München, Univ., Diss., 2002. / Computerdatei im Fernzugriff.

Thesis (Ph. D.)--University of Wisconsin, 1966. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Düsseldorf, Universiẗat, Diss., 2004.

Freiburg (Breisgau), University, Diss., 2001. / Erscheinungsjahr an der Haupttitelstelle: 2001.

Hamburg, Universiẗat, Diss., 2004.

Hannover, University, Diss., 2003.

Thesis (Ph. D.)--Illinois State University, 1983. / Title from title page screen, viewed May 5, 2005. Dissertation Committee: Mathew J. Nadakavukaren, Derek A. McCracken (co-chairs), Jerome R. Cain, Anthony E. Liberta, David F. Weber. Includes bibliographical references (leaves 108-117) and abstract. Also available in print.

Made available in DSpace on 2014-06-11T19:31:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-12-09Bitstream added on 2014-06-13T18:41:23Z : No. of bitstreams: 1 cupertino_fb_dr_araiq.pdf: 10818363 bytes, checksum: 34f272c80573854af968c2e298ba0e61 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / 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... / 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)

Circadian rhythms in biological processes occur in a wide range of organisms and are generated by endogenous oscillators. In Neurospora crassa, the FRQ-oscillator (comprised of FRQ, WC-1 and WC-2) is essential for rhythms in asexual sporulation and gene expression. How this oscillator signals to the cell to control rhythmicity is unknown. Furthermore, under certain growth conditions, rhythms are observed in FRQ-null strains, indicating the presence of one or more FRQ-less oscillators (FLOs). Interestingly, while circadian rhythms are observed in the related Aspergillus spp., they lack the frq gene, leading to the hypothesis that a FLO is responsible for rhythms in Aspergillus. Thus, Aspergillus provides a useful organism to investigate the components of the FLO. To investigate how an oscillator controls circadian output, we characterized the role of N. crassa NRC-2. The nrc-2 gene is under control of the clock and encodes a putative serine-threonine protein kinase. In a NRC-2-null strain cultured in low glucose conditions, FRQ-oscillator-dependent outputs are arrhythmic, but are rhythmic in high glucose. Our data suggests a model whereby NRC-2 relays metabolic information to the FRQ-oscillator to control rhythmic output. To understand the role of FLO(s) in the N. crassa circadian system, we examined regulation of the ccg-16 gene. We show that ccg-16 transcript rhythmicity is FRQ-independent, but WC-1-dependent. Furthermore, in contrast to current models for the FRQ-oscillator, we observed that rhythms in WC-1 protein accumulation persist in the absence of FRQ. These data support a new model involving two oscillators that are coupled through the WC-1 protein and that regulate different outputs. One approach to identify components of the FLO involved characterizing circadian rhythms in Aspergillus spp, which lacks FRQ. We find that A. flavus and A. nidulans, display circadian rhythms in sporulation and gene expression, respectively. Together, these findings provide a foundation for the identification of FLO components in both Aspergillus and N. crassa, that will ultimately lead to an understanding of how a multi-oscillator system can generate and coordinate circadian rhythmicity.

The Varkud satellite (VS) plasmid is found in the mitochondria of some strains of Neurospora, and exhibits properties that may allow it to be developed as a genetic transformation vector to study mitochondrial molecular biology. An ideal transformation vector would not confer a phenotype. The overall goal of my thesis was to examine interactions of VS with its Neurospora host to identify possible phenotypes, using biochemical and proteomic approaches. Biochemical experiments provided evidence consistent with the plasmid transcript, VS RNA, being present as a ribonucleoprotein particle that can be separated from ribosomal subunits by sucrose density gradient centrifugation; however, no VS-specific proteins were identified under the purification conditions examined. During the analyses of proteomic data I obtained new insights into the consequences of the statistical methods commonly used to normalize quantitative 2D gel data. However, irrespective of the method used, the fraction of the proteome amenable to 2D gel-based proteomics revealed, at most, subtle effects of VS on the abundance of a few proteins. I also observed no differences in growth rate between strains differing by the presence or absence of VS when grown in the presence of inhibitors and stressors affecting a wide range of mitochondrial and other cellular functions. Overall, despite VS RNA being as abundant as the large and small mitochondrial ribosomal RNAs, my genetic, biochemical and proteomic investigations of the effect of VS on its host strain provides evidence that VS is a phenotypically neutral element.