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Orientador: Glaucia Maria Pastore / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-07-27T00:57:25Z (GMT). No. of bitstreams: 1 Brigido_BereniceMandel_M.pdf: 30516224 bytes, checksum: ec9027bfcb519c38a90217d12c0ce686 (MD5) Previous issue date: 2000 / Resumo: Muitas espécies de microrganismos produzem compostos agradáveis de aroma em meio líquido de cultura. Neste trabalho foram estudados parâmetros do processo fermentativo para a produção de compostos voláteis de aroma por novas linhagens de Neurospora sp, isoladas de beiju (massa de mandioca naturalmente fermentada), de várias regiões do Maranhão Brasil. As três linhagens deste estudo, Neurospora spl, Neurospora sp2 e Neurospora sp5 produziram aroma bastante agradável de frutas e de fungo, variando apenas sua intensidade, dependendo do meio de cultura utilizado. Os parâmetros considerados foram: composição do meio de cultura, concentração inicial de esporos, temperatura e adição de precursor. Nos ensaios para produção de compostos voláteis pelas três linhagens de Neurospora sp foram selecionados oito meios de cultura: Caldo Extrato de Malte5%, Yeast Malt Broth, Czapeck modificado, Vogel mínimo sacarose, Vogel mínimo maltose, Com Steep sem glicose ,Com Steep com glicose e Frutose/Extrato de Levedura. Foi utilizada temperatura de 30°C, com agitação de 200rpm e a formação dos compostos foi acompanhada por 144 horas de fermentação. Através da utilização da técnica de extração dos voláteis por "Purge and Trap" acoplada a cromatografia gasosa e cálculo do Índice de Retenção, foram identificados 3 classes de compostos: aldeídos ( acetaldeido), ésteres ( acetato de etila, butirato de etila e 'hexanoato de etila) e álcoois (álcool isoamílico e l-octen-3-ol). Os diferentes compostos apresentaram suas melhores concentrações produzidos por linhagens distintas, em tempos de fermentação e meios de cultura diversos. O acetaldeido teve sua melhor concentração (98ppm) produzido pela linhagem Neurospora spl em meio Frutose/Extrato de Levedura com 144horas de fermentação. Os compostos acetato de etila e butirato de etila foram produzidos nas suas concentrações máximas pela linhagem Neurospora sp2, 165ppm em meio Vogel sacarose com 72 horas de fermentação e 17,9ppm em meio Frutose/Extrato de Levedura com 96 horas de fermentação respectivamente. A linhagem Neurospora sp5 produziu as melhores concentrações de : álcool isoamílico, 213 ppm em meio Frutose/Extrato de Levedura com 96 horas de fermentação; hexanoato de etila, 2,6ppm em meio Vogel sacarose com 48 horas de fermentação e de 1-octen-3-ol, lO,3ppm em meio Yeast Malt Broth em 48 horas de fermentação. No estudo das temperaturas feito com a linhagem Neurospora spl em meio Caldo Extrato de Malte 5%, a temperatura de 25°C produziu melhores concentrações dos compostos em relação a 30°C; já no meio Yeast Malt Broth a temperatura de 30°C produziu melhores resultados. Na temperatura de 35°C não houve desenvolvimento de aroma. Os resultados revelaram que as linhagens de Neurospora sp deste estudo apresenUJm potencialidade para produção de compostos voláteis de aroma sendo importante o ajuste das condições de fermentação para melhoria da produtividade e aplicação em industrias de aromas / Abstract: Severa1microbia1 species produce a p1easantodour in liquid culture medium. It was studied some fermentative processes parameters for the production of vo1atile compounds of fiavour by new strains Neurospora sp, iso1ated from beiju (naturally fermented cassava mass), in the Brazilian State ofMaranhão. The three se1ected strains, Neurospora sp1, Neurospora sp2 and Neurospora sp5 have produced a very p1easant fruity and mushrooms 1ike aroma, whose intensity varied dependent on the culture medium used. The parameters used were: cu1ture medium composition, initia1 spores concentration, temperature and precursor' s addition. Eight cu1ture medium culture medium composition were se1ected:5% Malt Extract, Yeast Malt Broth, Czapeck, Voge1 sucrose, Voge1 maltose, Com Steep Liquor G1ucose and Frutose/Yeast Extract. The temperature chosen was 30°C with agitation of 200 rpm; the production of vo1atilecompounds was followed by 144hours of fermentation., Using Purg and Trap concentration system, coup1ed with gas chromatography and calcu1ation of retention Index, three classes of compounds cou1d be identified: a1dehyds (acetaldehyde), esters (ethy1 acetate, ethy1 butyrate and ethy1 hexanoate) and alcoho1 (isoami1icalcoho1and 1-octen-3-01). The different compounds presented their best concentrations produced by distinct strains, under different fermentation times and cu1turemediums. The "aceta1deido" had its best concentration (98 rpm) produced by Neurospora sp1's strain in Frutose /Yeast Extract medium in 144 hours of fermentation. The compounds ethy1 acetate and ethy1 butyrate were produced under their maximum concentration by Neurospora sp2's strain, 165 ppm in Voge1 Sucrose medium in 72 hours offermentation and 17,9 ppm in FrutoselYeast in 96 hours offermentation respective1y. The Neurospora sp5's strain had produced the best concentrations of: isoamilic alcoho1, 231 ppm in Frutose/Yeast Extract medium in 96 hours of fermentation; ethy1hexanoate, 2,6 ppm in Voge1 Sucrose medium in 48 hours of fermentation and 1-octen-3-01,10,3 ppm in Yeast Malt Broth medium in 48 hours of fermentation. In the temperature studied, with the Neurospora sp1's strain in 5% Malt Extract Brqth medium, under a temperature of 25°C has produced better concentrations of compounds than in 30°C; in Yeast Malt Broth medium the 30°C temperature has produced better results. Under 3SoCit wasn't found flavour development. The results have revealed that Neurospora sp's strain in this study presented potential for the production of volatile compounds of flavour, being important the adjustment of fermentation conditions to make productivity and application in flavour better / Mestrado / Mestre em Ciência de Alimentos

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The study of DNA sequence variation is a powerful approach to study genome evolution, and to reconstruct evolutionary histories of species. In this thesis, I have studied genetic variation in the fungus Neurospora tetrasperma and other closely related Neurospora species. I have focused on N. tetrasperma in my research because it has large regions of suppressed recombination on its mating-type chromosomes, had undergone a recent change in reproductive mode and is composed of multiple reproductively isolated lineages. Using DNA sequence data from a large sample set representing multiple species of Neurospora I estimated that N. tetrasperma evolved ~1 million years ago and that it is composed of at least 10 lineages. My analysis of the type of asexual spores produced using newly described N. tetrasperma populations in Britain revealed that lineages differ considerably in life history characteristics that may have consequences for their evolution. A comparative genomic analysis using three genomes of N. tetrasperma and the genome of N. crassa revealed that the mat a chromosomes in the lineages examine have been introgressed from other Neurospora species and that this introgression has reduced levels of molecular degeneration on the mating-type chromosomes. Finally, I generated a population genomic dataset composed of 92 N. tetrasperma genomes and two genomes of other Neurospora species. Analysis of these genomes revealed that all strains of N. tetrasperma have large regions of suppressed recombination on their mating-type chromosomes ranging from 69-84% of the chromosome and that the extent of divergence between mating-type chromosomes within lineages varies greatly (from 1.3 to 3.2%). I concluded that the source of this great divergence mating-type chromosome is large-scale introgression from other Neurospora species, and that these introgressed tracts have become fixed within N. tetrasperma lineages. I also discovered that genes within non-recombining introgressed regions of the mating-type chromosome have severely reduced levels of genetic variation as compared to the autosomes, and exhibit signatures of reduced molecular degeneration. My analysis of variation in coding regions revealed that positive selection on the introgressed regions has resulted in the removal of deleterious mutations and is responsible for the reductions in molecular degeneration observed.

The primary research aims of this thesis were the identification of novel cell fusion mutants of Neurospora crassa and the subsequent functional characterization of selected candidate proteins during conidial anastomosis tube (CAT)-mediated cell fusion by means of genetic, molecular, biochemical and live-cell imaging analysis. Chapter 1 provides a general introduction of the model organism and the cell fusion processes studied during different stages of the fungal lifecycle. Chapter 2 summarizes the materials and methods used. Chapter 3 introduces the comparative genomics screen conducted between appressorium-mediated plant infection by the rice blast fungus Magnaporthe oryzae and hyphal fusion in Neurospora crassa. Novel cell fusion mutants were identified in MAP kinase signalling, redox-signalling and Rho-type GTPase signalling pathways, whereas no functional overlap in the cAMP response pathway between both species could be found. Chapter 4 demonstrates how newly developed fluorescent reporters for F-actin and activated Rho GTPases in filamentous fungi lead to novel insights into the dynamic rearrangement of the F-actin cytoskeleton and cortical activation of Rho GTPases during cell symmetry breaking, polarized tip growth and cell fusion. Chapter 5 focuses on the role of the cell wall integrity (CWI) MAP kinase pathway during cell fusion, and in particular, on the function of the terminal MAP kinase MAK-1 during CAT homing and fusion pore formation. Inhibitor studies indicated that MAK-1 kinase activity is required for its own recruitment to the fusion site already during homing and for cell wall remodelling during fusion pore enlargement between interacting cells. Chapter 6 presents ultrastructural scanning electron microscope (SEM) studies which indicate that defects in hyphal attachment, extracellular matrix deposition and cell wall remodelling prematurely abort morphogenesis of the female fruitbody. These findings are put into context with defects observed in mutants of components acting in related signalling pathways which appear to regulate non-self fusion events at later stages of sexual development leading to fertilization in N. crassa. Chapter 7 provides the first evidence for a role of NADPH-oxidase (NOX)-generated reactive oxygen species (ROS) in the regulation of morphogenetic changes required for CAT-mediated cell fusion. Redox-modification of signalling proteins might be involved in cell-cell chemoattraction. Chapter 8 provides a summary of the key findings and discusses future directions.

Orientador: Maria Célia Bertolini / Resumo: Glicogênio representa um dos principais carboidratos de reserva em muitos organismos e seu metabolismo está sob o controle de um complexo mecanismo envolvendo o balanço de nutrientes e sinais ambientais. A proteína glicogenina constitui a molécula iniciadora do processo de síntese de glicogênio, sendo as etapas seguintes efetuadas pelas enzimas glicogênio sintase e enzima ramificadora. Glicogênio sintase é a enzima limitante no processo e é regulada alosterismo e fosforilação reversível. Neste trabalho foi realizada uma caracterização do metabolismo de glicogênio no fungo N. crassa enfocando as enzimas glicogenina, glicogênio sintase e glicogênio sintase quinase-3. A proteína glicogenina (GNN) foi caracterizada do ponto de vista molecular, bioquímico e funcional. O cDNA codificando para esta proteína foi isolado e a seqüência polipeptídica deduzida mostrou uma proteína de 664 aminoácidos, uma das maiores proteínas glicogenina já isoladas. A inativação do gene gnn resultou em uma linhagem mutante incapaz de acumular glicogênio. A produção da proteína GNN em E. coli resultou em um polipeptídeo altamente susceptível à proteólise e formas truncadas da proteína mostraram ser mais estáveis e igualmente ativas nos processos de auto- e trans-glicosilação, além de servirem de substrato para ação da glicogênio sintase. Tais formas também foram capazes de complementar funcionalmente mutantes de S. cerevisiae. Além disso, a expressão do gene gnn foi mostrado ser regulado durante crescimento vegetativo e deprivação de carbono. Os resíduos Tyr196 e Tyr198 foram identificados como os sítios de glicosilação, os quais contribuem diferencialmente para este processo. Análise das interações entre GNN e a proteína glicogênio sintase de N. crassa (GSN) demonstrou que a região C-terminal da GNN é a mais importante para a interação. Entretanto, o...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Glycogen represents one of the main reserve carbohydrates in many organisms and its metabolism is under control of a complex mechanism involving the balance of nutrients and environmental signals. The protein glycogenin is the initiator molecule in glycogen biogenesis and the subsequent steps are carried out by the enzymes glycogen synthase and branching enzyme. Glycogen synthase is the rate-limiting enzyme in the process and is regulated both by alosterism and reversible phosphorylation. In this work we performed a characterization of the glycogen metabolism in the filamentous fungus Neurospora crassa, focusing on the enzymes glycogenin, glycogen synthase and glycogen synthase kinase-3. The protein glycogenin (GNN) was characterized under the molecular, biochemical and functional aspects. The cDNA encoding for this protein was isolated and the deduced polypeptide sequence showed a protein with 664 residues, one of the largest glycogenins isolated so far. The inactivation of the gnn gene rendered a mutant strain that was no longer able to accumulate glycogen. The production of GNN protein in E. coli cells resulted in a polypeptide highly susceptible towards proteolysis and truncated forms were more stable and equally active, judged by their abilities to self- and trans-glucosylate, and to serve as substrate for glycogen synthase elongation. These proteins were also able to recover the glycogen deficiency phenotype in a S. cerevisae mutant strain. Moreover, the gnn gene expression was shown to be ...(Complete abstract, click electronic access below) / Doutor

Orientador: Maria Célia Bertolini / Banca: Marcia Aparecida Silva Graminha / Banca: Marcos Roberto Mattos Fontes / Resumo: 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... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: 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) / Mestre

M.S. / Molecular Biology / Neurospora crassa arg-2 encodes the small subunit of Arg-specific carbamoyl phosphate synthetase and is negatively regulated by arginine. This regulation is mediated by a 24-codon upstream open reading frame (uORF). The sequence of this uORF is critical for Arg-specific regulation. Six mutated templates were used to examine which residues of the uORF are important for this regulation. Mutations were created using megaprimer PCR and a luciferase gene was used as a reporter in the in vitro translation studies. Mutations of Asp 12, Asp 16, and Ser 10 eliminate Arg-specific regulation. Leaky scanning is thought to be involved, and a hypothetical ribosome stalling model that mediates Arg-specific attenuation of translation is proposed.

MAP kinases and transcription factors homologous to Saccharomyces cerevisiae Fus3p/Kss1p and Ste12p have been identified in several plant pathogenic fungi and found to be required for pathogenicity and sexual reproduction. A gene encoding the homolog of Fus3p/Kss1p in Neurospora crassa was isolated previously and named mak- 2 (mitogen activated kinase -2). This dissertation describes the isolation of the Ste12p homolog, pp-1 (protoperithecia-1) and the comparison of the phenotypes of the mak-2 and pp-1 mutants. The similar phenotypes of the mak-2 and pp-1 null mutants suggest that these proteins are part of the same MAP kinase signaling cascade in N. crassa. In addition to reduced growth rate, the phenotypes of the mutants demonstrate that this pathway is required for female fertility, contributes to aerial hyphal development and repression of conidiophore development. The mak-2 MAP kinase pathway also regulates several genes putatively involved in secondary metabolism during the mating process. Among these is a gene cluster that is likely to be involved in the production of a polyketide secondary metabolite. An orthologous gene cluster was also identified in M. grisea, and the structural and functional homology of these two related gene clusters was characterized. Microarray analysis was used to extend the analysis of gene expression in mak-2 and pp-1 mutants, and a number of downstream target genes of the MAP kinase pathway were identified and called mak-2 kinase-regulated genes (mkr). A model of this MAP kinase pathway in N. crassa was developed. Since N. crassa is a saprophytic fungus but closely related to several plant pathogens, this research may provide an important perspective on the evolution of a major regulatory pathway governing fungal pathogenesis.

Thesis (Ph. D.)--University of Oregon, 2001. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 161-174). Also available for download via the World Wide Web; free to University of Oregon users.