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.

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.

L'objectif de ce travail est de concevoir une nouvelle interface haptique en vue de son exploitation pour la chirurgie mini-invasive. La technique d'anastomose ciblée consiste à réunir deux parties désolidarisées d'une artère par des sutures et des nœuds. Ceci est effectué par des outils chirurgicaux introduits à travers de petites incisions. Une étude expérimentale de cette tâche a été effectuée en collaboration avec des chirurgiens afin de caractériser leurs gestes. L'enregistrement de l'opération par un système de capture de mouvement a permis d'identifier la nature et les gestes canoniques de cette technique. Une structure parallèle sphérique a été ensuite adoptée comme base de l'interface haptique. Cette architecture présente un centre fixe de rotation semblable au point d'incision réel et offre les trois degrés de liberté de rotation nécessaires autours de ce point. Une étude détaillée de cette architecture suivie d'une phase d'optimisation, a permis d'adapter la structure à l'application chirurgicale. L'optimisation, basée sur un algorithme générique, a porté dans un premier lieu sur l'espace de travail de la tâche. La dextérité de la structure a été ensuite prise en compte. Une phase de conception basée sur les paramètres résultants de cette optimisation a aboutie à la réalisation d'un premier prototype. L'influence des erreurs de fabrication sur l'orientation de la plateforme a été aussi traitée dans ce travail afin de déterminer les plages des défauts admissibles. Une modélisation, utilisant les torseurs de petits déplacements, a été élaborée.La dernière partie de ce travail porte sur la commande a retour d'effort de l'interface. Un banc d'essais à 1 degré de liberté a été réalisé afin de tester les différents schémas de contrôle pour la téléopération. Les essais en simulation on permit de dresser une vue comparative de ces schémas. / The aim of this work is to develop a new haptic interface to perform a minimally invasive surgery. The targeted anastomosis technique consists of the surgical binding of a ruptured blood vessel, using sutures and knots. This task is performed by surgical tools inserted through small incisions. An experimental study of this task was conducted in collaboration with surgeons in order to characterize their gesture. The recording of the operation by a motion capture system helped identifying the nature and the canonical actions of this technique. A spherical parallel mechanism (SPM) was then adopted as a basis for the haptic interface. This architecture has a fixed center of rotation similar to the real incision point and offers the three required degrees of freedom of rotation around that point. A detailed study of the architecture followed by an optimization procedure led to a suitable mechanism for the surgical application. The optimization, which is based on a generic algorithm, used the workspace of the task as a criterion. Then the dexterity of the structure was taken into account. A design phase based on the parameters resulting from this optimization led to building the first prototype.The influence of manufacturing errors on the orientation of the platform was also addressed in this work to determine the ranges of allowable defects. The manufacturing errors are modeled by screws of small displacement in order to determine their effect on the orientation error of the end effector. The last part of this work focuses on the command of the force feedback interface. An experimental setup made out of a one degree of freedom system, was built to test different control schemes for teleoperation. Simulation trials allowed developing a comparative view of these schemes.

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.

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.

L'encéphalopathie hépatique (EH) est un syndrome neuropsychiatrique dû à une dysfonction hépatique où l'ammoniaque est un facteur central. Il a déjà été rapporté que l’intoxication aiguë d'ammoniaque induise le stress oxydatif/nitrosatif. La présente étude cible à évaluer le rôle du stress oxydatif/nitrosatif dans 2 modèles de l’EH chronique : (1) l’anastomose portocave (PCA) et (2) la ligation de la voie biliaire (BDL). Ces 2 modèles sont caractérisés par une hyperammoniémie et une augmentation d’ammoniaque centrale, cependant l’œdème cérébral est trouvé seulement chez les rats BDL. Des marqueurs du stress oxydatif/nitrosatif ont été évaluées dans le plasma et cortex frontal. Un stress nitrosatif central a été observé chez les rats PCA; tandis qu’un stress oxydatif/nitrosatif systémique a été démontré seulement chez les rats BDL. Ces résultats suggèrent (1) que l’hyperammoniémie chronique n’induise pas le stress oxydatif/nitrosatif systémique et (2) qu’un synergisme existe entre l’ammoniaque et le stress oxydatif/nitrosatif, en association avec l’œdème cérébral. / Hepatic encephalopathy (HE) is a neuropsychiatric complication due to liver failure where ammonia is believed to be central in the pathogenesis. Acute ammonia intoxication has demonstrated to induce oxidative/nitrosative stress in both in vivo and in vitro models. The present study was aimed to assess the role of oxidative/nitrosative stress in 2 models of chronic liver failure/HE; 1. portacaval anastomosis (PCA) and 2. bile duct ligation (BDL). Both models are characterised with hyperammonemia and increased brain ammonia however cerebral edema is only found in BDL rats. Oxidative/nitrosative stress markers were evaluated in plasma and frontal cortex of both animal models. Central nitrosative stress was observed in PCA rats, but systemic oxidative/ntrosative stress was demonstrated only in BDL rats. The results of our study suggest i) chronic hyperammonemia does not induce oxidative stress and ii) a synergistic effect between ammonia and systemic oxidative/nitrosative stress is associated with cerebral edema.

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.