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Orientador: Celso Eduardo Benedetti / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T07:02:38Z (GMT). No. of bitstreams: 1 Camillo_LucianaRodrigues_M.pdf: 5919875 bytes, checksum: 5cf571ac0a143a68d6340c3ad3c97f49 (MD5) Previous issue date: 2006 / Resumo: A doença cancro cítrico, causada pela bactéria Xanthomonas axonopodis pv. citri (Xac), emergiu como uma das principais ameaças à citricultura brasileira pois afeta todas as variedades comerciais de citros, diminuindo a produção e qualidade dos frutos e podendo se dispersar rapidamente em áreas de cultivo de citros. Entre os gêneros de Xanthomonas, foram encontrados muitos genes associados com patogenicidade e virulência, entretanto, pouco se conhece sobre os mecanismos envolvidos nas interações entre Xac e laranjeira e os genes envolvidos no desenvolvimento dos sintomas do cancro cítrico em folhas de citros (Citrus sinensis) em resposta à infecção por Xac. Neste trabalho, foi analizada a expressão diferencial de genes envolvidos no desenvolvimento do cancro cítrico em folhas de laranja, em resposta à infecção por Xac. Para tanto foram construídas duas bibliotecas de Hibridização Subtrativa Suprimida (SSH) com mRNAs de folhas infiltradas com Xac ou H20 após 10 dias de inoculação. O "screnning" das bibliotecas foi feito por dot blot e 17 genes diferencialmente expressos foram sequenciados e identificados por homologia no banco de dados ncbi-BLAST contra o banco de dados de EST de citros. A expressão gênica diferencial foi analizada por Northern Blot e PCR em tempo real (qPCR). Para complementar nossos dados, a expressão dos 17 genes diferenciais foi aI).alisada a partir de cDNAs de folhas de citros que foram infiltradas com Xac, H20 ou Xanthomonas axonopodis pv. aurantifolii (Xaa), que não é patogênica à laranja, mas causa cancro no limão galego. Nossos resultados demonstraram que Xac e Xaa induzem e reprimem o mesmo grupo de genes, porém em diferentes níveis. Nós identificamos que ambos patóg;enos alteram as vias de transdução de sinal de auxina, tráfico e fusão de vesículas, resposta de defesa e doença, síntese de proteínas e ciclo celular, metabolismo de carbono-nitrogênio e metabolismo secundário. A análise desses genes poderá ser de grande importância para o entendimento dos eventos que levam ao desenvolvimento dos sintomas do cancro / Abstract: The citrus canker disease, caused by Xanthomonas axonopodis pv. citri (Xac), has emerged as one of the major threats to the Brazilian citriculture because it affects all commercial citrus varieties, decreases the production and quality of the fruits and can spread rapidly in the citrus growing areas. The symptoms include canker lesions, leading to abscission of fruits and leaves and general tree decline. Within the genus Xanthomonas, several genes have been found associated with pathogenicity and virulence. However, little is known about the mechanisms involved in the Xac- citrus interaction and the development of the canker disease. In this work we analyzed the differential expression of genes involved in the development of the canker disease in sweet orange (Citrus sinensis) leaves in response to Xac infection. Therefore we constructed two Supression Subtracted Hybridization (SSH) libraries with mRNA from leaves infiltrated with Xac or water after 10 days of inoculation. The libraries were screnned by dot blot and the 17 differentially expressed genes were sequenced and identified through BLAST searches against the Citrus EST and protein databases. The differential gene expression was evaluated by Northem blot and Real Time PCR (qPCR). To complement our analysis, the expression of 17 genes was compared in cDNA samples from citrus leaves that had been infilt:r:ated with Xac, water or X axonopodis pv. aurantifolii (Xaa), wich is not pathogenic to orange but causes canker in key lime. Our results show that Xac and Xaa induce and repress a similar set of genes, however at different leveis. We found that both pathogens altered the pathways of auxin transport and signaling, vesicle trafficking and transport, cell cycle and protein synthesis, photorespiration and disease response. Further analysis of these genes will be of great importance to understand the events triggering the development of the canker symptoms / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

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Les anévrysmes intracrâniens (AIC) sont des malformations artérielles développées au dépend des vaisseaux qui vascularisent le parenchyme cérébral. Leur rupture provoque une hémorragie intracrânienne, appelée hémorragie sous-arachnoïdienne, responsable d'une mortalité importante ou de séquelles fonctionnelles lourdes. Le traitement préventif de ces lésions est fréquemment réalisé lors d'une procédure endovasculaire (appelée coiling), par implantation, au sein de la poche artérielle pathologique, de spires métallique en platine à détachement contrôlé (les coils). La présence de ce matériel provoque une thrombose de la poche ce qui entraine secondairement une exclusion de l'anévrysme de la circulation artérielle. Une modalité de traitement endovasculaire plus récente fait appel à un dispositif implantable innovant appelé stent "flow diverter" (FD) que l'on déploie en regard de l'orifice qui fait communiquer l'artère et l’anévrysme : le collet anévrysmal. Ces stents FD, au design particulier, associant une faible porosité à une densité de pores élevée, agissent comme des "déflecteurs" et diminuent le flux sanguin entrant et sortant de l'anévrysme. L'objectif du traitement demeure toujours l'exclusion de l'anévrysme mais celle-ci est obtenue indirectement en agissant sur la "porte d'entrée" de l'anévrysme (le collet) et non plus directement sur la poche anévrysmale elle-même. Il ne s'agit plus alors de remplir le sac anévrysmal avec des coils mais de provoquer une thrombose stable et pérenne en altérant uniquement le flux sanguin qui le pénètre. Cette modalité thérapeutique novatrice a suscité un engouement important de la part des neuroradiologues interventionnels depuis 2007, date des premières implantations en Europe. Cependant, bien que reposant sur les capacités d'un tel dispositif à modifier le flux, on constate qu'il existe très peu d'outils d'imagerie disponibles actuellement et capables de quantifier ces modifications en un délai raisonnable pour pouvoir être exploité lors du traitement endovasculaire. De cette constatation clinique est né un projet collaboratif dont la finalité était le développement d'un outil logiciel basé sur les séquences d'angiographie numérisées soustraites et capable de mesurer au moins un des aspects du flux sanguin (et donc de ses modifications). La démarche de recherche mise en œuvre s'est effectuée en trois étapes. Premièrement, une étape expérimentale portant sur la réalisation d'un modèle "optimisé" d'AIC permettant le recueil de données hémodynamiques et d'imagerie. Puis, une étape de recherche plus fondamentale comprenant deux parties: d'une part des simulations numériques réalisées dans le cadre d'un modèle 3D réaliste d'AIC et d'autre part l'analyse d'images angiographiques. Au cours de cette étape, nous avons utilisé des outils de traitement d'images existants et développé certains algorithmes, puis les avons validés avant de les implémenter sous JAVA pour créer un outil logiciel d'analyse de flux. Enfin, la dernière étape du projet a consisté en l'exploitation du logiciel pour étudier une série clinique de patients traités d'un AIC par stent FD. Elle a permis de mettre en évidence certains facteurs prédictifs d'exclusion de l'anévrysme à long terme susceptible d'avoir un impact, en temps réel, sur le traitement des AIC par stent FD. / Intracranial aneurysms treatment based on intra aneurismal flow modification tend to replace traditionally coiling in many cases and not only complex aneurysms for which they were initially designed. Dedicated stents (low porosity, high pores density stents) called “flow diverter” stents are deployed across the neck of the aneurysm to achieve this purpose. The summation of three different mechanisms tend to lead to the healing of the aneurysm: immediate flow alteration due to the mechanical screen effect of the stent, physiological triggering of acute or progressive thrombus formation inside the aneurysm’s pouch and long term biological response leading in neointima formation and arterial wall remodeling. This underlying sequence of processes is also supposed to decrease the recanalization rate. Scientific data supporting the flow alteration theory are numerous and especially computational flow dynamics (CFD). These approaches are very helpful for improving biomechanical knowledge of the relations between blood flow and pathology, but they do not fit in real-time treatments. Neuroendovascular treatments are performed under dynamic x-ray modality (digital subtracted angiography a DSA-).However, in daily practice, FD stents are sized to the patient’s 3D vasculature anatomy and then deployed. The flow modification is then evaluated by the clinician in an intuitive manner: the decision to deploy or not another stent is based solely on a visual estimation. The lack of tools available in the angioroom for quantifying in real time the blood flow hemodynamics should be pointed out. It would make sense to take advantage of functional data contained in contrast bolus propagation and not only anatomical data. Thus, we proposed to create flow software based on angiographic analysis. This software was built using algorithms developed and validated on 2D-DSA sequences obtained in a swine intracranial aneurysm model. This intracranial animal model was also optimized to obtain 3D vascular imaging and experimental hemodynamic data that could be used to realize realistic computational flow dynamic. In a third step, the software tool was used to analyze flow modification from angiographic sequences acquired during unruptured IA from patients treated with a FD stent. Finally, correlation between flow change and aneurysm occlusion at long term follow-up with the objective of identifying predictive markers of long term occlusion was performed.