Made available in DSpace on 2014-06-11T19:23:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-01-16Bitstream added on 2014-06-13T19:29:00Z : No. of bitstreams: 1 corrocher_fa_me_araiq.pdf: 1976492 bytes, checksum: 9e6bb08376e3b4ea3c3817e9d3fe792a (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O fungo Neurospora crassa é um organismo amplamente utilizado como organismo modelo na compreensão de aspectos fundamentais da biologia dos eucariotos. Nosso laboratório tem utilizado este fungo para estudar os mecanismos bioquímicos e moleculares envolvidos na regulação do metabolismo do glicogênio. A avaliação de uma coleção de linhagens mutantes individualmente nocauteadas em genes que codificam fatores de transcrição permitiu identificar várias proteínas potencialmente envolvidas na regulação do metabolismo do glicogênio neste organismo modelo. As linhagens mutantes selecionadas apresentaram alterações no perfil de acúmulo de glicogênio e expressão do gene que codifica a enzima glicogênio sintase (gsn) durante a situação de estresse térmico. Entre estas, as linhagens mutantes nas ORFS NCU03043, NCU01629 e NCU04731, anotadas como proteínas hipotéticas no banco de dados do genoma do fungo, foram selecionadas para o presente estudo. Através de análises de Blast, a proteína codificada pela ORF NCU04731 mostrou ser homóloga ao grupo de fator de transcrição SREBPs (Sterol Regulatory Element Binding Protein) de mamíferos, que atuam como principal regulador da síntese de colesterol. Estas proteínas possuem domínio transmembrana e são ativadas após clivagem. Uma proteína ortóloga a SREBP (Sre1) foi identificada em Schizosaccharomyces pombe, entretanto, enquanto a habilidade de resposta a esteróis é conservada, as SREBPs de fungo regulam genes envolvidos na resposta transcricional à hipóxia, sendo necessárias para o crescimento em baixas concentrações de oxigênio. A proteína codificada pela ORF NCU03043 mostrou homologia a proteínas FlbC e FLE1 de fungos, as quais estão envolvidas na conidiação e desenvolvimento asexual. A linhagem flbCKO de N. crassa apresentou defeitos na progressão... / The fungus Neurospora crassa has been widely used as a model organism for fundamental aspects of eukaryotic biology. We have been studying the biochemical and molecular mechanisms involved in glycogen metabolism regulation in this fungus. The screening of a set of knocked-out strains in genes encoding transcription factors was previously performed as an attempt to identify transcription factors regulating glycogen metabolism in N. crassa. Mutant strains presenting changes in glycogen accumulation and glycogen synthase gene (gsn) expression under normal growth temperature and under heat shock stress were selected. Among them, the mutant strains in the ORFs NCU03043, NCU01629 and NCU04731, annotated as hypothetical proteins in the fungus genome database were studied in the present work. By Blast analysis, the proteins NCU04731 showed homology to a group of mammalian transcription factors SREBPs (Sterol Regulatory Element Binding Protein), which act as regulators of cholesterol synthesis and requiring cleavage from the membrane for activation. A SREBP orthologue was identified in Schizosaccharomyces pombe (Sre1), however while the ability to respond to sterol is conserved, fungal SREBPs regulates genes involved in the transcriptional response to hypoxia and are required for growth under low-oxygen conditions. The proteins encoded by the ORF NCU03043 showed homology to the fungal proteins FlbC and FLE1 involved in conidiation and asexual development. The N. crassa flbCKO strain presented defects in cell cycle progression and morphology. FLBC protein is necessary for proper induction of conidiation and is important for growth and development in N. crassa. flbC gene expression was highly induced in the early times of conidia germination confirming the importance of this protein to the fungus... (Complete abstract click electronic access below)

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The study of membrane assembly has, until now, consisted of fractionating a membrane into its components or of assembling model membranes from non-physiological components. A native precursor to membranes was obtained as a quasi-crystalline material. It was the purpose of this work to develop the appropriate techniques for characterizing the material as well as to carry out the characterization. The results of the characterization indicate the quasi-crystalline material contains a single protein, structural protein. This protein is in two forms which may reflect two sequential steps in membrane assembly. One form is solubilized in 2-chloroethanol and isolated by gel filtration. The other form may be isolated on gel filtration only after previous release by alkaline hydrolysis. This work forms the basis of future study of membrane assembly in that it has developed solvents and compatible techniques for the preparation, identification and characterization of a native precursor of membranes.

<p> Nitrate assimilation is the process by which nitrate is converted into ammonia, and ultimately into organic nitrogenous compounds, which are then made available to organisms which require an exogenous supply of organic nitrogen. Nitrite is an intermediate in this process and the mechanism of its conversion to ammonia, which is catalyzed by the enzyme nitrite reductase, needs clarification. </p> <p> The purpose of this investigation was to find a suitable assay system for nitrite reductase in N. crassa and to examine some aspects of the metabolic control of the enzyme. A new assay system for nitrite reductase is described and evidence suggesting that the enzyme is derepressible is presented. </p> / Thesis / Master of Science (MSc)

<p> Nitrite is an intermediate in the pathway of nitrate assimilation. Several questions about reduction beyond the level of nitrite remain to be answered. </p> <p> The purpose of this investigation was to induce mutants deficient in nitrite reduction and to characterize these mutants phenotypically and genetically in an attempt to answer the following questions: </p> <p> (a) How many enzymes are required in vivo for nitrite reduction? </p> <p> (b) How many genes control nitrite reduction? </p> <p> (c) Is nitrite reductase localized within a particle? </p> <p> The results of this investigation showed that nitrite reductase is controlled by at least three genes and three cistrons on two linkage groups. None of the 'nitrite-mutants' were allelic with nitrate reductase mutants. There appeared to be more than one type of nitrite reductase activity in extracts of repressed wild type mycelia. Only one of these nitrite reductase species seemed to be necessary for the reduction of nitrite in vivo. </p> / Thesis / Master of Science (MSc)

<p> The investigation was carried out in order to determine the nature of some aberrant asci obtained, at a high frequency, from a single cross in Neurospora crassa. The subsequent investigations shed light on an aspect of pyridoxine metabolism and the nature of its genetic control, as well as showing the existence of dicentric chromosome and its great instability when present in the disomic condition. </p> / Thesis / Master of Science (MSc)

<p> Evidence of gene conversion and inter=allelic complementation from earlier work on the pdx-1 locus of Neurospora crassa suggested that a more detailed analysis of this locus should lead to a better understanding of these genetic phenomenao The work described in this thesis is concerned with the derivation of complementation maps for seven alleles of the pdx-1 locus, and with a study of recombination between these alleles through the isolation of prototrophs from suitably marked inter-allelic crosses together with the isolation of asci including those exhibiting the phenomenon of gene conversione In additioni the relationships betwean the pdx-1 alleles and a gene, en-pdx-1, affecting the excretion of a pigment into the medium on which the pdx-1 strains were grown were studied. The effect of desoxypyridoxine hydrochloride 9 and the extent to which the pdx-1 mutants respond to various normal forms of the vitamin, were also studied. </p> / Thesis / Doctor of Philosophy (PhD)

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