Submitted by Simone Souza (simonecgsouza@hotmail.com) on 2018-05-02T17:09:56Z No. of bitstreams: 1 DISS_2015_Mayara Inácio Vincenzi da Silva.pdf: 1467868 bytes, checksum: 1589e777267d83f74d5b55da57e6a5db (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2018-05-16T15:13:07Z (GMT) No. of bitstreams: 1 DISS_2015_Mayara Inácio Vincenzi da Silva.pdf: 1467868 bytes, checksum: 1589e777267d83f74d5b55da57e6a5db (MD5) / Made available in DSpace on 2018-05-16T15:13:07Z (GMT). No. of bitstreams: 1 DISS_2015_Mayara Inácio Vincenzi da Silva.pdf: 1467868 bytes, checksum: 1589e777267d83f74d5b55da57e6a5db (MD5) Previous issue date: 2015-08-07 / CAPES / Ferro (Fe) é um elemento essencial e a capacidade de adquiri-lo in vivo têm sido descrita em diversos agentes patogênicos através de fatores de virulência. Análises globais de transcritos durante a privação de ferro tem sido descritos através da técnica de “microarray”, entretanto através da técnica de RNA-seq recentemente tem demonstrado resultados superiores. O isolado de Pasteurella multocida (Pm 16759) altamente patogênica em suínos foi cultivado em duas condições com diferentes concentrações de Fe (controle e privação) com o objetivo de analisar transcritos diferencialmente expressos. O RNA total das duas condições foi extraído e sequenciado através da plataforma de nova geração Ion Torrent. Os dados foram analisados no Software Ion Reporter™ e processados no programa Rockhopper. Foram obtidas 1.341.615 leituras com tamanho médio de 81pb, com 96% de alinhamento com o genoma de Pasteurella multocida subsp. multocida 3480 e 98,8% de acurácia. No mapeamento das leituras das duas condições, observou-se 2,652 transcritos e destes, 177 (6,7%) foram diferencialmente expressos, sendo 93 na condição controle (Fe+) e 84 na condição de privação (Fe-). Na condição de privação de ferro, o perfil de transcritos foram associados a função de transporte celular (fbpABC, high-affinity Fe2+/Pb2+ permease e periplasmic protein probably involved in hight-affinity Fe2+), reguladores transcricionais e proteínas hipotéticas. O perfil na condição controle (Fe+) apresentou transcritos diferencialmente expressos associados ao RNAs anti-sense (asRNA) e genes do metabolismo energético (fructose-1,6-bisfosfatase). A técnica de RNA-seq mostrou-se eficiente na identificação de transcritos diferencialmente expressos por P. multocida em condições de privação de Fe, o que pode auxiliar nos estudos de patogenicidade. / Iron (Fe) is an essential element and the ability to acquire it, in vivo, have been described in several pathogens such as virulence factors. Global analyses of transcripts during iron deprivation have been described by microarray studies, however, recently RNA-seq analysis shows superior results. The high pathogenic swine strain of Pasteurella multocida (BRMSA 1113) was grown in the two conditions with different concentrations of Fe (control and deprivation) in order to analyze the differentially expressed transcripts. The total RNA of the two conditions was extracted and sequenced by new generation Ion Torrent plataform. Data were analyzed in Ion Reporter ™ Software and processed in Rockhopper software. Sequence analysis shows 1,341,615 readings with median length of 81pb, with 96% of alignment to the reference genome Pasteurella multocida strain 3489, and 98.8% accuracy. Reads mapping to genome of P. multocida in these two conditions, detected 2,652 transcripts, which , 177 (6.7%) were differentially expressed, with 93 in the control condition (Fe +) and 84 provided with iron deprivation condition (Fe-). In condition (Fe-), differential expressed transcript profile were associated to function of cellular transport (fbpABC,, high-affinity Fe2+/Pb2+ permease and periplasmic protein probably involved in hight-affinity Fe2+), transcptional regulators and hypothetical proteins. The control condition (Fe+) shows differential expressed transcripts profile associated to RNA anti-sense (asRNA) energetic metabolism genes (fructose-1,6-bisphosphatase). The technique of RNA-seq proved effective in identifying novel differentially expressed transcripts by P. multocida (BRMSA 1113) under conditions of Fe deprivation, which can assist in pathogenicity studies.

Le chêne-liège (Quercus suber) est une essence forestière d’une grande importance écologique et socio-économique pour les habitants de Méditerranée. Cependant, au cours des dernières décennies, ces subéraies et particulièrement les subéraies marocaines ont été soumises à de fortes contraintes climatiques, environnementales et humaines entrainant une accélération des processus de dégradation. La conservation de ces écosystèmes est fortement dépendante de notre capacité à prédire les changements induits par ces différentes pressions ainsi que du développement d’approches durables pour leur réhabilitation. Dans ce contexte, l’identification d’indicateurs biologiques de l’état de santé des subéraies et l'intensification des processus de facilitation entre plantes (arbres/arbustes) apparaissent comme des stratégies écologiques prometteuses. Le succès de ces approches est cependant assujetti à notre compréhension des interactions entre les communautés végétales et les champignons du sol, notamment les champignons mycorhiziens, éléments clés du fonctionnement des écosystèmes forestiers. Ce travail a visé le décryptage des réseaux fongiques, notamment mycorhiziens associés au chêne-liège et la végétation du sous-bois dans trois subéraies marocaines (Maâmora, Benslimane, Chefchaoun) caractérisées par différents niveaux de dégradation. La diversité fongique associée aux racines du chêne-liège et à plusieurs plantes arbustives représentatives des subéraies (Cistus salviifolius, Cistus monpeliensis et Lavandula stoechas) a été étudiée en combinant les méthodes traditionnelles basées sur l’aspect morphologique des mycorhizes et les nouvelles technologies de séquençage haut-débit par identification moléculaire des communautés fongiques.Les résultats obtenus représentent la plus vaste enquête de la diversité fongique du sol, notamment mycorhizienne, au sein des subéraies marocaines. Différents niveaux de structuration des communautés de champignons du sol ont été révélés, fonction de l’habitat, du type de plantes et de l'état de dégradation. Une large gamme d’indicateurs fongiques de l’état de dégradation de la subéraie, en lien avec la plantes hôte, ont pu être mise en évidence au sein des différents habitats, soulignant l’importance de plusieurs champignons ectomycorhiziens (notamment Cenococcum, Russula, Terfezia et Tomentella) mais aussi des champignons mycorhiziens éricoïdes (Cladophialophora, Oidiodendron) et à arbuscules (Rhizophagus, Redeckera, Racocetra, Paraglomus). Ce travail a permis d’établir une base de données majeure sur l’écologie des champignons du sol dans les subéraies marocaines, et de proposer un nouvel éclairage sur leur potentiel pour le suivi de l’état de santé des subéraies, ainsi que pour la mise en place de programmes de conservation adaptés tenant compte aussi des champignons associés. L’application des approches proposées à une plus large diversité d’écosystèmes forestiers devrait constituer un atout important pour la meilleure compréhension du fonctionnement biologique des écosystèmes forestiers et leur sauvegarde face à l’aggravation des pressions humaines et climatiques au niveau mondial. / The Cork oak (Quercus suber) forests play an important role in terms of ecological services and socio-economic development for the Mediterranean populations. However, the cork oak forests, notably in the Southern Mediterranean basin are highly threatened by increasing human and climate pressures, which accelerates desertication. The conservation of this ecosytem is strongly dependent of our ability to predict the environmental changes induced by these pressures as well as to develop sustainable approach for their restoration. In this context, the identification of biological indicators of cork oak health and the intensification of plant-plant facilitation processes appears as promisising ecological strategies. Their success is however subjected to our understanding of plant-fungal interactions, notably with fungal mycorrhiza, key factors of forest ecosystem functionning. The current work aimed at deciphering plant-fungal networks, notably mycorrhizal networks with cork oak and its understory shrub vegetation in three Moroccan cork oak habitats (Maâmora, Benslimane, Chefchaoun) characterized by different degradation levels. The root-fungal diversity associated to cork oak and major components of its understory shrub vegetation (Cistus salviifolius, Cistus monpeliensis et Lavandula stoechas) has been analysed by combining traditional methods based on morphological identification, and new generation high- throughput DNA sequencing methods to characterize communities at the molecular level.The study represents the most extensive survey of soil fungal diversity, notably mycorrhizal diversity, in Moroccan cork oak ecosystems. Different fungal community structures were revealed, depending on habitat, plant host type, and degradation forest status. A wide range of fungal indicators of plant type × forest status has been identified, highlighting the importance of several ectomycorrhizal fungi (notably Cenococcum, Russula, Terfezia and Tomentella) as well as ericoid mycorrhizal fungi (Cladophialophora, Oidiodendron) and arbuscular mycorrhizal fungi (Rhizophagus, Redeckera, Racocetra, and Paraglomus). The current work provides an extensive database on the ecology of soil fungi related to the Moroccan cork oak forest, offers new insights into the potential of soil fungi for monitoring the health of the cork oak forest, and for the developement of efficient conservation programs of this ecosystem by taking into account the soil fungal communties associated. The use of proposed appoaches to a larger diversity of forest ecosystems are promising to better understand the biological fonctionning of forest ecosystem and their conservation in response to the worsening of worldwide human and climate pressures.

INTRODUÇÃO: A doença de Chagas é causada pelo protozoário flagelado Trypanosoma cruzi (T. cruzi) e ainda hoje representa um grande problema de saúde pública tendo infectado mais de oito milhões de pessoas. A patogênese da cardiomiopatia chagásica ainda não é completamente compreendida. A inflamação no miocárdio é intensa em relação ao número de parasitos presentes e também se observa um dano progressivo em outros órgãos como esôfago e cólon em 30% a 40% dos casos em que se diagnosticou a doença. Alguns estudos começaram a mostrar que a resposta imunológica a um parasita pode depender da microbiota intestinal; porém, ainda não existem estudos com a tecnologia de sequenciamento de nova geração (NGS) que descrevam a microbiota intestinal em doença de Chagas. É possível que uma pequena alteração do peristaltismo intestinal, decorrente da infecção por T. cruzi possa alterar a colonização de algumas bactérias as quais podem causar mudanças na reatividade do sistema imune como aumentar a resposta autoimune, gerando maior dano ao coração. OBJETIVO: Este trabalho objetivou descrever a microbiota intestinal de acordo com a forma clínica da doença de Chagas, através da amplificação do gene 16s RNA ribossomal e avaliar seu papel na patogênese da doença. MÉTODO: Foram selecionados 114 indivíduos, sendo 30 portadores da forma cardíaca da doença, 11 com a forma digestiva (megacólon), 32 com a indeterminada e 31 indivíduos saudáveis (controles). De cada um deles, foram coletadas amostras de fezes para a análise da microbiota por meio de técnicas de sequenciamento de nova geração Ion Torrent. Os resultados obtidos foram analisados pelo software QIIME para determinar a população de bactérias presentes nas amostras. A análise estatística foi realizada utilizando-se os testes não paramétricos de Kruskal-Wallis e Mann-Whitney U-test. RESULTADOS: A frequência relativa do filo Verucomicrobia foi significantemente menor no grupo cardíaco em comparação ao grupo controle e as outras formas clínicas: indeterminada e digestiva. Apesar da abundância relativa desse filo ser menor do que 1%, a diferença observada se manteve significante, mesmo após a correção de Bonferroni. CONCLUSÕES: Nosso estudo sugere que uma menor proporção do filo Verrucomicrobia possa estar relacionada ao processo inflamatório na forma cardíaca; porém, ainda pouco se conhece sobre este grupo de bactérias e seus componentes, que nos permita afirmar o seu efetivo papel na forma cardíaca da doença de Chagas / INTRODUCTION: Chagas disease is caused by the flagellate protozoan Trypanosoma cruzi (T.cruzi) and still represents a major public health problem with more than eight million people infected. Chagas cardiomyopathy pathogenesis is still not completely understood. Inflammation in the myocardium is intense in relation to the number of parasites present and progressive damage is also observed in other organs such as the esophagus and colon in 30% to 40% of the cases. Some studies are beginning to show that the immune response to a parasite may depend on the intestinal microbiota. However, there are no studies using NGS technology that describes the intestinal microbiota of Chagas disease. It is possible that a small change in intestinal peristalsis due to T cruzi infection may alter the colonization of some bacteria. These changes could cause changes in the reactivity of the immune system such as increasing the autoimmune response causing greater damage to the heart. OBJECTIVE: This study aimed to describe the intestinal microbiota according to the clinical form of Chagas disease, through amplification of the 16s ribosomal RNA gene and to evaluate its role in the pathogenesis of the disease. METHODS: A total of 114 individuals were selected, 30 of cardiac form of the disease, 11 with the digestive form (megacolon), 32 with indeterminate form and 31 healthy individuals (controls). Stool samples were collected and analysed for the microbiota using Ion Torrent sequencing technique. The results were analyzed by the QIIME software to determine the population of bacteria present in the samples. Statistical was performed using Kruskal-Wallis non-parametric test and Mann-Whitney U-test. RESULTS: The relative frequency of the Verrucomicrobia phylum was significantly lower among the cardiac group when compared to control, indeterminate and digestive form. Our study suggest that the phylum Verrucomicrobia may play a role in the miocardio inflammation process in Chagas disease, however little is known about these bacteria to infer the mechanism

An important goal of biology is to understand the key mechanisms of evolution underlying the diversity of living organisms on Earth. In that respect, the recent innovations in the field of new generation sequencing technologies (NGS) are bringing new and exciting opportunities. This thesis presents results obtained with these tools in the specific context of the study of two sister species of cold-adapted leaf beetles, Gonioctena intermedia and G. quinquepunctata. More specifically, this work is focused around four research directions: the two first explore methods of statistical inference using a spatially explicit model of coalescence, by (1) evaluating the potential of various summary statistics to discriminate phylogeographic hypotheses, and (2) investigating the dispersal abilities of a montane leaf beetle, G. quinquepunctata, using an original method that avoids using summary statistics. The third research direction focuses on the adaptation to cold conditions in this montane leaf beetle, by testing the association between genetic polymorphism across tens of thousands of genetic markers and altitude in samples collected at various elevation levels in the Vosges (France). Finally, the fourth, and last, research axis presents the discovery of mitochondrial heteroplasmy, i.e. the presence in an individual of multiple copies of the mitochondrial genome, in natural populations of G. intermedia. We illustrate, here, how NGS technologies could help identify this phenomenon, probably underestimated in animals, on a large scale. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Russians actions in Ukraine came as a chock to the west, who have been striving to conceptualize Russian warfare since. This has resulted in several conflicting definitions involving different means and methods. This might be problematic, both for a country and its military, in trying to define and counter the threat of Russian warfare. It might also be problematic for scientific enquiry, since the definitions might become too broad and neglect vital aspects of Russian warfare. This study there-fore compare and test two rivalling theories about Russian warfare, New-Generation Warfare (NGW) and Full-Spectrum Conflict (FSC), by analysing Russia’s actions in the Ukrainian conflict. By doing so it aims to shed light on which of the two theories offers the best approach to analysing the case. The study uses a qualitative text research method to examine secondary sources describing the conflict and transliterations of Putin’s speeches. Several indicators for each theoretical framework are confirmed and made the analysis possible. The results of this analysis show that Russia in Ukraine used their means and methods synchronously rather than applying a diachronic course of action as advocated by NGW. Results therefore suggest that FSC and its mix of means and methods better describes Russia’s actions in Ukraine.

Until 2008, almost two decades since the end of the cold war, Russia still retained old Soviet military structures. Steady opposition by the Russian generals against military modernization and reforms was finally broken in relation with the poor performance of Russian forces during the Russia-Georgian war in 2008. The process of modernization commenced in the following years has important security implications for post-Soviet countries. We could observe the new abilities of the Russian military during the seizure of the Crimean peninsula in 2014. Russian forces had been considered an ineffective institution with outdated military technology where corrupt practices were the rule rather than the exception. As we could see in 2008 Georgia Russian forces depended on numerical superiority and blunt military force. On the other hand during the Crimean events of 2014 the Russian military conducted the whole operation with high level of professionalism, without any bloodshed, and with modern weaponry. This research is based on the theory of neorealism which defines key parameters for performing a military modernization analysis. These include numbers, effectivity and strategy of military forces that help us to determine the current strength and power of each state. Therefore this research focuses on the...

Comme le Wi-Fi est devenu de plus en plus important dans les réseaux actuels, ainsi que dans les réseaux du futur, de nouvelles exigences " opérateur " se sont apparues afin de supporter les attentes des utilisateurs et de fournir des réseaux Wi-Fi de haute performance. Dans ce contexte, nous étudions plusieurs problèmes liés à la conception et l'optimisation des réseaux Wi-Fi opérateur de nouvelle génération. Dans la première étape, notre objectif est d'améliorer l'expérience utilisateur Wi-Fi et de lui offrir un accès personnalisé et transparent aux réseaux et services Wi-Fi. Pour cela, nous proposons une extension des trames de gestion IEEE 802.11 pour activer la découverte des services locaux avant l'association Wi-Fi, tout en évitant la surcharge du canal. Nous définissons également un ensemble d'étiquettes de service pour identifier d'une manière standardisée les services les plus connus. Dans la deuxième étape, nous adressons les problèmes liés à l'architecture et la gestion du réseau dans un environnement Wi-Fi opérateur de nouvelle génération. Plus précisément, nous proposons, tout d'abord, une nouvelle architecture Wi-Fi qui exploite les concepts de NFV et du Edge Cloud Computing. Nous visons à travers cette architecture à apporter plus d'agilité et d'adaptabilité et d'améliorer la QoS perçue par l'utilisateur en plaçant des fonctions réseau et certains services à proximité de lui. Pour faire face à certains problèmes de gestion dans cette architecture, nous proposons ensuite des stratégies de placement et de provisionnement des fonctions de réseau virtuelles en s'appuyant sur des exigences de QoS. / As Wi-Fi is gaining a lot of momentum in today’s networks as well as in future networks, new carrier-grade requirements are emerging to support future user expectations and provide high-performance Wi-Fi networks. In this context, we investigate several problems surrounding the design and optimization of carrier-grade next-generation Wi-Fi networks. In the first stage, our objective is to improve the Wi-Fi user experience and offer to him a personalized and seamless access to Wi-Fi networks and services. For this, we propose an extension to the IEEE 802.11 management frames to enable venue service discovery prior to Wi-Fi association while avoiding channel overhead. We define also a set of extensible service labels to uniquely and globally identify the most known venue-based services. In the second stage, we deal with network architecture and management issues in next-generation carrier Wi-Fi environment. More specifically, we first propose a novel carrier-managed Wi-Fi architecture that leverages NFV and Edge Cloud Computing concepts. We aim through this architecture to bring more agility and adaptability and improve user perceived QoS by placing network functions and certain services close to end-users. To address some major management issues in this proposed architecture, we then propose placement and provisioning strategies of Virtual Network Functions based on QoS requirements. These strategies can also be applied to any edge-central wireless carrier architecture, since they do not make any assumption about the underlying wireless technology.

Les corticostéroïdes inhalés (ICs) utilisés dans le traitement de l'asthme ont un effet sur le microbiome pulmonaire. Il est toutefois difficile de séparer leurs effets sur les communautés bactériennes via leur rôle immunomodulateur, de leurs effets indirects via l’amélioration de la ventilation. Notre objectif est de déterminer si les ICs altèrent le microbiote pulmonaire indépendamment de leurs effets sur la fonction pulmonaire. Douze chevaux atteints d’asthme en exacerbation ont reçu des bronchodilatateurs à longue durée d'action (LABA, salmétérol) ou une combinaison ICs/LABA (fluticasone/salmétérol) pendant deux semaines. Des tests de fonction pulmonaire et des lavages broncho-alvéolaires (LBA) ont été effectués avant et après traitement. La quantification par PCR et le séquençage du gène codant pour l’ARNr 16S via une plateforme MiSeq-Illumina ont été réalisés sur les LBA. Les données ont été analysées avec mothur. La fonction pulmonaire s’est améliorée dans les deux groupes. Les abondances relatives de familles et genres appartenant au phylum Bacteroidetes ont augmenté suite au traitement ICs/LABA, tandis que les phyla Verrucomicrobia et Actinobacteria se sont appauvris avec les LABA. Une modification de la β-diversité et une diminution de la charge bactérienne ont été observées seulement après le traitement LABA. L’α-diversité n’a en revanche pas varié dans les deux groupes. Les différences observées entre les groupes ICs/LABA et LABA suggèrent que les changements au sein des communautés bactériennes ne sont pas seulement dus à l’amélioration de la ventilation. Il reste toutefois difficile de trancher quant à un effet positif ou bien néfaste des ICs sur le microbiote pulmonaire. / Inhaled corticosteroids (ICs) affect the respiratory microbiome in asthma. It remains difficult to separate the immunomodulatory effects of ICs on bacterial communities from their indirect effects via improvement of ventilation. Our objective was to determine if ICs alter the lung microbiota independently from their effects on lung function. Twelve horses with equine asthma in exacerbation were assigned to receive a long-acting bronchodilator (LABA, salmeterol) or the combination ICs/LABA (fluticasone/salmeterol) for 2 weeks. Lung function and bronchoalveolar lavages (BAL) were performed before and after treatment. 16S rRNA gene quantification and sequencing were performed on BAL fluid, using PCR and the Illumina MiSeq platform. Data were processed using the software package mothur. Lung function improved with both treatments. The relative abundances of families and genera belonging to the Bacteroidetes phylum increased with ICs/LABA, while the Verrucomicrobia and the Actinobacteria phyla decreased with LABA. Furthermore, β-diversity differed from baseline and the bacterial load decreased after LABA only. On the contrary, α-diversity indices did not vary in both groups. The differences observed between the ICs/LABA and LABA groups suggest that the changes in bacterial communities are not only due to improved ventilation. However, it remains difficult to clarify if the effects of ICs are positive or detrimental to the lung environment.

"This document was submitted as a dissertation in December 2009 in partial fulfillment of the requirements of the doctoral degree in public policy analysis at the Pardee RAND Graduate School." / Title from PDF title screen (viewed Jan. 21, 2010). Includes bibliographical references (p. 343-360).

Contexte: Le carcinome hépatocellulaire (CHC) est la plus fréquent des tumeurs primitives du foie. Près de 50% des CHC sont causés par une infection par le virus de l'hépatite B (VHB). Au cours des différents stades de l’infection par le VHB, des multiples altérations génétiques et/ou chromosomiques s'accumulent et favorisent ainsi le développement tumoral. Objectives: a) étudier, in vitro et in vivo, le rôle potentiel d’un nouveau gène susceptible d’être impliqué dans la carcinogénèse hépatique : IRF-2 (Interferon regulatory factor 2). Ce gène a été identifié comme fréquemment délété par analyse CGH-SNP dans les CHC liés à l’infection par le VHB. b) caractériser une série de CHC liés au VHB en étudiant le statut viral, les altérations génétiques et l’expression de différents gènes afin de mieux comprendre le rôle du VHB dans l’hépato-carcinogenèse et comparer ces différents paramètres avec une série de CHC non liés au VHB. Résultats : a) Dans une série de 125 CHC, Sandrine Imbeaud, au sein du laboratoire, a effectué une analyse CGH-SNP microarray et a identifié une région commune de délétion homozygote localisée en 4q34.3-35 comprenant le gène IRF-2 dans 4 échantillons. Nous avons ensuite mis en évidence par séquençage des mutations somatiques inactivatrices dans 2 autres tumeurs. In vitro, la sousexpression d’IRF-2 a entrainé une augmentation de la prolifération cellulaire et sa sur-expression a induit une promotion de l'apoptose cellulaire. In vivo, l’extinction d’IRF-2 était responsable de la formation de tumeurs de grande taille. Les 6 tumeurs inactivées pour IRF-2 étaient associées au VHB (p = 0.0003), et les mutations de TP53 et d’IRF-2 étaient mutuellement exclusives. La sousexpression de IRF-2 induisait une sous-expression de TP53 et une forte corrélation entre les expressions protéiques de p53 et de IRF-2 a été observée (r2 = 0,72, p = 0,004). En plus, on a observé que l’expression des gènes cibles directs de TP53 était modulée par le niveau d‘expression d’IRF-2. Nous avons émise l'hypothèse que IRF-2 pourrait altérer la fonction de p53 car IRF-2 est connue pour se lier à MDM2, un régulateur négatif de l’expression de p53. Le traitement des cellules inactivées pour IRF-2 avec MG132, un inhibiteur du protéasome, a induit la restauration de l'expression de p53. In vivo, le traitement avec bortézomib, un inhibiteur du proteasome utilisé en oncologie, a entrainé une régression des tumeurs inactivées pour IRF-2. b) Nous avons caractérisé sur le plan clinique et moléculaire une série de CHC liés au VHB et, ensuite, nous avons comparé nos résultats avec ceux d’une série de CHC liés à autres étiologies. Nous avons montré que les CHC liés au VHB présentaient des caractéristiques cliniques et pathologiques différentes de celles des CHC non liés au VHB : ils survenaient chez des patients plus jeunes (P < 0.0001), d'origine Africaine ou Asiatique (P < 0.0001), avec un taux sérique d'alpha-foeto protéine élevé (P = 0.008) et étaient sur le plan histologique des tumeurs peu différenciés (P = 0.04). Nous avons identifié des mutations inactivatrices du gène HBX dans 71% des tumeurs et dans 33% des tissus non tumoraux adjacents (P < 0.0001). Dans 63% des cas, le nombre de copies virales dans les tumeurs était plus faible que dans les tissus non tumoraux adjacents (P < 0.0001). Le gène TP53 était le gène le plus fréquemment muté dans les CHC liés au VHB (41%, P = 0.0002), avec des mutations R249S présentes dans 14 échantillons (16%, p < 0.0001). Ce type de mutation est classiquement associé à l'aflatoxine B1. Nous avons observé que les mutations de TP53 étaient un prédicteur indépendant de survie uniquement pour les patients infectés par le VHB. Enfin, ... / Pas de résumé en anglais / Introduzione: Il carcinoma epatocellulare (HCC) è il tumore primitivo più comune del fegato. Nel mondo, quasi il 50% di tutti gli HCC sono causati dal virus dell'epatite B (HBV). Durante le fasi dell’ infezione da HBV, si possono accumulare alterazioni genetiche e / o cromosomiche e quindi promuovere lo sviluppo del tumore. Obiettivi: a) analizzare in vitro e in vivo il ruolo potenziale di un nuovo gene potenzialmente coinvolto nella carcinogenesi epatica: IRF-2 (Interferon regulatory factor 2). Questo gene è stato identificato mediante l’analisi CGH-SNP come frequentemente deleto negli HCC correlati all’ HBV. b) caratterizzare una cohorte di HCC correlati all’HBV studiandone lo stato virale, le alterazioni genetiche e l’espressione di differenti geni al fine di comprendere meglio il ruolo di HBV nella carcinogenesis epatocellulare e confrontare questi parametri con una cohorte di HCC a diversa eziologia. Risultati: a) In laboratorio, Sandrine Imbeaud ha condotto un'analisi SNP-CGH microarray su una cohorte di 125 HCC che ha evidenziato una regione deleta in maniera omozigote localizzata sul braccio lungo del cromosoma 4 (4q34.3-35) in 4 campioni tumorali. La regione comprende un unico gene: IRF2. In altri due campioni sono state identificate mutazioni somatiche inattivatrici mediante sequenziamento della regione codante di IRF-2. In vitro, la soppressione di IRF-2 ha indotto un aumento della proliferazione cellulare, al contrario, la sua sovra-espressione ha causato un aumento dell’apoptosi cellulare. In vivo, la soppressione di IRF-2 è responsabile della formazione di tumori più grandi nei topi nude. I 6 tumori mutati per IRF2 sono tutti correlati all’ HBV (p = 0,0003. Nella cohorte di tumori studiati, le mutazioni di TP53 e di IRF-2 erano vicendevolmente esclusive. Inoltre, la soppressione dell’espressione della proteina IRF-2 induceva una riduzione dell’espressione della proteina p53 ed una stretta correlazione tra l’espressione delle due proteine è stata osservata (r2 = 0,72, p = 0,004). Inoltre, abbiamo dimostrato che il livello di espressione di IRF-2 è in grado di modulare l'espressione di alcuni geni target di TP53. Abbiamo, quindi, ipotizzato che IRF2 possa alterare la funzione di p53. Come è noto IRF2 può legarsi a MDM2, un regolatore negativo di p53 che induce la sua degradazione proteasomica. Il trattamento di cellule inattivate per IRF2 con MG132, un inibitore del proteasoma, induceva il restauro dell’espressione di p53. In vivo, il trattamento con bortezomib, chemioterapico inibitore del proteasoma, ha determinato la regressione del tumore inattivato per IRF2. b) 86 HCC correlati all’HBV sono stati caratterizzati dal punto di vista clinico e molecolare ed in seguito sono stati confrontati una serie di 90 HCC correlati ad altre eziologie. Gli HCC correlati all’HBV hanno delle caratteristiche cliniche e patologiche diverse da quelle degli HCC d’altra eziologia: insorgenza in pazienti più giovani (p <0,0001), di origine africana o asiatica (P <0.0001), alfa-fetoproteina sierica elevata (P = 0.008) e scarsa differenziazione istologica (P = 0,04). Mutazioni inattivatrici del gene HBX sono state identificate nel 71% dei tumori e il 33% dei tessuti non tumorali adiacenti (P <0.0001). Nel 63% dei casi, il numero di copie virali nel tessuto tumorale era inferiore rispetto al tessuto non tumorale adiacente (p <0,0001). Il gene TP53 è stato il gene più frequentemente mutato nella serie di HCC correlati a HBV (41%, p = 0,0002), con una considerevole presenza di mutazioni al codone 249 (R249S) (16%, p <0,0001). Questo tipo di mutazione è associate classicamente all’ aflatossina B1. Abbiamo osservato, inoltre, che TP53 mutato era un predittore indipendente di sopravvivenza solo per i pazienti infetti da HBV. Infine, ...