Os mexilhões são alimentos marinhos freqüentemente ingeridos crus, ou parcialmente cozidos, e o hábito de aferventar estes bivalves somente até que abram as valvas, é insuficiente para eliminar os microrganismos patogênicos eventualmente presentes neste molusco. Após levantamento inicial, e visando melhorar a qualidade do mexilhão Perna perna cultivado e comercializado no município de Ubatuba, SP, esta pesquisa estudou o crescimento de Staphylococcus aureus e Bacillus cereus em mexilhões in natura e pré-cozidos, e a eliminação dos mesmos por meio de tratamentos térmicos, avaliando, posteriormente, as características físico-químicas e sensoriais dos produtos. Em ambos os casos, lotes de 1 kg de mexilhão foram inoculados, individualmente, com cepas de S. aureus e B. cereus e mantidos, por 10 horas, a temperatura ambiente (25ºC±1ºC) e sob refrigeração (7ºC±1ºC). Posteriormente, foram estabelecidos seis tipos de tratamentos térmicos, sendo três sob vapor (5, 10 e 15 min) e três por imersão em água (5, 10 e 15 min), buscando estabelecer o binômio que proporcionasse a eliminação dos mesmos, e avaliando o rendimento, os aspectos físico-químicos e sensoriais. Para ambos microrganismos, ocorreu crescimento durante as 10 horas de estudo, sendo este mais evidente, nos tratamentos mantidos a temperatura ambiente. No mexilhão pré-cozido ocorreram as maiores contagens microbianas, se comparado ao mexilhão in natura. Com relação aos tratamentos térmicos, todos foram eficientes, eliminando os microrganismos da ordem de, pelo menos 2 ciclos logarítmicos, no entanto, os tratamentos térmicos por imersão em água, permitiram melhores resultados do que os tratamentos sob vapor. As análises fisico-químicas e sensoriais, não apresentaram diferença estatística entre os tratamentos térmicos estudados. Com o emprego de altas temperaturas por um determinado período, obteve-se perda de alguns minerais, como Potássio e Boro, tendo outros, não apresentado alteração com relação ao tempo de exposição ao calor. Já, quanto ao rendimento, houve diferença, em nível de 5%, sendo os melhores rendimentos alcançados nos menores tempos de exposição ao calor e, os tratamentos por imersão, apresentaram resultados melhores que os tratamentos sob vapor. Concluiu-se que o tratamento térmico, binômio tempo-temperatura, de 10 min em água à ebulição, é suficiente para reduzir os microrganismos, permitindo a retenção dos nutrientes e um rendimento de 54,36%, podendo, portanto, ser recomendado para os produtores, visando melhorar a qualidade do mexilhão, via adequação do manejo atualmente empregado. / Mussels are seafood frequently ingested raw or partially cooked and the habit of boiling bivalves only to open the valves, is insufficient to eliminate several species of pathogenic bacteria. Seeking to improve the quality of the cultivated and marketed Perna perna mussel in the district of Ubatuba, SP, this research studied the microbiological growth of Staphylococcus aureus and Bacillus cereus in fresh and pre-cooked mussels, and the elimination by thermal treatments, being evaluated its physicochemical and sensorial characteristics. For such, lots of 1 kg of mussel was inoculated individually with strains of S. aureus and B. cereus, and maintained by 10 hours at environmental temperature (25ºC±1ºC) and under refrigeration (7ºC±1ºC). Six thermal treatments were established, 3 in steam (5, 10 and 15 min) and 3 in boiling water (5, 10 and 15 min), being looked in the elimination of B. cereus and S. aureus, and also evaluating the performance, and physical-chemical and sensorial aspects. Microbiological growth was verified after 10 hours for both microorganisms, and this being more evident in the treatments maintained at environmental temperature. Pre-cooked mussel obtained the largest microbial developments, if compared to fresh mussels. About the thermal treatments, everyone was efficient, eliminating at least 2 logarithmic cycles, however, thermal treatments in boiling water obtained better results than the steam treatments. The physical-chemical and sensorial analyses, didn't present statistical difference among the thermal treatments studied. Use of high temperatures for a determinate period, obtained lost in some minerals, like potassium and boron, and others minerals not presented alteration in relation to the heat time exposure. Already in the performance, it was obtained statistical difference, being the best performances reached in the smallest times of heat exposition, and the treatments in boiling water presented better results than the steam treatments. The thermal treatment, binomial time-temperature, of 10 min in boiling water, is enough to reduce the microorganisms, allowing the retention of the nutrients and performance of 54.36%, could be recommended for the producers seeking to improve the traditional handling.

A Xylella fastidiosa é uma bactéria gram.negativa, fastidiosa, que vive limitada ao xilema de plantas causando várias doenças de importância econômica como a doença de Pierce em videiras nos Estados Unidos e a Clorose Variegada dos Citros (CVC) no Brasil. A CVC tem afetado severamente a citricultura do estado de São Paulo pondo em risco milhares de empregos e milhões de dólares em geração de divisas. O sequenciamento do genoma de X. fastidiosa revelou genes envolvidos em possíveis mecanismos de patogenicidade dessa bactéria, entre eles um operon possivelmente envolvido na produção de um exopolissacarídeo extracelular denominado goma fastidiana. Supõe.se que esse exopolissacarídeo seja o responsável pela manutenção dos biofilmes bacterianos que causam a oclusão dos vasos xilemáticos levando ao surgimento dos sintomas da CVC. Para estudar esse operon, denominado operon gum, foram construídos vetores para a inativação dos genes gumB, gumD e gumF por duas estratégias: mutagênese por inserção.deleção e mutagênese por troca alélica. A mutagênese por inserção.deleção envolve a integração via recombinação homóloga com uma permuta.de um plasmídeo contendo uma cópia truncada do gene alvo. A mutagênese por troca alélica, por sua vez, envolve duas permutas e se caracteriza pela troca do gene alvo selvagem por uma cópia interrompida por um marcador de seleção. Nenhum mutante gum foi obtido usando.se a estratégia de troca alélica, todavia, mutantes para os genes gumB e gumF foram obtidos com sucesso pela estratégia de mutagênese por inserção.deleção. Nenhum mutante para o gene gumD foi obtido, sugerindo que essa mutação possa ser letal para a célula. A análise de células e colônias desses mutantes crescidos em meio sólido ou em suspensão não mostrou diferenças morfológicas em relação a linhagem selvagem. A inativação dos genes gumB e gumF não influenciou a capacidade de X. fastidiosa se aderir a vidro. Com o uso do gene repórter CAT, que codifica para a enzima clorafenicol acetil transferase a qual confere à bactéria resistência ao antibiótico clorafenicol foi possível verificar que a glicose não influencia na expressão desse operon ao nível de transcrição. Com o uso desse gene reporter, também foi possível identificar uma região transcrita a partir de um promotor não caracterizado, localizada na fita antisenso do operon gum. A comparação do perfil cromatográfico de proteínas solúveis totais dos mutantes e da linhagem selvagem mostrou diferenças significativas nesses pefis, indicando um efeito pleiotrópico dessas mutações. O estudo da função dos genes gumB e gumF na patogenicidade de X. fastidiosa foi impossibilitado por se ter verificado recentemente que a linhagem usada na construção dos mutantes não coloniza a planta eficientemente para a indução de sintomas em citros e tabaco em condições experimentais após inoculação mecânica. / Xylella fastidiosa is a fastidious, xylem restricted, gram.negative bacteria, that causes several economically important diseases as Pierce's disease of grapevine in USA and the Citrus Variegated Chlorosis (CVC) in Brasil. CVC affects severely the São Paulo State citriculture jeopardizing thousands of jobs and millions of dollars of incomes. The genome sequence of X. fastidiosa has revealed several genes possibly involved in the pathogenicity mechanisms of this bacterium, among them, an operon containing nine genes possibly involved in the synthesis of an exopolisaccharide named fastidian gum. This gum is possibly involved in the bacterial biofilm maintenance that causes the xylem occlusion leading to CVC symptoms development. To study this operon, named gum operon, vectors were constructed to inactivate the gumB, gumD and gumF genes by two strategies, insertion.duplication mutagenesis and allelic exchange mutagenesis. The insertion.duplication mutagenesis involves the integration a whole plasmid containing a truncated copy of the target gene by homologous recombination with one crossing over. The allelic exchange mutagenesis involves homologous recombination with two crossing overs that substitutes the wild.type copy of the target gene by a truncated copy interrupted by a selectable marker gene. No gum mutant was obtained using the allelic exchange strategy; however gumB and gumF mutants were obtained by insertion-duplication mutagenesis strategy. GumD mutant was not obtained, suggesting that the mutation in this gene is lethal to the cell. Analysis of cells and colonies of these mutants growing in solid media and in suspension hasn't reveal any morphological difference to the wild.type strain. The disruption of the gumB and gumF genes does not influenced the adhesion capacity of X. fastidiosa to the glass, used as a substrate. Using the reporter gene CAT, wich codes for cloramphenicol acetil transferase enzime confering resistance to cloramphenicol, we verified that glucose has no influence in the expression of this operon at the transcription level. Using this reporter gene, we also identified a transcribed region directed by a non characterized promoter, localized in the antisense strand of the gum operon. A comparison between the soluble protein profile of the mutants and the wild.type strain, obtained by liquid chromatography, showed significative differences, indicating a pleiotropic effect of these mutations. The study of the function of the gumB and gumF genes in the pathogenicity of X. fastidiosa was not concluded because we verified recently that the strainm, used to generate the mutants, do not colonize the plants efficiently to induce symptoms in citrus and tobacco plants after mechanical inoculation.

Os antibióticos têm sido utilizados como aditivos na alimentação animal para aumentar o desempenho e manter a saúde dos animais, como suínos e frangos, destinadas à produção de alimentos de origem animal. No entanto, desde 2006, a Comunidade Europeia proibiu o uso de antibióticos para esse proposito devido ao desenvolvimento de resistência bacteriana aos antibióticos. Como resultado, várias alternativas foram estudadas e propostas para substituir os antibióticos utilizados na alimentação animal. Os óleos essenciais têm recebido considerável atenção devido às suas propriedades antimicrobianas. Portanto, o objetivo do presente trabalho foi avaliar in vitro a atividade antibacteriana dos óleos essenciais contra a microbiota patogênica e probiótica de ocorrência no trato gastrointestinal de suínos e aves, destinadas à produção de alimentos de origem animal. A atividade antibacteriana seletiva, a qual, significou uma alta atividade antibacteriana contra bactérias patogênicas e reduzida ou nenhuma atividade sobre bactérias probióticas, foi avaliada como característica fundamental dos óleos essenciais com alto desempenho. Esta característica foi avaliada nos óleos essenciais usados individualmente e em combinações binarias. Inicialmente, no Capítulo 2, uma triagem de vinte e oito óleos essenciais (OEs) através do método de difusão em disco mostrou que Eucalyptusglobulus, E. exserta, Pimenta pseudocaryophylllus, Orange Oil Phase Essence, e CitrusTerpens (Os dois últimos OEs foram subprodutos do processamento da laranja para obtenção de suco) tiveram uma atividade antibacteriana seletiva sobre a bactéria patogênica Salmonella Enteritidis e a bactéria probiótica Lactobacillus plantarum. Numa fase posterior, esses cinco óleos foram avaliados individualmente e em misturas binárias, contra cinco bactérias patogênicas e três bactérias probióticas. Os melhores resultados foram observados quando os OEs foram avaliados isoladamente e não em misturas. Assim, Orange Oil Phase Essence e Citrus Terpens destacaram-se por ter a melhor atividade antibacteriana seletiva contra essas bactérias. No Capítulo 3, uma análise mais detalhada da atividade antibacteriana dos óleos essenciais foi realizada utilizando Orange Oil Phase Essence e a mistura composta pelos óleos de E. globulus e P. pseudocaryophyllus. Estes dois óleos foram selecionados com base nos resultados do Capitulo 2 e da disponibilidade de OEs em nosso estoque. Ambos, óleo e a mistura foram avaliados sobre a bactéria patogênica mais resistente, E. faecalis, e a bactéria probiótica menos resistente L. rhamnosus, como observado no Capitulo 2. A avaliação da Concentração Inibitória Mínima e Concentração Bactericida Mínima do Orange Oil Phase Essence e da mistura mostrou que não tiveram um efeito antibacteriano seletivo sobre E. faecalis e L. rhamnosus. Finalmente, no Capitulo 4, foi avaliada a atividade antibacteriana individual e combinada dos óleos de E. globulus e P. pseudocaryophyllus sobre E. faecalis e L. rhmanosus. Os resultados mostraram que a combinação destes dois OEs, avaliadas pelo método checkerboard, não potencializou a atividade antibacteriana seletiva dos dois OEs. Portanto, observou-se que o óleo de E. globulus isoladamente apresentou a melhor atividade antibacteriana seletiva contra E. faecalis e L. rhamnosus. Em conclusão, este trabalho permitiu identificar óleos essenciais com perfil antibacteriano seletivo para eles serem possíveis alternativas botânicas aos antibióticos utilizados na alimentação animal. / Antibiotics have been used in animal feed to maintain health and increase performance, as in the case of pigs and poultry intended for food production of animal origin. However, since 2006 the European Community has banned the use of antibiotics for this purpose due to the emergence and increase of antibiotic-resistant bacteria. As a result, several alternatives have been studied and proposed to substitute antibiotics used in animal feed. Essential oils have received considerable attention due to their antimicrobial properties. Therefore, the objective of this work was to evaluate in vitro the antibacterial activity of essential oils against pathogenic and probiotic bacteria that occur in the gastrointestinal tract of swine and poultry, intended for food production of animal origin. The selective antibacterial activity, which means high antibacterial activity on pathogenic bacteria and reduced or no activity on probiotic bacteria, was evaluated as a fundamental feature of the highest-performance essential oils. This feature was evaluated in essential oils used individually and in binary combinations. Initially, in Chapter 2, the screening of twenty-eight essential oils (EOs) by disk diffusion method showed that Eucalyptus globulus, E. exserta, Pimenta pseudocaryophylllus, Orange Oil Phase Essence, and Citrus Terpens (the last two EOs were by-products of orange juice production) had a selective antibacterial activity against the pathogenic Salmonella Enteritidis and probiotic Lactobacillus plantarum. At a later stage those five oils were evaluated, individually and in binary blends, against five pathogenic bacteria and three probiotic bacteria. Better results were observed when the EOs were checked alone and not in blends. Orange Oil Phase Essence and Citrus Terpens stood out for having the two best selective antibacterial activities against those bacteria. In Chapter 3, a more detailed analysis of essential oil antibacterial activities was perfomed using Orange Oil Phase Essence and the blend composed of E. globulus and P. pseudocaryophyllus. These two oils were selected based on the results of Chapter 2 and from the availability of our EO stock. Both oil and blend were checked on the most resistant pathogenic bacterium, E. faecalis, and on the less resistant probiotic bacterium of the Lactobacillus genus, L. rhamnosus, as observed in Chapter 2. The evaluation of Minimal Inhibitory Concentration and Minimal Bactericidal Concentration for Orange Oil Phase Essence and the blend showed that there was not a selective antibacterial effect against E. faecalis and L. rhamnosus. Finally, in Chapter 4, the individual and combined antibacterial activities of E. globulus and P. pseudocaryophyllus essential oils on E. faecalis and L. rhmanosus were evaluated. The results showed that the combination of two EOs evaluated by checkerboard method did not potentiate the selective antibacterial activity of the two EOs. Therefore, it was observed that the E. globulus essential oil alone had the best selective antibacterial activity against E. faecalis and L. rhamnosus. In conclusion, this work enabled the identification of essential oils with selective antibacterial profile that can become possible botanical alternatives to antibiotics used in animal feed.

La gestion de populations de rongeurs est importante car ils peuvent causer divers problèmes économiques, écologiques et sanitaires. Cette gestion s'appuie sur des méthodes de prévention, celles-ci étant des mesures architecturales, hygiéniques et des pratiques agricoles. Elle s'appuie également sur des méthodes de lutte biologique, chimique et mécanique. La lutte chimique se fait principalement avec l'utilisation de rodenticides anticoagulants qui agissent plusieurs heures après ingestion permettant d'éviter l'éviction alimentaire. L'utilisation excessive de ces molécules a entrainé la sélection de résistance chez certains individus rendant leur utilisation peu efficace. Il y a alors eu le développement de nouvelles molécules, mais celles-ci sont plus écotoxiques. Nous nous sommes intéressés dans ces travaux de thèse à la gestion de populations de rongeurs dans un cadre de risque sanitaire leptospirosique. Les rongeurs sont les principaux réservoirs de leptospires pathogènes, des bactéries Spirochètes responsables de la leptospirose. La leptospirose est une zoonose ré-émergeante à répartition mondiale dont le nombre de cas annuel est évalué à approximativement un million et dont il y a eu une augmentation du nombre de cas ces dernières années. Elle est endémique en régions tropicales. C'est une maladie dont l'incidence réelle est sous-estimée en raison notamment de la grande variabilité de symptômes possibles allant du syndrome grippal à des formes rénales. Le taux de mortalité est élevé aux alentours de 10% mais pouvant aller jusqu'à 50% dans certains cas avec des complications pulmonaires. De nombreux facteurs influent sur le cycle complexe de transmission de la leptospirose. Il peut y avoir un risque sanitaire lié à la présence de rongeurs porteurs de leptospires pathogènes, qui peut être difficile à contrôler si les rongeurs sont résistants aux rodenticides anticoagulants utilisés pour réguler leurs populations. De plus, il peut y avoir des problèmes d'exposition à des risques écotoxiques lors de mise en place de traitements chimiques. Nous avons étudié plusieurs cas concrets montrant la difficulté dans la prise de décision liée à la gestion des rongeurs dans des contextes divers. Dans un contexte de problèmes économiques, nous avons analysé le portage de leptospires pathogènes par des campagnols des champs et des campagnols terrestres ainsi que la résistance de cible à la bromadiolone, molécule utilisée en lutte chimique à basse densité de population. Nous nous sommes intéressés à l'influence de la perception sociétale sur le risque sanitaire lié à des souris dans un élevage bovin dans lequel il y a des cas de leptospirose humaine et de leptospirose bovine. Dans le cadre d'un projet visant à étudier l'efficacité de gestion de populations de rongeurs commensaux en Martinique dans un contexte leptospirosique, nous avons regardé l'exposition des rongeurs aux AVKs et la résistance de cible aux AVKs. Pour le portage de leptospires pathogènes par les campagnols nous avons trouvé des prévalences allant de 3% à 53% selon la zone étudiée. Il y avait 3 mutations sur le gène vkorc1 amenant peu de résistance de cible à la bromadiolone. Dans l'élevage bovin nous avons notamment trouvé que la perception sociétale de l'éleveur sous-estimait le risque lié aux souris dont 41% étaient porteuses de leptospires pathogènes et 50% étaient porteuses de mutations du gène vkorc1 amenant de la résistance de cible aux AVKs. En Martinique nous avons trouvé que 54% des individus analysés avaient été exposé à au moins une molécule AVKs et qu'il y avait plusieurs mutations du gène vkorc1 dont certaines pas encore caractérisées amenant de la résistance de cible aux AVKs. La gestion des rongeurs est un problème complexe avec plusieurs facteurs à prendre en compte que ce soient les risques liés à l'exposition aux rongeurs ou ceux liés à l'utilisation de rodenticides anticoagulants [etc...] / Management of rodent populations is important because they can cause various economic, ecologic and health problems. This management relies on prevention methods, these ones being architectural, hygienic and agricultural practices. It relies on biologic, chemical and mechanic control methods. Chemical control methods are made mainly with the use of anticoagulant rodenticide acting several hours after ingestion to provide food eviction. Excessive use of these molecules led to resistance selection of some individuals yielding their use underperforming. Then there has been new molecules development, but they are more ecotoxic. In these thesisworks we are interested in management of rodent populations within a framework of health risk of leptospirosis. Rodents are main reservoirs of pathogenic leptospires, Spirochete bacterias responsible of leptospirosis. Leptospirosis is a worldwide re-emerging zoonosis of which there are approximately one million of cases each year and of which there is an increasing number of cases these last years. It is endemic in tropical regions. It is a disease of which real incidence is underestimated because of especially large variability of possible symptoms ranging from flu syndrome to renal forms. Mortality rate is high around 10% and up to 50% in certain cases with pulmonary complications. Numerous factors influence on complex transmission cycle of leptospirosis. There can be health risk associated with presence of rodent carriers of pathogenic leptospires, which can be difficult to control if rodents are resistant to anticoagulant rodenticides used to regulate their populations. Furthermore, there can have exposition problems to ecotoxic risks throughout establishment of chemical treatment. We studied several concrete cases showing the difficulty in decision-making relied with rodent management in various contexts. In a context of economic problems, we analysed pathogenic leptospires carriage by meadow voles and ground voles as well as target resistance to bromadiolone, used molecule in chemical control of low population density. We are interested in influence of societal perception on health risk relied with mice in bovine farming in which there are human and bovine leptospirosis cases. In framework of a project leading to study efficiency of commensal rodent populations management in Martinique in a leptospirosis context, we looked rodent exposition to AVKs and target resistance to AVKs. For pathogenic leptospires carriage by voles we found prevalence ranging from 3% to 53% according to studied area. There were 3 mutations on vkorc1 gene leading to few target resistances to bromadiolone. In the bovine farming we found especially that societal perception of the breeder underestimated risk relied with mice of which 41% were carriers of pathogenic leptospires and 50% were carriers of vkorc1 gene mutations leading to target resistance to AVKs. In Martinique we found that 54% of analysed individuals have been exposed to at least one AVKs molecules and that there were several vkorc1 gene mutations in which certain were not characterised yet leading to target resistance to AVKs. Rodent management is a complex problem with several factors to take into account whether risks relied with rodent exposition or those relied with the use of anticoagulant rodenticides. Furthermore, the concept of risk is scientific but the perception which is subjective participates in decision-making of a way of management

Les chimiothérapies conventionnelles anticancéreuses ont été développées dans le but de traiter le cancer par élimination directe et/ou par inhibition de croissance les cellules tumorales en division. Les cellules endothéliales en prolifération à l’origine de la vascularisation intra-tumorale sont également connues pour être sensibles aux effets cytotoxiques des agents anticancéreux. Depuis, de nombreuses études ont montré que certaines thérapies conventionnelles peuvent être exploitées pour leurs capacités anti-angiogéniques (Browder et al. Cancer Research 2000). La stratégie mise en place consiste à suivre des protocoles où la thérapie est administrée à des doses faibles non myéloablatives et plus fréquemment que les thérapies conventionnelles, appelés dosages métronomiques (Hanahan et al. JCI 2000, Gasparini et al. Lancet Oncology 2001). Le cyclophosphamide (CTX) est un agent alkylant communément utilisé en chimiothérapie dans des protocoles à dosage métronomique. Dans les années 1980, 2 études ont montré que le CTX utilisé à dose métronomique pouvait avoir aussi un rôle sur l’immunité en réduisant la fonction suppressive d’une population de lymphocytes T CD4+ dans un modèle expérimental de tumeur (Awwad et al. Cancer Research 1989) et chez des patients atteints de cancer (Berd et al. Cancer Research 1987). Depuis, les connaissances ont progressé et à présent le CTX métronomique est reconnu pour pouvoir limiter l’expansion et les fonctions des lymphocytes T régulateurs (Treg) (Ghiringhelli et al. EJI 2004, Lutsiak et al. Blood 2005) conduisant à une polarisation des cellules T auxiliaires vers un profil Th1 (Matar et al. Eur J cancer 2000 et Cancer Immunol Immunother 2002). Utilisé en association, le CTX métronomique s’avère donc être un outil intéressant dans le traitement anticancéreux (Hermans et al. Cancer Research 2003, Taieb et al. JI 2006). Nos résultats montrent l’importance des lymphocytes T CD4+ sécréteurs d’IL-17 et d’IFNg dans les effets du CTX. / Conventional cancer chemotherapies were developed to target cancer cells either by directly eliminating them or by inhibiting the growth of dividing tumor cells. Proliferating endothelial cells at the origin of intratumoral vascularization are known to be sensitive to the cytotoxic effects of antineoplastic agents. Many studies have shown that some conventional therapies can be exploited for their anti-angiogenic capabilities (Browder et al. Cancer Research 2000). The adopted strategy, called metronomic chemotherapy, consists of administering low doses of drug that do not induce myelosuppression, on a more frequent schedule as compared to conventional therapies (Hanahan et al. JCI 2000, Gasparini et al. Lancet Oncology 2001). Cyclophosphamide (CTX) is an alkylating agent commonly used as a metronomic chemotherapy. In the 1980s, two studies demonstrated that when used at a metronomic dosing, CTX could impact the immune response particularly in reducing the suppressive function of a CD4+ T lymphocyte population in an experimental tumour model (Awwad et al. Cancer Research 1989) and in cancer patients (Berd et al. Cancer Research 1987). Since then, knowledge has evolved and now CTX used as a metronomic or low-dose therapy is administered to limit expansion and functions of regulatory T cells (Treg) (Ghiringhelli et al. EJI 2004, Lutsiak et al. Blood 2005), leading to a helper T cell polarization toward a Th1 profile (Matar et al. Eur J cancer 2000 et Cancer Immunol Immunother 2002). When used in combination, CTX turns out to be a potent drug in the antineoplastic treatments armamentarium (Hermans et al. Cancer Research 2003, Taieb et al. JI 2006). Our results demonstrate the importance of CTX effects on IL-17 and IFNg secreting CD4+ T lymphocytes.

Les ARNs non-codants régulateurs (ARNnc) regroupent des acteurs majeurs de la régulation de l'expression des gènes, retrouvés de manière ubiquitaire dans l'ensemble des domaines du vivant. Chez les bactéries ils sont également appelés sRNAs, jouent des rôles clefs dans de nombreuxprocessus physiologiques et adaptatifs. Ces sRNAs ont été mis en évidence par des méthodes expérimentales haut-débit (microarray, tilling-array...) dans plusieurs espèces bactériennes d'intérêt. Ils agissent majoritairement au niveau post-transcriptionnel via une interaction physique avec un ou plusieurs ARNs messagers(s) cibles(s). Cependant, les informations sur les ARNmet les fonctions potentielles de ces sRNAs restent très parcellaires à ce jour. De plus, les profils d'évolution des sRNAs ont été peu étudiés chez les bactéries pathogènes. Le travail réalisé dans cette thèse repose sur l'hypothèse que l'évolution des sRNAs et leur coévolution avec d'autres éléments fonctionnels dans un ensemble de génomes, peut permettre de mieux comprendre leurs histoires évolutives, mais également de caractériser leurs fonctions potentielles et peut-être d'aider à identifier le ou les ARNm cibles avec lesquels ils interagissent. Dans ce but, nous avons conçu et implémenté une stratégie de phylogénomique robuste et géné- rique permettant d'analyser l'évolution et la coévolution des sRNAs et des ARNm cibles dans un ensemble de génomes bactériens annotés, à partir de leur profil de présence-absence. Cette méthode a été appliquée à l'analyse de l'évolution et de la coévolution de 154 sRNAs trans régulateurs de Listeria monocytogenes EGD-e. Elle nous a permis d'identifier 52 sRNAs accessoires dont la majo- rité étaient présents dans l'ancêtre commun des souches de Listeria et ont été perdus au cours de l'évolution. Nous avons ensuite détecté une coévolution significative entre 23 sRNAs et 52 ARNm et nous avons reconstruit le réseau de coévolution des sRNAs et ARNm de Listeria. Ce réseau contient un hub principal de 12 sRNAs qui coévoluent avec des ARNm codant pour des protéines de la paroi ainsi que des facteurs de virulence. Parmi eux nous avons pu identifier 4 sRNAs coévoluant avec 7 gènes codant pour des internalines qui sont connues pour regrouper d'importants facteurs de virulence chez Listeria. De plus, l'ARN rli133, qui coévolue avec plusieurs gènes impliqués dans le pouvoir pathogène de Listeria, contient des régions compatibles avec des interactions physiques directes inhibitrices pour la majorité de ses partenaires de coévolution. / Non coding RNAs (ncRNA) are main actors of gene expression regulation and are found ubiquitously in all domains of life. In bacteria, ncRNAs play key roles in a wide range of physiological and adaptive processes. These "small non coding RNAs" (sRNAs) are identified by high-throughput experimental methods (microarray, tilling-array, ...) in several bacteria species of interest. They mainly act at post-transcriptional level through physical interactions with one or several mRNA(s). Nevertheless, the available informations about mRNA targets and sRNAs functions, remain very limited. In addition, evolutionary patterns of sRNAs have been poorly studied in pathogenic bacteria. The main hypothesis of my PhD work is therefore that analysis of evolution and coevolution between sRNAs and other functional elements in a given genomes set, may allow to understand their evolutionary histories, to better characterize their putative functions, and may also help to identify their potential mRNA(s) target(s). For this purpose, we designed and developed a robust and generic phylogenomic approach to analyze evolution and coevolution between sRNAs and mRNA from their presence-absence profiles, in a set of annotated bacterial genomes. This method was thereafter used to analyze evolution and coevolution of 154 Listeria monocytogenes EGD-e trans regulatory sRNAs in 79 complete genomes of Listeria. This approach allowed us to discover 52 accessory sRNAs, the majority ofwhich were present in the Listeria common ancestor and were subsequently lost during evolution of Listeria strains. We then detected significant coevolutions events between 23 sRNAs and 52 mRNAs and reconstructed the coevolving network of Listeria sRNA and mRNA. This network contains a main hub of 12 sRNAs that coevolves with mRNA encoding cell wall proteins and virulence factors. Among them, we have identified 4 sRNAs coevolving with 7 internalin-coding genes that are known to group important virulence factors of Listeria. Additionaly, rli133, a sRNA that coevolve with several genes involved in Listeria pathogenicity, exhibits regions compatible with direct translational inhibitory physical interactions for most of its coevolution partners.

No presente trabalho, foi utilizada uma coleção de 14 isolados de Sporisorium scitamineum coletados em diferentes regiões canavieiras, para estudar a diversidade genética por RFLP da região telomérica de maneira comparativa a marcadores AFLP. Os teliósporos, esporos de resistência do fungo, foram coletados a partir do sintoma mais característico da planta infectada que é formação do chicote. Os teliósporos são diplóides e quando germinam dão origem ao probasídio, onde, por meiose formam-se os esporídios haplóides. Estes quando compatíveis sexualmente voltam a se fundir formando um micélio dicariótico infectivo. A fase do ciclo de vida escolhida para as análises foram os derivados haplóides de cada linhagem dicariótica obtida a partir dos teliósporos. Na técnica de AFLP foram encontrados 40 loci polimórficos (3%) entre 1311 analisados obtidos a partir de 2 enzimas de corte raro, 1 de corte frequente e 19 combinações de primers. A técnica de RFLP da região telomérica foi comparativamente mais eficiente, no qual foram utilizadas três enzimas de restrição que geraram 102 loci, sendo 36 polimórficos (34,3%). O agrupamento com base nos coeficientes de similaridade e os resultados de atribuição pelo programa Structure revelaram dois grupos genotípicos homogêneos, tanto quando os marcadores foram analisados separadamente como na análise conjunta. Não houve agrupamento por localidade, mas ficou claro que o fluxo desses isolados é baixo. Os derivados haplóides de tipos de reação sexual opostos de cada linhagem dicariótica (teliósporo) permaneceram dentro do mesmo grupo, com exceção de uma única linhagem. Desta forma, de maneira geral na natureza, a fusão entre os esporídios deve acontecer entre aqueles que estão mais próximos. Uma análise molecular de diversidade genética em isolados obtidos em diferentes regiões do mundo por outros autores revelou uma alta homogeneidade entre eles, de forma que somente em localidades da Ásia foi possível revelar, com os marcadores utilizados, alguma diversidade genética. Nossos resultados indicam que a escolha do marcador foi fundamental para revelar diversidade entre os isolados de S. scitamineum, sendo que o RFLP-tel revelou um fingerprinting de DNA quase que específico para cada isolado, sendo assim mais apropriado do que os descritos anteriormente. A quantidade de loci AFLP necessária para revelar polimorfismo foi mais alta do que para RFLP-tel. Uma segunda contribuição deste trabalho foi a detecção de heterozigotos quando consideramos a análise conjunta de derivados haplóides por teliósporo. Um alto grau de homozigosidade foi detectado quando as análises foram realizadas considerando o comportamento dicariótico como diplóide, no entanto, loci heterozigotos puderam ser encontrados. Esta é a primeira vez que um estudo desta natureza foi realizado com isolados de Sporisorium scitamineum do Brasil. / The genetic diversity of Sporisorium scitamineum, the sugarcane smut agent, was characterized in a 14 isolates collection. The isolates were obtained from various sugarcane growing areas and RFLP of the telomeric region was used as molecular marker, compared with AFLP. Teliospores were collected from the whip of infected plants. Teliospores are diploids and germinate producing a probasidium where meiosis takes place originating four haploid cells. These cells if sexually compatible can fuse and a dikaryotic mycelium is formed. The haploid phase, derived from each dikaryotic line obtained from teliósporos, was used to perform the analysis. Our results showed that 40 polymorphic loci (3%) were described among 1,311 analyzed. These were obtained with two rare-cutter restriction enzymes, one frequent-cutter restriction enzyme and 19 primers combinations. The RFLP markers were more efficient when compared to AFLP to reveal polymorphisms. Three restriction enzymes produced 102 loci, from which 36 were polymorphic (34.3%). Clustering using similarity coefficients and results obtained by Structure software revealed two genotypic groups. The analyses were performed with individual markers and combining RFLP and AFLP markers. Locations were not responsible for clustering, and low flux of isolates was evident. The opposite sexual types haploid derivatives originated from the same teliospore were clustered together, with only one exception. This implies that sporideos that are located close together are more likely to fuse. Our data suggest that choosing RFLP as markers were the key for unrevealing diversity among isolates of S. scitamineum. RFLP-tel revealed almost unique DNA fingerprintings to various isolates. A second contribution of this work was that heterozygous were detected when considering a combined analyses of haploids derivatives from the same teliospore. This is the first time a study of this nature was organized with Brazilian isolates of S. scitamineum.

A bactéria Xylella fastidiosa causa a clorose variegada dos citros, também conhecida como amarelinho. Em 2000, foi concluído o seqüenciamento do genoma deste organismo, o primeiro de uma bactéria fitopatogênica, um fato que estimulou o estudo dos possíveis mecanismos de patogenicidade empregados pela bactéria para causar a doença em citros. X. fastidiosa é uma bactéria limitada ao xilema, sendo transmitida de planta a planta através de insetos vetores. Especula-se que a bactéria produza enzimas que degradem a membrana da pontuação, permitindo a colonização de novos vasos do xilema. A identificação de genes com similaridade de seqüência a genes de celulases, xilanases, pectinases e proteases, fomentou o presente estudo visando caracterizar os genes Xf - 810, Xf - 818 e Xf - 2708, similares a endoglicanases. Estes genes foram clonados em vetores de expressão e as respectivas proteínas foram produzidas em Escherichia coli. Através de ensaios enzimáticos as proteínas foram caracterizadas como endoglicanases (EC 3.2.1.4), que são celulases com mecanismo endoglicolítico de ataque às moléculas de celulose. Estas celulases hidrolisam carboximetil celulose, Avicel e xilana, enquanto as enzimas Xf - 810 e Xf - 818 hidrolisam a celulose amolecida com ácido. Estas celulases degradam mais eficientemente a carboximetil celulose em pH ácido (entre 5,2 e 5,6) e na temperatura de 65 °C. Coletivamente, estas celulases são capazes de degradar polímeros solúveis e insolúveis, enquanto a enzima Xf - 818, é capaz de degradar oligossacarídeos derivados da celulose, como celotetraose e celopentaose, apresentando ampla variação catalítica. Esta celulase possui capacidade de ligação à celulose microcristalina, denotando a funcionalidade de seu domínio ligador de celulose. Desenvolvemos um protocolo, empregando cromatografia de troca aniônica, afinidade por metal (níquel) e filtração em gel, eficiente na purificação da proteína Xf - 818 expressa heterologamente em E. coli com fusão hexahistidina à extremidade N-terminal. A caracterização enzimática destas proteínas, com a confirmação da atividade celulásica, fornece subsídios para uma eventual função das celulases durante a colonização do hospedeiro pela bactéria, pois são cataliticamente funcionais. Ademais, corrobora a similaridade destes genes, verificada durante o seqüenciamento do genoma de X. fastidiosa. / Xylella fastidiosa is the causal agent of citrus variegated chlorosis, also known as "amarelinho". The recent sequencing of its genome, achieved in 2000, was the first of a plant pathogen, a fact that stimulated the search for putative pathogenicity factors employed by this bacterium while infecting citrus trees. X. fastidiosa inhabits exclusively the xylem vessels, being transmitted by sharpshooter vectors. Several authors argue that the bacterium produces enzymes to degrade plant cell, as a way to colonize new xylem vessels through pit membrane degradation. The identification of putative cellulases, xylanases, pectinases and proteases on X. fastidiosa genome, led us to carry out the present work to characterize the putative products of the endoglucanase genes Xf - 810, Xf - 818 and Xf - 2708. These genes were cloned into expression vectors and the proteins were produced in Escherichia coli. Based upon enzymatic assays, those proteins were characterized as endoglucanases (EC 3.2.1.4), which are cellulases able to promote the endo-hydrolysis of cellulose chains. These cellulases degraded carboxymethylcellulose, Avicel and xylan, while only Xf - 810 and Xf - 818 degraded acid swollen cellulose. The hydrolysis of carboxymethylcellulose was higher at acidic pH between 5.2 and 5.6) and at a temperature of 65 °C. As a group, these enzymes were able to degrade soluble and insoluble cellulose derivatives, while only the cellulase Xf - 818 could hydrolyze the cello-oligosaccharides cellotetrose and cellopentose, thus showing a high catalytic diversity. This protein also has the capacity to bind microcristalline cellulose, confirming the presence of a functional cellulose-binding domain. We set a protocol, employing anion exchange, metal affinity and gel filtration chromatography, to purify the Xf - 818 enzyme expressed in E. coli as a N-hexahistine fusion tag. The endoglucanase activity studied gives support for an eventual role of such cellulases during host colonization by the bacterium. Besides that, it agrees with similarities searches observed on the sequencing of X. fastidiosa genome.

O meloeiro (Cucumis melo L.) é uma cultura de grande importância econômica para o comércio de exportação brasileiro e é cultivada principalmente na região Nordeste. A produção da cultura pode ser limitada por uma doença das partes aéreas, denominada oídio, sendo no Brasil, causada pelo fungo Podosphaera xanthii. Este patógeno apresenta diversas raças definidas com base na reação de um conjunto de cultivares diferenciadoras de meloeiro. Dentre estes genótipos, o acesso PI 414723 é resistente à maior parte das raças e a linhagem Védrantais é suscetível. O presente trabalho teve como objetivos: (i) estudar a herança da resistência às raças 1, 3 e 5 de P. xanthii em indivíduos da geração F2 do cruzamento PI 414723 x Védrantais e (ii) mapear os genes de resistência a estas raças com base em marcadores de polimorfismo de comprimento de fragmentos amplificados (AFLP), de repetições de sequências simples (SSR) e análogos de genes de resistência (RGA) também nesta população. A herança da resistência foi analisada em 87 indivíduos F2 cultivados em condições de casa-de-vegetação. As três raças foram inoculadas em seis regiões eqüidistantes da nervura central em quatro folhas de cada planta. Plantas foram classificadas como resistentes ou suscetíveis com base em avaliações visuais do desenvolvimento do fungo nas folhas. As plantas foram classificadas como suscetíveis quando houve reprodução abundante de conídios e resistentes quando a reprodução foi inexistente ou escassa. Frequências de indivíduos resistentes e suscetíveis indicaram que a resistência às três raças é controlada por um gene dominante de efeito maior. Um mapa genético foi construído compreendendo 1.469 cM, consistindo de 207 marcadores (139 AFLP, 47 SSR, 18 RGA e três fenotípicos) e com uma distância média de 7,4 cM entre marcadores distribuídos em 12 grupos de ligação. Análises de co-segregação com marcadores indicaram que os genes de resistência estão localizados no grupo de ligação II. Em adição a isto, as análises indicaram ligação completa entre os genes de resistência às raças 1 e 5, sendo este gene denominado Pm-x1.5. Já o gene de resistência à raça 3 (Pm-x3) foi localizado a 5,1 cM dos demais. Um marcador AFLP (H35M75_156) foi localizado entre os dois genes a 1,3 cM de Pm-x1.5 e 3,8 cM de Pm-x3. Este é o primeiro relato da localização genética de genes de resistência às raças 3 e 5 em PI 414723 e também o primeiro relato do mapeamento de marcadores RGA gerados pela técnica TRAP em meloeiro. / Melon (Cucumis melo L.) is a crop of great economic importance for the export trade in Brazil and is cultivated mainly in the Northeast. Crop yield can be affected by a disease of the aerial parts, called powdery mildew that in Brazil is caused by the fungus Podosphaera xanthii. This pathogen has several races characterized based on the reaction of a set of differential melon cultivars. Among these genotypes, the plant introgression PI 414723 is resistant to most races and the breeding line Védrantais is susceptible. This study aimed to: (i) study the inheritance of resistance to races 1, 3 and 5 of P. xanthii in the F2 generation from the cross PI 414723 x Védrantais, and (ii) map resistance genes to these races in this same population based on amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR) and resistance gene analog (RGA) markers. The inheritance of resistance was analyzed on 87 F2 individuals grown under greenhouse conditions. The three races were inoculated simultaneously on four leaves of each plant. Plants were classified as resistant or susceptible based on visual assessments of fungal growth on the leaves. Plants were considered susceptible when there was abundant production of conidia and resistant when the production was scarce or non-existent. The frequencies of resistant and susceptible individuals indicated that resistance to all three races is controlled by a dominant major gene. A genetic map was constructed comprising 1469 cM, consisting of 207 markers (139 AFLP, 47 SSR, 18 RGA, and three phenotypic) with an average distance of 7.4 cM between markers distributed in 12 linkage groups. Co-segregation analysis with markers indicated that the resistance genes are located on linkage group II. Moreover, the analysis indicated complete linkage between resistance to races 1 and 5, and this gene was denominated Pm-x1.5. The gene for resistance to race 3 (Pm-x3) was located at 5.1 cM from Pmx1.5. An AFLP marker (H35M75_156) was located between the two genes at 1.3 cM from Pmx1.5 and 3.8 cM from Pm-x3. These is the first report on the location of resistance genes to races 3 and 5 in PI 414723, and also the first report of RGA markers mapping using the TRAP technique in melon.

Chlamydia trachomatis serovars D-K are the leading cause of bacterially-acquired sexually transmitted infections in the United States. As an obligate intracellular pathogen, C. trachomatis infects columnar epithelial cells of the genital mucosae and can cause deleterious sequelae such as pelvic inflammatory disease, infertility, and ectopic pregnancy. Several chlamydial antigens reach the host cell cytosol prior to the natural release of chlamydiae at the end of the developmental cycle. While some of these extra-inclusion antigens traffic to the host cell surface, others remain intracellular where they are proposed to influence vital host cell functions and antigen trafficking and presentation. The research herein examines the escape and trafficking of the immunodominant chlamydial antigens MOMP, LPS, and cHsp60 within C. trachomatis serovar E-infected polarized human endometrial epithelial cells. Studies using high-resolution transmission electron microscopy (TEM) and immuno-TEM report the novel escape mechanism of chlamydial antigens via vesicles everted/pinched off from the inclusion membrane, an occurrence observed both in the presence and absence of the antibiotic azithromycin. These extra-inclusion vesicles were differentiated from Golgi vesicles and were shown to deliver chlamydial heat shock protein 60 (cHsp60)-homologs 2 and 3, but not homolog 1, to the infected cell surface. Examination of the iron-responsiveness of the three cHsp60 homologs by immuno-TEM revealed a significant increase in cHsp60-2 following iron deprivation. Further investigation of the trafficking of chlamydial MOMP and LPS antigens enveloped within the protective everted inclusion membrane vesicles within host cells involved density gradient centrifugation for the separation of epithelial secretory pathway components followed by SDS-PAGE and Western blot to determine whether the chlamydial antigen-containing vesicles could fuse with and deliver the antigens to host cell organelles. Coupled with immuno-TEM, these data confirmed the presence of major chlamydial antigens within the endoplasmic reticulum of infected host cells. Additionally, chlamydial lipopolysaccharide (LPS) was co-localized with CD1d, a lipid antigen-presenting molecule. Collectively, these studies (i) establish a novel escape mechanism for chlamydial antigens, (ii) identify cHsp60-2 as a marker of iron stress response in C. trachomatis, and (iii) define for the first time the host cell ER as a destination for selected chlamydial antigens during infection.