Key words: adenocarcinoma of the esophagus and esophagogastric junction, neoadjuvant chemotherapy, PET/CT, histopathological response, technique of esophagogastric anastomosis, anastomotic leak, anastomotic stricture Previous studies have shown that preoperative chemotherapy of locally advanced AEG is beneficial only for patients with a good histopathological response, the so-called responders. The aim of the first part of the thesis was to prospectively verify whether positron emission tomography (PET/CT) could be used for early identification of histopathological non- responders, who could be spared ineffective neoadjuvant treatment. Our study did not prove that the early metabolic response, expressed as the percentage change of the consumption of glucosis on PET/CT performed before (PET1) and 12 to 22 days after the start of the first cycle of preoperative chemotherapy (PET2) correlated with the histopathological response in the resection specimen in the entire population of 90 patients. In a post hoc explorative analysis we found the correlation between metabolic and histopathological response in a subgroup of patients with PET2 performed ≤16 days after the start of the therapy, but this hypothesis needs to be prospectively validated. Our study suggests that PET/CT performed after the first...

O domínio da técnica de microanastomose vascular é um pré-requisito essencial para a realização de procedimentos microcirúrgicos reconstrutivos, como reimplantes e transferência livre de tecidos. Até hoje, a técnica de sutura convencional é a mais aceita na prática clínica, por sua segurança e versatilidade. Apesar disso, ela apresenta alguns problemas por ser tecnicamente difícil, consumir tempo considerável e causar traumatismo adicional à parede do vaso. O objetivo deste estudo, foi testar um método alternativo de microanastomose arterial, reduzindo o número de pontos de sutura com aplicação do adesivo de fibrina. Sessenta ratos da raça Wistar foram submetidos a microanastomose vascular nas artérias femorais ou carótidas. Os animais foram divididos em quatro subgrupos de acordo com a artéria operada e a técnica de sutura empregada: FSC (femoral - sutura convencional), FAF (femoral - sutura mínima com adesivo de fibrina), CSC (carótida - sutura convencional) e CAF (carótida - sutura mínima com adesivo de fibrina). As duas técnicas de anastomose foram comparadas através de análise estatística dos parâmetros clínicos e histopatológicos. A média de pontos de sutura por anastomose nos subgrupos FSC e CSC foi de 7,7 e 9,5, respectivamente. No subgrupo FAF, as anastomoses foram realizadas com apenas quatro pontos de sutura e no subgrupo CAF, com apenas seis. O tempo de anastomose foi, em média: 15,81 minutos no subgrupo FSC, 13,62 minutos no subgrupo FAF, 18,87 minutos no subgrupo CSC e 17,33 minutos no subgrupo CAF. A aplicação do adesivo de fibrina reduziu, significativamente, o número de pontos e o tempo necessário para realização das anastomoses, nos subgrupos FAF e CAF. A intensidade do sangramento anastomótico também foi reduzida de maneira significativa nestes subgrupos. A freqüência da permeabilidade imediata e tardia foi de 100% em todos os subgrupos, exceto no subgrupo FAF, onde a permeabilidade tardia foi de 93,33%. Não foram observadas diferenças significativas entre as duas técnicas, em relação aos parâmetros histopatológicos avaliados (processo inflamatório, fibrose da camada média e hiperplasia subintimal). O autor concluiu que a técnica de sutura mínima com aplicação do adesivo de fibrina foi mais fácil e rápida que a técnica de sutura convencional, sem aumento da trombogenicidade das anastomoses, no modelo experimental utilizado. / Mastering of the microvascular anastomosis technique is an essencial requirement to perform reconstructive microsurgical procedures, such as replantation surgery and free tissue transfers. Until now, the conventional suture technique is the most widely accepted in the clinical setting, for its safety and versatility. However, this technique presents some problems for being technically difficult, time consuming and causes additional trauma to the vessel wall. The aim of this study was to test an alternative method of microvascular arterial anastomosis, by reducing the number of sutures with application of fibrin adhesive. Sixty Wistar rats underwent to microvascular anastomosis at the femoral or carotid arteries. The animals were divided into four subgroups, according to the operated artery and the employed suture technique: FCS (femoral - conventional suture), FFA (femoral - minimal suture with fibrin adhesive), CCS (carotid - conventional suture) and CFA (carotid - minimal suture with fibrin adhesive). Both anastomosis techniques were compared by means of statistical analisys of the clinical and histopathological parameters. The mean number of sutures required to complete the anastomosis was 7,7 in subgroup FCS and 9,5 in subgroup CCS. In subgroup FFA, the anastomosis was performed with only four sutures and in subgroup CFA, with only six. The mean anastomotic time was 15,81 minutes in subgroup FCS, 13,62 minutes in subgroup FFA, 18,87 minutes in subgroup CCS and 17,33 minutes in subgroup CCS. The application of fibrin adhesive, significantly reduced the number of sutures and the time taken to perform the anastomosis, in subgroups FFA and CFA. The amount of anastomotic bleeding was also significantly reduced in these subgroups. The immediate and late patency rates were 100% in all subgroups, except in subgroup FFA where it was 93,33%. No significant differences were observed among the two techniques, concerning the evaluated histopathological parameters (inflammatory process, medial fibrosis and subintimal hyperplasia). The author concluded that, the fibrin adhesive application with minimal suture technique was faster and easier than the conventional suture technique, without increasing the trombogenicity of the anastomosis, in this experimental model.

O transplante hepático com a técnica convencional envolve a ressecção da veia cava inferior retrohepática como parte da hepatectomia do receptor e conseqüente clampeamento da mesma acima e abaixo do fígado, bem como o clampeamento da veia porta durante a fase anepática. Este procedimento pode se caracterizar por alterações hemodinâmicas importantes, disfunção renal e maior sangramento em áreas de dissecção submetidas a um regime de maior pressão. Pacientes idosos, previamente instáveis do ponto de vista hemodinâmico ou muito graves, gealmente, não toleram esta situação. Para evitar estes distúrbios, um \"bypass\" venovenoso, é habitualmente utilizado, permitindo com que o sangue da veia porta e da veia cava inferior retorne à circulação sistêmica através da veia axilar. O \"bypass\" venovenoso, foi adotado por muitos centros transplantadores como procedimento de rotina, enquanto outros empregavam este apenas quando o clampeamento da veia cava inferior determinasse grave alteração hemodinâmica, buscando assim, redução do custo, do tempo de operação e das complicações em função do uso \"bypass\", tais como: hipotermia, coagulopatia e fenômenos tromboembólicos. Outra alternativa técnica, que consiste na preservação da veia cava inferior durante a hepatectomia foi descrita com o intuito de preservar o fluxo na veia cava inferior durante a fase anepática. Além disto, uma anastomose porto-cava temporária pode ser confeccionada para evitar o clampeamento da veia porta e conseqüente congestão esplâncnica. O objetivo deste estudo foi comparar os resultados imediatos do transplante hepático com preservação da veia cava inferior e anastomose porto-cava temporária, ou com o uso do \"bypass\" venovenoso. De outubro de 1999 a outubro de 2001, 104 pacientes submetidos ao transplante hepático foram analisados retrospectivamente. O \"bypass\" venovenoso foi utilizado em 50 pacientes e a técnica de \"piggy back\" em 54. Ambos os grupos foram comparáveis em termos de idade, sexo, diagnóstico e grau de função hepática pré-operatória. Nosso estudo demonstrou não haver diferença entre as duas técnicas em relação à duração da hepatectomia, à duração da operação e à necessidade de transfusão de hemoderivados. Observou-se uma fase anepática mais breve e um menor tempo de permanência na unidade de terapia intensiva no período pós operatório dos pacientes submetidos à técnica que reserva a veia cava inferior, além disto o estudo aponta para uma tendência à redução no tempo de internação, no índice de insuficiência renal pós-operatória e para um melhor funcionamento do enxerto quando os fluxos da veia cava inferior e da veia porta são preservados. / The liver transplant with conventional technique involves the resection of the retrohepatic inferior vena cava as part of the recipient hepatectomy and consequent clamping of the same above and below the liver, as well as the clamping of the portal vein during the anhepatic phase. This procedure can be characterized by critical hemodynamic alterations, renal disfunction and greater bleeding in dissection areas submitted to higher pressure conditions. Elderly patients, previously unstable on a hemodynamic point of view or in very serious conditions, usually do not tolerate this situation. In order to avoid these disturbances, a venovenous bypass is commonly applied, allowing blood from the portal vein and inferior vena cava to return to the systemic circulation through the axilar vein. The venovenous bypass has been adopted by many transplant centers as a routine procedure, while others have applied this procedure only when clamping of the inferior vena cava determines a serious hemodynamic alteration, eeking, in this way, cost cutting, reduction of operation time and complications caused by usage of the bypass, such as: hypothermy, coagulopathy and romboembolic phenomenae. Another technical alternative, which consists of preservation of the inferior vena cava during the hepatectomy was described focusing preservation of the inferior vena cava flow during the anhepatic phase. Besides, a temporary portocaval shunt can be made to avoid clamping of the portal vein and consequently splanchnic congestion. The objective of this study was compare the immediate results of the liver transplantation with inferior vena cava preservation and temporary portocaval shunt or using the venovenous bypass. From October 1999 to October 2001, 104 patients submitted to an liver transplantat were analyzed retrospectively. The venovenous bypass was applied in 50 patients and the \"piggy back\" technique in 54. Both groups were comparable in terms of age, gender, diagnosis and preoperative hepatic conditions. Our study has demonstrated that there was no difference between the two techniques in relation to the hepatectomy and operation time and the need of hemoderivate transfusion. As a result, a shorter anhepatic phase was observed, as well as a shorter stay in intensive care unit during the postoperative period, in relation to patients submitted to the technique that preserves the inferior vena cava. Additionally, the study points out a trend towards reduction of hospital stay, postoperative renal insufficiency, and a better graft function when the inferior cava and portal veins are preserved.

Η μικροχειρουργική τεχνική και η μεταφορά ελεύθερων αγγειούμενων κρημνών για την αποκατάσταση εκτεταμένων και σύνθετων ελλειμμάτων ογκολογικής ή τραυματικής αιτιολογίας της κεφαλής και του τραχήλου αποτελεί μέθοδο επιλογής. Με την τεχνική αυτή επιτυγχάνεται στον ίδιο χρόνο με την εκτομή, η λειτουργική και αισθητική αποκατάσταση με μειωμένο ποσοστό επιπλοκών και επίπτωση από την δότρια περιοχή. Στην παρούσα μελέτη καταγράφουμε και αναλύουμε την εμπειρία μας σε ασθενείς που αντιμετωπίστηκαν με μικροχειρουργική τεχνική και ελεύθερους αγγειούμενους κρημνούς. Την περίοδο 2003 έως 2010, σε 48 ασθενείς πραγματοποιήθηκαν 56 ελεύθεροι αγγειούμενοι κρημνοί. Οι 34 ασθενείς από τους 48 υπεβλήθηκαν συγχρόνως σε ογκολογική εκτομή λόγω Ca και σε αποκατάσταση, ενώ σε 14 από τους 48 ασθενείς αφορούσε έλλειμμα τραυματικής αιτιολογίας. Η τοπογραφία του ελλείμματος αφορούσε το τριχωτό της κεφαλής και του μετώπου σε 12 ασθενείς (25%), το μέσο τριτημόριο του προσώπου και τα παραρίνια σε 9 ασθενείς (18.7%), το κάτω τριτημόριο του προσώπου και τραχήλου σε 27 ασθενείς (56.25%). Σε 7 ασθενείς χρησιμοποιήθηκαν διπλοί ελεύθεροι αγγειούμενοι κρημνοί και σε 41 ασθενείς μονήρεις κρημνοί. Χρησιμοποιήθηκαν συνολικά 56 ελεύθεροι αγγειούμενοι κρημνοί. Σε 16 ασθενείς χρησιμοποιήθηκε φλεβικό μόσχευμα (33.3%). Οι κρημνοί επιλογής ήταν ο κερκιδικός κρημνός 28.5%, ο κρημνός της περόνης 17.8%, ο προσθιοπλάγιος κρημνός του μηρού 14.2%, ο μυοδερματικός κρημνός του ορθού κοιλιακού(VRAM) 12.5% και εγκάρσιος(ΤΡΑΜ) 5.3%, ο κρημνός του πλατέως ραχιαίου 8.9%, ο κρημνός του ισχνού προσαγωγού 7.1% και ο πρόσθιος οδοντωτός 1.7%. Οι μικροαγγειακές αναστομώσεις έγιναν στα αγγεία του τραχήλου σε ποσοστό 96.2%. Η επιβίωση των κρημνών ήταν σε ποσοστό 92.8% (52/56) και 4 κρημνοί απορρίφθηκαν (7.1%) λόγω θρόμβωσης. Ένας ασθενής απεβίωσε στην μετεγχειρητική περίοδο λόγω σοβαρών συστηματικών επιπλοκών. Ο σωστός σχεδιασμός, η επιλογή του κατάλληλου κρημνού, η εμπειρία στην μικροχειρουργική και η συνεργασία των εμπλεκόμενων ιατρικών ομάδων είναι αυτά που διασφαλίζουν το υψηλό ποσοστό επιτυχίας στην επανορθωτική μικροχειρουργική με πολύ καλό λειτουργικό και αισθητικό αποτέλεσμα. / Microsurgical free tissue transfer considered as the best choice for the reconstruction of head and neck extended and complex tissue defects due to tumor resection or trauma. A total of 48 patients underwent free tissue transfer between 2003-2010. There were 34 patients underwent one stage tumor resection and microsurgical free flap reconstruction and 16 patients for free flap reconstruction due to head and neck trauma. The defect in 12 patients 25% was on the scalp and forehead, the middle third in 9 patients 18.7% lower third in neck in 27 patients 56.25%. We used a combination of double free flaps for reconstruction in 7 cases and in 41 patients a single free flap. Vane grafts were used in 16 cases (33.3%). We used in total 56 free flaps with success rate 92.8% (52/56). Four flaps were lost due to anastomotic thromboses. Work horse flaps in our series include the radial forearm 28.5%, fibula 17.8%, ALT 14.2%, VRAM 12.5%, TRAM 5.3%, latissimus dorse 8.9%, gracilis 7.1% and serratus anterior 1.7%. The neck recipient vessels were used in 96.2%. One patient died in post surgical period after systemic complications. Preoperative surgical and reconstruction plan, flaps selection, high microsurgical experience and team collaboration are essential for the good functional and aesthetic results in microsurgery reconstruction of head and neck tissue defects.

L’œdème cérébral est une complication associée à l’encéphalopathie hépatique (EH) lors d’une insuffisance hépatique chronique (cirrhose du foie). Présentement, l’origine de sa pathogenèse, vasogénique (rupture de la barrière hémato-encéphalique (BHE)) ou cytotoxique (prise anormale d’ions), n’a pas encore été déterminée. Il a été démontré que le co-transporteur Na-K-Cl (NKCC1) du côté luminal des microvaisseaux sanguins cérébraux (CMV) joue un rôle dans le développement de l’œdème cérébral dans des modèles d’ischémie où la bumetanide, un inhibiteur de NKCC, atténue l’œdème cérébral. Deux modèles d’EH ont été utilisés pour cette étude i) la ligature de la voie biliaire (BDL) qui présente l’hyperammoniémie chronique, l’œdème cérébral et le stress oxydatif systémique ; ii) l’anastomose portocave (PCA) qui présente de l’hyperammoniémie chronique seulement. Les buts du projet étaient de: i) définir l’origine du développement de l’œdème chez les rats BDL en étudiant l’extravasation de macromolécules, les jonctions serrées et l’activation des métalloprotéinases matricielles de la BHE; ii) observer les effets de l’hyperammoniémie chronique indépendamment sur la BHE chez les rats PCA; iii) évaluer le rôle de l’hyperammoniémie et du stress oxydatif et iv) étudier le rôle du NKCC1 dans les CMV dans la pathogenèse de l’œdème cérébral. Les résultats du projet démontrent que l’œdème est d’origine cytotoxique chez les rats BDL et que l’intégrité de la BHE est conservée chez les rats PCA malgré l’hyperammoniémie. L’expression génique du NKCC1 est associée à l’œdème mais pas son expression protéique et sa phosphorylation. Enfin, l’étude démontre que l’hyperammoniémie et le stress oxydatif indépendant ne jouent pas un rôle dans la pathogenèse de l’œdème mais suggère qu’ils y aient un effet synergique. / Brain edema is a complication associated with hepatic encephalopathy (HE) due to chronic liver failure (cirrhosis). It is unclear whether brain edema is of vasogenic (blood brain barrier (BBB) breakdown) or cytotoxic (abnormal cellular uptake of ions) origin. It has been demonstrated that the Na-K-Cl cotransporter (NKCC1) located on the luminal side of the cerebral microvessels (CMV) is implicated in the pathogenesis of brain edema in animal models of ischemia and that the administration of bumetanide, an inhibitor of NKCC, attenuates brain water increase. Two distinct animal models of chronic liver failure and HE are used in the present study; 1) bile duct ligation (BDL) where brain edema, chronic hyperammonemia and systemic oxidative stress are observed; 2) portacaval anastomosis (PCA) where only chronic hyperammonemia is observed. The aims of the study were to: i) determine the origin of brain edema in BDL rats measuring brain extravasation, tight junctions expression and matrix metalloproteinase activation; ii) observe the effects of chronic hyperammonemia on the BBB in PCA rats; iii) study the role of oxidative stress and hyperammonemia; iv) evaluate the role of NKCC in CMV in the pathogenesis of brain edema. The results of the study determined that brain edema in BDL rats is of cytotoxic origin and chronic hyperammonemia independently has no effect on the BBB. An increase of NKCC1 mRNA is associated with brain edema but protein expression and phosphorylation are not. Furthermore, hyperammonemia and oxidative stress independently are not implicated in the development of brain edema however a synergistic effect between the two pathogenic factors in BDL rats remains a possibility.

Contexte: Les champignons mycorhiziens à arbuscules (AMF) établissent des relations symbiotiques avec la plupart des plantes grâce à leurs réseaux d’hyphes qui s’associent avec les racines de leurs hôtes. De précédentes études ont révélé des niveaux de variation génétique extrêmes pour des loci spécifiques permettant de supposer que les AMF peuvent contenir des milliers de noyaux génétiquement divergents dans un même cytoplasme. Si aucun processus de reproduction sexuée n’a jusqu’ici été observé chez ces mycorhizes, on constate cependant que des niveaux élevés de variation génétique peuvent être maintenus à la fois par l’échange de noyaux entre hyphes et par des processus fréquents de recombinaison entre noyaux. Les AMF se propagent par l’intermédiaire de spores qui contiennent chacune un échantillon d’une population initiale de noyaux hétérogènes, directement hérités du mycélium parent. À notre connaissance les AMF sont les seuls organismes qui ne passent jamais par un stade mononucléaire, ce qui permet aux noyaux de diverger génétiquement dans un même cytoplasme. Ces aspects singuliers de la biologie des AMF rendent l’estimation de leur diversité génétique problématique. Ceci constitue un défi majeur pour les écologistes sur le terrain mais également pour les biologistes moléculaires dans leur laboratoire. Au-delà même des problématiques de diversité spécifique, l’amplitude du polymorphisme entre noyaux mycorhiziens est mal connue. Le travail proposé dans ce manuscrit de thèse explore donc les différents aspects de l’architecture génomique singulière des AMF. Résultats L’ampleur du polymorphisme intra-isolat a été déjà observée pour la grande sous-unité d’ARN ribosomal de l’isolat Glomus irregulare DAOM-197198 (précédemment identifié comme G. intraradices) et pour le gène de la polymerase1-like (PLS) de Glomus etunicatum isolat NPI. Dans un premier temps, nous avons pu confirmer ces résultats et nous avons également pu constater que ces variations étaient transcrites. Nous avons ensuite pu mettre en évidence la présence d’un goulot d’étranglement génétique au moment de la sporulation pour le locus PLS chez l’espèce G. etunicatum illustrant les importants effets d’échantillonnage qui se produisaient entre chaque génération de spore. Enfin, nous avons estimé la différentiation génétique des AMF en utilisant à la fois les réseaux de gènes appliqués aux données de séquençage haut-débit ainsi que cinq nouveaux marqueurs génomiques en copie unique. Ces analyses révèlent que la différenciation génomique est présente de manière systématique dans deux espèces (G. irregulare et G. diaphanum). Conclusions Les résultats de cette thèse fournissent des preuves supplémentaires en faveur du scénario d’une différenciation génomique entre noyaux au sein du même isolat mycorhizien. Ainsi, au moins trois membres du genre Glomus, G. irregulare, G. diaphanum and G. etunicatum, apparaissent comme des organismes dont l’organisation des génomes ne peut pas être décrit d’après un modèle Mendélien strict, ce qui corrobore l’hypothèse que les noyaux mycorhiziens génétiquement différenciés forment un pangenome. / Background: Arbuscular mycorrhizal fungi (AMF) are root-inhabiting fungi whose hyphal networks form symbioses with plants. Previous studies have revealed extremely high levels of genetic variation for some loci, which has lead to the proposition that AMF contain thousands of genetically divergent nuclei that share the same cytoplasm, i.e. they are heterokaryotic coenocytes. No reproductive stage has as yet been observed in AMF, yet evidence is accumulating that the observed high levels of diversity could be maintained by the exchange of nuclei between hyphal systems and (meiotic) recombination. AMF spores contain varying fractions of this heterogeneous population of nuclei, which migrate directly from the parent mycelium. To our knowledge, AMF are the only organisms that never pass through a single nucleus stage in their life cycle, which allows nuclei to diverge into genetically distinct nuclei within the same cytoplasm. Thus, estimating genetic diversity in arbuscular mycorrhizal fungi (AMF) is a major challenge, not only for ecologists in the field but also for molecular biologists in the lab. It is unclear what the extent of polymorphism is in AMF genomes. The present thesis investigates different aspects of this peculiar genome organization. Results The second chapter in this thesis confirms the extensive intra-isolate polymorphism that was previously observed for large subunit rDNA (in G. irregulare DAOM-197198) and the polymerase1-like gene, PLS (in G. etunicatum), and shows that this polymorphism is transcribed. In the third chapter I report the presence of a bottleneck of genetic variation at sporulation for the PLS locus, in G. etunicatum. Analyses in the fourth chapter, based on a conservative network-based clustering approach and five novel single copy genomic markers, reveal extensive genome-wide patterns of diversity in two different AMF species (G. irregulare and G. diaphanum). Conclusions The results from this thesis provide additional evidence in favor of genome differentiation between nuclei in the same isolate for AMF. Thus, at least three members of the Glomus genus, G. irregulare, G. diaphanum and G. etunicatum appear to be organisms whose genome organization cannot be described by a single genome sequence: genetically differentiated nuclei in AMF form a pangenome.

L’encéphalopathie hépatique (EH) est un syndrome neuropsychiatrique découlant des complications de l'insuffisance hépatique. Les patients souffrant d'une insuffisance hépatique chronique (IHC) présentent fréquemment une EH minimale (EHM) caractérisée par des dysfonctions cognitives subtiles qui affectent leur qualité de vie. L'insuffisance hépatique entraîne une hyperammoniémie, le facteur central dans la pathogenèse de l'EH. Pourtant, les taux d'ammoniaque sérique ne sont pas corrélés avec la sévérité de l'EH lors d'une IHC, suggérant que d'autres facteurs y contribuent. L'oedème cérébral est une caractéristique neuropathologique décrite chez les patients souffrant d'une EHM et plusieurs facteurs dont le stress oxydatif, les altérations du métabolisme énergétique et l'augmentation de la glutamine cérébrale pourraient contribuer à la pathogenèse de l'oedème cérébral lors d'une EHM induite par une IHC. Les mécanismes sous-jacents exacts ainsi que les relations entre ces facteurs et l'ammoniaque ne sont pas connus. Présentement, le seul traitement efficace de l'IHC est la transplantation hépatique, une option thérapeutique très limitée. Le but de cette thèse est de contribuer à l'avancement des connaissances sur les mécanismes sous-jacents liés au rôle du stress oxydatif, de la glutamine et du lactate dans la pathogenèse de l'oedème cérébral lors d'une EHM induite par une IHC afin d'envisager de nouvelles options thérapeutiques. Les objectifs précis étaient: 1. Établir le rôle de l’ammoniaque et sa relation avec le stress oxydatif dans la pathogenèse de l'oedème cérébral lors d'une EHM induite par une IHC. 2. Établir le rôle du stress oxydatif dans la pathogenèse de l'oedème cérébral, sa relation avec l'ammoniaque et l'effet du traitement avec des antioxydants. 3. Confirmer l'effet synergique entre l'ammoniaque et le stress oxydatif dans la pathogenèse de l'oedème cérébral. 4. Établir le rôle du lactate et de la glutamine dans la pathogenèse de l'oedème cérébral et leur relation avec l’ammoniaque. Pour atteindre ces objectifs, 2 modèles animaux d'EHM obtenus par microchirurgie chez le rat ont été utilisés: 1) la ligature de voie biliaire, un modèle d'IHC et 2) l'anastomose porto-cave, un modèle d'hyperammoniémie induite par la dérivation portosystémique. Nos résultats démontrent que l'ammoniaque et le stress oxydatif indépendamment n'induisent pas l'oedème cérébral lors d'une EHM. Pourtant, lorsque les 2 facteurs agissent ensemble ils présentent ii un effet synergique qui entraîne le développement de l'oedème cérébral, le stress oxydatif étant une première insulte, qui est suivie par l'hyperammoniémie comme deuxième insulte. En plus, le stress oxydatif a été mis en évidence seulement au niveau systémique, et non au niveau central dans notre modèle d'IHC en association avec l'oedème cérébral, suggérant que le stress oxydatif systémique est une conséquence de la dysfonction hépatique et que l'hyperammoniémie n’induit pas le stress oxydatif ni systémique ni central. Nous avons démontré qu’une augmentation du lactate cérébral est une conséquence directe de l'hyperammoniémie et joue un rôle important dans la pathogenèse de l'oedème cérébral lors d'une EHM induite par une IHC, tandis qu’une augmentation de la glutamine au niveau cérébral n'est pas un facteur clé. La compréhension de ces mécanismes a entraîné la proposition de 3 nouvelles stratégies thérapeutiques potentielles pour l'EHM. Elles ciblent la diminution de l'ammoniaque sérique, la réduction du stress oxydatif et l'inhibition de la synthèse du lactate. / Hepatic encephalopathy (HE) is a metabolic neuropsychiatric syndrome which occurs as a complication of liver failure/disease. Patients with chronic liver disease (CLD) present often with minimal HE (MHE) characterized by subtle cognitive dysfunction which impairs their quality of life. Impaired liver function leads to hyperammonemia which is a central factor in the pathogenesis of HE. However, ammonia alone is poorly correlated with the severity of HE during CLD, strongly suggesting other factors may contribute. Brain edema is a neuropathological feature described in MHE patients and several factors such as oxidative stress, energy metabolism alterations and an increase in glutamine may to contribute to the pathogenesis of brain edema during HE related to CLD. However the exact underlying mechanisms and the relationships between these factors and ammonia are poorly understood. To date, the only effective treatment of CLD remains liver transplantation, a limited therapeutic option. The aim of this thesis is to advance the knowledge into the mechanisms underlying the role of oxidative stress, glutamine and lactate in the pathogenesis of brain edema during MHE associated with CLD in order to uncover new therapeutic options. The study objectives were: 1. Define the role of ammonia and its relationship with oxidative stress in the pathogenesis of brain edema in CLD. 2. Define the role of oxidative stress in the pathogenesis of brain edema, its relationship with ammonia as well as the effect of antioxidant treatment. 3. Confirm a synergistic role of ammonia and oxidative stress in the pathogenesis of brain edema. 4. Define the role of lactate and glutamine in the pathogenesis of brain edema and their relationship with ammonia. To achieve these objectives, we used 2 microsurgical rat models: 1) bile-duct ligation, a cirrhosis model and 2) portacaval anastomosis, a hyperammonemia model following portal-systemic shunting. Our findings demonstrate that ammonia and systemic oxidative stress independently do not induce brain edema in MHE related to CLD. However, when both factors are present, they exert a synergistic effect leading to the development of brain edema with oxidative stress presenting as a “first hit”, followed by hyperammonemia as a “second hit”. Moreover, solely systemic and not central oxidative stress was observed in our CLD rat model in relation to brain edema implying that systemic oxidative stress is a consequence of liver dysfunction and that central oxidative stress is not a direct iv effect of hyperammonemia in the setting of CLD. Moreover, we revealed that increased cerebral lactate is a direct consequence of hyperammonemia and also plays an important role in the pathogenesis of brain edema, while increased cerebral glutamine does not. The understanding of these mechanisms led to the proposal of three different strategies as potential HE therapies. These are directed towards lowering ammonia, reducing oxidative stress and inhibiting lactate synthesis.

Les champignons mycorhiziens arbusculaires (CMA) sont très répandus dans le sol où ils forment des associations symbiotiques avec la majorité des plantes appelées mycorhizes arbusculaires. Le développement des CMA dépend fortement de la plante hôte, de telle sorte qu'ils ne peuvent vivre à l'état saprotrophique, par conséquent ils sont considérés comme des biotrophes obligatoires. Les CMA forment une lignée évolutive basale des champignons et ils appartiennent au phylum Glomeromycota. Leurs mycélia sont formés d’un réseau d’hyphes cénocytiques dans lesquelles les noyaux et les organites cellulaires peuvent se déplacer librement d’un compartiment à l’autre. Les CMA permettent à la plante hôte de bénéficier d'une meilleure nutrition minérale, grâce au réseau d'hyphes extraradiculaires, qui s'étend au-delà de la zone du sol explorée par les racines. Ces hyphes possèdent une grande capacité d'absorption d’éléments nutritifs qui vont être transportés par ceux-ci jusqu’aux racines. De ce fait, les CMA améliorent la croissance des plantes tout en les protégeant des stresses biotiques et abiotiques. Malgré l’importance des CMA, leurs génétique et évolution demeurent peu connues. Leurs études sont ardues à cause de leur mode de vie qui empêche leur culture en absence des plantes hôtes. En plus leur diversité génétique intra-isolat des génomes nucléaires, complique d’avantage ces études, en particulier le développement des marqueurs moléculaires pour des études biologiques, écologiques ainsi que les fonctions des CMA. C’est pour ces raisons que les génomes mitochondriaux offrent des opportunités et alternatives intéressantes pour étudier les CMA. En effet, les génomes mitochondriaux (mt) publiés à date, ne montrent pas de polymorphismes génétique intra-isolats. Cependant, des exceptions peuvent exister. Pour aller de l’avant avec la génomique mitochondriale, nous avons besoin de générer beaucoup de données de séquençages de l’ADN mitochondrial (ADNmt) afin d’étudier les méchanismes évolutifs, la génétique des population, l’écologie des communautés et la fonction des CMA. Dans ce contexte, l’objectif de mon projet de doctorat consiste à: 1) étudier l’évolution des génomes mt en utilisant l’approche de la génomique comparative au niveau des espèces proches, des isolats ainsi que des espèces phylogénétiquement éloignées chez les CMA; 2) étudier l’hérédité génétique des génomes mt au sein des isolats de l’espèce modèle Rhizophagus irregularis par le biais des anastomoses ; 3) étudier l’organisation des ADNmt et les gènes mt pour le développement des marqueurs moléculaires pour des études phylogénétiques. Nous avons utilisé l’approche dite ‘whole genome shotgun’ en pyroséquençage 454 et Illumina HiSeq pour séquencer plusieurs taxons de CMA sélectionnés selon leur importance et leur disponibilité. Les assemblages de novo, le séquençage conventionnel Sanger, l’annotation et la génomique comparative ont été réalisés pour caractériser des ADNmt complets. Nous avons découvert plusieurs mécanismes évolutifs intéressant chez l’espèce Gigaspora rosea dans laquelle le génome mt est complètement remanié en comparaison avec Rhizophagus irregularis isolat DAOM 197198. En plus nous avons mis en évidence que deux gènes cox1 et rns sont fragmentés en deux morceaux. Nous avons démontré que les ARN transcrits les deux fragments de cox1 se relient entre eux par épissage en trans ‘Trans-splicing’ à l’aide de l’ARN du gene nad5 I3 qui met ensemble les deux ARN cox1.1 et cox1.2 en formant un ARN complet et fonctionnel. Nous avons aussi trouvé une organisation de l’ADNmt très particulière chez l’espèce Rhizophagus sp. Isolat DAOM 213198 dont le génome mt est constitué par deux chromosomes circulaires. En plus nous avons trouvé une quantité considérable des séquences apparentées aux plasmides ‘plasmid-related sequences’ chez les Glomeraceae par rapport aux Gigasporaceae, contribuant ainsi à une évolution rapide des ADNmt chez les Glomeromycota. Nous avons aussi séquencé plusieurs isolats de l’espèces R. irregularis et Rhizophagus sp. pour décortiquer leur position phylogénéque et inférer des relations évolutives entre celles-ci. La comparaison génomique mt nous montré l’existence de plusieurs éléments mobiles comme : des cadres de lecture ‘open reading frames (mORFs)’, des séquences courtes inversées ‘short inverted repeats (SIRs)’, et des séquences apparentées aux plasimdes ‘plasmid-related sequences (dpo)’ qui impactent l’ordre des gènes mt et permettent le remaniement chromosomiques des ADNmt. Tous ces divers mécanismes évolutifs observés au niveau des isolats, nous permettent de développer des marqueurs moléculaires spécifiques à chaque isolat ou espèce de CMA. Les données générées dans mon projet de doctorat ont permis d’avancer les connaissances fondamentales des génomes mitochondriaux non seulement chez les Glomeromycètes, mais aussi de chez le règne des Fungi et les eucaryotes en général. Les trousses moléculaires développées dans ce projet peuvent servir à des études de la génétique des populations, des échanges génétiques et l’écologie des CMA ce qui va contribuer à la compréhension du rôle primorial des CMA en agriculture et environnement. / Arbuscular mycorrhizal fungi (AMF) are the most widespread eukaryotic symbionts, forming mutualistic associations known as Arbuscular Mycorrhizae with the majority of plantroots. AMF are obligate biotrophs belonging to an ancient fungal lineage of phylum Glomeromycota. Their mycelia are formed by a complex network made up of coenocytic hyphae, where nuclei and cell organelles can freely move from one compartment to another. AMF are commonly acknowledged to improve plant growth by enhancing mineral nutrient uptake, in particular phosphate and nitrate, and they confer tolerance to abiotic and biotic stressors for plants. Despite their significant roles in ecosystems, their genetics and evolution are not well understood. Studying AMF is challenging due to their obligate biotrophy, their slow growth, and their limited morphological criteria. In addition, intra-isolate genetic polymorphism of nuclear DNA brings another level of complexity to the investigation of the biology, ecology and function of AMF. Genetic polymorphism of nuclear DNA within a single isolate limits the development of efficient molecular markers mainly at lower taxonomic levels (i.e. the inter-isolate level). Instead, mitochondrial (mt) genomics have been used as an attractive alternative to study AMF. In AMF, mt genomes have been shown to be homogeneous, or at least much less polymorphic than nuclear DNA. However, by generating large mt sequence datasets we can investigate the efficiency and usefulness of developing molecular marker toolkits in order to study the dynamic and evolutionary mechanisms of AMF. This approach also elucidates the population genetics, community ecology and functions of Glomeromycota. Therefore, the objectives of my Ph.D. project were: 1) To investigate mitochondrial genome evolution using comparative mitogenomic analyses of closely related species and isolates as well as phylogenetically distant taxa of AMF; 2) To explore mt genome inheritance among compatible isolates of the model AMF Rhizophagus irregularis through anastomosis formation; and 3) To assess mtDNA and mt genes for marker development and phylogenetic analyses. We used whole genome shotgun, 454 pyrosequencing and HiSeq Illimina to sequence AMF taxa selected according to their importance and availability in our lab collections. De novo assemblies, Sanger sequencing, annotation and comparative genomics were then performed to characterize complete mtDNAs. We discovered interesting evolutionary mechanisms in Gigaspora rosea: 1) we found a fully reshuffled mt genome synteny compared to Rhizaphagus irregularis DAOM 197198; and 2) we discovered the presence of fragmented cox1 and rns genes. We demonstrated that two cox1 transcripts are joined by trans-splicing. We also reported an unusual mtDNA organization in Rhizophagus sp. DAOM 213198, whose mt genome consisted of two circular mtDNAs. In addition, we observed a considerably higher number of mt plasmidrelated sequences in Glomeraceae compared with Gigasporaceae, contributing a mechanism for faster evolution of mtDNA in Glomeromycota. We also sequenced other isolates of R. irregularis and Rhizophagus sp. in order to unravel their evolutionary relationships and to develop molecular toolkits for their discrimination. Comparative mitogenomic analyses of these mtDNAs revealed the occurrence of many mobile elements such as mobile open reading frames (mORFs), short inverted repeats (SIRs), and plasmid-related sequences (dpo) that impact mt genome synteny and mtDNA alteration. All together, these evolutionary mechanisms among closely related AMF isolates give us clues for designing reliable and efficient intra- and inter-specific markers to discriminate closely related AMF taxa and isolates. Data generated in my Ph.D. project advances our knowledge of mitochondrial genomes evolution not only in Glomeromycota, but also in the larger framework of the Fungal kingdom and Eukaryotes in general. Molecular toolkits developed in this project will offer new opportunities to study population genetics, genetic exchanges and ecology of AMF. In turn, this work will contribute to understanding the role of these fungi in nature, with potential applications in both agriculture and environmental protection.

Introdução: A incidência de obesidade é crescente e alarmante, principalmente no mundo ocidental. De acordo com o National Center for Health Statistics, cerca de 61% da população adulta nos Estados Unidos está acima do peso e 30% é obesa, sendo que 5 a 6% está classificada na faixa de obesidade Grau III. No Brasil, o Ministério da Saúde aponta que 32,9% dos brasileiros estão fora da faixa de peso ideal, e 4,8% dos homens e 11,7% das mulheres encaixam-se na faixa de obesidade Grau III. Devido a alta prevalência da Doença Hepática Gordurosa Não Alcoólica (DHGNA) em pacientes portadores de obesidade grave e os escassos conhecimentos acerca de sua evolução para doença crônica do fígado após cirurgias bariátricas, foram objetivos deste estudo avaliar os efeitos da cirurgia gastrorredutora com derivação intestinal em Y de Roux Cirurgia de Fobi-Capella) sobre DHGNA após 24 meses. Métodos: Dentre 40 pacientes com IMC > 40 kg/m2 submetidos à cirurgia bariátrica (cirurgia de Fobi-Capella) no período de 2001 a 2003, 18 pacientes foram seguidos por aproximadamente 24 meses (700 ± 42 dias) e incluídos no estudo, realizando-se exames laboratoriais, tais como enzimas hepáticas, perfil lipídico e glicêmico; e a biopsia hepática no perioperatório e 24 meses após a cirurgia. O diagnóstico histológico de DHGNA e Esteatohepatite Não Alcoólica (ENA) foi determinado segundo a classificação padronizada por meio da revisão pelo Pathology Committee of the NASH Clinical Research Network Americano, que designou e validou as características histológicas e um sistema de escore de atividade para DHGNA para estudos clínicos. esultados: O IMC médio inicial dos 18 pacientes foi de 51,7 ± 7 kg/m2 e na segunda biopsia, após 24 meses de seguimento foi de 32,3 ± 6 kg/m2, com excesso do índice de massa corpórea perdida de 72,56%. DHGNA foi constatada no exame histológico inicial em 100% dos pacientes, sendo steatohepatite em 67% (10 pacientes com escore de atividade da DHGNA maior ou igual a 5 e dois pacientes com escore 4 com algum grau de fibrose) e 33% com esteatose isolada. Dos pacientes com ENA, 8,3% apresentavam cirrose. Após cerca de 24 meses houve desaparecimento da esteatose em 89% e manutenção da esteatose Grau I em 11% (p < 0,001). Em relação à fibrose, observada inicialmente em 10 (55%) dos pacientes, somente 4 (22,22%) dos pacientes mantiveram algum grau de fibrose (p = 0,020). No que se refere ao infiltrado inflamatório, 78% mantiveram discreto infiltrado lobular (Grau I) não relacionado à degeneração gordurosa. A balonização hepatocelular desapareceu em 50% dos pacientes e manteve-se discreta (Grau I) em 50% (p < 0,001). Não houve diferença estatística no que se refere às aminotranferases no pré e pós-operatório tardio. Houve redução significativa dos lípides e glicemia em quase a totalidade dos pacientes. Conclusão: A correção da síndrome metabólica obtida pela acentuada perda de peso após cirurgia de Fobi-Capella promoveu melhora da esteatose, fibrose, e os escores de atividade da DHGNA menores que 5, respectivamente em 89%, 75% e 100%dos pacientes previamente portadores de DHGNA, não se observando efeito deletério na histologia hepática nesta série. / Background: The incidence of obesity is increasing in western countries at an alarming rate. The National Center for Health Statistics of United Stated estimated in adult population 61% the prevalence of overweight or obesity, and 30% has obesity, and 5 to 6% were classified in severe obesity. In Brazil, the Ministry of Health reported 32.9% the prevalence of overweight or obese in adult brazilian population, and severe obesity 4.8% were men and 11.7% were women. Although nonalcoholic fatty liver disease (NAFLD) has been proved very frequent among morbidly obese patients and the effect of weight loss after bariatric surgery in inflammation and fibrosis related NAFLD is still a matter of debate. The aim of this study was to evaluate the impact of Fobi-Capella surgery in NAFLD in a follow up of 24 months. Methods: Forty patients with body mass index (BMI) IMC > 40 kg/m2 were submitted to Roux-en-Y gastric bypass with intraoperatory liver biopsies between 2001 a 2003, and 18 patients were followed and selected to underwent a liver biopsies after 24 months (700 ± 42 days). Blood biochemical tests and liver histology were compared before and after weight loss. The histological diagnosis of Nonalcoholic fatty liver disease (NAFLD) and Nonalcoholic steatohepatitis (NASH) was analyzed using the classification proposed by Pathology Committee of the NASH Clinical Research Network, which designed and validated a histological feature scoring system that address the characteristics of NASH lesions and a NAFLD activity score (NAS) for use in clinical trials. Eighteen patients with body mass index >40 kg/m2 submitted to Roux-n-Y gastric bypass were enrolled, and wedge liver biopsy was obtained at the operation. After 24 months, patients agreed to be submitted to a percutaneous liver biopsy. Results: The initial average BMI of 18 patientes were 51.7 ± 7 kg/m2. After following 24 months, average BMI was 32.3 ± 6 kg/m2. The average of percent excess body mass index loss was 72.56%. NAFLD was present in all 18 patients at the initial biopsy, NASH in 67% (10 patient had score of NAS ? 5 and two patients with score 4 had some degree of fibrosis) and 33% with steatosis only; 8.3% of patients with NASH has cirrhosis. After 24 months steatosis disappeared in 89% (p < 0,001) and fibrosis disappeared in 60% of the patients (p = 0.020). Hepatocellular ballooning disappeared in 50% (p < 0.001). A slight lobular inflammatory infiltrate remained in 78%, apparently unrelated to fatty degeneration. Since liver biochemical variables AST and ALT had been found within normal limits in 88% and 89%, respectively of patients at initial biopsy, no difference was found 24 months later (p = 1.000). Lipid profile and blood sugar plasma concentration were closer to normal in all patients after 24 months of follow up (p < 0.05). Conclusions: The improvement of metabolic syndrome related a severe obesity after sustained weight loss surgery promoted significant improvement in liver histology. The steatosis, fibrosis and NAS ? 5 were decreased in 89%, 75% and 100% of patients, respectively. None patient had progression of hepatic fibrosis in this series.

INTRODUÇÃO: A secreção do hormônio de crescimento (GH) está diminuída em obesos. Existem controvérsias se esta diminuição é conseqüência ou um dos fatores causais da obesidade. Perda de peso leva a alguma recuperação da secreção de GH. Não há estudos publicados sobre o efeito da derivação gástrica (gastrojejunal) com anastomose em Y-de-Roux (BPG) sobre o perfil de secreção de 24 h de GH. Por outro lado, a ghrelina é um peptídeo secretagogo de GH produzido no estômago, orexigênico, lipogênico e adipogênico, cujos níveis oscilam ao longo do dia e estão diminuídos na obesidade. As variações circadianas de ghrelina têm papel no controle da homeostase energética e secreção de GH. O nível de ghrelina eleva-se com perda de peso induzida por dieta, mas os dados são controversos sobre mudanças desses níveis após cirurgias bariátricas. Este estudo tem por objetivo caracterizar os perfis de secreção de GH e ghrelina em mulheres com obesidade grau III antes e após BPG e suas correlações com variáveis metabólicas. MÉTODOS: Coletas de sangue a cada 20 minutos por 24 horas foram realizadas em obesas mórbidas não diabéticas na pré-menopausa antes e seis meses após BPG. O procedimento foi realizado em balanço calórico neutro por quatro dias. Foram dosados glicose e insulina; GH em todas as amostras e ghrelina às 08:00h, 10:00h, 12:00h, 19:00h e 02:00h. A taxa metabólica de repouso (TMR) foi avaliada por calorimetria indireta e as massas adiposa (MA) e magra (MM) foram medidas por DEXA. RESULTADOS: Houve uma redução de 27% do peso corporal e IMC (de 55,9 ± 6,2 kg/m2 para 40,7 ± 5,8 kg/m2, p<0,001) com elevação de vários parâmetros de secreção de GH (GH basal, GH médio, p<0,05; área, amplitude e número de picos, p<0,001); redução de glicemia (p = 0,03), insulinemia de jejum (p = 0,005) e HOMA (p = 0,004). Não houve diferença nos níveis de ghrelina basal, pós-prandial e médio. O GH médio apresentou correlação negativa com as mudanças no peso (p = 0,003; r = -0,631), IMC (p <0,001; r = -0,731), MA (p = 0,003; r = -0,635), MM (p = 0,02; r = -0,507), circunferência abdominal (p = 0,01; r = -0,555), TMR (p = 0,01; p = -0,539), insulina de jejum (p = 0,014, r = -0,538) e HOMA (p = 0,01; r = -0,560), mas não com a glicemia de jejum (p = 0,13; r = -0,354) e a ghrelina (p = 0,6; r = 0,118). O melhor determinante da secreção de GH foi o IMC sendo responsável por 54% da variação do GH médio (r2 = 0,54). CONCLUSÕES: Há uma recuperação parcial da secreção de GH, reduzida no pré-operatório em obesas mórbidas, após perda de peso induzida seis meses após a cirurgia, indicando que a secreção reduzida não é um fator primário ou causal da obesidade, mas sim uma conseqüência da obesidade e essa recuperação é independente do perfil de secreção de ghrelina / INTRODUCTION: Growth hormone (GH) concentration is decreased in obesity. It is not clear if reduced GH secretion is consequence or cause of the obese state. GH secretion is partially restored by weight loss. There are no published studies about the effect of Roux-en-Y gastric bypass (RYGBP) on GH secretory profile. Ghrelin is a GH releasing peptide produced by stomach, with orexigenic, lipogenic and adipogenic actions. Ghrelin levels oscillate throughout the day and are low in obesity. Circadian changes in ghrelin levels have a role both in energy homeostasis control and GH secretion. Ghrelin levels rise after diet-induced weight loss, but results are controverse in relation to changes in ghrelin levels after bariatric surgeries. In this study, we analyzed GH and ghrelin concentrations in morbidly obese women before and after RYGBP and its relationships with metabolic parameters. METHODS: Blood was sampled at 20-minute intervals during 24 hours in non diabetic pre-menopausal morbid obese women before and six months after RYGBP. The study was done after four days in neutral caloric balance. Fasting glucose and insulin were determined in basal samples. GH concentrations were measured in all samples and ghrelin in serum collected at 08:00h, 10:00h, 12:00h, 19:00h e 02:00h. Resting metabolic rate (RMR) was evaluated by indirect calorimetry and fat mass (FM) and free-fat mass (FFM) were measured by DEXA. RESULTS: A 27% drop in body weight and BMI (55.9 ± 6.2 kg/m2 to 40.7 ± 5.8 kg/m2, p<0.001), augmentation of spontaneous GH secretory episodes (basal and mean levels, p <0.05; area, amplitude and peak frequency, p <0.001); and reduction of fasting glucose (p = 0.03), insulinemia (p = 0.005) and HOMA (p = 0.004) were observed. Neither basal, post-prandial or mean ghrelin were changed. A negative correlation was found between mean GH levels and weight changes (p = 0.003, r = -0.631), BMI (p <0.001, r = -0.731), FM (p = 0.003, r = -0.635), FFM (p = 0.02, r = -0.507), waist (p = 0.01, r = -0.555), RMR (p = 0.01, p = -0.539), fasting insulin (p = 0.014, r = -0.538), as well as HOMA (p = 0.01, r = -0.560), but not between mean GH levels and glucose (p = 0.13, r = -0.354) or ghrelin (p = 0.6, r = 0.118). BMI accounted for 54% of the mean GH variation (r2 = 0.54). CONCLUSIONS: There is a partial recovery of GH secretion after weight loss induced by RYGBP, suggesting that a blunted secretion is not a primary or causal factor of obesity, but a consequence of the obese state. This recovery is independent of ghrelin secretory profile