Thesis (Ph. D.)--University of Wisconsin--Madison, 1970. / Vita. Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

University, Diss., 2004--München.

Hannover, Universiẗat, Diss., 1996.

Universiẗat, Diss., 2005--Heidelberg.

Made available in DSpace on 2014-06-11T19:23:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-03-08Bitstream added on 2014-06-13T20:29:46Z : No. of bitstreams: 1 savassa_sm_me_araiq_parcial.pdf: 560908 bytes, checksum: 7322d6c1907899887286e7c9514807e4 (MD5) Bitstreams deleted on 2015-07-02T12:36:14Z: savassa_sm_me_araiq_parcial.pdf,. Added 1 bitstream(s) on 2015-07-02T12:37:34Z : No. of bitstreams: 1 000719185_20180101.pdf: 541449 bytes, checksum: 71b832c9a74978e0b6356b081a51d520 (MD5) Bitstreams deleted on 2018-01-02T17:04:40Z: 000719185_20180101.pdf,. Added 1 bitstream(s) on 2018-01-02T17:05:45Z : No. of bitstreams: 1 000719185.pdf: 3296921 bytes, checksum: 3209de8217c724ff260135d1572d7752 (MD5) / O fungo filamentoso Neurospora crassa é um organismo modelo muito utilizado para estudos de diversos aspectos da biologia dos eucariotos. Nosso laboratório tem utilizado este fungo para o estudo dos mecanismos moleculares e bioquímicos da regulação do metabolismo de glicogênio. A presente proposta de trabalho é uma consequência de experimentos anteriores realizados com o objetivo de identificar proteínas (fatores de transcrição ou não) que se ligam à região promotora do gene gsn, o qual codifica a enzima glicogênio sintase, regulatória do processo de síntese do carboidrato. Esses estudos combinaram experimentos de ensaios de retardamento em gel utilizando fragmentos do promotor gsn e proteínas do extrato total do fungo, acoplados à análise proteômica e identificação das proteínas por espectrometria de massas. Os experimentos resultaram na identificação de algumas proteínas do fungo, as quais podem ou não estar envolvidas na regulação da expressão do gene. Alguns estudos preliminares com estas proteínas foram anteriormente realizados no laboratório e apontaram um provável papel das mesmas na regulação do metabolismo do carboidrato em N. crassa. Duas dessas proteínas, as codificadas pelas ORFs NCU3482 e NCU06679 foram objeto de estudo neste trabalho. Portanto, o objetivo deste trabalho foi realizar a caracterização das linhagens mutantes nestas ORFs, além da produção e purificação das proteínas na forma recombinante. Foram realizadas análises morfológicas da linhagem mutante na ORF NCU06679, tais como: crescimento colonial e radial, crescimento linear e análise microscópica das extremidades das hifas. Esses experimentos foram realizados em comparação com a linhagem selvagem do fungo e, mostraram esta proteína está envolvida no processo de desenvolvimento do... / 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 present work is a consequence of previous experiments performed in the laboratory to identify proteins that bind to the promoter region of the gsn gene which encodes glycogen synthase, the regulatory enzyme in glycogen synthesis. Previous studies were performed by using a combination of DNA gel shift assay coupled to proteomic analysis, followed by identification of proteins by mass spectrometry. The assays resulted in the identification of proteins likely involved in the regulation of gene expression. Preliminary studies with these proteins have previously been carried out and suggested that they might have a role in the regulation of glycogen metabolism in N. crassa. Two of them, the ORFs NCU3482 and NCU06679 gene products were object of study in this work. The main objective was to characterize the mutant strains in both proteins and to produce and purify the recombinant proteins. Morphological analyzes were performed in the ORF NCU06679 mutant strain such as colony and radial linear growth and microscopic examination of the ends of the hyphae. These experiments showed that this protein is involved in the fungus development since growth and ability to conidiate were deficient when compared to the wild-type strain. The expression of gsn and gpn (encodes glycogen phosphorylase, the regulatory enzyme in glycogen degradation) genes were analyzed by qPCR and the results showed differences in gene expression of both genes during vegetative growth of the NCU06679 mutant strain when compared to the wild-type strain. The protein encoded by ORF NCU06679 was produced as a recombinant protein and the purification... (Complete abstract click electronic access below)

Orientador: Maria Célia Bertolini / Banca: Jesus Aparecido Ferro / Banca: Ana Paula Ulian de Araújo / Banca: Fábio Marcio Squina / Banca: Rolf Alexander Prade / Resumo: Este trabalho descreve a caracterização funcional de alguns fatores de transcrição possivelmente envolvidos na regulação do metabolismo do glicogênio no fungo filamentoso Neurospora crassa. Em uma análise sistemática realizada com 69 linhagens mutantes em genes codificadores para fatores de transcrição, foram identificadas e selecionadas sete linhagens que apresentaram perfis diferentes de acúmulo de glicogênio, em relação à linhagem selvagem, tanto durante o crescimento vegetativo (30°C) como no estresse térmico (45°C). Com o objetivo de verificar o envolvimento dos fatores de transcrição selecionados na expressão dos genes gsn (glicogênio sintase) e gpn (glicogênio fosforilase), experimentos de Northern blot foram realizados e revelaram variações ao nível transcricional em algumas linhagens. A análise por Blast revelou que alguns fatores de transcrição haviam sido previamente estudados em N. crassa (CSP-1, RCO-1, NIT2) ou em outros organismos (PacC, FlbC, Fkh1) e participam na regulação de diferentes processos biológicos. O fator de transcrição PACC e a influência do pH externo sobre a regulação do metabolismo do glicogênio foram investigados mais detalhadamente neste trabalho. Os resultados mostraram que na linhagem selvagem o acúmulo de glicogênio e a expressão do gene gsn foram reprimidos em condições de pH alcalino, enquanto que o gene pacC foi superexpresso nesta condição. A linhagem mutante pacCKO mostrou perder a regulação sobre o acúmulo de glicogênio e expressão do gene gsn, tanto durante o crescimento em pH fisiológico (5,8) como alcalino (7,8). A análise de ligação DNA-proteína mostrou que a proteína PACC de N. crassa recombinante produzida em E. coli foi capaz de se ligar ao motif de DNA para a proteína PACC, presente na região promotora do gene gsn... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This work describes the functional characterization of transcription factors likely involved in glycogen metabolism regulation in the filamentous fungus Neurospora crassa. In a systematic analysis performed with 69 strains knocked-out in genes encoding transcription factors, seven strains were identified and selected by presenting profiles of glycogen accumulation different from that existent in the wild-type strain during vegetative growth (30°C) and under heat stress (45°C). In order to verify the involvement of the selected transcription factors in regulation of gsn (codes for glycogen synthase) and gpn (codes for glycogen phosphorylase) genes, Northern blot assays were performed. Differences in gene expression were observed in some strains compared to the wild-type strain. Blast analysis showed that transcription factors have been previously studied either in N. crassa (CSP-1, RCO-1, NIT2) or in other organisms (PacC, FlbC, Fkh1) participating in the regulation of different biological processes. The transcription factor PACC and the influence of the external pH under the regulation of the glycogen metabolism were further investigated. The results showed that in the wild-type strain the glycogen content and the gsn gene expression were repressed under alkaline conditions, while the pacC gene was overexpressed in this condition. The pacCKO strain showed impairments in the glycogen accumulation and gsn gene expression under normal pH (5.8) and alkaline (7.8) conditions. Protein-DNA binding analysis showed that N. crassa PACC recombinant protein produced in E. coli cells was able to bind to the pacC motif present in the gsn promoter. Binding specificity was confirmed by competition assays using an oligonucleotide containing the DNA motif and by binding to a DNA fragment containing the motif mutated by site-directed mutagenesis... (Complete abstract click electronic access below) / Doutor

<p> This thesis proposed to analyse the recombination, complementation and biosynthetic implications of a series of hitherto unstudied carotenoidless mutant strains of Neurospora crassa and to confirm the reports of previous authors through analysis of a number of their mutant strains. A new selective technique permitted the fine structure analysis of the locus. Complementation studies with an extensive range of mutants including several recently discovered phenotypes permitted the resolution of new cistronic limits within the locus. A speculative model of the gene products was proposed to embrace the recombination, complementation and biochemical paradigms.</p> / Thesis / Doctor of Philosophy (PhD)

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