Synthetic vascular grafts are useful to bypass diseased arteries. The long-term failure of synthetic grafts is primarily due to intimal hyperplasia at the anastomotic sites. The accelerated intimal hyperplasia may stem from a compliance mismatch between the host artery and the graft since commercially available synthetic conduits are much stiffer than an artery. The objective of this thesis is to design a method for fabricating a vascular graft that mechanically matches the patients native artery over the expected physiologic range of pressures. The creation of an optimized mechanical graft will hopefully lead to an improvement in patency rates. The mechanical equivalency between the graft and the host artery is defined locally by several criteria including the diameter upon inflation, the elasticity at mean pressure, and axial force. A single parameter mathematical for a thin-walled tube is used to describe of the final mechanical behavior of a synthetic graft. For the general problem, the objective would be to fabricate a mechanics-matching vascular graft for each host artery. Typically, fabrication parameters are set initially and the properties of the fabricated graft are measured. However, by modeling the entire fabrication process and final mechanical properties, it is possible to invert the situation and let the typical output mechanical values be used to define the fabrication parameters. The resultant fabricated graft will then be mechanically matching. As a proof-of-concept, several prototype synthetic grafts were manufactured and characterized by a single Invariant to match a canine artery. The resultant graft equaled the diameter upon inflation, the elasticity at mean pressure, and axial force of the native canine artery within 6%. An alternative to making an individual graft for each artery is also presented. A surgeon may choose the best graft from a set of pre-manufactured grafts, using a computer program algorithm for best fit using two parameters in a neighborhood. The design optimization problem was solved for both canine carotid and human coronary arteries. In conclusion, the overall process of design, fabrication and selection of a mechanics matching synthetic vascular graft is shown to be reliable and robust.

Σκοπός: Διεξάγαμε την παρούσα μελέτη για να καταδείξουμε την ύπαρξη των βρογχικο-στεφανιαίων αναστομώσεων στο πειραματικό μοντέλο του χοίρου. Επιπλέον διερευνήσαμε την επίπτωση της αρτηριοσκλήρυνσης στις βρογχικές αρτηρίες. Υλικό – Μέθοδος: Χρησιμοποιήθηκαν τα παρασκευάσματα καρδιάς και πνευμόνων από 6 χοίρους. Επιπλέον, δείγματα βρογχικών αρτηριών ελήφθησαν από 40 ασθενείς που υποβάλλονταν σε θωρακοτομή. Σημειώθηκαν αναλυτικά οι κλινικοί και εργαστηριακοί παράγοντες κινδύνου για ανάπτυξη αρτηριοσκλήρυνσης. Αποτελέσματα: Με υπολογιστική τομογραφία, ψηφιακή αγγειογραφία και χορήγηση χρωστικής ρητίνης καταδείξαμε το αναστομωτικό δίκτυο μεταξύ των βρογχικών και κυρίως των αριστερών στεφανιαίων αρτηριών σε 5 από τα 6 παρασκευάσματα. Η μικροσκοπική εξέταση των δειγμάτων δεν στοιχειοθέτησε ύπαρξη αθηροσκλήρυνσης, παρά μόνο ύπαρξη ασβεστοποιού σκλήρυνσης του μέσου χιτώνα σε ποσοστό 2.5%, που δεν συσχετίστηκε με τους παράγοντες κινδύνου αρτηριοσκλήρυνσης. Συμπεράσματα: Με δεδομένο ότι βρογχικές αρτηρίες παρουσιάζουν ελάχιστο βαθμό ασβεστοποιού σκλήρυνσης του μέσου χιτώνα., υποθέτουμε ότι θα μπορούσαν να συνδράμουν στη στεφανιαία κυκλοφορία μέσω των προαναφερθεισών αναστομώσεων σε καταστάσεις εκσεσημασμένης στεφανιαίας νόσου. Η μελέτη μας υπογραμμίζει την σπουδαιότητα των βρογχικών αρτηριών και των βρογχικο-στεφανιαίων αναστομώσεων σε περιπτώσεις εμβολισμού των βρογχικών αρτηριών, μεταμοσχεύσεων καρδιάς-πνευμόνων και αντιμετώπισης ανευρυσμάτων θωρακικής αορτής. / Aim of the study: We conducted this study to demonstrate the coronary-bronchial anastomotic routes in a porcine model. Additionally, we estimated the incidence of bronchial arteries arteriosclerosis. Material and Methods: Six heart-lung porcine blocks were used. Furthermore, 40 bronchial arteries were obtained from patients who underwent thoracotomy. Detailed clinical and laboratory atherosclerotic risk factors of the patients were documented. Results: Using CT-scan, Digital Subtraction Angiography and colored latex, we demonstrated communications between the bronchial and coronary circulation in 5 of 6 subjects. Histology revealed no established atherosclerotic lesion and narrowing of the lumen, but medial calcific sclerosis in 2.5%, that was independent from the arteriosclerotic risk factors. Conclusions: As evidence suggests that bronchial arteries only exhibit medial calcific sclerosis, we hypothesize that bronchial arteries can contribute to the coronary flow through the broncho-coronary anastomoses in cases of severe coronary artery disease. Our study emphasizes their importance and their anastomoses to coronaries in cases of embolization, heart-lung transplantation and thoracic aorta aneurysms repair.

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’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.

Ophiostoma novo-ulmi is the causal agent of Dutch elm disease (DED) which has had a severe impact on the urban landscape in Canada. This research program focused on developing molecular genetic strategies to control this pathogenic fungus. The first strategy involved the development of RNA interference (RNAi) for the down-regulation of genes involved in pathogenicity. An efficient RNAi cassette was developed to suppress the expression of the endopolygalacturonase (epg1) locus which encodes a cell-wall degrading enzyme. This epg1-RNAi cassette significantly reduced the amount of polygalacturonase activity in the fungus and resulted in almost complete degradation of epg1 mRNA. The need for a native promoter to selectively down-regulate specific gene loci was addressed by developing a carbon-catabolite regulated promoter (alcA) to drive the expression of the epg1-RNAi cassette. The expression of an alcA-driven epg1-RNAi cassette resulted in the down-regulation of epg expression under glucose starvation but normal levels of expression in high glucose. The expression could therefore be controlled by culture conditions. The second strategy explored the potential of using dsRNA viruses to vector disruptive RNAi cassettes. An isolate of O. novo-ulmi strain 93-1224 collected in the city of Winnipeg, was infected by two dsRNA mitoviruses which upon sequence characterization were named OnuMV1c and OnuMV7. To assess the transmissibility of this dsRNA virus the infected isolate 93-1224 was paired with three naive isolates of the related fungi O. ulmi and O. himal-ulmi. Through the use of nuclear and mitochondrial markers it was determined that the virus OnuMV1c may not rely on mitochondrial fusion for transmission but may have a cytoplasmic transmission route. This investigation of gene expression and manipulation has provided tools to help understand gene regulation in O. novo-ulmi. It has also added to our knowledge of mitoviruses, their transmission and potential use as a biological control. By enhancing our understanding of transmissible hypovirulence this work contributes to efforts to develop a new approach to target DED as well as a potential model for the control of other fungal diseases. / Graduate / 0307 / 0306 / 0369 / jscarneiro@hotmail.com

Made available in DSpace on 2015-07-22T20:49:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-06-29 / TEDE / BV UNIFESP: Teses e dissertações

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

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.

Les champignons mycorhiziens à arbuscules (CMA) sont des organismes microscopiques du sol qui jouent un rôle crucial dans les écosystèmes naturels et que l’on retrouve dans tous les habitats de la planète. Ils vivent en relation symbiotique avec la vaste majorité des plantes terrestres. Ils sont des biotrophes obligatoires, c'est-à-dire qu'ils ne peuvent croître qu'en présence d'une plante hôte. Cette symbiose permet entre autres à la plante d'acquérir des nutriments supplémentaires, en particulier du phosphore et du nitrate. Malgré le fait que cette symbiose apporte des services importants aux écosystèmes, la richesse des espèces, la structure des communautés, ainsi que la diversité fonctionnelle des CMA sont mal connues et l'approfondissement des connaissances dans ces domaines dépend d’outils de diagnostic moléculaire. Cependant, la présence de polymorphisme nucléaire intra-isolat combiné à un manque de données génomiques dans différents groupes phylogénétique de ces champignons complique le développement de marqueurs moléculaires et la détermination de l'affiliation évolutive à hauts niveaux de résolution (c.a.d. entre espèces génétiquement similaires et/ou isolats de la même espèce). . Pour ces raisons, il semble une bonne alternative d’utiliser un système génétique différent en ciblant le génome mitochondrial, qui a été démontré homogène au sein d'un même isolat de CMA. Cependant, étant donné le mode de vie particulier de ces organismes, une meilleure compréhension des processus évolutifs mitochondriaux est nécessaire afin de valoriser l'utilisation de tels marqueurs dans des études de diversité et en génétique des populations. En ce sens, mon projet de doctorat consistait à investiguerétudier: i) les vecteurs de divergences inter-isolats et -espèces génétiquement rapprochéesphylogénétiquement apparentées, ii) la plasticité des génomes mitochondriaux, iii) l'héritabilité mitochondriale et les mécanismes potentiels de ségrégation, ainsi que iv) la diversité mitochondriale intra-isolat in situ. À l'aide de la génomique mitochondriale comparative, en utilisant le séquençage nouvelle génération, on a démontré la présence de variation génétique substantielle inter-isolats et -espèces, engendrées par l'invasion d'éléments mobiles dans les génomes mitochondriaux des CMA, donnant lieu à une évolution moléculaire rapide des régions intergéniques. Cette variation permettait de développer des marqueurs spécifiques à des isolats de la même espèce. Ensuite, à l'aide d'une approche analytique par réseaux de gènes sur des éléments mobiles, on a été en mesure de démontrer des évènements de recombinaisons homologues entre des haplotypes mitochondriaux distincts, menant à des réarrangements génomiques. Cela a permis d'ouvrir les perspectives sur la dynamique mitochondriale et l'hétéroplasmie dans un même isolatsuggère une coexistence de différents haplotypes mitochondriaux dans les populations naturelles et que les cultures monosporales pourraient induirent une sous-estimation de la diversité allélique mitochondriale. Cette apparente contradiction avec l'homogénéité mitochondriale intra-isolat généralement observée, a amené à investiguer étudier les échanges génétiques à l'aide de croisements d'isolats génétiquement distincts. Malgré l'observation de quelques spores filles hétéroplasmiques, l'homoplasmie était le statut par défaut dans toutes les cultures monosporales, avec un biais en faveur de l'un des haplotypes parentaux. Ces résultats suggèrent que la ségrégation opère durant la formation de la spore et/ou le développement de la coloniedu mycélium. De plus, ils supportent la présence d'une machinerie protéique de ségrégation mitochondriale chez les CMAAMF, où l'ensemble des gènes impliqués dans ce mécanisme ont été retrouvé et sont orthologues aux autres champignons. Finalement, on est revenue aux sources avecon a étudié le polymorphisme mitochondrial intra-isolat à l'aide d'une approche conventionnelle de PCR en utilisant une Taq polymérase de haute fidélité, suivie de clonage et de séquençage Sanger, sur deux isolats de R. irregularis. Cela a permis l'observation d'hétéroplasmie in situ, ainsi que la co-expression de variantes de variantes de protéines'ARNm dans une souche in vitro. Les résultats suggèrent que d'autres études basées sur le séquençage nouvelle génération aurait potentiellement ignorée cette variation, offrant ainsi plusieurs nouveaux arguments permettant de considérer les CMA comme des organismes possédant une population de génomes mitochondriaux et nucléaires distincts. / The association between arbuscular mycorrhizal fungi (AMF) and plant roots is one of the most widespread symbioses involving plants, and thus has an important role in terrestrial ecosystems. In exchange for carbohydrates, AMF improve plant fitness by enhancing mineral nutrient uptake, especially in particular phosphate and nitrate. Although this symbiosisDespite the fact that these symbioses contribute provides to important services toin ecosystems, the species richness, community structure and functional diversity of AMF is not well understood due to a lack of reliable molecular tools. The intra-isolate genetic polymorphism of nuclear DNA observed in AMF, combined with a lack of genomic data in a broad range of phylogenetic groups, has made it difficult to develop molecular markers and to determine evolutionary relatedness at high levels of resolution (i.e. between genetically-similar species and/or isolates). For these reasons, it seems a good alternative to use a different genetic system by targeting the mitochondrial genome, which have been shown to be homogeneous within AMF isolates. However, given the peculiar lifestyle of these organisms, a better understanding of the mitochondrial evolutionary processes and dynamics were is necessary in order to validate the usefulness of such markers in diversity and population genetics studies. In that regard, the objectives of my PhD project were to investigate: i) the divergence between closely related species and isolates, ii) mitochondrial genomes plasticity, iii) mitochondrial heritability and potential segregation mechanisms and iv) in situ mitochondrial intra-isolate allelic diversity. With Using comparative mitochondrial genomics using and next generation sequencing (NGS) sequencing, we found substantial sequence variation in intergenic regions caused by the invasion of mobile genetic elements. This variation gives risecontributes to rapid mitochondrial genome evolution among closely related isolates and species, which makes it possible to design reliable intra- and inter-specific markers. Also, an extensive gene similarity network-based approach allowed us to provide strong evidence of inter-haplotype recombination in AMF, leading to a reshuffled mitochondrial genome. These findings suggest the coexistence of distinct mtDNA haplotypes in natural populations and raise questions as to whether AMF single spore cultivations artificially underestimates mitochondrial genetic diversity in natural population.. This apparent contradiction with the intra-isolate mtDNA homogeneity usually observed in these fungi, led to the investigation of mitochondrial heritability in the spore progeny resulting from crossed-cultures. Although an heteroplasmic state was observed in some daughter spores, we found that homoplasmy was the dominant state in all monosporal cultures, with an apparent bias towards one of the parental haplotypes. These results strongly support the presence of a putative mitochondrial segregation proteic machinery in AMF, whose complete set of genes were orthologous with those found in other fungi. Our findings suggest that segregation takes place either during spore formation or colony mycelium development. Finally, we performed a conventional PCR based approach with a high fidelity Taq polymerase, followed by downstream cloning and Sanger sequencing using the model organism Rhizophagus irregularis. We found in situ heteroplasmy along with substantial intra-isolate allelic variation within the mtDNA that persists in the transcriptome. Our study also suggest that genetic variation in Glomeromycota is higher than meets the eye and might be critically underestimated in most NGS based-AMF studies both in nuclei and mitochondria.