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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
281

Caractérisation de nouveaux gènes et polymorphismes potentiellement impliqués dans les interactions hôtes-pathogènes / Finding novel gene candidates and polymorphisms involved in host-pathogen interactions

Abou-Khater, Charbel 05 July 2017 (has links)
La coévolution ainsi que les différentes interactions entre hôte et pathogène contribuent à former la diversité génétique de ces deux organismes. Dans le cadre de cette thèse, nous nous sommes intéressés à l’étude de la variabilité génétique de 1760 gènes immunitaires choisis suivant des critères définis, pour essayer d’expliquer pourquoi il existe une variation individuelle face aux infections. L’objectif principal de ce projet était alors de caractériser et d'analyser de nouveaux gènes et polymorphismes immunitaires pouvant expliquer le contrôle ou la susceptibilité à certaines infections. Deux études pilotes nous ont permis de développer le pipeline de détection de polymorphismes. Pour la première, le polymorphisme des 3 gènes CD28, CTLA4, et ICOS a été caractérisé. Dans la deuxième, nous avons caractérisé le polymorphisme de 10 gènes impliqués dans la réponse immunitaire contre M. tuberculosis. Ces gènes ne sont pas très polymorphes et trois d’entre eux sont très conservés. Ces deux études nous ont aidés à préparer l’analyse à grande échelle avec les mises au point et l’amélioration du pipeline. Nous avons sélectionné 1760 gènes en se basant sur des critères définis. La variabilité génétique a été étudiée dans les populations humaines par une analyse minutieuse in silico de données de séquençage d’exomes générées par différents projets et consortiums pour plus de 700 individus représentant 20 populations à travers le monde. 30 gènes les plus polymorphes ont été ainsi identifiés. Ces gènes pourront être entièrement caractérisés et les données produites pourraient être comparées avec des données de résistance/sensibilité de certaines maladies infectieuses. / Host-pathogen co-evolution and interactions contribute in shaping the genetic diversity of both organisms. The objective of this thesis is to define the genetic basis of variability in disease resistance/susceptibility through the development of large-scale in silico screens to identify novel gene candidates implicated in host-pathogen interactions (such as tuberculosis).A pilot study was conducted on CD28, CTLA4, and ICOS to investigate their polymorphism. As a first step in our study based on data available in the literature, we selected a set of ten genes relevant for the immune response against M. tuberculosis. Seven of these genes were moderately polymorphic, while three of them were highly conserved. This analysis was used to prepare and setup the large scale analysis using the same developed pipeline for polymorphism detection and allele reconstruction. For our in silico, we used sequence data from several projects and consortiums to isolate most polymorphic human genes amongst a list of over 1760 candidates selected based on already established relevance for infections and on evolutionary considerations. A first screen of 64 individuals from eight different populations from several regions of the world was performed and most variable genes were selected for further extensive analyses on a larger panel (715 individuals). 30 most polymorphic genes were thus identified. The extent of polymorphism and the allelic worldwide variants of each of these 30 genes are ready to be fully characterized. The data generated could be compared against infectious disease resistance/susceptibility data to identify potentially relevant gene variation.
282

Rôle de la clathrine dans le processus infectieux du champignon phytopathogène Botrytis cinerea / Role of clathrin in infection process of fungal plant pathogen Botrytis cinerea

Souibgui, Eytham 04 May 2017 (has links)
Les champignons sont les principaux agents pathogènes des plantes. Leur étude est donc essentielle pour contrôler les maladies et maintenir un bon rendement de production agricole. La nutrition de ces pathogènes est basée sur l'absorption de nutriments, préalablement dégradés par un arsenal d'enzymes lytiques secrétées. La sécrétion des protéines est assurée par le trafic intracellulaire mettant en jeu de nombreuses vésicules. Chez les champignons filamenteux, ces vésicules ont été visualisées en microscopie électronique mais le processus mis en jeu pour leur biogénèse n'est toujours pas élucidé. L'identification de ce mécanisme est un donc un prérequis pour comprendre la sécrétion de facteurs de virulence. Dans ce but, un mutant non pathogène altéré au niveau de l'expression du gène codant la chaine lourde de la clathrine a été sélectionné parmi une banque de mutants générés chez le champignon nécrotrophe Botrytis cinerea. Le gène codant pour la chaine lourde de la clathrine est essentiel chez de nombreux organismes, ainsi un mutant dominant négatif de la chaine lourde de la clathrine a été généré et confirme la perte de pathogénicité. La caractérisation du mutant par une approche de protéomique a mis en évidence un défaut de sécrétion de 82 protéines incluant des facteurs de virulence connus. Un défaut de production de vésicules intracellulaires a également été constaté. Par ailleurs, le marquage de la clathrine à la GFP a permis de préciser sa localisation dans les cellules fongiques. Enfin, de façon surprenante, aucun défaut d'endocytose n'a été constaté au sein des mutants déficients en clathrine. Cette étude met en évidence pour la première fois le rôle essentiel de la clathrine dans le processus infectieux d'un champignon pathogène ainsi que son rôle dans a sécrétion de facteurs de virulence / Fungi are the most important plant pathogens on agricultural and horticultural crops. Study of fungal pathogens remains essential to understand pathogenic process and control plant diseases. These organisms secrete high amount of degrading enzymes involved in plant decomposition and they feed by absorption of degraded nutriments. Secretory proteins were described to be transported form Endoplasmic Reticulum and Golgi apparatus to extracellular space through intracellular vesicles. In filamentous fungi, intracellular vesicles were observed using electron microscopy but their biogenesis process is still unknown. Therefore, elucidation of the process and the identification of proteins involved in secretory vesicles biogenesis remains a challenge to understand virulence factors delivery. A nonpathogenic mutant altered in the expression of the gene coding for clathrin heavy chain was selected in a random mutant library generated in the necrotrophic pathogen Botrytis cinerea,. This gene is essential in many organisms, thus a clathrin dominant negative mutant was generated and confirming the nonpathogenic phenotype observed on several host plant. In eukaryotic cells, clathrin heavy chain is mainly described to be involved in endocytosis, but it is also essential for high density secretory vesicles formation in yeast. Characterization of the mutants using a proteomic approach revealed a secretion defect of 82 proteins including known virulence factors, as Plant Cell Wall Degrading Enzymes and elicitors. Furthermore, the clathrin mutant revealed a strong reduction of intracellular vesicles production. Clathrin was also localized in living cells using fluorescent GFP-tag protein. Endocytosis was also studied and surprisingly, any observable defect was observed for clathrin mutants. This study demonstrated for the first time the essential role of clathrin in the infectious process of a fungal pathogen and its role in virulence factors secretion
283

Ocorrência de Arcobacter spp. em carne de frango / Occurence of Arcobacter spp. in poultry meat

Padovani, Nicolle Ferraz de Arruda 11 December 2018 (has links)
Arcobacter spp., anteriormente conhecido como Campylobacter aerotolerante, é considerado um gênero bacteriano que inclui espécies consideradas patógenos emergentes que podem ser veiculados por alimentos. O gênero Arcobacter tem sido associado a gastroenterites, diarreia persistente e bacteremia em humanos. É uma bactéria Gram negativa, termosensível, embora possa sobreviver à 4°C. No Brasil, há poucos estudos de ocorrência de Arcobacter em alimentos, inclusive os de origem animal, especialmente os mais consumidos, como as carnes de frango e suína. Existem estudos pontuais de sua ocorrência em produtos resfriados, como cortes e em carcaças de frango resfriadas do varejo. O objetivo deste estudo foi avaliar a ocorrência de Arcobacter spp. em cortes e carcaças de frango refrigeradas e congeladas do varejo e em coxas de frango livres de antibiótico e orgânico refrigeradas provenientes de abatedouro, por técnica de isolamento convencional com posterior confirmação do gênero por reação de polimerase em cadeia (PCR). Foram analisadas 153 amostras de carne de frango, das quais 39,21% (59/153) resultaram positivas para o gênero Arcobacter. Foi obtido o total de sessenta e quatro isolados positivos para Arcobacter spp., que corresponderam a 89,06% (57/64) de A. lacus, 4,7% (3/64) de A. thereius, 3,12% (2/64) de A. butzleri, e 3,12% (2/64) de Arcobacter spp. espécie não identficada até o momento. Foram realizadas análises fenotípicas de resistência a 12 antibióticos com 34 isolados, previamente selecionados, de quatro diferentes fontes de carnes de frango obtidos nesse trabalho, e de três linhagens utilizadas como controle positivo de Arcobacter. A resistência fenotípica frente aos antimicrobianos foi de 100% para ácido nalidíxico e clindamicina, 29,73% para eritromicina, 24,32% para canamicina, 21,62% para tetraciclina, 18,42% para cloranfenicol, 13,51% para gentamicina, 8,11% para estreptomicina, 5,41% para azitromicina e ciprofloxacina, 2,10% para vancomicina e 0,00% para ampicilina. / Arcobacter spp., previously known as aerotolerant Campylobacter, is considered a bacterial genus that includes species considered emerging pathogens that can be transmitted by food. Arcobacter has been associated with gastroenteritis, persistent diarrhea and bacteremia in humans. It is a gram negative, thermosensitive bacterium, although it can survive at 4 ° C. In Brazil, there are few studies of the occurrence of Arcobacter in animal products including those of animal origin, especially those most consumed, such as poultry and pork. There are occasional studies of their occurrence in cooled products, such as cuts and in refrigerated chicken carcasses. The objective of this study was to evaluate the occurrence of Arcobacter spp. in refrigerated chicken cuts and carcasses of the retail and in chicken thighs free of antibiotic and organic refrigerated from slaughterhouse by conventional isolation and genotyping by polymerase chain reaction (PCR). A total of 153 chicken meat samples were analyzed, of which 39.21% (59/153) were positive for the Arcobacter genus. A total of sixty-four isolates positive for Arcobacter spp., corresponding 89.06% (57/64) of A. lacus, 4.7% (3/64) of A. thereius, 3.12% (2/64) of A. butzleri, and 3.12% (2/64) of Arcobacter spp. species not yet identified. Phenotypic resistance analyzes were performed on 12 antibiotics with 34 isolates, previously selected from four different sources of chicken meat obtained in this study, and three strains used as positive control of Arcobacter spp. Phenotypic resistance to antimicrobials were 100% for nalidixic acid and clindamycin , 29.73% for erythromycin, 24.32% for kanamycin, 21.62% for tetracycline, 18.42% for chloramphenicol, 13.51% for gentamycin, 8.11% for streptomycin, 5.41% for azithromycin and ciprofloxacin, 2.10% for vancomycin and 0.00% for ampicillin.
284

Identificação in silico e perfil transcricional de genes candidatos a efetores de Austropuccinia psidii / In silico identification and transcriptional profile of effector candidate genes from Austropuccinia psidii

Lopes, Mariana da Silva 06 November 2017 (has links)
Austropuccinia psidii (sin Puccinia psidii) é um fungo biotrófico que infecta diversos gêneros de mirtáceas. É uma ferrugem (Puccinialles) nativa da América do Sul que apresenta ampla distribuição e rápida dispersão geográfica, alcançando atualmente a Austrália, centro de diversidade das mirtáceas. Este patógeno tem alarmado a comunidade científica devido à vasta capacidade de dispersão e por causar perdas econômicas consideráveis em culturas de interesse comercial, com destaque na eucaliptocultura. Apesar de sua importância, estudos relacionados ao patossistema A. psidii - Eucalyptus ainda são incipientes, sendo que o entendimento das bases moleculares envolvidas na interação planta-patógeno é essencial para adoção de medidas de controle eficazes e duradouras. Atualmente, a busca por candidatos a efetores tem crescido consideravelmente, pois são moléculas capazes de alterar a fisiologia do hospedeiro, suprimindo ou ativando os mecanismos de defesa. Dessa forma, o objetivo do presente estudo foi identificar in silico genes candidatos a efetores e validá-los por meio de análise de expressão por RT-qPCR. Para predição dos genes candidatos a efetores foi utilizado o genoma parcial de A. psidii e uma pipeline específica, baseada em diversos parâmetros, tais como: presença do peptídeo sinal, ausência de domínio transmembrana e superfície de ancoragem e pequeno tamanho. Foram identificados 2.886 candidatos, dos quais 13% apresentaram similaridade com Puccinialles. As categorias mais importantes de localização subcelular preditas foram apoplasto (87,3%), cloroplasto (7%) e núcleo (4,1%). Oito candidatos foram selecionados para análise de expressão após mineração em dados de RNA-seq. Os ensaios foram realizados in vitro com uredósporos de duas populações distintas de A. psidii, uma proveniente de eucalipto (MF-1) e outra de S. jambos (GM-J1) e as amostras foram coletadas em 6, 12 e 24 horas após inoculação (h.a.i). O perfil de expressão dos candidatos variou entre os tempos investigados, sendo que dois candidatos mostraram altos valores de expressão em todos os tempos, ao passo que quatro foram mais expressos nos tempos iniciais (6 e 12 h.a.i). Os tempos iniciais correspondem às fases de germinação e pré-penetração, fortalecendo a predição desses quatro candidatos como apoplásticos. Foi observado um perfil diferencial de expressão entre as duas populações do patógeno, o que sugere uma provável modulação pelo hospedeiro de origem na expressão dos candidatos a efetores, fato ainda pouco abordado na literatura e que deve ser melhor investigado. Embora o trabalho tenha sido realizado in vitro, foi possível selecionar potenciais candidatos para continuidade dos estudos, incluindo a validação in planta e caracterização funcional. Os dados são relevantes em vista a escassez de informações biológicas desse patossistema e fornecem uma visão inicial de como esses efetores atuam na interação planta-patógeno. / Austropuccinia psidii (sin Puccinia psidii) is a biotrophic fungus that infects several genera of Myrtaceae. It is a rust (Puccinialles) native from South America that displays a broad distribution after have shown a quick geographic dispersion, being currently present even in Australia, which is Myrtaceae\'s diversity center. This pathogen has alarmed the scientific community due to its wide dispersion ability and because of considerable economic losses occurring in crops of commercial interest, especially eucalyptus cultures. Despite its importance, studies related to A. psidii-Eucalyptus pathosystem are still incipient and the understanding of the molecular basis involved in the plant-pathogen interaction is essential to develop effective and durable control mechanisms. Currently, the search for effector candidates has increased considerably since they are molecules that alter the host physiology, suppressing or activating the defense mechanisms. In this study, we aimed to identify in silico effectors candidate genes and also to validate them by expression analysis (RT-qPCR). To predict the effector candidate genes, we used the partial genome of A. psidii and a specific pipeline based on several parameters, such as small size, presence of signal peptide, absence of transmembrane domain and anchorage surface. A total of 2,886 candidates were identified, from which 13% are similar to Puccinialles. The most important categories of predicted subcellular localization were apoplast (87,3%), chloroplast (7%) e nucleus (4,1%). Eight candidates were selected for expression analysis after mining in RNA-seq data. The in vitro assays were carried out with uredospores from two distinct populations of A. psidii: one from eucalyptus (MF-1) and other from S. jambos (GM-J1); the samples were collected 6, 12 and 24 hours after inoculation (h.a.i). The expression profile of the candidates was distinct among the times investigated. The results showed that two candidates had high expression values in all times evaluated, while four were more expressed at 6 and 12 h.a.i. These times correspond to the germination and pre-penetration phases, corroborating with the prediction of these four candidates as apoplastics. A differential expression profile was found between the two pathogen populations, suggesting a probable modulation by the origin host in the expression of effectors candidates, what has not been extensively investigated and discussed in the literature on the topic. Although this study has been performed in vitro, it was possible to select potential candidates for further investigations, including in plant validation and functional characterization. The data presented here are relevant considering the scarcity of biological information about this pathosystem, besides providing an initial insight on how these effectors act in the plant-pathogen interaction.
285

Identification of candidate resistance metabolites to Leifsonia xyli subsp. xyli in sugarcane through metabolomic profiling / Identificação de metabólitos candidatos em cana-de-açúcar para resistência à Leifsonia xyli subsp. xyli através da análise de perfil metabólico

Moretti, Fernanda Raquel Rezende de Castro 30 November 2017 (has links)
Ratoon stunting disease (RSD) is a serious disease that affects all sugarcane producing countries. The major symptom of RSD is plant growth reduction, which is only seen in ratoon plants, causing up to 80% biomass reduction depending on environmental conditions. The disease is due to Leifsonia xyli subsp. xyli (Lxx), a gram-positive and nutritionally fastidious bacterium that so far has been found to specifically colonize the xylem vessels of sugarcane. However, the successful early detection of this pathogen is currently the main challenge for RSD prevention. Breeding for resistance to RSD, although not in practice, is a viable control measure. Since sugarcane varieties differ in relation to their degree of colonization by Lxx and losses are directly related to population densities of the pathogen in the plant, a promising breeding strategy would be to select for genotypes that are resistant to bacterial multiplication. Thus, knowledge on the responses of sugarcane to RSD at the \"omics\" level is an essential starting step to identify key metabolic targets for breeding resistant varieties. The overall goal of this study is to determine the metabolic profiles of a susceptible (CB49-260) and resistant (SP80-3280) variety inoculated or not with Lxx and to compare the results with existing proteomic and transcriptomic data to define a core of targets (proteins, genes, and metabolites) that can be tested as markers of resistance in a collection of sugarcane varieties. Bacterial titers were quantified by Real-Time PCR (qPCR). The metabolites were profiled from the leaves and from the xylem saps collected at 30 and 120 days after inoculation (DAI). Untargeted analysis were performed with Gas Chromatography - Mass Spectrometry (GC-MS) and were carried out on leaves and sap from 120 DAI. Targeted analysis was executed with Liquid Chromatography - Tandem Mass Spectrometry (LC-MS/MS) on both tissues at both timepoints. To validate metabolomics results, a set of metabolites was chosen to be tested in vitro, in order to detect growth alterations caused to Lxx. qPCR confirmed the susceptibility of CB49-260 as it had higher titers than SP80-3280. Global analysis revealed that both varieties and tissues have different metabolic profiles but that those differences are more quantitative than qualitative. The targeted approach identified more amino acids, sugars, organic acids and phosphorylated compounds in the non-inoculated susceptible genotype, while the resistant one had higher abundance of phenolics. It was also shown that inoculation with Lxx results in more relative abundance of amino acids, organic acids, phosphorylated compounds and phenolics. Furthermore, a key amino acid for Lxx survival was related to inoculation on both varieties, as well as a known phenolic compound related to plant defense. Distinguished phenolics resulting from the targeted analysis were selected to evaluate their effect on Lxx growth in vitro. Although some compounds caused inhibition, further optimization of the methodology is needed to confirm these results. / O Raquitismo-da-soqueira (RSD) é uma grave doença que afeta todos os paises produtores de cana-de-açúcar. O principal sintoma do RSD é tamanho reduzido das plantas, observado apenas nas plantas-soca, o que pode resultar em perdas de biomassa em até 80%, dependendo das condições climáticas. A doença é causada por Leifsonia xyli subsp. xyli (Lxx), uma bactéria gram-positiva e fastidiosa, descrita até o presente momento como hospedeira natural apenas da cana-de-açúcar, colonizando principalmente os vasos do xilema. Todavia, a detecção precoce deste patógeno é o principal desafio para prevenção do RSD. O melhoramento genético para resistência ao RSD, apesar de viável, não é uma medida de controle adotada na prática. Como existe diferenças entre as variedades de cana em relação ao grau de colonização por Lxx e as perdas estão diretamente relacionadas ao título bacteriano, uma estratégia de melhoramento promissora é a seleção de genótipos que apresentam resistência à multiplicação bacteriana. Portanto, o conhecimento das respostas da cana-de-açúcar ao RSD em termos \"ômicos\" é um passo inicial primordial para a identificação de alvos-chave para melhorar variedades resistentes. O objetivo geral deste estudo foi determinar os perfis metabólicos de duas variedade, uma suscetível (CB49-260) e uma resistente (SP80-3280) inoculada ou não com Lxx e comparar os resultados com dados já existentes de proteômica e transcriptômica para definir um núcleo de alvos (proteínas, genes e metabólitos) que possam ser testados como marcadores de resistência em uma coleção de cana-de-açúcar. Os títulos bacterianos foram quantificados por PCR em tempo real (qPCR). Os perfis metabólicos foram elaborados a partir de folhas e fluído xilemático coletados aos 30 e 120 dias após inoculação (DAI). A análise não-direcionada foi realizada por cromatografia gasosa acoplada à espectrometria de massas (GC-MS), usando folhas e extratos coletados aos 120 DAI. Já a análise direcionada foi efetivada via cromatografia líquida acoplada à espectrometria de massas em tandem (LC-MS/MS), em ambos tecidos e tempos de coleta. Para validar os resultados de metabolômica, um grupo de metabólitos destacado nas análises de metabolômica foi escolhido para testes in vitro e por fim detectar alterações no crescimento de Lxx. O resultado do qPCR confirmou a suscetibilidade da CB49-260, pois esta continha títulos superiores à SP80-3280. A análise global revelou que ambos variedades e tecidos possuem perfis metabólicos distintos, porém essas diferenças foram mais quantitativas que qualitativas. A análise direcionada identificou mais aminoácidos, açúcares, ácidos organicos e compostos fosforilados no genótipo suscetível não-inoculado, enquanto que o resistente apresentou maior abundância de compostos fenólicos. Também foi demonstrado que a inoculação com Lxx resultou em maior quantidade de aminoácidos, ácidos orgânicos, compostos fosforilados e fenólicos. Ademais, um aminoácido essencial à sobrevivência de Lxx foi relacionado à inoculação de ambas variedades, assim como um composto fenólico relacionado a defesa de plantas. O teste in vitro mostrou que, apesar de alguns compostos causarem inibição, é necessário aprimorar a metodologia utilizada para confirmar os resultados obtidos.
286

Aeromonas do grupo A. hydrophila em amostras de hortaliças comercializadas na cidade de São Paulo / Motile Aeromonas spp. in retail vegetables from São Paulo, Brazil

Saad, Susana Marta Isay 17 May 1993 (has links)
Em um total de 90 amostras de hortaliças, incluindo 30 de alface, 30 de agrião e 30 de escarola, foi verificada a ocorrência de Aeromonas do grupo A. hydrophila, empregando-se os métodos de semeadura direta em ágar amido-amplicilina (contagem) e após enriquecimento em caldo tripticase-soja adicionado de ampicilina (teste de presença/ausência). As incubações foram feitas a 28&#186;C, durante 24 horas. A presença dessas bactérias foi detectada em 43 (47,8%) das amostras analisadas, com contagens variando de < 102 a 2, 0x 106UFC/g. As amostras de agrião foram as que revelaram, na contagem, com maiores números de Aeromonas spp. Das 43 amostras positivas para Aeromonas spp. 9 (21,0%) revelaram-se com números superiores a 104 UFC/g. sendo que 7 eram de agrião . Dentre as amostras de hortaliças analisadas, as de agrião revelaram-se com positividade para Aeromonas do grupo A. hydrophila (70,0%) significativamente maior em relação às de alface (43,3%) e de escarola (30,0%) a nível de 5%. Não foram observadas diferenças significativas entre as positividades obtidas através do método de semeadura direta em placas e do teste de presença/ausência para as amostras de alface e de agrião. Para as amostras de escarola, a positividade foi significativamente mais alta no teste de presença/ausência. Do total de 143 cepas confirmadas como sendo do gênero Aeromonas, 138 (96,5%) eram de A. caviae, 4 (2,8%) de A. hydophila e 1 (0,7%) que, pelas suas características, foi considerada como Aeromonas atipica. Dos resultados obtidos, pode-se depreender que as hortaliças dos tipos analisados, alface, agrião e escarola, dado os níveis de contaminação observados, podem representar risco aos consumidores. / A total of 90 retail vegetable samples, including 30 of lettuce, 30 of water-cress and 30 of escarole were examined for the presence of Aeromonas of the A. hydrophila group, using two different isolation methods. One of the methods envolved direct plating on starch-ampicinin agar for the purpose of enumeration and the other one, after enrichment in trypticase-soy broth with ampicillin, for detection, both using 24 hour incubation at 28&#176C. Aeromonas spp. Were detected in 43 (47.8%) samples and their numbers varied from less than 102 up to 2.0x106 CFU/g. The water-cress samples were the ones to show greater numbers of Aeromonas spp. The counts of 9 (21%) of the 43 positive samples exceeded 104 CFU/g, 7 of them consisting of water-cress. The number of water-cress positive samples (70.0%) was significantly higher at 5% than those of lettuce (43.3%) and those of escarole (30.0%). No significant differences were found in relation to positivity for Aeromonas spp. Between both isolation methods used, regarding the lettuce and the water cress samples. On the other hand, with respect to the escarole samples, positivity was significantly superior for the isolation method envolving enrichment. In therms of species level identification, among 143 strains confirmed as being Aeromonas spp., 138 (96.51%) were A, cavia, 4 (2.81%) were A, hvdrophila and 1 (O,7%) was considered as atypical due to its different biochemical profile. The results show that the vegetables examined may represent risk to consumers in terms of presence and numbers of Aeromonas of the A, hvdrophila group.
287

The antiviral siRNA interactome in Drosophila melanogaster / L'interactome antivirale de la voie des siARNs de Drosophila melanogaster

Majzoub, Karim 23 September 2013 (has links)
La voie de l’ARN interférence (ARNi), en particulier celle des siRNA, constitue la défense antivirale majeure chez les plantes,les nématodes et les insectes. Le génome de l’organisme modèle Drosophila melanogaster code pour trois protéines, Dcr-­‐2, AGO2 et R2D2, indispensables à cette voie. Les mouches mutantes pour une de ces trois protéines sont plus susceptibles et succombent plus rapidement aux infections virales comparées aux mouches sauvages. Beaucoup d’études biochimiques ont permis d’obtenir une image assez précise de la fonction moléculaire de ces trois protéines in vitro. Cependant, plusieurs études in vivo ont révélé une réalité plus complexe, probablement liée à l’association de ces molécules avec des cofacteurs. Ce manuscrit décrit les approches adoptées afin d’identifier les partenaires protéiques de la voie des siRNA et d’étudier leurs rôles, notamment dans un contexte infectieux. Dcr-­‐2, AGO2 et R2D2 ont été étiquetés par génie génétique avec un tag de 16 acides aminés, reconnu par la biotin-­‐ligase BirA, qui permet leur biotinylation après leurs transfections dans les cellules S2. Les cellules transfectées ont été ensuite soumises à différentes infections virales,notamment avec le virus C de la Drosophile (DCV) (Dicistroviridae), le virus de la stomatite vésiculaire (VSV) (Rhabdoviridae) ou le Flock House virus (FHV) (Nodaviridae). Les cellules ont été ensuite lysées au pic de l’infection et les complexes protéiques purifiés et analysés par spectrométrie de masse.[...] / Fighting viral infections is hampered by the scarcity of viral targets and their variability resulting in development of resistance. Viruses depend on cellular molecules for their life cycle, which are attractive alternative targets, provided that they are dispensable for normal cell fonctions. Using the mode! organism Drosophila melanogaster, we identify the ribosomal protein RACK1 as a cellular factor required for infection by the internai ribosome entry site (IRES) containing virus Drosophila C virus (DCV). We further demonstrate that inhibition of RACK1 in human liver cells impairs hepatitis C virus (HCV) IRES-mediated translation and infection. Inhibition of RACK1 in Drosophila and hurnan cells does not affect cell viability and proliferation, and RACK1-silenced adult flies are viable, indicating that this protein is not essential for general translation. Our findings demonstrate a specific function for ribosomal protein RACK 1 in selective mRNA translation and uncover a promising targe! for the development of broad antiviral intervention.
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Étude du rôle de l'autophagie dans l'infection par le virus de la rougeole : mécanismes d'induction et conséquences sur le cycle viral / Role of autophagy in measles virus infection : mechanisms of autophagy induction and consequences on measles virus life cycle

Richetta, Clémence 07 October 2013 (has links)
La macroautophagie, appelée ici autophagie, est un processus de dégradation lysosomale qui joue un rôle clé dans l'immunité en dégradant des micro-organismes intracellulaires mais également en activant des réponses immunitaires. Cependant, de nombreux virus ont développé des stratégies pour inhiber, utiliser voire détourner l'autophagie à leur propre bénéfice. L'objectif de cette thèse a été d'analyser la place de l'autophagie dans l'infection par le virus de la rougeole (VR). Ce travail démontre que les souches atténuées du VR utilisent plusieurs voies moléculaires successives d'induction d'autophagie dans les cellules infectées. En effet, elles sont capables d'induire une première vague d'autophagie très précoce mais transitoire par l'engagement de l'une des isoformes de leur récepteur d'entrée CD46. Après un bref retour à l'état basal, une deuxième vague d'autophagie est induite par l'interaction de la protéine virale non structurale C avec la protéine cellulaire autophagique IRGM. La formation de syncytia conduit à une troisième voie d'induction d'autophagie qui permet de maintenir l'autophagie mise en place. Cette autophagie soutenue est exploitée par le VR afin de limiter la mort des cellules infectées, ce qui promeut la production de particules virales. Les souches virulentes du VR, incapables de lier CD46, et utilisant comme récepteur d'entrée la protéine CD150, n'induisent que l'autophagie tardive qui est également utilisée pour favoriser la production de particules virales infectieuses. Ce travail de thèse montre donc que l'induction d'une autophagie soutenue lors de l'infection par le VR promeut l'infection, principalement en limitant la mort cellulaire / Macroautophagy, thereafter referred to as autophagy, is a lysosomal degradation which plays a key role in immunity by directly degrading intracellular pathogens but also by favouring innate and adaptive immune responses. However, several viruses have evolved strategies to inhibit, exploit or even hijack autophagy for their own benefit. The aim of this thesis was to analyse the role of autophagy in the course of measles virus (MeV) infection. This work demonstrates that attenuated strains of MeV induce successive autophagy signalling in infected cells, via distinct and uncoupled molecular pathways. First, attenuated MeV strains are able to induce a first early and transient wave of autophagy through the engagement of one of the isoform of their cellular receptor CD46. Soon after infection, a new autophagy signalling is initiated by the interaction of the non-structural MeV protein C with the cellular autophagic protein IRGM. Strikingly, this second autophagy signalling can be sustained overtime within infected cells via a third autophagy input resulting from cell-cell fusion and the formation of syncytia. Sustained autophagy is exploited by MeV to limit the death of infected cells and to improve infectious viral particle formation. Interestingly, virulent strains of MeV, which do not use CD46 as a cellular receptor but use CD150, are unable to induce the early autophagy wave, whereas they induce and exploit the late and sustained autophagy. Thus, this work demonstrates that the induction of a sustained autophagy during MeV infection promotes infectivity, mostly by limiting cell death. Overall, this work describes an unusual and complex interplay between autophagy and MeV
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Turnip crinkle virus Coat Protein Suppresses the Hypersensitive Response in Plants

Jyoti, Jyoti 09 January 2007 (has links)
Turnip crinkle virus (TCV) has been implicated in the suppression of the hypersensitive response (HR), a type of programmed cell death induced during active resistance in Arabidopsis thaliana. In order to investigate the involvement of individual viral components in mediating suppression, TCV genes were cloned for use in an Agrobacterium tumefaciens mediated transient expression in Nicotiana benthamiana. Agroinfiltration of the HR-inducing avrPto/Pto system in conjunction with individual TCV genes has identified the p38 gene, which encodes the viral coat protein, as the gene responsible for the cell death suppression phenotype. The extent of cell death suppression by coat protein was quantified and found to be equal to the level of suppression by the whole virus and AvrPtoB, another cell death inhibitor from bacteria. Thus, the coat protein alone is sufficient to inhibit the HR in plants. Further, the effect of TCV on HR initiation by an avirulence factor from an unrelated bacterial pathogen was investigated. The presence of TCV does not affect the production, secretion or cellular processing of the bacterial avirulence factor.
290

Viral Suppression of Host Defenses

Mahadevan, Geetha B. 07 May 2004 (has links)
Upon detection of a pathogen, plants initiate specific signaling events designed to prevent host colonization and pathogen proliferation. Appearance of the hypersensitive response (HR), a type of programmed cell death signifies activation of active defenses in response to a one-to-one recognition of host, Resistance or R gene, and pathogen, avirulence or avr gene, encoded products. Turnip crinkle virus (TCV), however, has been shown to suppress the ability of Col-0 Arabidopsis thaliana plants to produce the HR in response to an avirulence factor. The extent of suppression was quantified by measuring cellular electrolyte leakage resulting from programmed cell death. Interestingly, cellular ion leakage levels were significantly lower in TCV-infected plants when challenged with bacteria expressing either of two bacterial effectors avrRpt2 or avrRpm1, suggesting that TCV can suppress the HR to a range of HR-inducing avirulence factors. In order to determine the viral component(s) responsible for mediating this suppression, each of the five TCV open reading frames (ORFs) was tested using an Agrobacterium tumefaciens-mediated transient expression assay in Nicotiana benthamiana. Though sequencing of the five TCV clones revealed mutations in the p28, p88, and p9 clones, Agro infiltration of an HR-inducing system in conjunction with individual TCV ORFs, or combinations of, was used to gather data to determine the role each may possess in the suppression phenotype. Full-length TCV was also expressed in the presence of AvrPto/Pto to establish suppression phenotype in Nicotiana. To assay for suppression of cell death in a heterologous system, both the mutant and wild-type clones were also tested in yeast for cell-death suppression induced by hydrogen peroxide exposure.

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