadas para as vias HR ou MMEJ, ao invéz de NHEJ canônica. O envolvimento da via MMEJ sujeita a erros na reparação de lesões induzidas por DTDF pode explicar a indução de mutação frameshift, observada para este agente. Além disso, investigou-se a citotoxicidade induzida pelas drogas antitumorais cisplatina (CDDP), 5-fluorouracil (5-FU) e sua combinação, em S. cerevisiae. Os resultados obtidos com os mutantes de reparação do DNA mostraram que a sensibilidade à CDDP + 5-FU parece refletir a sensibilidade de linhagens específicas a cada droga utilizada individualmente. Este resultado sugere que o efeito intensificado do tratamento combinado na terapia do câncer pode ser devido à ação de cada agente isoladamente, conforme observado em clones diferentes da população heterogênea de células transformadas. Os resultados referentes aos mutantes em remodelamento da cromatina demonstraram que o tratamento com CDDP + 5-FU provoca uma resposta mais eficaz na indução de morte celular, nos mutantes deficientes em CR (ino80Δ), HATs (elp3Δ, gcn5Δ, hat2Δ e hpa2Δ), HDACs (sin3Δ, sir2Δ, hos2Δ , hst1Δ, hst2Δ, e hst3Δ), HML (dot1Δ) e HMT (erg6Δ), enquanto CDDP induz sensibilidade em mutantes HAT, além do mutante de HDAC hos3Δ, e o tratamento com 5-FU induz sensibilidade em HAT (gcn5Δ) e HDAC (sin3Δ e hos3Δ). Esses resultados apontam para o envolvimento de proteínas de remodelamento da cromatina como importantes alvos terapêuticos em resposta a agentes genotóxicos. Portanto, os resultados obtidos podem abrir novas perspectivas para a compreensão dos mecanismos envolvidos na resposta ao tratamento com CDDP + 5-FU, bem como auxiliar na escolha dos melhores protocolos terapêuticos. / Interstrand DNA crosslinks (ICLs) are important genotoxic lesions that can cause cell death if not properly repaired. Several antitumoral drugs act by inducing ICLs in DNA, and the resistance to these drugs is often related to an increased capacity of tumor cells to repair such lesions. The ICLs are complex lesions requiring involvement of different pathways for repair, such as BER, NER, TLS and DSB repair pathways (HR and NHEJ). In addition, Pso2p was shown to play an important role in ICLs repair. Many studies also point to the epigenetic changes as essential for cell survival in response to genotoxic damage, through induction of cell cycle checkpoints and modulating the efficiency (and possibly, the choice) of the DNA repair pathways. So, the first part of this Thesis gives a review of the mechanisms involved in the cytotoxicity of the ICLs induced by the bifunctional furocumarin 8-methoxypsoralen (8-MOP) in Saccharomyces cerevisiae. The adopted approach discusses the integrated action of proteins that work in the DNA repair pathways involved in ICLs removing in combination with factors involved in ATP-dependent chromatin remodeling. In order to investigate the role of the chromatin remodeling in response to DNA damage induced by treatment with mono- and bifunctional photoactivated furocumarins and diphenyl ditelluride (DPDT), we used S. cerevisiae strains deficient in different proteins related to DNA repair and chromatin remodeling. DPDT is organotellurium compound proposed as a potential prototype for development of new antitumoral drugs. The sensitivity of pso2Δ to DPDT, similar to that observed for 8-MOP, indicates that this compound could induce lesions able to block DNA replication, such as crosslinks or hairpin-capped DSBs. The obtained results showed that the double mutants pso2Δswr1Δ and rad52Δswr1Δ are not more sensitive to the tested DPDT, 3-CPS and 8-MOP agents than the more sensitive single mutant (pso2Δ and rad52Δ, respectively). Having in mind that Rad52p and Pso2p have non-epistatic interaction (additive or synergistic) in the repair of 3-CPS and 8-MOP photoinduced lesions, we suggest that Pso2p could provide substrate for repair pathway different from HR, which could be canonical NHEJ and/or MMEJ. The fact that swr1Δ (deficient in NHEJ) do not enhance the sensitivity of the double-mutants, in addition to the observed low sensitivity of the yku80Δ mutant to DPDT and psoralens photoaddition, suggests that the induced lesions could be directed to HR or MMEJ pathways rather than to canonical NHEJ. The involvement of the error-prone MMEJ in repair of DPDT-induced lesions could explain the observed frameshift mutation induction by this agent. Moreover, we investigated the cytotoxicity induced by the antitumoral drugs cisplatin (CDDP), 5-fluorouracil (5-FU), and their combination in S. cerevisiae. The results obtained with DNA repair mutants showed that the sensitivity to cisplatin + 5-FU appears to reflect the sensitivity of the specific strains to each individual drug used. This finding suggests that the enhanced effect of the combined treatment in cancer therapy could be due to the action of each agent alone, as observed on different clones from the heterogeneous population of transformed cells. The results concerning chromatin remodeling mutants demonstrated that CDDP + 5-FU treatment causes a more effective response in the induction of cell death, in CR (ino80Δ), HATs (elp3Δ, gcn5Δ, hat2Δ and hpa2Δ), HDACs (sin3Δ, sir2Δ, hos2Δ , hst1Δ, hst2Δ, and hst3Δ), HML (dot1Δ) and HMT (erg6Δ) mutants, while CDDP induces sensitivity in HAT mutants, besides the HDAC mutant hos3Δ, and 5-FU treatment induces sensitivity in HAT (gcn5Δ) and HDACs (sin3Δ and hos3Δ). These results point to involvement of chromatin remodeling proteins as important therapeutic targets in response to genotoxic agents. Therefore, our findings could provide new insights for understanding the mechanisms involved in the response to treatment with CDDP + 5-FU, as well as to help in the choice of optimal therapeutic protocols.

Meiosis is the unique process in which diploid cells undergo two consecutive divisions to produce haploid daughter cells. It is indispensable for sexual reproduction in all eukaryotic organisms and maintains proper chromosome number through generations. An integral step in the meiotic program is genetic recombination; recombination is required for a successful reductional division. In the yeast Saccharomyces cerevisiae, recombination is initiated by DNA double strand breaks (DSBs) that are created by ten recombination initiation proteins. Similar phenotypes are observed when any of these genes is mutated. This has made the mechanism by which these proteins function to initiate recombination difficult to unravel. One hypothesis is that these proteins form a functional complex for activity, in which all (or most) of them physically interact. The work described in Chapter 2 contributes to understanding the putative DSB-producing recombination initiation complex, suggesting there is substantial flexibility among initiation protein interactions. The results are also consistent with the view that the proteins assemble on the DNA. Studies in Chapter 3 examined the recombination initiation protein interactions during DSB formation in more detail using a novel experimental approach. While the initial experiments using this approach produced unexpected results, the assay is a promising tool for the future. In addition to creating DSBs, a subset of the initiation proteins perform a second function during early meiosis; they create a recombination initiation signal (RIS) to delay the onset of the reductional division in wild-type cells. Although the signal and the downstream target are well-defined, less is known about how the RIS is transduced to the downstream target. The work in Chapter 4 contributes to defining this transduction, and therefore enhances our understanding of the relationship between the recombination initiation proteins and the reductional division.

No description available.

The glycine decarboxylase complex (GDC) of Succharomyces cerevisiae composed of four subunits (P, H, T and L) and plays an important role in the interconversion of serine and glycine and balancing the one-carbon unit requirements of the cell. It also enables the cell to use glycine as sole nitrogen source. This study was concerned with characterising the molecular mechanism of transcriptional regulation of the GCVgenes encoding the subunits of the GDC. The important findings of this work can be summarised as follows: i) Transcription of the GCV genes are regulated by glycine and rich nitrogen sources, which are mediated by different cis-acting elements. The LPDl gene did not show a glycine response since its transcriptional regulation is distinct from that of the other genes encoding the GDC subunits. ii) Glycine analogues or serine did not affect expression of GCV2, and therefore glycine probably needs to be metabolised to effect the glycine response of the GCV genes. iii) The repression of the GCV2 gene expression by rich nitrogen sources is mediated by a sequence between -227 and -205 of GCV2, and NCR-regulatory mutant studies showed that repression is not directly controlled by the known NCR system. iv) The glycine response of GCV2 is mediated by a motif (the glycine regulatory region; GRR; 5'-CATCN7CTTCTT-3') with CTTCTT at its core. Additional sequence immediately 5' of this motif (between -310 to -289) plays a minor role for the gene's full glycine response. v) The GRR of the GCV genes can mediate the glycine response by either activation or repression, indicating that the transcription factor(s) mediating the glycine response is/are dual-functional in nature. vi) Studies of GCV2 gene expression using different regulatory mutants showed that expression of the gene is further modulated by other transcription factors such as and Baslp which are distinct from the glycine response and possibly involved in setting up the basal expression level. vii) I n vitro studies of the GRR-protein interaction revealed THF affects the affinity of the DNA-binding protein(s) for the GRR. The importance of THF in regulation of the GCV2 gene was also shown in vivo using a foll mutant that is unable to synthesise any folates. THF or a C1-bound derivative of it acts as a ligand for the transcription factor, thus influencing transcription of the GCV genes in the appropriate physiological manner. viii) Using heparin-Sepharose chromatography fractions, four complex formations (complex I to IV) were observed with the GRR. The protein responsible for one of these was separable from the others. EMSA profiles using the GRR of the GCVI and GCV2 genes (in the presence or absence of THF) were very similar, indicating that these genes bind the same proteins and are regulated in a similar manner. ix) Mutation of the CTTCTT motif within the GRR caused significant reduction in in vitro DNA-protein complex formation, however, THF addition overcame this reduction. x) Only complex II formation was observed with a DNA fragment spanning -322 to -295, and THF affected this complex formation. xi) Footprinting analyses of complex I revealed that the binding protein protected the GRR of the GCV2 gene from DNaseI activity. This protein is an excellent candidate for the glycine response regulatory protein. Titration experiments using EMSA showed that this protein can dimerise. A preliminary genome-wide analysis of the S. cerevisiae transcriptome was carried out using miniarray membrane hybridisation. This investigated the global transcriptional changes within the cell in response to the addition of glycine into the medium. Identification of genes related to various cellular processes including onecarbon metabolism gave an insight into the regulation of the cellular metabolic flow, especially that of one-carbon metabolism. The results indicated that: xii) Glycine is transported into mitochondria to be used as substrate for the GDC which (with mitochondria1 SHMT) produces serine that is subsequently utilised for the various one-carbon metabolic pathways, such as methionine synthesis and purine synthesis. xiii) A gene of unknown function (YER183C) which showed homology to the gene for human 5,lO-CH-THF synthetase was identified from gene-array analysis to be upregulated on glycine addition, indicating the protein encoded by this gene may be involved in balancing the metabolic flow between methionine and purine synthesis when THF pools are disturbed by glycine addition. xiv) Addition of glycine to the medium also triggers the expression of other metabolic genes related to amino acid biosynthetic pathways and that of many other genes which are not directly related to one-carbon metabolism. This may be due to prolonged culturing with glycine in the medium resulting in altered expression of genes mediated by one or more secondary factors. These may reflect an adaptive response rather than a direct consequence of glycine induction. On the basis of the above data, a model for the mechanisms regulating glycine response is presented.

This thesis is concerned with cellular responses to stress including the adaptive response to H2O2, and the cellular roles of sumoylation in stress responses. 286 H2O2-sensitive Saccharomyces cerevisiae deletion mutants were screened and YAP1, SKN7, GAL11, RPE1, TKL1, IDP1 were identified to be important for adaptation to H2O2. The mutants fell into two groups based on their responses to acute and chronic doses of H2O2. Transcription factors Yap1p, Skn7p and Gal11p were important for both acute and chronic responses to H2O2. Yap1p and Skn7p were needed for up-regulation of anti-oxidant functions rather than generation of NADPH or glutathione. Adaptation was reduced in strains deleted for GPX3 and YBP1, which are involved in sensing H2O2 and activating Yap1p, but to a lesser extent than YAP1 deletion. RPE1, TKL1 and IDP1 deletants affected in NADPH production were chronically sensitive to H2O2, but resistant to an acute dose and other mutants affected in NADPH generation were also affected in adaptation. These mutants overproduced reduced glutathione (GSH) but maintained normal cellular redox homeostasis. Over-production of GSH was not regulated by transcription of the gene encoding -glutamylcysteine-synthetase. The Skn7p transcription factor is therefore important for the adaptive response to oxidative stress-induced by H2O2, and NADPH generation is also required for adaptation. The roles of sumoylation in stress responses and transcriptional regulation were examined by deleting the SUMO ligases Siz1p and Siz2p. Siz1p is required for tolerance to copper ions and DNA damage repair. Siz2p is involved in repression of stress responses, particularly oxidative stress and is required for activation of nucleotide and RNA metabolism, DNA processing and cell division. Both Siz1p and Siz2p act in parallel in the repressing heat-shock responses and in reducing chronological life span. Genome-wide transcriptional analysis showed that Siz1p and Siz2p repress the mitochondrial retrograde pathway and arginine biosynthesis, while activating some carbon and nitrogen metabolism genes. Sumoylation of proteins in the wild type was induced by nitrogen starvation or mitochondrial inhibition during the initial treatment. However, nitrogen starvation led to some protein degradation, while the SUMO-conjugated proteins were recycled in cells with disrupted mitochondrial functions.

Thesis (M.S.) -- Worcester Polytechnic Institute. / Keywords: IAA; S. cerevisiae; quorum sensing. Includes bibliographical references (p.57-59).

Thesis (Ph. D.)--Ohio State University, 2006. / Full text release at OhioLINK's ETD Center delayed at author's request