RESUMO: Determinadas espécies de fungos são responsáveis por diversas doenças no ser humano. Estudos epidemiológicos avaliam essas infecções tanto superficiais quanto profundas, e alguns destes avaliam em relação ao trabalho e condição de vida e saúde de trabalhadores de diversas áreas. Entretanto a contaminação por fungos filamentosos na região da cavidade oral com relação à atividade laboral rural não apresenta referência. OBJETIVOS: Avaliar a incidência de contaminação fúngica na região orofaringe normal de trabalhadores agrícolas das culturas de cana-de-açúcar, laranja e abacaxi do município de Frutal, Minas Gerais. MÉTODOS: Esse é um trabalho longitudinal, prospectivo, tipo coorte, não randomizado, em que os participantes eram migrantes vindos para trabalho temporário nas culturas referidas. Foram selecionados 60 participantes no momento das contratações após realizados os exames pré-admissionais. Avaliou-se as características sociodemográficas dos participantes. A coleta de material investigativo seguiu a ordem: região do sulco gengivo - labial, próximo à região do freio superior e freio inferior; material da mucosa jugal direita e esquerda, com um swab para cada região e dispostos na placa de Petri, respeitando a anatomia descrita. A coleta e análise do material dos trabalhadores e do meio ambiente seguiram os tempos de cada cultura, determinados de (t0), (t1), (t2). Também foram usados para caracterizar cada fase de coleta, cultura e análise das amostras. As amostras foram semeadas em meio Agar Sabouraud Dextrose. Após o crescimento dos isolados, as culturas filamentosas foram submetidas às técnicas de colônias gigantes e microcultivo. Os fungos foram identificados no microscópio com objetiva de 40 vezes. RESULTADOS: Não houve diferença entre idade e sexo nos três grupos analisados. Houve predomínio de homens afrodescendentes nas culturas de cana e laranja quando comparados ao abacaxi, que apresentou um predomínio de caucasianos. Trabalhadores de origem oriental foram detectados apenas na cultura de abacaxi. Quanto à renda, os trabalhadores de abacaxi recebem salários mais elevados do que os outros dois grupos de trabalhadores. Não houve diferença em relação ao tabagismo e a ingestão de álcool entre os três grupos analisados. Em relação à contaminação do meio ambiente, tanto na planta quanto no ar, encontrou-se um maior índice de placas de Petri contaminadas nas plantações de abacaxi e cana-de-açúcar, respectivamente. Observou-se um predominio de F. moniliforme na cultura de cana-de-açúcar e de F.subglutians nas culturas de laranja e de abacaxi nos tempos definidos dessa pesquisa. A contaminação dos trabalhadores ocorreu no segundo tempo da pesquisa. A respeito dos voluntários das plantações de abacaxi, 13,3% dos trabalhadores foram infectados entre os sessenta analisados; todos os trabalhadores foram contaminados com F.subglutinans. Na cultura de cana-de-açúcar, 8,3% deles foram encontrados infectados, sendo 5% por A. niger e 3,3% por F.moniliforme. Dois voluntários infectados pelo A. niger apresentaram infecção concomitante com C. albicans. Na lavoura de laranja 1,6% foram infectados por F.subglutians. CONCLUSÕES: Esse estudo demonstrou que trabalhar na cultura do abacaxi se mostrou um fator de risco para infecção fúngica na mucosa oral quando comparado às atividades na cultura da laranja. Houve também contaminação fúngica na cultura de cana-de-açúcar quando comparado ao grupo de referência. Outros fatores como idade, ingestão de álcool, tabagismo, renda familiar ou etnia não se mostraram estatisticamente significativos para incidência da infecção / ABSTRACT: Certain species of fungi are responsible for several diseases in humans. Epidemiologic studies rate those infections, both superficial and profound, and some of them rate regarding the occupation and life and health conditions of workers from several areas. However, the filamentous fungal infection in the oral cavity regarding laboral activity presents no reference. OBJETIVE: To rate the incidence of fungal contamination in the normal oropharynx of agricultural workers of sugar cane, orange and pineapple crops in the municipal district of Frutal, Minas Gerais. METHODS: This is a longitudinal, prospective and cohort work, in which the participants were migrants to temporary work in the aforesaid crops. Sixty participants were selected in the hiring moment after pre-admission examinations. The participants\' sociodemographic features were rated. The gathering of investigative materials followed the order: region of the gingival sulcus, close to the upper and lower lip curb region; material from the right and left jugal mucous with a swab for each region and arranged in a Petri dish, respecting the anatomy described. The gathering and analysis from workers and environment material followed the time of each culture, determined of (t0), (t1), (t2). They were also used to feature each stage of gathering, culture and analysis of samples. The samples were sown amid Sabouraud Dextrose Agar. After the growth of the isolates, the filamentous cultures were subjected to the techniques of giant colonies and microcultivation. The fungi were identified in the microscope with a 40x magnification. RESULTS: There was no difference between age and sex in the three groups studied. There was a predominance of African descent men in cane and orange crops compared to pineapple, which showed a predominance of Caucasians. Oriental workers were detected only in pineapple crop. As for income, the pineapple workers receive higher wages than the other two groups of workers. There was no difference regarding smoking and alcohol intake among the three groups analyzed. Regarding the environment contamination, both in the plant and in the air, a higher rate was found in Petri dishes contamined in pineapple and sugar cane crops, respectively. There was a predominance of F. moniliforme in the sugar cane crop and of F.subglutians in orange and pineapple crops in the times defined in this research. Contamination of workers occured in the second half of the research. Regarding the volunteers of pineapple crops, 13,3% workers were infected among the sixty analyzed; all workers were contamined with F. subglutinans. In the sugar cane crop, 8,3% of them were found infected, 5% by A. niger and 3,3% by F. moniliforme. Two volunteers infected by the A. niger presented concomitant infection with C. albicans. A worker of the orange crop was infected (1,6% ) by F. subglutians. CONCLUSIONS: This study demonstrated that working in the pineapple crop showed a risk factor for the fungal infection in the oral mucosa when compared to the activities in the orange crop. Also, there was a fungal contamination in the sugar cane crop when compared to the reference group. Other factors such as age, alcohol intake, smoking, family income or ethnicity were not statistically significant for the incidence of infection

Proteases ácidas pertencem a um importante grupo de enzimas industriais produzidas por fungos filamentosos, com aplicações na indústria de alimentos, de couro, farmacêutica e de cosméticos. O objetivo principal deste trabalho foi avaliar a produção de proteases ácidas extracelulares de fungos filamentosos isolados do solo do cerrado do centro-oeste brasileiro. Inicialmente, foi realizada uma triagem para avaliar a capacidade de 17 linhagens de fungos quanto à produção de protease em meio de cultura contendo Agar-leite. O fungo Aspergillus foetidus foi selecionado como melhor produtor de protease ácida extracelular. Visando à otimização da produção de proteases pelo fungo selecionado, avaliou-se a influência de diversos fatores no cultivo (pH, temperatura, agitação e diferentes fontes de nitrogênio e carbono). Após essa etapa, um planejamento experimental estatístico foi realizado com as variáveis independentes temperatura, pH inicial do meio e fonte de carbono e nitrogênio. A produção máxima de protease foi encontrada (63,7 U/mL) nas condições: pH inicial do meio igual a 7,0 a 28 ºC, 150 rpm em peptona 2% (p/v). Os estudos em biorreator demonstraram produção de protease nas condições de agitação e aeração iguais à 300 rpm e 1,0 vvm, após 120 h de cultivo. Os ensaios com diferentes temperaturas para a estimativa dos parâmetros termodinâmicos demonstraram que a protease ácida produzida pelo fungo é altamente estável apresentando máxima atividade em pH 5,0 e temperatura ótima igual a 55ºC. E, finalmente, para a purificação da enzima foi realizada cromatografia de gel-filtração. A enzima apresentou massa molecular de 50,6 kDa, e a análise do zimograma confirmou a atividade proteolítica. Além disso, a protease purificada foi inibida pelo composto pepstatina, indicando uma característica de protease ácida. Esses resultados obtidos demonstram um fungo filamentoso produtor de uma nova protease ácida com potencial aplicação para indústria farmacêutica e de cosméticos. / The acid proteases belong to the most important group of industrial enzymes produced by filamentous fungi, with applications in the food, leather, pharmaceutical and cosmetics industries. This study aimed the evaluation of extracellular acid proteases production from filamentous fungi isolated from different samples of the midwestern Brazil cerrado. Initially, a screening was performed to assess the ability of the 17 strains of yeast for production of protease-agar medium containing milk culture. The Aspergillus foetidus was selected as the best producer. Aimed at optimizing the production of proteases by the selected fungus, first evaluated the influence of various factors on the cultivation (pH, temperatura, agitation and different sources of nitrogen and carbon). After this step, a statistical experimental design was carried out with the independent variables temperatura, initial pH of the medium and source of carbon and nitrogen. The best conditions for protease production were (63.7 U / mL): initial pH values greater than 7.0, at 28 °C, 150 rpm peptone 2% (w/v). Aiming future production of this protease in industrial scale, studies have shown better in bioreactor protease production under the conditions of agitation and aeration equal to 300 rpm and 1.0 vvm, after 120 h of cultive. The tests at different temperaturas to estimate the thermodynamic parameters showed that the acid protease produced by the fungus is highly stable with maximum activity at pH 5.0 and optimum temperatura of 55 °C. And finally, for the purification of the enzyme were performed gel-filtration chromatography. The enzyme had a molecular mass of 50.6 kDa, and the analysis of the zymogram showed a proteolytic band. Furthermore, the purified protease was inhibited by pepstatin compound, indicating a feature of acid protease. These results demonstrate a new filamentous fungus producing acid protease with potential application to pharmaceuticals and cosmetics.

Submitted by Celso Magalhaes (celsomagalhaes@ufrrj.br) on 2017-05-12T11:13:08Z No. of bitstreams: 1 2016 - Michele Rodrigues Francisco.pdf: 1285744 bytes, checksum: 598c2fe8a98038b9745debaf13625959 (MD5) / Made available in DSpace on 2017-05-12T11:13:08Z (GMT). No. of bitstreams: 1 2016 - Michele Rodrigues Francisco.pdf: 1285744 bytes, checksum: 598c2fe8a98038b9745debaf13625959 (MD5) Previous issue date: 2016-05-20 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Trichoderma spp. They are not pathogenic fungi and develop in substrates and diverse environmental conditions. Identification based only on morphological characteristics is considered complex, due to the genetic diversity of the species. Therefore, the taxonomy of the genus Trichoderma is performed with the aid of other analyzes such as biochemical and molecular changes. Some species are capable of producing high amounts of extracellular enzymes involved in the hydrolysis of polysaccharides are thus considered fungi with high potential in the production of hydrolytic enzymes. The aim of this study was to select and identify the species of the genus Trichoderma spp. And evaluate the production of industrial enzymes these isolates. They identified 58 isolates of Trichoderma spp fungi. soil and plants by morphological and molecular analysis and was also evaluated the enzymatic production capacity. Enzyme technology is a promising area in new technologies for synthesis of compounds with high added value, where biocatalisados industrial processes have a lower environmental impact, lower energy cost, because the enzymes are biodegradable substances and have specificity to minimize the undesirable effects. In this work, we used PDA culture medium for development of fungi. Morphological identification is achieved using the identification key according to Samuels et al. (2015) Molecular and held by DNA extraction from the mycelium of fungi in accordance with CTAB protocol. For enzyme analysis, qualitative assays were performed, used specific means of solid culture to evaluate the production of hydrolytic enzymes: Where the culture media containing as a substrate for development and enzyme production of Trichoderma spp. skimmed milk production peptidase; soluble starch for the production of amylase, cellulase production carboxymethylcellulose, citrus pectin for the production of pectinase and yeast extract, dextrose, ferrous sulfate, ammonium, magnesium, manganese and potassium chloride for the production of phytase. According to the results, the morphological analysis identified 4 species such as T. atroviride, T .asperelloide, T. harzianum, T. longibrachiatum, these species were verified by molecular analysis. In relation to the enzyme potential, the result was negative for the activity of peptidase enzymes, cellulase and pectinase and positive for amylase, and the activity of the enzyme phytase, only an isolated was positive, this was identified according to morphological analysis as It is the species T. longibrachiatum / Os Trichoderma spp. n?o s?o fungos patog?nicos e se desenvolvem em diversificados substratos e condi??es ambientais. Sua identifica??o baseada apenas em caracter?sticas morfol?gicas ? considerada complexa, por conta da diversidade gen?tica das esp?cies. Portanto, a taxonomia do g?nero Trichoderma ? realizada com o aux?lio de outras an?lises, tais como bioqu?micas e moleculares. Algumas esp?cies s?o capazes de produzir elevadas quantidades de enzimas extracelulares envolvidas na hidr?lise de polissacar?deos, sendo assim considerados fungos com alto potencial na produ??o de enzimas hidrol?ticas. O objetivo deste estudo foi selecionar e identificar as esp?cies do g?nero Trichoderma spp. e avaliar a produ??o de enzimas industriais destes isolados. Foram identificados 58 isolados de fungos Trichoderma spp. de solo e plantas por an?lise morfol?gica, molecular e tamb?m foi avaliado a capacidade de produ??o enzim?tica. A Tecnologia enzim?tica ? uma ?rea promissora dentro das novas tecnologias para s?ntese de compostos que possuem alto valor agregado, onde os processos industriais biocatalisados apresentam menor impacto ambiental, menor custo de energia, pois as enzimas s?o subst?ncias biodegrad?veis e possuem especificidade que minimizam os efeitos indesej?veis. Neste trabalho, foi utilizado meio de cultura BDA para desenvolvimento dos fungos. A identifica??o morfol?gica foi realizada com aux?lio da chave de identifica??o de acordo com Samuels et al. (2015) e molecular, realizada atrav?s da extra??o de DNA do mic?lio dos fungos, de acordo com protocolo CTAB. Para as an?lises enzim?ticas, foram realizados ensaios qualitativos, utilizado meio de cultura s?lido espec?fico para avaliar a produ??o de enzimas hidrol?ticas: Onde os meios de cultura continham como substrato para desenvolvimento e produ??o enzim?tica dos isolados de Trichoderma spp. leite desnatado para produ??o de peptidase; amido sol?vel para produ??o de amilase, carboximetilcelulose para produ??o de celulase, pectina c?trica para produ??o de pectinases e extrato de levedura, dextrose, sulfato ferroso, am?nio, magn?sio, mangan?s e cloreto pot?ssio para produ??o de fitase. De acordo com os resultados, a an?lise morfol?gica identificou 4 esp?cies, tais como: T. atroviride, T. asperelloide, T. harzianum, T. longibrachiatum, estas esp?cies foram confirmadas por an?lise molecular. J? com rela??o ao potencial enzim?tico, o resultado foi negativo para a atividade das enzimas peptidase, celulase e pectinase e positivo para amilase, e para a atividade da enzima fitase, somente um isolado apresentou resultado positivo, este foi identificado de acordo com an?lise morfol?gica como sendo da esp?cie T. longibrachiatum

RESUMO: Determinadas espécies de fungos são responsáveis por diversas doenças no ser humano. Estudos epidemiológicos avaliam essas infecções tanto superficiais quanto profundas, e alguns destes avaliam em relação ao trabalho e condição de vida e saúde de trabalhadores de diversas áreas. Entretanto a contaminação por fungos filamentosos na região da cavidade oral com relação à atividade laboral rural não apresenta referência. OBJETIVOS: Avaliar a incidência de contaminação fúngica na região orofaringe normal de trabalhadores agrícolas das culturas de cana-de-açúcar, laranja e abacaxi do município de Frutal, Minas Gerais. MÉTODOS: Esse é um trabalho longitudinal, prospectivo, tipo coorte, não randomizado, em que os participantes eram migrantes vindos para trabalho temporário nas culturas referidas. Foram selecionados 60 participantes no momento das contratações após realizados os exames pré-admissionais. Avaliou-se as características sociodemográficas dos participantes. A coleta de material investigativo seguiu a ordem: região do sulco gengivo - labial, próximo à região do freio superior e freio inferior; material da mucosa jugal direita e esquerda, com um swab para cada região e dispostos na placa de Petri, respeitando a anatomia descrita. A coleta e análise do material dos trabalhadores e do meio ambiente seguiram os tempos de cada cultura, determinados de (t0), (t1), (t2). Também foram usados para caracterizar cada fase de coleta, cultura e análise das amostras. As amostras foram semeadas em meio Agar Sabouraud Dextrose. Após o crescimento dos isolados, as culturas filamentosas foram submetidas às técnicas de colônias gigantes e microcultivo. Os fungos foram identificados no microscópio com objetiva de 40 vezes. RESULTADOS: Não houve diferença entre idade e sexo nos três grupos analisados. Houve predomínio de homens afrodescendentes nas culturas de cana e laranja quando comparados ao abacaxi, que apresentou um predomínio de caucasianos. Trabalhadores de origem oriental foram detectados apenas na cultura de abacaxi. Quanto à renda, os trabalhadores de abacaxi recebem salários mais elevados do que os outros dois grupos de trabalhadores. Não houve diferença em relação ao tabagismo e a ingestão de álcool entre os três grupos analisados. Em relação à contaminação do meio ambiente, tanto na planta quanto no ar, encontrou-se um maior índice de placas de Petri contaminadas nas plantações de abacaxi e cana-de-açúcar, respectivamente. Observou-se um predominio de F. moniliforme na cultura de cana-de-açúcar e de F.subglutians nas culturas de laranja e de abacaxi nos tempos definidos dessa pesquisa. A contaminação dos trabalhadores ocorreu no segundo tempo da pesquisa. A respeito dos voluntários das plantações de abacaxi, 13,3% dos trabalhadores foram infectados entre os sessenta analisados; todos os trabalhadores foram contaminados com F.subglutinans. Na cultura de cana-de-açúcar, 8,3% deles foram encontrados infectados, sendo 5% por A. niger e 3,3% por F.moniliforme. Dois voluntários infectados pelo A. niger apresentaram infecção concomitante com C. albicans. Na lavoura de laranja 1,6% foram infectados por F.subglutians. CONCLUSÕES: Esse estudo demonstrou que trabalhar na cultura do abacaxi se mostrou um fator de risco para infecção fúngica na mucosa oral quando comparado às atividades na cultura da laranja. Houve também contaminação fúngica na cultura de cana-de-açúcar quando comparado ao grupo de referência. Outros fatores como idade, ingestão de álcool, tabagismo, renda familiar ou etnia não se mostraram estatisticamente significativos para incidência da infecção / ABSTRACT: Certain species of fungi are responsible for several diseases in humans. Epidemiologic studies rate those infections, both superficial and profound, and some of them rate regarding the occupation and life and health conditions of workers from several areas. However, the filamentous fungal infection in the oral cavity regarding laboral activity presents no reference. OBJETIVE: To rate the incidence of fungal contamination in the normal oropharynx of agricultural workers of sugar cane, orange and pineapple crops in the municipal district of Frutal, Minas Gerais. METHODS: This is a longitudinal, prospective and cohort work, in which the participants were migrants to temporary work in the aforesaid crops. Sixty participants were selected in the hiring moment after pre-admission examinations. The participants\' sociodemographic features were rated. The gathering of investigative materials followed the order: region of the gingival sulcus, close to the upper and lower lip curb region; material from the right and left jugal mucous with a swab for each region and arranged in a Petri dish, respecting the anatomy described. The gathering and analysis from workers and environment material followed the time of each culture, determined of (t0), (t1), (t2). They were also used to feature each stage of gathering, culture and analysis of samples. The samples were sown amid Sabouraud Dextrose Agar. After the growth of the isolates, the filamentous cultures were subjected to the techniques of giant colonies and microcultivation. The fungi were identified in the microscope with a 40x magnification. RESULTS: There was no difference between age and sex in the three groups studied. There was a predominance of African descent men in cane and orange crops compared to pineapple, which showed a predominance of Caucasians. Oriental workers were detected only in pineapple crop. As for income, the pineapple workers receive higher wages than the other two groups of workers. There was no difference regarding smoking and alcohol intake among the three groups analyzed. Regarding the environment contamination, both in the plant and in the air, a higher rate was found in Petri dishes contamined in pineapple and sugar cane crops, respectively. There was a predominance of F. moniliforme in the sugar cane crop and of F.subglutians in orange and pineapple crops in the times defined in this research. Contamination of workers occured in the second half of the research. Regarding the volunteers of pineapple crops, 13,3% workers were infected among the sixty analyzed; all workers were contamined with F. subglutinans. In the sugar cane crop, 8,3% of them were found infected, 5% by A. niger and 3,3% by F. moniliforme. Two volunteers infected by the A. niger presented concomitant infection with C. albicans. A worker of the orange crop was infected (1,6% ) by F. subglutians. CONCLUSIONS: This study demonstrated that working in the pineapple crop showed a risk factor for the fungal infection in the oral mucosa when compared to the activities in the orange crop. Also, there was a fungal contamination in the sugar cane crop when compared to the reference group. Other factors such as age, alcohol intake, smoking, family income or ethnicity were not statistically significant for the incidence of infection

Le champignon filamenteux, Podospora anserina, possède deux types sexuels, mat+ et mat-, caractérisés chacun par une séquence spécifique. La séquence mat+ contient un seul gène FPR1; la séquence mat- contient trois gènes : FMR1, SMR1 et SMR2. La fonction moléculaire de SMR1 est inconnue, les autres gènes codent des facteurs de transcription qui contrôlent la fécondation (reconnaissance intercellulaire), et le passage d’un syncytium à un hyphe spécialisé binucléé contenant un noyau mat+ et un noyau mat- (reconnaissance internucléaire). Il n’y a pas eu d’analyse exhaustive des gènes impliqués dans la reconnaissance intercellulaire et le mécanisme de la reconnaissance internucléaire est encore inconnu. Afin de déterminer les cibles de FPR1 et FMR1, et les différents mécanismes impliqués, nous avons utilisé une approche microarray. Le profil transcriptomique des souches mat+ et mat- compétentes pour la fécondation a permis d’identifier 157 gènes cibles, et l’analyse transcriptomique des souches mutantes fpr1- et fmr1- a révélé que ces cibles peuvent être soit réprimées, soit activées par FMR1 ou FPR1, ou être sous le contrôle de ces deux facteurs. Ces expériences ont aussi détecté l’existence de 10 gènes activés ou réprimés au même niveau dans mat+ et mat-. La délétion de 32 gènes choisis parmi ces 167 gènes cibles n’a permis de mettre en évidence que deux gènes impliqués dans la fécondation. Les comparaisons des gènes cibles des facteurs de transcription MAT de Gibberella moniliformis et Sordaria macrospora avec ceux de P. anserina révèlent un nombre significatif de gènes cibles communs entre ces espèces, mais ces gènes ont des profils transcriptomiques différents, soulevant la question du rôle de ces gènes cibles. La recherche des gènes cibles de FPR1, FMR1 et SMR2 impliqués dans la reconnaissance internuléaire a été effectuée en comparant le transcriptome des périthèces issus de deux croisements, l’un n’exprimant que les gènes spécifiques mat+, l’autre que les gènes spécifiques mat-. Les résultats ont été interprétés selon le modèle d’identité nucléaire et le modèle de ségrégation aléatoire. Le premier modèle a conduit à l’identification de 27 gènes cibles, tandis que 154 gènes cibles ont été identifiés en appliquant le deuxième modèle. Au total 46 souches mutantes ont été construites. Cependant aucune délétion n’a affecté le développement sexué. En parallèle de ces expériences transcriptomiques, nous avons invalidé tous les gènes à HMG-box de P. anserina. Les résultats montrent que ces derniers ont un rôle très important dans le développement sexué, particulièrement Pa_1_13940 qui code un régulateur des gènes des types sexuels, le premier identifié chez les Pezizomycotina. / The filamentous fungus, Podospora anserina, has two mating-type idiomorphs, mat+ and mat-. The mat+ sequence contains one gene FPR1, while mat- contains three genes: FMR1, SMR1 and SMR2. The molecular function of SMR1 is unknown, FPR1, FMR1 and SMR2 encode transcriptional regulators which control the fertilization (intercellular recognition) and the transition from a syncytium to a specialized dikaryotic hypha which contains one mat+ and one mat- nucleus (internuclear recognition). No exhaustive analysis is available for the genes involved in the intercellular recognition, while the mechanism of the internuclear recognition is unknown. In order to understand the mechanism of these events and to identify the target genes of mating-type transcription factors, we used a microarray approach. The transcriptomic profiles of the mat+ and mat- strains that are competent for fertilization revealed 157 differentially transcribed genes, and transcriptomic analysis of fmr1- and fpr1- mutant strains was used to determine the regulatory actions exerted by FMR1 and FPR1 on these differentially transcribed genes. All possible combinations of transcription repression and/or activation by FMR1 and/or FPR1 were observed. Furthermore, 10 additional mating-type target genes were identified that were up- or down-regulated to the same level in mat+ and mat- strains. Of the 167 genes identified, 32 genes were selected for deletion, which resulted in the identification of two genes essential for the sexual cycle. A comparison with similar data set from the two ascomycetes, Gibberella moniliformis and Sordaria macrospora, reveals significant numbers of orthologous pairs, although transcriptional profiles were not conserved between species, questioning the function of these target genes. Internuclear recognition was investigated by the transcriptomic analysis of perithecia from two crosses expressing mat+ and mat- genes, respectively. The tow internuclear recognition models: nuclear identity and random segregation, were used to interpret our results. According to the former model, 27 target genes have been identified, while 154 target genes were identified with the latter model. A total of 46 mutant strains were constructed. However, these strains showed no defects in sexual development. Besides this microarray experiences, we have invalidated all HMG-box genes of P. anserina. The results show that the HMG-box genes have a very important role in sexual development, especially Pa_1_13940 which encodes the first identified regulator of Pezizomycotinan mating-type genes.

Les objectifs de cette thèse sont, d’une part, de développer un système d’agitation performant en termes de transfert d‘oxygène et de forces de cisaillement afin qu’il soit adapté aux cultures de levures, de bactéries et de champignons filamenteux. D’autre part, de mettre au point un procédé de production des spores de champignons en milieu liquide pour remplacer la culture sur milieu solide (boîte de roux) utilisée au démarrage de la thèse par la société DSM. Ces travaux ont permis de sélectionner le mobile type « oreilles d’éléphant » comme système d’agitation. Son efficacité de transfert d’oxygène a été caractérisée dans une large gamme de débits d’air et de vitesses d’agitation. Les données obtenues ont été modélisées pour développer un outil de prédiction du KLa et d’extrapolation de procédé d’agitation. L’approche utilisée pour l’extrapolation est basée sur la similitude non géométrique et le maintien du coefficient de transfert d’oxygène (KLa) constant entre les échelles. L’efficacité du nouveau mobile d’agitation a été validée à l’échelle pilote en présence des cultures de bactéries, de levures et de champignons. Parallèlement au choix du mobile d’agitation, un procédé de production de spores de champignons filamenteux en milieu liquide a été développé. Ainsi, le milieu de culture et les conditions opératoires de chaque étape de la fermentation (germination, croissance végétative et sporulation) ont été optimisés. Ce nouveau procédé permet d’obtenir jusqu’à 200.106 cellules par millilitre de culture aussi bien à l’échelle du laboratoire que pilote (900L). Il a été aussi validé en présence de cultures de levures et de bactéries. Sur le même principe, les données obtenues à l’échelle pilote ont permis de développer un outil d’extrapolation à l’échelle industrielle de 10 m3 / The objectives of this thesis are, on one hand, to develop an efficient system of agitation in terms of oxygen transfer and shear stress, suited for the fermentation of yeasts, bacteria and filamentous fungi, on the other hand, to develop a process of spore production in a liquid medium. This new process will be used instead of the solid culture in Roux flasks used by DSM at the beginning of the PhD program. In a first step, we have selected “Elephant Ears” impellers as stirrers; its effectiveness of oxygen transfer was characterized in a broad range of stirring speeds and airflow rates. The data obtained were modeled and a predictive tool for KLa and process extrapolation was developed. The extrapolation is based on the non-geometrical similarity and the coefficient of oxygen transfer (KLa) was kept constant between scales. The effectiveness of the new stirrer was validated in a pilot-scale in the presence of the cultures of bacteria, yeasts and filamentous fungi. Besides the studies on the stirrer, a process in a liquid medium of filamentous fungi was developed. Thus, the fermentation medium and the operating conditions of each steps of the fermentation (germination, vegetative growth, and sporulation) were optimized. This new process makes possible to obtain up to 200.106 cells per milliliter of culture on both laboratory and pilot scale (900L). Finally, the data gathered on the pilot scale was used to extrapolate this process to the industrial-scale of 10m3

Les dispersions de virus filamenteux présentent une succession d'états cristallins liquides comprenant les phases nématique, smectique (ou lamellaire) et colonnaire. L’auto-organisation de ces particules colloïdales en forme de bâtonnet s’est révélée être essentiellement pilotée par l’entropie dont résulte un potentiel d’interaction entre particules purement répulsif. Dans cette thèse, les propriétés structurales et dynamiques de bâtonnets présentant une interaction attractive directionnelle fortement localisée (interaction dite à « patch ») à l'une des extrémités des particules ont été étudiées. L’interaction attractive locale a été obtenue en fonctionnalisant les extrémités des virus filamenteux par greffage régiosélectif de colorants fluorescents hydrophobes qui jouent le rôle de « patch » enthalpique. La force d'attraction peut être modulée en faisant varier le nombre de molécules de colorant liées. Nous avons montré que cette interaction à « patch » stabilise la phase smectique au détriment de la phase nématique, laissant les autres phases cristallines liquides essentiellement inchangées. En outre, la présence de molécules de colorant fluorescent sur les extrémités des virus permet l'observation de structures lamellaires cristal-liquides avec un contraste et une résolution exacerbés. La visualisation in situ de défauts topologiques en phase smectique, telle des dislocations de type coin et vis, a été réalisée à l'échelle de la périodicité du réseau. Le champ de déplacement autour d’une dislocation coin a été établi expérimentalement et comparé au profil prédit par les théories élastiques. Des dislocations de type vis ont également été mises en évidence, pour lesquelles la taille du cœur et l'helicite ont été déterminées.La dynamique des virus « patchy » et de ceux non fonctionnalisés a été étudiée par suivi temporel du déplacement des particules individuelles en microscopie de fluorescence. Dans toutes les phases cristallines liquides, la diffusion de particules « patchy » s'est avérée être entravée. En particulier dans la phase smectique, les bâtonnets « patchy » ont tendance à résider dans les couches diffusant principalement dans la direction perpendiculaire à l'axe principal du virus, tandis que les bâtonnets non fonctionnalisés présentent une diffusion entre couches beaucoup plus prononcée. Ce comportement peut s’explique par la plus grande valeur du potentiel smectique associé et mesuré expérimentalement dans les deux types de dispersion.Nous avons combiné des effets de « patch » entropique et enthalpique en ajoutant des polymères non-absorbants à la dispersion virale fonctionnalisée. Dans ce cas, les bâtonnets s’auto-assemblent latéralement par déplétion en des clusters. La diffusion de rayons X et la microscopie optique ont été utilisées pour comparer les propriétés structurales et dynamiques des dispersions virales fonctionnalisées - ou pas - mélangées à des polymères non absorbants, et pour établir les diagrammes de phases correspondants.En résumé, nous avons démontré un nouveau moyen efficace de contrôler la structure de fluides complexes par la modifications régio-sélective des particules constituantes. / Dispersions of filamentous viruses exhibit a plethora of liquid crystalline states including nematic, smectic (or lamellar), and columnar phases. Self-organization of these rod-shaped colloidal particles has been shown to map the hard-core behavior for which the interaction potential is purely repulsive. In this thesis, the structural and dynamical properties of rods with highly localized directional attractive interaction (or “patchiness”) between one of the ends of the particles have been studied. Local attraction has been achieved by functionalizing the filamentous virus tips via regioselective grafting hydrophobic fluorescent dyes which act as enthalpic patch. The single tip attraction strength can be tuned by varying the number of bound dye molecules. We have shown that increasing attraction interaction stabilizes the smectic phase at the cost of nematic phase leaving all other liquid crystalline transitions unchanged. Furthermore, the fluorescent dye molecules on the viral tips enable the observation of liquid crystalline lamellar structures with improved contrast and resolution. In situ visualization of topological defects in the smectic phase such as edge and screw dislocations has been thus performed at the lattice periodicity level. The displacement field around an edge dislocation has been experimentally established and compared to the profile predicted by elastic theory. Screw dislocations have been also evidenced, for which the core size and handedness have been determined.Dynamics of patchy and pristine viruses has been investigated by tracking individual rod displacements. In all liquid crystalline phases, the self-diffusion of patchy rods has been found to be hindered compared to the self-diffusion of pristine rods. Particularly in the smectic phase, patchy rods tend to reside within the layers mainly diffusing in the direction perpendicular to the main virus axis, contrary to pristine rods whose self-diffusion between layers is far more pronounced. This behavior is explained by the higher unidimensional smectic ordering potential experimentally measured in the dispersions of patchy rods compared to that obtained for pristine rods.We have combined both entropic and enthalpic patchinesses by adding non-adsorbing polymers into tip-functionalized viral dispersions. In this case, rod sides act as entropic patchy sites due to attractive depletion interaction between them. Small angle X-ray scattering and optical microscopy techniques have been used to compare the structural and dynamical properties of pristine and tip-functionalized viral dispersions mixed with hydrophilic polymers acting as depletants agent. We have determined and compared the phase diagrams obtained for the two types of virus-polymer systems.In summary, we have demonstrated a new and efficient way to control the structure of complex fluids by implementing site-specific modifications of building blocks.

In eukaryotes, the defined loci on each chromosome, the centromeres, accomplish the critical task of correct cell division. In some organisms, centromeres are composed of a euchromatic central core region embedded in a stretch of heterochromatin and the inheritance and maintenance of centromeres are controlled by dynamic epigenetic phenomena. Although the size of centromeres differs between organisms, its organization, and the placement of euchromatic and heterochromatic regions is conserved from the fission yeast, Schizosaccharomyces pombe, to humans, Homo sapiens. However, relatively little is known about centromeres in the filamentous fungi from the Ascomycota, representing the largest group of fungi and fungal pathogens. Further, studies from humans, flies, yeast and plants have shown that the inheritance of centromeres is not strictly guided by centromeric DNA content, which is highly AT-rich, repetitive and constantly evolving. Therefore, it is difficult to align ans assemble the sequenced contigs of centromeric regions of higher eukaryotes, including most filamentous fungi. A genetic technique, tetrad (or octad) analysis has helped to map the centromeres of the filamentous fungus Neurospora crassa early on. The research presented in this dissertation used N. crassa as a model to focus on characterizing different features of centromeres with an emphasis on the centromere-specific histone H3 (CenH3) protein. Data included here represent the first study on centromere-specific proteins in Neurospora, and demonstrate that the central core of the centromeres are heterochromatic, showing enrichment of silent histone marks, which is in contrast to the centromere arrangement in fission yeast. The CenH3 protein, whose deposition on the genome licenses formation or maintenance of centromeres, shows highly divergent N-terminal regions and a conserved histone fold domain (HFD) in all eukaryotes. This bipartite nature of CenH3 is also observed in the Ascomycota, which provides an opportunity for functional complementation assays by replacing Neurospora CenH3 (NcCenH3) with CenH3 genes from other species within the Ascomycota. The results from this experimental approach provide good measures for (1) determining the specific regions of CenH3 required for the assembly of centromeres during meiotic and mitotic cell divisions and (2) analyzing the resistance to changes in the organization of centromeres in N. crassa. The genetic analysis showed that the divergent N-terminal region is essential for the proper assembly of centromeres, and that the conserved carboxy-terminus of CenH3 is important for the process of meiosis but not mitotic cell division. ChIP-seq analyses suggest that the observed loss of Podospora anserina CenH3 (PaCenH3- GFP) from certain N. crassa centromeres does not result in obvious phenotypic defects, e.g. diminished growth or evidence for aneuploidy. Further, the low enrichment of PaCenH3-GFP at certain centromeres is possibly predetermined during meiosis, which results in irreversible and progressive decreases in enrichment. It remains to be determined if this process is random as far as selection of centromeres is concerned. Together the results presented here suggest that during meiosis more stringent structural requirements for centromere assembly apply and that these are dependent on CenH3, and that depletion of CenH3 from centromeres does not critically affect mitosis in the asynchronously dividing nuclei of Neurospora hyphae. / Graduation date: 2013

No description available.

No description available.