Orientador: Maria Célia Bertolini / Banca: Ana Paula Ulian de Araujo / Banca: Julio Cesar Borges / Resumo: Trabalhos anteriores realizados pelo nosso grupo levaram à identificação da proteína RVB-1 de Neurospora crassa como capaz de se ligar a um fragmento de DNA contendo o motif STRE (Stress Responsive Element). Este elemento de DNA, em Saccharomyces cerevisiae, é descrito estar presente na região promotora de genes responsivos a estresse, incluindo o estresse térmico. Uma busca nos bancos de dados de proteínas mostrou que a RVB-1 apresenta homologia estrutural à proteína RuvBL1 de humanos. Além disso, esta proteína é descrita possuir uma proteína paráloga, RuvBL2 ou Rvb2 de humano e S. cerevisiae, respectivamente, cuja proteína ortóloga em N. crassa foi denominada RVB-2. As proteínas RuvBLs foram encontradas estarem associadas a vários processos celulares, muito provavelmente devido as suas capacidades de formar grandes complexos proteicos e possuírem atividade ATPásica. Neste trabalho, estas proteínas foram parcialmente caracterizadas do ponto de vista funcional, bioquímico e biofísico. Os resultados obtidos por microscopia de fluorescência mostraram que ambas apresentam localização nuclear quando o fungo foi exposto a estresse térmico. A análise da expressão da proteína RVB-V5 mostrou estar aumentada, nessa mesma condição ambiental, quando analisada por Western blot. As duas proteínas foram produzidas na forma recombinante em Escherichia coli, tanto isoladamente quanto juntas, e a análise da expressão mostrou alta estabilidade em solução quando ambas foram produzidas em uma me... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Previous work by our group identified the Neurospora crassa RVB - 1 protein as able of binding to a DNA fragment containing the Stress Responsive Element (STRE). In Saccharomyces cerevisiae, this element is present in the promoter region of genes responsive to stress, including heat stress. RVB - 1 shows structural homology to human RuvBL1 protein, and is described to have a paralog, the RuvBL2 or Rvb2 protein in human and S. cerevisiae, respect ively. The N. crassa orthologous protein was identified and named RVB - 2. The RuvBLs proteins have been found to be associated with diverse cellular processes, most likely due to their ability to form large protein complexes and to have ATPase activity. In this work, these proteins were functional, biochemical and biophysically characterized. The fluorescence microscopy results showed that both proteins present nuclear localization in the fungus exposed to heat stress. Analyses of protein expression by Weste rn blot showed an increased expression of the RVB - 1 - v5 protei n in this same condition . The two proteins were produced in Escherichia coli, and expression analyses showed higher stability in solution when both were produced together. Both proteins showed in vitro interaction by pulldown analysis. The RVB - 1/2 complex has the secondary structure mostly formed by α - helices as analysis by CD. The size - exclusion chromatography suggested that the complex present different oligomeric structures when analyzed in the absence a... (Complete abstract click electronic access below) / Mestre

The understanding of basic cellular processes has been greatly facilitated through investigation of the behaviour of mutant forms. In a similar way the mechanisms of genetic recombination may be clarified by a study of strains which are known to show inherited differences in recombination behaviour at meiosis. The haploid fungus Neurospora crassa is particularly well suited to such an investigation since recombination frequency heterogeneity has been extensively reported in that organism, and the differences are believed to be, to a large extent, under genetic control. Strains showing recombination frequency heterogeneity over a marked genetic region have been extensively analysed in the present work and the mode of action of the factors controlling recombination frequency has been investigated by combining differing strains in heterokaryons. / Thesis / Doctor of Philosophy (PhD)

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LOL is a novel secondary metabolite gene cluster associated with the production of loline alkaloids (saturated 1-aminopyrrolizidine alkaloids with an oxygen bridge) exclusively in closely related grass-endophyte species in the genera Epichloë and Neotyphodium. In this study I characterize the LOL cluster in E. festucae, including the presentation of sequence corresponding to 10 individual lol genes as well as defining the boundaries of the cluster and evaluation of the genomic DNA region flanking LOL in E. festucae. In addition to characterizing the LOL cluster in E. festucae, I present LOL sequence from two additional species, Neotyphodium coenophialum and Neotyphodium sp. PauTG-1. Together with two recently published LOL clusters from N. uncinatum, these data allow for a powerful phylogenetic comparison of five clusters from four closely related species. There is a high degree of microsynteny (conserved gene order and orientation) among the five LOL clusters, allowing us to predict potential transcriptional co-regulatory binding motifs in lol promoter regions. The relatedness of LOL clusters is especially interesting in light of the history of interspecific hybridizations that generated the asexual, Neotyphodium lineages. In fact, three of the clusters appear to have been introduced to different Neotyphodium species by the same ancestral Epichloë species, for which present day isolates are no longer able to produce lolines. To address the evolutionary origins of the cluster we have investigated the phylogenetic relationships of particular lol ORFs to their paralogous primary metabolism genes (and gene families) from endophytes, other fungi and even other kingdoms. I present extensive evidence that at least two individual lol genes have evolved from primary metabolism genes within the fungal ancestors of endophytes, rather than being introduced via horizontal gene transfer. I also present complementation studies in Neurospora crassa exploring the functional divergence of one lol gene from its primary metabolism paralog. While it is clear that these insecticidal compounds should convey a selective advantage to the fungus and its host, thus explaining preservation of the trait, this analysis provides an exploration into the evolutionary origin and maintenance of the genes that comprise the LOL and the cluster itself.