Il existe un certain nombre de situations au cours desquelles l’intégrité du génome cellulaire est mise en danger. Ceci se produit notamment lors de mouvements de gènes (translocation, éléments mobiles), lors d’infections virales parfois intégratives (AAV, HBV, HPV) ou lors d’infections rétrovirales. En effet, le cycle de réplication des rétrovirus nécessite une étape d’intégration du génome viral dans l’ADN génomique de la cellule infectée. Malgré les nombreuses études menées sur les infections par le VIH, les cancers viro-induits ou non et les thérapies géniques basées sur les rétrovirus, aucune donnée n’est actuellement disponible sur les modifications cellulaires induites par de telles perturbations chromosomiques. L’objectif du projet était d’identifier des mécanismes cellulaires induits par des atteintes à l’intégrité du génome, plus précisément par l’insertion de molécules d’ADN non cellulaire dans les chromosomes. L’intégration de matériel génétique additionnel a été induite par des vecteurs lentiviraux dérivés du VIH-1. Les modifications cellulaires uniquement dues à l’intégration ont été isolées par comparaison de vecteurs intégratif et non intégratif. Des cellules primaires du derme humain ont été sélectionnées pour l’étude. Les temps post infection et les doses virales les plus adéquates ont été sélectionnés grâce à des expériences de cinétiques d’intégration couplées à une quantification des ADN viraux intégrés. L’analyse des modifications cellulaires induites par l’intégration de l’ADN étranger a portée sur l’ensemble du transcriptome et sur l’ensemble du protéome cellulaire. Dans le but d’effectuer une analyse transcriptomique, des puces à ADN, représentant le génome humain complet, ont été utilisées. Cette étude a démontré une forte répression transcriptionnelle induite par l’intégration. De plus, toutes les fonctions cellulaires sont perturbées par le processus. Finalement, une classification par interactions et fonctions biologiques a mis en évidence cinq processus cellulaires majoritairement affectés par l’intégration de l’ADN étranger, qui correspondent au cycle et à la mort cellulaire, au remodelage et à la réparation de la chromatine et à la réponse immunitaire ou au stress. Dans le but de compléter cette analyse transcriptomique, une étude protéomique a été réalisée. Les protéines cellulaires ont été séparées sur des gels bi-dimensionnels. Parmi les neuf protéines identifiées en spectrométrie de masse, certaines appartiennent au cytosquelette et d’autres aux mécanismes de réponse au stress. L’intégration d’un ADN étranger au sein du génome provoque donc bien des perturbations cellulaires. L’intégration de matériel génétique additionnel ne concernant pas seulement les rétrovirus, les données obtenues lors de cette étude pourront permettre (i) de développer des stratégies de défenses contre les rétrovirus ou contre les autres maladies caractérisées par des atteintes à l’intégrité du génome, (ii) d’évaluer les risques encourus par l’intégration d’un vecteur thérapeutique dans le génome cellulaire lors des thérapies géniques ou des expériences de transfert de gènes / In numerous situations, cell genome integrity could be in danger. This is the case during gene movements (translocations, mobiles elements), during viral infections that could be sometimes integrative (AAV, HBV, HPV) or during retroviral infections. Indeed, the replication cycle of retroviruses requires a viral genome integration step. In spite of the studies on HIV infections, viral or non viral cancers and retrovirus-based gene therapy, no data are available concerning the cellular modifications induced by such chromosomal disruptions. The aim of work was to identify cellular mechanisms induced by the insertion of a non cellular DNA into the chromosomes. The integration of additional DNA was provoked by HIV-1-based lentiviral vectors. Cellular modifications only due to the integration step were isolated by comparison of integrative and non integrative vectors. Primary human dermal fibroblasts cells were selected for the study. Optimal times post infection and viral quantities were defined using kinetic integration experiments and integrated viral DNA quantifications. The study of cellular modifications induced by the integration of the foreign DNA was applied on the cellular global transcriptome and proteome. In order to perform a transcriptomic analyses, DNA microarray corresponding to the whole human genome, were used. This study revealed a strong transcriptional repression induced by the integration. Moreover, every cellular function are disturbed by the process. Finally, a network based on molecular interactions and biological functions underlined five cellular processes mostly affected by the foreign DNA integration and corresponding to the cell cycle and death, the remodelling and repair of chromatin and the immunity or stress responses. To complete this transcriptomic analyses, a proteomic study was realized. Cellular proteins were separated on 2D gels. Among the nine proteins identified by mass spectrometry, some are linked to the cytoskeleton and other to the cellular stress. Thus, integration of a foreign DNA into the genome provoked cellular perturbations. As additional DNA integration do not only concern retroviruses, data obtained during this study could allow (i) the development of defensive strategies against retroviruses or other diseases implicating the genome integrity and (ii) the evaluation of risks linked to the integration of a therapeutic vector into the genome during gene therapy and gene transfer experiments

Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2015-11-30T16:56:25Z No. of bitstreams: 2 Tese - Sheyla Maria Rondon Caixeta BONFIM - 2014.pdf: 8198505 bytes, checksum: 0f84b8e65f24477c5179e31a085ce655 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-12-01T06:39:29Z (GMT) No. of bitstreams: 2 Tese - Sheyla Maria Rondon Caixeta BONFIM - 2014.pdf: 8198505 bytes, checksum: 0f84b8e65f24477c5179e31a085ce655 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-12-01T06:39:29Z (GMT). No. of bitstreams: 2 Tese - Sheyla Maria Rondon Caixeta BONFIM - 2014.pdf: 8198505 bytes, checksum: 0f84b8e65f24477c5179e31a085ce655 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-06-06 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Paracoccidioides is a fungal human pathogen that causes systemic mycosis paracoccidioidomycosis (PCM). Infection occurs via inhalation of fungal propagules by the host and macrophages are important in the initial contention of the fungus through innate mechanisms. In this study, analysis of proteomic profile of yeast cells of Paracoccidioides, isolate Pb18, recovered from infection in macrophages J774 A1 so as to identify molecules that are expressed in this condition and could represent targets for new antifungal therapies. The analysis of differential protein expression could be detected 181 proteins with induction of expression and decreased expression was observed in 245 proteins in Pb18. The data show up regulation of proteins involved in the processes on the alternative carbon metabolism such as gluconeogenesis, beta-oxidation of fatty acids and amino acids catabolism. Proteins with decreased levels of expression include those related to protein synthesis and glycolysis. Aditionally proteins involved in oxidative stress response and cell defense exhibit increased levels of expression such as superoxide dismutase, heat shock proteins, cytochrome c peroxidase and thioredoxins. A mutant was generated to assess the importance of the enzyme cytochrome c peroxidase during infection suggesting involvement in a complex system of protection against oxidative stress, reinforcing role in the survival of the fungus. The proteomic profile of Paracoccididioides sp in response to internalization in macrophages, first described, reflects significant remodeling of fungal metabolism against oxidative stress and highlighting the versatility of the fungus to adapt to the hostile environment of the macrophage seeking new strategies survival. / O fungo Paracoccidioides é um patógeno humano causador da micose sistêmica paracoccidioidomicose (PCM). A infecção ocorre através da inalação de propágulos do fungo pelo hospedeiro e os macrófagos são importantes na contenção inicial do fungo por meio de mecanismos inatos. Neste estudo realizamos a análise do perfil proteômico de células leveduriformes de Paracoccidioides, isolado Pb18, recuperadas da infecção em macrófagos J774 A1 para identificar moléculas que são expressas nesta condição e poderiam representar alvos para novas terapias antifúngicas. Nas análises de expressão diferencial de proteínas foi possível detectar 181 proteínas com indução da expressão e a diminuição da expressão foi observada em 245 proteínas em Pb18. Os dados obtidos revelam a regulação positiva de proteínas nos processos envolvidos no metabolismo alternativo do carbono como gliconeogênese, beta-oxidação de ácidos graxos e catabolismo de aminoácidos. As proteínas com diminuição nos níveis de expressão incluem aquelas relacionadas à glicólise e síntese proteica. Ainda proteínas envolvidas na resposta ao estresse oxidativo e defesa celular apresentam aumento nos níveis de expressão tais como superóxido dismutase, proteínas de choque térmico, tioredoxinas e citocromo C peroxidase. Um mutante foi gerado para avaliar a importância da enzima citocromo c peroxidase durante a infecção sugerindo o envolvimento em um complexo sistema de proteção contra o estresse oxidativo, reforçando papel na sobrevivência do fungo. O perfil proteômico de Paracoccididioides spp em resposta à internalização em macrófagos, descrito pela primeira vez, reflete significativo remodelamento do metabolismo do fungo frente ao estresse oxidativo e ressalta a versatilidade do fungo em adaptar-se ao ambiente hostil do macrófago buscando novas estratégias de sobrevivência.

Made available in DSpace on 2016-06-02T20:21:25Z (GMT). No. of bitstreams: 1 3253.pdf: 2155477 bytes, checksum: 03c92d367d865f20f90059d887400f1f (MD5) Previous issue date: 2010-08-30 / Financiadora de Estudos e Projetos / Citrus canker is an economically important disease that affects many citrus growing areas around the world, and there is no effective control or cure. The causative agents are bacteria from the genus Xanthomonas, recently classified as two novel species, X. citri e X. fuscans. The most virulent strain is X. citri subsp. citri (Xac). In this study, we did a differential proteomic analysis of a periplasmatic proteins enriched fraction from Xac, by comparison of the protein profiles after cellular growth in pathogenicity inducing medium (XAM1) or non-inducing medium (Nutrient Broth). We performed Bidimensional Electrophoresis (2D-PAGE) in triplicate and statistical analyses were done with the software Image Master Platinum (GE). At least 80 spots showed significant differences on protein expression (p<0.05) between the two conditions tested (induction/ non-induction) and had their proteins digested by trypsin. The peptides were analyzed by mass spectrometry (LCESI- Q-Tof) and the results were compared with proteins from genome databases using the Mascot tool. Among Xac proteins identified with higher expression in in vitro pathogenicity inducing situation, two are classified as proteins involved with Pathogenicity, Virulence, and Adaptation (in according to the genome annotation): superoxidase dismutase (XAC2386) e phosphoglucomutase (XAC3579). A highly differential spot (46), increased in the inducing condition, had their proteins identified as lytic murein transglycosilase (XACb0007) and/or transglycosylase (XAC3225), oxidoreductase (XAC1160) and DNA polymerase III subunit beta (XAC0002). The spot 46 was isolated from several 2D gels and used to produce antibody in mouse. The expressions of these proteins were analyzed by Western blot in others citrus canker genome strains, which differ from Xac in the virulence and host types. This analysis showed that proteins from the spot 46 had different expression pattern among these strains, with higher expression in Xac growing in inducing condition. We conclude that the proteomic study of proteins from periplasmic fraction is an important strategy to detect Xac proteins involved with pathogenicity and virulence, even including proteins transported outside the cell. Proteins found in this fraction could be used as biomarkers, however more experiments are still necessary. / O cancro cítrico é uma doença economicamente importante para a citricultura, não existindo atualmente medidas preventivas e curativas eficazes para combatê-la. Os agentes etiológicos são bactérias do gênero Xanthomonas, classificadas em duas espécies, X. citri e X. fuscans. A variedade mais agressiva aos citros é a bactéria X. citri subsp. citri (Xac). Pelo presente trabalho, realizamos a análise proteômica diferencial de uma fração celular de Xac enriquecida em proteínas periplasmáticas, após indução in vitro da sua patogenicidade, comparativamente à condição de não indução de patogenicidade (controle). As proteínas dessa fração foram separadas por eletroforese bidimensional (2D-PAGE), sendo a análise estatística das intensidades dos spots realizada por meio do software Image Master Platinum (GE). Aproximadamente 80 spots apresentaram expressão diferencial significativa (p<0,05) entre as duas condições estudadas, os quais foram isolados do gel e sua(s) proteína(s) constituinte(s) digerida(s) com tripsina. Os peptídeos resultantes da digestão foram analisados por espectrometria de massas (LC-ESI-QTof) e os dados obtidos utilizados na identificação da proteína por pesquisa em bancos de Xac e/ou de outros organismos com o software Mascot. Dentre as proteínas de Xac identificadas como mais expressas após indução de patogenicidade in vitro duas estão classificadas como proteínas relacionadas com Patogenicidade, Virulência e Adaptação (segundo anotação do genoma de Xac), sendo elas: superoxidase dismutase (XAC2386) e fosfoglicomutase (XAC3579). Proteínas pertencentes a um spot com notável aumento de expressão em situação de indução de patogenicidade (spot 46) foram identificadas por espectrometria de massas, sendo elas: proteína lytic murein transglycosilase (XACb0007) e/ou transglycosylase (XAC3225), oxidoredutase (XAC1160) e subunidade beta da DNA polimerase III (XAC0002). O spot 46 foi isolado a partir de vários géis 2D e utilizado para produção de anticorpos em camundongos. A análise da expressão da(s) proteína(s) pertencente(s) ao spot 46 foi realizada por meio de Western blot nas linhagens-genoma B e C do cancro cítrico, as quais diferem de Xac na virulência e tipo de citros- hospedeiro. Por esta análise foi verificado que proteínas do spot 46 possuem expressão muito distinta entre as linhagens genoma do cancro cítrico, sendo sua expressão preponderante em Xac, especialmente em situação de indução de patogenicidade. Concluímos que o estudo das proteínas utilizando a fração periplasmática como objeto de estudo é de grande interesse para investigação de proteínas envolvidas com patogenicidade e virulência em Xac, e que tenham passagem pelo periplasma, inclusive de proteínas transportadas para o meio extracelular. Além disso, algumas proteínas desta fração possuem potencial para serem utilizadas como biomarcadores ou como possível alvo de combate ao cancro cítrico.

Rice (Oryza sativa L.) is an important crop cultivated worldwide, and abiotic stresses limit its productivity. Different approaches were carried out to understand the mechanisms of rice defense responses against abiotic stresses, mainly drought. The NADPH-generating enzymes engaged in response to dehydration and salt stresses during the seedling stage of Nipponbare cultivar were analyzed. Enzyme activities of 6-phosphogluconate dehydrogenase (6PGDH), NADP-dependent aldehyde dehydrogenase, NADP-malic enzyme (NADP-ME) in leaves, and 6PGDH in roots were significantly increased in response to dehydration stress. NADP-ME and NADP-glutamate dehydrogenase activities in roots increased significantly in response to salt stress. These results suggest the involvement of NADPH-generating enzymes in plant responses to dehydration and salinity stresses, and the increased demands of NADPH in plants under abiotic stress can be furnished by enhanced activities of NADPH-producing enzymes. Also, a dehydration-induced protein was detected and identified as serine-hydroxymethyltransferase. This result indicates that serine-hydroxymethyltransferase can play a key role in regulating dehydration response in rice. Moreover, comparative proteomic analyses of CL163 (drought-tolerant), Cheniere (drought-sensitive), and Rex (moderately-drought-sensitive) rice varieties were performed. Drought-responsive proteome changes were profiled in leaves and roots at the seedling stage in response to drought stress imposed by polyethylene glycol (PEG-6000). Eighteen significantly differentially expressed proteins were identified by mass spectrometry. Elongate factor1 alpha and 17.9-kDa classI heat shock protein appear to have different expression patterns between CL163 and Cheniere, which may be attributable to the difference in drought response of the two rice varieties. Furthermore, a compendium of 103 drought resistance genes in rice was compiled to construct and analyze networks formed by associations between genes/proteins and to identify the most significant genes, biological processes/pathways. Genes were classified based on gene ontology and protein class into 26 groups. Forty-two genes were classified as transcription factors. Proteins encoded by the genes were localized in 8 subcellular locations and classified into three classes. Two pathways from KEGG whose genes were overrepresented in the compendium were identified. Gene expression, network presenting pairwise interactions between genes/proteins, and co-expression network were constructed. This study provides a systematic view of the crucial genes that can be contributing collectively to drought tolerance.

La maladie de Lyme est la maladie à transmission vectorielle la plus répandue de l’hémisphère Nord. La peau est un organe clef dans cette maladie, car c’est à cet endroit qu’interagissent les cellules de l’hôte, le pathogène, le microbiote cutané et le vecteur. Nous avons développé un modèle murin d’infection disséminée de borréliose de Lyme, qui nous a permis de développer une méthode spécifique de détection de protéines de Borreliella dans le tissu cutané murin par SRM-MS, pouvant aboutir à une méthode de diagnostic chez l’homme. Dans un deuxième temps, nous nous sommes intéressés aux relations entre le microbiote cutané, les cellules résidentes de la peau, kératinocytes et fibroblastes, avec Borreliella. Les sécrétomes de trois bactéries commensales, S. epidermidis, P. acnes et C. striatum possèdent un effet synergique sur l’expression de gènes inflammatoires par les kératinocytes et les fibroblastes. Les sécrétomes de P. acnes et de C. striatum se sont également montrés capables d’inhiber une partie de la réponse inflammatoire des kératinocytes, pouvant aider le pathogène lors de la transmission/dissémination précoce. / Lyme disease is the most common vector-borne disease in the Northern Hemisphere. Skin is a key organ in the disease, since it is the key interface where host cells, pathogen, skin microbiota and vector interact early during pathogen transmission. We developed a late lyme borreliosis model on mice. This model allowed us to develop a specific detection method of Borreliella proteins in the mouse skin by SRM-MS that might be used to develop a human diagnosis of disseminated Lyme disease. In a second part, we analysed the relationship between skin microbiota, resident skin cells (keratinocytes and fibroblasts), in the presence or absence of Borreliella. The secretome of three commensals bacteria, S. epidermidis, P. acnes and C. striatum was shown to have a synergistic activity with Borreliella in pro-inflammatory gene expressions by keratinocytes and fibroblasts. P. acnes and C. striatum secretomes were also able to inhibit partially the inflammatory response of keratinocytes that might help the transmission/dissemination of the pathogen.

Ce projet de recherche porte sur l’étude de la régulation des systèmes cannabinoïdes et vanilloïdes chez Caenorhabditis elegans (C. elegans), dans le but d’évaluer les effets antinociceptifs du tétrahydrocannabinol (THC) et du cannabidiol (CBD). C. elegans est un modèle largement utilisé pour étudier la nociception, visant principalement à caractériser les réponses nociceptives induites par le THC et le CBD, ainsi qu’à identifier les mécanismes et les cibles moléculaires impliqués. Les résultats des études sur l’utilisation du cannabis dans le traitement de la douleur chronique chez les mammifères sont controversés. Cette recherche vise à étudier l’effet du CBD et du THC sur la réponse nociceptive chez C. elegans et à approfondir la compréhension des mécanismes pharmacologiques sous-jacents. La méthodologie consiste à quantifier l’effet antinociceptif du CBD et du THC chez C. elegans par la méthode de la thermotaxie. Les nématodes sauvages (N2) étaient exposés à des concentrations croissantes de phytocannabinoïdes pour évaluer la relation concentration-effet. D’autres tests étaient effectués sur des souches mutantes exprimant des récepteurs cannabinoïdes et vanilloïdes afin d’identifier préalablement leurs cibles. Enfin, les analyses protéomiques et bioinformatiques seront effectuées pour identifier les voies de signalisation et les processus biologiques induits par l’interaction entre les phytocannabinoïdes et leurs cibles. Cette étude démontre l’activité antinociceptive du CBD et du THC chez C. elegans avec des effets rémanents pour THC, en ciblant respectivement le vanilloïde pour le CBD et le cannabinoïde pour les systèmes THC. Les analyses protéomiques et bio-informatiques mettent en évidence des différences significatives dans leurs voies de signalisation et leurs processus biologiques. / The objective of this research project was to focus on studying the regulation of cannabinoid and vanilloid systems in Caenorhabditis elegans (C. elegans) to evaluate the anti-nociceptive effects of tetrahydrocannabinol (THC) and cannabidiol (CBD). C. elegans is a widely used model for studying nociception, with the main objective being to characterize nociceptive responses induced by THC and CBD, as well as identify the underlying molecular mechanisms and targets involved. Recent studies on the use of cannabis for the treatment of chronic pain in mammals have shown controversial results. This research aims to investigate the effect of CBD and THC on the nociceptive response in C. elegans and understand the underlying pharmacological mechanisms. The methodology consisted in quantifying the antinociceptive effect of CBD and THC in C. elegans using the thermotaxis method. WT(N2) were exposed to decreasing concentrations of phytocannabinoids to evaluate the dose and effect relationship. Further tests performed on mutant expressing cannabinoid and vanilloid receptors allowed preliminarily identification of their targets. Finally, proteomic and bioinformatics analyses were used to identify the signaling pathways and biological processes induced by these phytocannabinoids. The result of this study confirmed the antinociceptive effect of CBD and THC in C. elegans, with a remanent effect of THC. This effect is mediated by the vanilloid system for CBD and the cannabinoid system for THC, respectively. Also, proteomics and bioinformatics analyses revealed significant differences in signaling pathways and biological processes.