Made available in DSpace on 2014-06-11T19:31:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-07-23Bitstream added on 2014-06-13T19:19:49Z : No. of bitstreams: 1 rossetto_db_dr_araiq_parcial.pdf: 291749 bytes, checksum: ae8637199d26161742ff3d85e6251821 (MD5) Bitstreams deleted on 2014-08-22T14:57:08Z: rossetto_db_dr_araiq_parcial.pdf,Bitstream added on 2014-08-22T15:02:09Z : No. of bitstreams: 1 000713136.pdf: 2398877 bytes, checksum: 8df0c65c21bdaeccecb3792c6d29b28f (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Ácido retinóico (AR) regula a transcrição de uma série de genes envolvidos em proliferação celular, diferenciação celular e apoptose através de sua ligação com o receptor de ácido retinóico (RAR) ligado com o receptor de retinóide X (RXR) na forma de heterodímero. A proteína ligante de ácido retinóico celular (CRABP2) é envolvida no transporte do AR do citoplasma para o núcleo, atuando assim como uma proteína coativadora dos receptores retinóides nucleares. Com o objetivo de melhor entender o mecanismo de sinalização celular por ácido retinóico envolvendo CRABP2, foi utilizado o sistema de duplo-híbrido em levedura como uma ferramenta para identificação de interações físicas proteína-proteína. Um total de 20 proteínas candidatas ligantes de CRABP2 foram identificadas no rastreamento de duplo-híbrido, das quais cinco são relacionadas com regulação da transcrição, mais especificamente com o processo de remodelagem da cromatina: polipeptídeo do complexo T (TCP1), histona H3/família 3A (H3F3A), histona H3/família 3B (H3F3B), tubulina beta (TUBB) e fator associado ao CTD relacionado com SR (SCAF1). Esses resultados sugerem um papel mais direto de CRABP2 na remodelagem de cromatina e poderá revelar novos aspectos da regulação da transcrição mediada por receptores de ácido retinóico. Além disso, também foi abordado neste trabalho um estudo dos níveis de expressão dos receptores de ácido retinóico em resposta a agentes desmetilantes / Retinoic acid (RA) regulates the transcription of a series of genes involved in cell proliferation, differentiation and apoptosis by binding to RA Receptor (RAR) and Retinoid X Receptor (RXR) heterodimers. The cellular retinoic acid-binding protein 2 (CRABP2) is involved in the transport of RA from the cytosol to specific RA receptors in the nucleus, acting as a coactivator of nuclear retinoid receptors. In order to have a better understanding of the mechanism of cellular signaling by retinoic acid involving CRABP2, we used the yeast two-hybrid system as a tool for the identification of physical protein-protein interactions. A total of twenty putative CRABP2-interacting proteins were identified in the two-hybrid screen, out of which five are related to transcription regulation, more specifically to the process of chromatin remodeling: t-complex 1 (TCP1); H3 histone, family 3A (H3F3A); H3 histone, family 3B (H3F3B); tubulin beta (TUBB) and SR-related CTD-associated factor 1 (SCAF1). These results suggest a more direct role for CRABP2 in chromatin remodeling and may reveal new aspects of the control of transcription by RA receptors. Furthermore, we have also investigated the expression levels of the retinoic acid receptors, in mammalian cells in response to demethylating agents

Made available in DSpace on 2014-06-11T19:31:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-07-24Bitstream added on 2014-06-13T20:41:04Z : No. of bitstreams: 1 batistote_m_dr_araiq.pdf: 1258136 bytes, checksum: f9f580d2a88ef74edcdd73a29908a401 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O presente trabalho teve como objetivo principal realizar estudos do efeito da complexidade estrutural de fontes de nitrogênio no fluxo metabólico do carbono em leveduras industriais utilizadas nas indústrias de panificação, de produção de vinhos e cervejas. Os resultados obtidos com os carboidratos trealose e glicogênio estão de acordo com o comportamento esperado para o acúmulo destes compostos, uma vez que foi observado que a quantidade destes carboidratos de reserva nas células sofrem acentuadas variações em resposta a diferentes alterações nutricionais experimentadas pelas leveduras durante o processo fermentativo, e isto ocorre como conseqüência do complexo sistema regulatório que controla a produção dos carboidratos. A maioria dos dados indica que a quantidade de trealose e glicogênio foram sempre maiores na suplementação com amônio e menor com peptona. A concentração de trealose e glicogênio produzidas pelas linhagens talvez reflita o processo de seleção a que foram submetidas as linhagens, o tipo a e concentração da fonte de carbono, o tempo de fermentação e também com a natureza estrutural da fonte de nitrogênio. / The present work had as main objective to carry out studies of effects of the structural complexity of nitrogen sources in the metabolic flux of carbon in industrial yeasts used in the production of bread, wines, and beers. The results obtained with the carbohydrates trehalose and glycogen are in accordance with the expected behavior for the accumulation of these compounds, once it was observed that the amount of these reserve carbohydrates of in the cells suffers accented variations in response to the different nutritional alterations experienced by the yeasts during the fermentative process. This may occurs as consequence of the complex regulatory system that controls the production of the carbohydrates. The majority of the data indicates that the amount of trehalose and glycogen was always higher under ammonium and casamino acids supplementation than with peptone. Perhaps the concentration of trehalose and glycogen produced by the strains reflects the process of selection that the strains were submitted, the type and concentration of the carbon source, the time of fermentation and also with the structural nature of the nitrogen source.

Meiosis is the process by which diploid cells undergo DNA synthesis, homologous recombination and pairing, followed by the reductional division then the equational division. I present work in this PhD thesis which furthers the understanding of the coordination of the initiation of meiotic recombination and the reductional division. Ten genes are required to initiate recombination in Saccharomyces cerevisiae. The presence of a subset of recombination initiation proteins creates a Recombination Initiation Signal (RIS) that delays the start of MI in wild type cells. I present experiments demonstrating the first division kinetics of the two remaining recombination initiation genes that our lab had not yet studied. Rec107 is part of the RIS, while Ski8 is not. The RIS is conserved in a divergent Saccharomyces strain background. rec102 and rec104 SK1 strains both start the first division earlier that wildtype SK1 strains. I present evidence that suggests that the RIS acts independently of the pathway that controls securin (PDS1) degradation. The work in this thesis expands our knowledge of the mechanism by which the RIS delays the reductional division. In this thesis I present experiments showing that the DNA damage, spindle and S phase checkpoints do not transduce the RIS. I establish the meiosis-specific candidate Mek1 as a candidate for relaying the RIS. Lastly, experiments described in these chapters show that the transcriptional activator of Middle Meiosis, NDT80, is the target of the RIS. NDT80 transcription and activity are both necessary and sufficient to affect an earlier reductional division, similar to the early MI seen in RIS mutants.

No description available.

No description available.

Thesis (Ph. D.)--University of Oregon, 2006. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 93-99). Also available for download via the World Wide Web; free to University of Oregon users.

Development of genome-wide reagents has allowed systematic analysis of gene function. The experimental accessibility of budding yeast makes it a test-bed for technology development and application of new functional genomic tools and resources that pave the way for comparable efforts in higher eukaryotes. In this Thesis, I describe a two-color GFP-RFP reporter system I developed to assess the consequences of genetic perturbations on a promoter of interest. The dual-reporter system is compatible with the synthetic genetic array methodology, an approach that enables marked genetic elements to be introduced into arrays of yeast mutants via an automated procedure. I use this approach to probe cell cycle-regulation of histone gene transcription by introducing an HTA1 promoter-GFP reporter gene construct into an ordered array of ~4500 yeast deletion mutants. I scored defects in reporter gene expression for each mutant, generating a quantitative analysis of histone promoter activity. The results of my screen motivated a number of follow-up experiments, including chromatin immunoprecipitation, transcript profiling and genome-wide analysis of nucleosome positions, which revealed a previously unappreciated pathway that specifies regions of repressed chromatin in a cell cycle-sensitive manner. A novel aspect of this pathway is that it involves histone chaperones and a chromatin boundary element. Specifically, we discovered that the histone chaperone Rtt106 works with two other chaperones, Asf1 and the HIR complex, to create a repressive chromatin structure at histone promoters which is bound by the protein Yta7. It was clear from previous work that Asf1 and HIR repress transcription at HTA1 and that HIR localizes to and functions through a specific element in histone promoters. However, there was no previous data demonstrating a role for Rtt106 in cell cycle-dependent gene transcription. In sum, I describe a new genomic screen that I used to discover a novel pathway regulating cell cycle-dependent transcription. While I examined histone gene expression as proof-of-principle, my screening system could be applied to virtually any pathway for which a suitable reporter can be devised. I anticipate this methodology will enable yeast researchers to collect quantitative data on hundreds of gene expression pathways.

Development of genome-wide reagents has allowed systematic analysis of gene function. The experimental accessibility of budding yeast makes it a test-bed for technology development and application of new functional genomic tools and resources that pave the way for comparable efforts in higher eukaryotes. In this Thesis, I describe a two-color GFP-RFP reporter system I developed to assess the consequences of genetic perturbations on a promoter of interest. The dual-reporter system is compatible with the synthetic genetic array methodology, an approach that enables marked genetic elements to be introduced into arrays of yeast mutants via an automated procedure. I use this approach to probe cell cycle-regulation of histone gene transcription by introducing an HTA1 promoter-GFP reporter gene construct into an ordered array of ~4500 yeast deletion mutants. I scored defects in reporter gene expression for each mutant, generating a quantitative analysis of histone promoter activity. The results of my screen motivated a number of follow-up experiments, including chromatin immunoprecipitation, transcript profiling and genome-wide analysis of nucleosome positions, which revealed a previously unappreciated pathway that specifies regions of repressed chromatin in a cell cycle-sensitive manner. A novel aspect of this pathway is that it involves histone chaperones and a chromatin boundary element. Specifically, we discovered that the histone chaperone Rtt106 works with two other chaperones, Asf1 and the HIR complex, to create a repressive chromatin structure at histone promoters which is bound by the protein Yta7. It was clear from previous work that Asf1 and HIR repress transcription at HTA1 and that HIR localizes to and functions through a specific element in histone promoters. However, there was no previous data demonstrating a role for Rtt106 in cell cycle-dependent gene transcription. In sum, I describe a new genomic screen that I used to discover a novel pathway regulating cell cycle-dependent transcription. While I examined histone gene expression as proof-of-principle, my screening system could be applied to virtually any pathway for which a suitable reporter can be devised. I anticipate this methodology will enable yeast researchers to collect quantitative data on hundreds of gene expression pathways.

ArsA is the catalytic component of an arsenite extrusion pump in E. coli that confers arsenite and antimonite resistance. YDL100cp is the ArsA homologous protein found in S. cerevisiae. Previous studies show that YDL100c gene is not directly related to arsenical resistance mechanism in S. cerevisiae but the YDL100c disrupted strain (KO) showed sensitivity to Zn2+ at 30oC and more pronounced sensitivity at 37oC. To study the role of YDL100c on Zn2+ sensitivity, wild type strain (WT) and KO were grown at 30oC and 37oC for 6 hr after adding Zn2+. Both strains were assayed for trehalose accumulation, intracellular oxidation level and GSH content. The results demonstrate that KO had a decreased growth and increased intracellular oxidation at 37oC when compared to WT. Addition of Zn2+ did not increase the intracellular oxidation in WT and KO grown at 30oC but to a greater extent in KO compared to WT grown at 37oC. Further assess the function of antioxidant genes shows that there is no significant difference in SOD1 expression between KO and WT grown at 37oC but CTT1 expression is low in KO. There is an increase in catalase activity for both WT and KO by adding Zn2+ at 30oC or 37oC, but the level of catalase activity to KO is still lower than that of WT.In conclusion, a defect of YDL100c results in a defect in the activation of general stress response at 37oC. Consequently, the cause of the increased level of intracellular oxidation of KO in the presence of Zn2+ grown at 37oC is most likely related to the decrease in cellular GSH content and trehalose accumulation in KO compared to that of WT. Therefore, the pronounced sensitivity to Zn2+ at 37oC is mainly due to a defect in general stress response in KO when grown at 37oC.

Thèse de doctorat : Sciences du vivant. Aspects moléculaires et cellulaires de la biologie : Strasbourg 1 : 2008. / Titre provenant de l'écran-titre. Bibliogr. p. 173-190.