Thesis (M. Sc.)--Carleton University, 2004. / Includes bibliographical references (p. 118-131). Also available in electronic format on the Internet.

Thesis (Ed. D.)--Illinois State University, 1990. / Title from title page screen, viewed November 28, 2005. Dissertation Committee: Herman E. Brockman (chair), Alan J. Katz, Lynne A. Lucher, Radheshyam K. Jayaswal, David F. Weber, Anthony E. Liberta. Includes bibliographical references (leaves 114-131) and abstract. Also available in print.

Thesis (Ph. D.)--University of Wisconsin, 1964. / Typescript. Vita. Abstracted in Dissertation abstracts, v. 25 (1965) no. 7, p. 3812. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 262-270).

No description available.

Orientador: Marcos Roberto de Mattos Fontes / Resumo: A comunicação entre o núcleo celular e o citoplasma acontece através de mecanismos de transporte que permitem a passagem de moléculas por poros presentes no envoltório nuclear. Na Via Clássica de Importação Nuclear, a proteína Importina-α (Impα) atua na identificação das proteínas a serem transportadas ao núcleo a partir do reconhecimento de sequências de localização nuclear (NLS). Os primeiros estudos de caracterização estrutural da Impα de Neurospora crassa (NcImpα) e a sua estrutura cristalográfica mostraram a presença de regiões que podem estar relacionadas a especificidades da proteína NcImpα no reconhecimento de NLSs de proteínas de fungos. Além disso, foram reconhecidos prováveis NLSs em proteínas reguladoras do metabolismo de carbono e nitrogênio em fungos. O objetivo desse trabalho é identificar e caracterizar o modo de interação de NLSs de proteínas fúngicas com a NcImpα e verificar possíveis especificidades da proteína NcImpα no reconhecimento de NLSs. Os potenciais peptídeos NLS de proteínas dos fungos filamentosos N. crassa e Aspergillus nidulans foram submetidos a experimentos de calorimetria de titulação isotérmica (ITC) com a proteína NcImpα, para calcular a afinidade entre as moléculas. Dentre os peptídeos testados, as sequências correspondentes ao potenciais NLS dos fatores de transcrição PAC-3, NIT-2, FLB-3, VOSA e VEA, apresentaram elevada afinidade com a NcImpα, conforme indicado pelos valores de Kd obtidos. Quando submetidos a experimentos de importação ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The communication between the cell nucleus and the cytoplasm happens through transport mechanisms that allow the passage of molecules through pores present in the nuclear envelope. In the classical nuclear import pathway, the protein Importin-α (Impα) acts in the identification of the proteins to be transported to the nucleus from the recognition of nuclear localization sequences (NLS). The first structural characterization studies of N. crassa Impα (NcImpα) and its crystallographic structure showed the presence of regions that may be related to protein specificities in the recognition of fungal NLSs. In addition, NLSs were recognized in proteins related to fungal metabolism. The objective of this work is to identify NLSs in fungal proteins and observe the specificities of the NcImpα protein. Potential NLS peptides of N. crassa were subjected to isothermal titration calorimetry (ITC) experiments with NcImpα to verify and calculate the affinity of the complexes. Among the peptides tested, the sequences corresponding to the potentials NLS of the transcription factors PAC-3, NIT-2, FLB-3, VOSA and VEA, showed high affinity with NcImpα, as indicated by the Kd values obtained. When subjected to functional experiments with HeLa cells, the peptide NIT2-NLS were efficiently transported into the cell nucleus. Crystallization tests were performed to elucidate the structure of the complexes Impα/NLS peptides. A set of data from the NcImpα/ NIT2NLS complex was collected, with 99.71% comp... (Complete abstract click electronic access below) / Doutor

Orientador: Maria Célia Bertolini / Banca: Flávio Henrique da Silva / Banca: Nilce Maria Martinez Rossi / Banca: Maria Isabel Nogueira Cano / Banca: Nádia Monesi / Resumo: As células eucarióticas são capazes de responder e de se adaptar a diferentes condições ambientais estressantes tais como o choque térmico, modulando sua atividade metabólica. Na levedura Saccharomyces cerevisiae o choque térmico é conhecido por induzir múltiplos genes relacionados a esta forma de estresse, através dos elementos transativadores Hsf1p (Heat Shock Protein) e Msn2/4p, os quais se ligam, respectivamente, às seqüências consensos HSE e STRE, ativando a transcrição. A seqüência nucleotídica do gene da glicogênio sintase (gsn) de Neurospora crassa foi previamente isolada em nosso laboratório e uma análise da expressão gênica mostrou que a transcrição do gene foi diminuída na situação de choque térmico (30 para 45ºC). Uma análise da região 5' flanqueadora revelou a presença dos elementos de DNA CRE, HSE e STRE tanto na região promotora do gene quanto na 5'-UTR. Neste trabalho, nós investigamos o envolvimento destes elementos de DNA na regulação da transcrição na condição de choque térmico, através de ensaios de mobilidade em gel (EMSA) usando como sondas, fragmentos de DNA contendo os elementos transcricionais citados anteriormente. Para isto, foram utilizados como fonte de proteínas extratos nucleares brutos ou fracionados em coluna de Heparina-Sepharose, preparados a partir de micélios coletados antes e após choque térmico (amostras HS30). Os resultados obtidos mostraram a presença de proteínas capazes de reconhecer e ligar aos elementos de DNA testados sob a condição estressante analisada. A especificidade das bandas de mobilidade reduzida foi confirmada por diferentes ensaios de competição, tais como 1) adição prévia de sondas de DNA não marcadas antes da reação de ligação, 2) adição de oligonucleotídeo de DNA dupla fita contendo o elemento STRE do promotor gsn como competidor específico, 3) utilização...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Eukaryotic cells respond and adapt to environmental stressing conditions such as heat shock, by modulating metabolic responses to counteract them. In the yeast Saccharomyces cerevisiae heat shock activates multiple stressing related genes by the trans acting elements Hsfp (Heat shock factors) and Msn2/4p, which bind to the cis regulatory elements HSE (Heat Shock Element) and STRE (Stress Responsive Element), respectively, and activates the gene transcription. We have previously isolated the gene encoding the Neurospora crassa glycogen synthase (gsn) and demonstrated that the gene transcription was decreased under heat shock (30 to 45ºC). An analysis of the gsn 5' flanking region showed the presence of the DNA elements CRE, HSE, and STRE, in the promoter region and in the 5'-untranslated region. In this work we investigated the involvement of these DNA elements in gene transcription regulation under heat shock condition by performing electrophoretic mobility shift assays (EMSA) using, as probes, DNA fragments containing the regulatory elements, and crude or Heparin-Sepharose fractionated nuclear extracts prepared from mycelia collected before and after 30 min of heat shock (HS30 sample). Ours results showed the presence of nuclear proteins capable to recognize and bind to the DNA regulatory elements under heat stress. The specificity of the DNA shifts were confirmed by using 1) unlabelled DNA probes as specific competitor, 2) the gsn promoter STRE double-stranded oligonucleotide as specific competitor, 3) a mutated gsn promoter STRE probe, and 4) the HSE unlabelled probe in a cross-reaction competition assay with the gsn promoter STRE probe. The involvement of the regulatory CRE elements in the modulation of the gsn gene transcription was investigated by using EMSA in the wild type strain, and also in two N. crassa mutant strains defective in the cAMP-signaling pathway: one having hyperactive...(Complete abstract click electronic access below) / Doutor

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)

No description available.

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)