Les hépatopathies médicamenteuses survenant sur un mode idiosyncrasique représentent un obstacle majeur au développement de médicaments et sont à l’origine du retrait du marché de nombreux d’entre eux. Un des mécanismes mis en cause est lié à la survenue d’un épisode inflammatoire aigu qui, lors d’un traitement médicamenteux, sensibiliserait le foie pour les effets indésirables des médicaments, mettant en évidence leur toxicité idiosyncrasique. A l’heure actuelle, la détection précoce de médicaments potentiellement hépatotoxiques dans un contexte inflammatoire avant leur mise sur le marché, reste encore difficile et leurs mécanismes sous-jacents ne sont pas clairement élucidés. Dans ce contexte, notre travail s’articule sur 3 axes : 1) Développer un nouveau modèle cellulaire in vitro humain, adaptable au criblage à haut débit prédictif d'hépatotoxicité idiosyncrasique liée à un stress inflammatoire, basé sur l’exposition synergique des cellules HepG2 cultivées dans des conditions particulières exposées à des médicaments potentiellement idiosyncrasiques et des médiateurs pro-inflammatoires(LPS et TNF- ).2) Elucider les mécanismes sous jacents de la toxicité de 4 médicaments idiosyncrasiques connus (trovafloxacine, nimésulide, télithromycine et néfazodone), en mettant l'accent sur le stress oxydatif, la stéatose et la cholestase 3) Etudier les mécanismes moléculaires sous-jacents de la mort cellulaire observée lors d’une hépatotoxicité idiosyncrasique liée à un stress inflammatoire. Ainsi, nous avons développé un modèle cellulaire humain prédictif d’hépatotoxicité idiosyncrasique liée à un stress inflammatoire sensible, spécifique et applicable au criblage en haut débit d’un grand nombre de médicaments. Pour cela, nous avons étudié dans ce modèle, les effets toxiques de quatre médicaments testés et élucidé leurs mécanismes. La trovafloxacine exerce un effet cholestatique par diminution de l’expression et de l’activité de MDR1 et MRP2. Le nimésulide favorise l'accumulation intracellulaire de radicaux superoxydes en plus de son potentiel cholestatique par inhibition de l’activité MRP2. La télithromycine favorise une hépatotoxicité principalement via un mécanisme cholestatique impliquant l’inhibition de MDR1. La néfazodone favorise l'accumulation des radicaux superoxydes en plus de son potentiel stéatosique important et de son effet inhibiteur sur les deux transporteurs MDR1 et MRP2. Bien que chaque médicament idiosyncrasique testé présente un mécanisme de toxicité différent, ils ont tous entraîné une mort hépatocellulaire amplifiée en présence de LPS et TNF- , via la voie apoptotique intrinsèque pour la trovafloxacine, extrinsèque pour la néfazodone et les deux voies de l'apoptose pour le nimésulide et la télithromycine. Le potentiel apoptotique amplifié des quatre médicaments s'est avéré être médié par la surexpression de Bax et de caspase 8 via un mécanisme dépendant de ERK ½. Nos résultats indiquent que notre modèle peut être utilisé non seulement comme un outil préclinique pour l'identification de nouveaux médicaments qui pourraient être potentiellement hépatotoxiques lors d’un stress inflammatoire, mais aussi pour l'élucidation de leurs mécanismes. / Idiosyncratic adverse drug reactions (IADRs) are considered as an important subset of ADRs, accounting for approximately 13% ofall acute liver failure cases and representing one of the leading causes for post-marketing drug withdrawal (Shaw et al. 2010). The lack of effective in vitro or in vivo models able to predict the hepatotoxic potential of idiosyncratic drugs before being approved formarketing on one hand, and the ambiguity of the mechanisms underlying their hepatic pathogenesis on the other hand render IADRs a perplexing human health problem (Shaw et al. 2010). Accordingly, the work presented in this thesis was based on three main objectives: 1) Development of a high throughput human-based cellular model for the prediction of inflammation associated idiosyncratic drug-induced hepatotoxicity; based on the synergistic exposure of HepG2 cells to potentially hepatotoxic drugs and proinflammatory mediators (LPS and TNF- ). 2) Elucidation of the hepatotoxic mechanisms underlying four known idiosyncratic drugs (trovafloxacin, nimesulide, telithromycin and nefazodone) with emphasis on oxidative stress, steatosis and cholestasis.3) Investigation of the molecular mechanisms underlying drug-inflammation synergistic induction of hepatocellular death Firstly, the results attained in this thesis demonstrated that the developed model is sensitive, specific and applicable to high throughputtoxicity screening of different categories of drugs. Secondly, our results demonstrated that the inflammation associated hepatotoxicpotentials of the four tested idiosyncratic drugs are mediated as follows: trovafloxacin exerts a cholestatic potential that involves thedown-regulation of both MDR1 and MRP2. Nimesulide promotes the intracellular accumulation of superoxide anions in addition topotently inhibiting MRP2. Telithromycin promotes hepatotoxicity predominately via a cholestatic mechanism that involves the downregulation of MDR1. Nefazodone favors the accumulation of superoxide anions in addition to its prominent steatotic potential and inhibitory effect on both MDR1 and MRP2. Although each of the idiosyncratic drugs exhibited a different mechanism of toxicity they all induced amplified hepatocellular death in presence of LPS and TNF- , which proved to be mediated via the intrinsic apoptotic pathway for trovafloxacin, the extrinsic for nefazodone and both apoptotic pathways for nimesulide and telithromycin. The amplified apoptotic potential of the four drugs proved to be based on the up-regulation of Bax and caspase 8 via an ERK½-dependent mechanism. These results indicate that the presented drug-inflammation model constitute an effective pre-clinical tool not only for the detection of inflammation-associated hepatotoxic drugs but also for the elucidation of their underlying mechanisms.

A esteatose hepática, que se caracteriza pelo acúmulo excessivo de gordura nas células do fígado, é um problema que vem preocupando a comunidade médico-científica, pois sua incidência vem aumentando a nível global, com expectativa de se tornar a doença crônica hepática de maior predominância em várias partes do mundo. Apesar de ser considerada uma doença benigna, a esteatose pode evoluir para doenças mais graves como cirrose, fibrose avançada, esteato hepatite (com ou sem fibrose) ou carcinoma. Entretanto, é potencialmente reversível, mesmo em quadros mais graves, o que reforça a urgência de se desenvolver métodos confiáveis para detecção e avaliação, inclusive ao longo de tratamento. Os métodos atuais para diagnóstico e quantificação da gordura hepática ainda são falhos: com a ultrassonografia não se é capaz de realizar quantificação; a tomografia computadorizada faz uso de radiação ionizante; a punção (biópsia), considerada o padrão ouro, é precisa, mas invasiva e pontual. A Ressonância Magnética (RM), tanto com espectroscopia (MRS) como com imagem (MRI), são alternativas completamente não invasivas, capazes de fornecer o diagnóstico e quantificação da gordura infiltrada no fígado. Entretanto, os trabalhos encontrados na literatura utilizam sequências de pulsos desenvolvidas especialmente para esse fim, com métodos de pós-processamento extremamente rebuscados, o que não é compatível com o estado atual dos equipamentos encontrados em ambientes clínicos nem mesmo ao nível de experiência e conhecimento das equipes técnicas que atuam em clínicas de radiodiagnóstico. Assim, o objetivo central do presente trabalho foi avaliar o potencial da RM como candidato a método de diagnóstico e de quantificação de gordura em ambientes clínicos, utilizando, para isso, sequências de pulsos convencionais, disponíveis em qualquer sistema comercial de RM, com protocolos de aquisição e processamento compatíveis com àqueles realizados em exames clínicos, tanto no que se refere à simplicidade como ao tempo total de aquisição. Foram avaliadas diferentes abordagens de MRS e MRI utilizando a biópsia hepática como padrão de referência. Foram avaliados pacientes portadores de diabetes tipo II, que apresentam alta prevalência de esteatose hepática não alcoólica, além de grande variabilidade nos percentuais de gordura. Foram realizadas medidas de correlação, acurácia, sensibilidade e especificidade de cada uma das abordagens utilizadas. Todos os métodos avaliados apresentaram alto grau de correlação positiva (> 87%) com os dados obtidos de maneira invasiva, o que revela que os valores obtidos utilizando RM estão de acordo com aquilo observado pela biópsia hepática. Muito embora os métodos de processamento utilizados não sejam tão complexos quanto seriam necessários caso uma quantificação absoluta fosse desejada, nossas análises mostraram alta acurácia, especificidade e sensibilidade da RM na avaliação da esteatose. Em conclusão, a RM se apresenta, de fato, como uma excelente candidata para avaliar, de forma não invasiva, a fração de gordura hepática, mesmo quando se considera as limitações impostas por um ambiente clínico convencional. Isso sugere que essas novas metodologias podem começar a migrar para ambientes clínicos sem depender das sequências complexas e dos processamentos exóticos que estão descritos na literatura mais atual. / Hepatic steatosis is a disease characterized by excessive accumulation of fat in liver cells. Its incidence is increasing and it is expected to become the chronic liver disease with the highest prevalence worldwide, which became a major concern to medical and scientific community. Although considered a benign disease, fatty liver can evolve to more serious diseases such as cirrhosis, advanced fibrosis, steatohepatitis (with or without fibrosis) or carcinoma. However, it is potentially reversible, even in the most severe cases, which reinforces the urgent need to develop reliable methods for detection and evaluation, including throughout treatment. Current methods for diagnosis and measurement of liver fat are still flawed: ultrasound is not able to perform quantification; CT uses ionizing radiation; biopsy, considered the gold standard, is accurate but invasive and focal. Magnetic resonance imaging (MRI) and spectroscopy (MRS) are completely non-invasive alternatives capable of providing diagnosis and quantification of infiltrated fat in the liver. However, studies in the literature frequently employ pulse sequences especially developed for this purpose, with extremely elaborate post-processing methods, which are not compatible with the current state of the art of typical clinical scanners or even with the level of experience and knowledge of technical staff working in radiology clinics. Thus, the main goal of this study was to evaluate the potential of MRI as a candidate for diagnosing and quantifying fat liver in clinical settings, using conventional pulse sequences, acquisition and post-processing protocol, compatible with those performed in clinical examinations in both simplicity and total acquisition time. We investigated different approaches of MRI and MRS using liver biopsy as reference standard. We evaluated patients with type II diabetes, which have a high prevalence of non-alcoholic fat liver disease, and great variability in the percentage of fat. Correlation, accuracy, sensitivity and specificity measurements of each of the approaches used were performed. All evaluated methods highly correlated (> 87%) with the data obtained from invasive measurement, which shows that the values obtained using MRI are consistent with that observed for liver biopsy. Although the processing methods used are not as complex as would be required if an absolute quantification was desired, our analyzes showed high accuracy, sensitivity and specificity of MRI in the evaluation of steatosis. In conclusion, MRI is an excellent candidate to assess liver fat fraction in a non-invasive way, even when considering the limitations imposed by conventional clinical setting. This suggests that these new methods may begin to migrate to clinical environments without relying on complex sequences and exotic post-processing techniques that are described in the current literature.

Introdução: Em relação ao fígado, a esteatose é a consequência mais comum do consumo abusivo do etanol e predispõe à doença hepática mais grave. Os mecanismos da doença hepática alcoólica não são plenamente conhecidos e as terapias são escassas. Os objetivos desta tese foram: 1) averiguar os efeitos do etanol no fígado, utilizando o zebrafish como modelo experimental; 2) avaliar o tratamento com o Lactobacillus rhamnosus GG (LGG) na esteatose hepática; 3) observar os efeitos do etanol e do tratamento com o LGG no comportamento do zebrafish. Métodos: Foram realizados três experimentos utilizando peixes zebrafish, adultos, wildtype. O primeiro experimento foi formado por dois grupos, Controle (C) e Etanol (E), com 52 animais em cada um. O grupo E foi exposto a 0,5% de etanol por quatro semanas. Foram conduzidas análises histológicas e moleculares dos genes il-1b, tnf-α, il-10, sirt1, adiponectina e adipor2 nos fígados dos animais. No 2°experimento foram avaliados quatro grupos: Controle (C), Probiótico (P), Etanol (E) e Probiótico + Etanol (P + E), com 220 animais respectivamente. Durante quatro semanas os grupos P + E e P foram alimentados com ração com o probiótico LGG e os grupos E e C com ração sem probiótico. Foram realizadas análises histológicas e morfométricas no tecido hepático, quantificações de lipídeos séricos e hepáticos. No 3° experimento, os grupos C, E, P e P+E foram formados (n=15 animais por grupo) e, após duas semanas, o comportamento dos animais foi analisado no teste open-tank com o programa ANYmaze ®. Resultados: No 1° experimento os animais do grupo E apresentaram intensa esteatose hepática, aumento de glicogênio plasmático associado às gotículas lipídicas, alterações no retículo endoplasmático rugoso e degeneração de canalículos biliares. Houve acentuação na expressão hepática de il-1b, tnf-α, sirt1 e do adipor2, indicando que o etanol desencadeou resposta inflamatória e de proteção hepática. No 2° experimento, o grupo E apresentou intensa esteatose após quatro semanas, ao contrário do grupo P + E. A morfometria celular mostrou um aumento de 14,8 vezes no tamanho dos hepatócitos do grupo E (4° semana) quando comparado com C (p <0,0001). Os triglicerídeos séricos diminuíram no grupo P + E em comparação com C, P (p <0,001) e E (p = 0,004). O colesterol sérico do grupo P diminuiu comparado aos grupos C e E na segunda semana (p = 0,002 e p = 0,007) e do grupo P + E diminuiu comparado aos grupos E e C (p<0,0001), na quarta semana. As concentrações de triglicerídeos hepáticos reduziram no grupo P + E na quarta semana em comparação com E (p = 0,006). No 3° experimento, os animais expostos ao etanol apresentaram menor ansiedade em relação ao novo ambiente, evidenciada pela maior exploração da área superior do aquário. O efeito desibinidor do etanol não foi significativamente atenuado pelo tratamento com o LGG. Conclusões: Os resultados do primeiro estudo indicaram que o etanol desencadeia uma série de eventos celulares e moleculares e que a inflamação desempenha papel significativo na esteatose hepática. No segundo, foi demonstrado que o tratamento com LGG diminuiu os níveis séricos de triglicerídeos e de colesterol, atenuando a esteatose hepática. O terceiro estudo mostrou que o etanol teve efeito significativo no comportamento do zebrafish, que não foi modificado pelo LGG. / Introduction: Regarding to the liver, hepatic steatosis is the most common consequence of abusive alcohol consumption and predisposes to more severe liver disease. The mechanisms of alcoholic liver disease are not fully known and therapies are scarce. The objectives of this thesis were: 1) to verify the effects of ethanol in the liver using the zebrafish as an experimental model; 2) to evaluate a treatment with the probiotic Lactobacillus rhamnosus GG (LGG) in hepatic steatosis; 3) to observe the effects of ethanol and treatment with LGG in zebrafish behavior. Methods: Three experiments were performed using zebrafish, adult, wildtype. For the 1st trial, two groups were formed: Control (C) and Ethanol (E), with 52 animals in each group. E group was exposed to 0.5% ethanol during four weeks. Histological and molecular analysis of genes il-1b, tnf-α, il-10, sirt1, adiponectin, and adipor2 were conducted in zebrafish livers. For the 2nd trial, four groups were evaluated: Control (C), Probiotic (P), Ethanol (E) and Probiotic + Ethanol (P + E). During four weeks, the P + E and P groups were fed with food supplemented with LGG and E and C groups received food without probiotic. Histological and morphometric analysis in liver tissue, measurements of serum and hepatic lipids were performed. In the 3rd trial, C, E, P and P + E groups were formed and after two weeks, the animals' behavior was analyzed in opentank test with ANYmaze ® program. Results: In the 1st trial, animals in E group developed severe liver steatosis and cell abnormalities were observed: increase of glycogen associated to lipid droplets, alterations in the rough endoplasmic reticulum, degeneration of biliary canaliculi with presence of myelin figures inside. Increased hepatic expression of il-1b, tnf-α, sirt1 and adipor2 possibly indicates that ethanol triggered both inflammatory and hepatic protection responses. In the 2nd trial, E group presented severe steatosis after four weeks, in contrast to the E + P group. Cell morphometry showed a 14.8 fold in hepatocytes size of E (4th week) compared to C group (p <0.0001). Serum triglycerides decreased in the P + E group compared with C, P (p <0.001) and E groups (p = 0.004). Serum cholesterol decreased in P group compared to C and E groups at second week (p = 0.002 and p = 0.007) and in E + P group decreased compared with E and C groups (p<0.0001) at fourth week. Liver triglycerides were reduced in the P + E group at the fourth week compared to E group (p = 0.006). In the 3rd trial, there was an alteration in the behavior of animals exposed to ethanol compared to that nonexposed, an effect not significantly attenuated by treatment with LGG. Conclusions: Results of the first study indicate that ethanol triggers a series of cellular and molecular events and inflammation plays a significant role in hepatic steatosis. Then, it was shown that treatment with LGG decreased serum levels of triglycerides and cholesterol, attenuating hepatic steatosis. The third study showed that the ethanol, but not LGG has a significant effect on zebrafish behavior.

A aterosclerose é um processo inflamatório que se inicia na infância e progride com a idade, sendo o principal processo patológico que culmina nas doenças cardiovasculares. Ácidos graxos ômega 3 (N-3 FA) reduzem triacilgliceróis plasmáticos e inflamação, enquanto fitosteróis possuem ação hipocolesterolêmica. Portanto, o objetivo deste estudo foi de aplicar a combinação dessas duas classes de lipídios bioativos na infância, visando reduzir a aterosclerose na fase adulta. O estudo foi dividido em duas etapas. Inicialmente, o efeito de três ingredientes contendo N-3 FA foi avaliado em termos de perfil lipídico e biomarcadores inflamatórios. Camundongos adultos LDLr Knockout receberam uma dieta hiperlipídica e foram simultaneamente suplementados com água (CON), óleo de algas (ALG), óleo de peixe (FIS) e óleo de Echium (ECH) através de gavagem durante 4 semanas. Os animais suplementados com óleo de Echium apresentaram os maiores índices de redução de triacilgliceróis e VLDL, além de inibição da esteatose causada pelo dieta hiperlipídica. Entretanto, diferente dos óleos marinhos, tais efeitos não envolveram ativação de fatores de transcrição envolvidos no metabolismo lipídico, como PPAR&#945; e LXR&#945; hepáticos. Na segunda etapa deste estudo, camundongos Knockout LDLr recém-desmamados foram suplementados durante 2 meses com emulsões preparadas com óleo de soja (CON), óleo de de Echium (ECH), óleo de alga (ALG), fitosteróis isolados (PHY), óleo de alga + fitosteróis (ALG+PHY) e óleo de Echium + fitosteróis (ECH+PHY). A seguir, dislipidemia e estresse oxidativo foram induzidos através de uma dieta hiperlipídica por mais 2 meses. Todos os animais apresentaram estrias gordurosas na aorta, sendo que a área de lesão foi maior naqueles suplementados com fitosteróis isoladamente (PHY). Entretanto, esse efeito negativo foi totalmente revertido pela co-suplementação com N-3 FA. Observou-se que os fitosteróis isolados agiram como agonistas de LXR&#945;, e que a reversão proporcionada pelos N-3 FA envolveu aumento da expressão do fator de transcrição PPAR&#945; e redução do aumento de LXR&#945;. Além disso, o óleo de Echium reduziu o estresse oxidativo no fígado através de mecanismos associados à modulação da atividade e expressão de enzimas antioxidantes. Nossos resultados sugerem que a melhor alternativa no desenvolvimento de alimentos funcionais para crianças, visando prevenção de aterosclerose na fase adulta, foi conferida pelos N-3 FA provenientes do óleo de Echium. / Atherosclerosis is an inflammatory process that begins in childhood and progresses with age, being the main pathological process that culminates in cardiovascular disease. Omega 3 fatty acids (n-3 FA) reduces triacylglycerol (TG) and inflammation, while phytosterols present hypocholesterolemic action. Therefore, the aim of this study was to apply the combination of these two classes of bioactive lipids in childhood to reduce atherosclerosis in adulthood. The study was divided into two steps. Initially, the effect of three ingredients containing n-3 FA was evaluated in terms of lipid and inflammatory biomarkers. LDLr knockout adult mice received a high-fat diet and were simultaneously supplemented with water (CON), algae oil (ALG), fish oil (FIS) and Echium oil (ECH) by gavage for 4 weeks. The animals supplemented with Echium oil presented higher reduction of TG and VLDL. Besides, Echium oil inhibited hepatic steatosis caused by high-fat diet. However, unlike of marine oils, this hypotriglyceridemic effect did not involve activation of transcription factors associated to lipid metabolism, such as PPAR&#945; and LXR&#945;. At the second step of this study, weaning LDLr knockout mice were supplemented with emulsions prepared with soybean oil (CON), Echium oil (ECH), algae oil (ALG), isolated phytosterols (PHY), algae oil + phytosterols (ALG + PHY) and Echium oil + phytosterols (PHY + ECH) for 2 months. Thereafter, dyslipidemia and oxidative stress were induced by a high fat diet for 2 months more. All animals showed fatty streaks in the aorta artery. The lesion area was greatest in mice supplemented with isolated phytosterol (PHY). However, this negative effect was completely reversed by co-supplementation with n-3 FA. It was observed that isolated phytosterols acted as LXR&#945; agonists, and that the reversal provided by the N-3 FA involved the increase of PPAR&#945; and reduction of LXR&#945; expression. Furthermore, the Echium oil reduced oxidative stress in liver through mechanisms associated with modulation of antioxidant enzymes activity and expression. Our results suggest that the best alternative to develop functional foods for children, aimed at preventing atherosclerosis in adulthood, was conferred by the N-3 FA from Echium oil.

Orientador: Licio Augusto Velloso / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T13:12:05Z (GMT). No. of bitstreams: 1 Cintra_DennysEsper_D.pdf: 2036070 bytes, checksum: 1c0ff9462c147443a4261b0699b9128d (MD5) Previous issue date: 2008 / Resumo: A obesidade se manifesta de forma pandêmica, acometendo milhões de pessoas, principalmente nos países ocidentais e industrializados. Além dos inúmeros aspectos comportamentais e sociais, o desenvolvimento dessa doença contribui consideravelmente para o aumento da morbidade e mortalidade em decorrência de sua freqüente associação com outras doenças que compõem a síndrome metabólica, tais como, diabetes mellitus, hipertensão arterial, dislipidemia, ovários policísticos, esteatose hepática, entre outras. Os distúrbios no metabolismo de ácidos graxos frequentemente relacionados à obesidade contribuem para a disfunção de múltiplos órgãos e sistemas. No fígado, o simples acúmulo de gordura no parênquima (esteatose hepática) pode ser um estado transitório e benigno, facilmente reversível. Entretanto, com a perpetuação do quadro, e dependendo de algumas características genéticas ainda pouco conhecidas, ativa-se uma resposta inflamatória que desempenhará papel central na progressão da esteatose para esteato-hepatite, fibrose e cirrose, sendo esta um estágio final da doença hepática. Durante a instalação da inflamação hepática, uma série de citocinas pró-inflamatórias são expressas e participam da indução da disfunção orgânica. Entretanto, alguns estudos recentes revelam que a interleucina-10 (IL-10), uma citocina antiinflamatória, também é produzida no fígado e que sua presença nesse contexto ameniza a progressão para graus avançados de lesão hepática. O objetivo desse trabalho foi investigar como a IL- 10 endógena modula alguns parâmetros inflamatórios e metabólicos no fígado de um modelo animal de obesidade, resistência à insulina e esteato-hepatite induzidos por dieta. Para tal, a IL-10 foi inibida in vivo por técnicas de inativação com anticorpo específico e oligonucleotídeos de cDNA modificado complementares ao RNAm dessa proteína, impedindo sua tradução. A inibição da IL-10 em animais que consumiram dieta rica em gordura acentuou a disfunção hepática, inibindo a atividade de importantes proteínas como as do receptor de insulina e seus substratos (IR, IRS-1 e IRS-2), fomentando o processo inflamatório local e ativando vias pró-apoptóticas. A partir desses resultados, conclui-se que a IL-10 endógena atua como um fator protetor, controlando vias inflamatórias durante a progressão da esteatose para esteato-hepatite não alcoólica. / Abstract: Obesity has grown as an epidemiological phenomenon that threatens millions of people worldwide, and particularly those living in western, developed countries. Besides its multiple behavioral and social problems, obesity contributes considerably to increase morbidity and mortality due to its common association with diseases that compose the metabolic syndrome, such as diabetes mellitus, hypertension, dyslipidemia, polycystic ovaries, hepatic steatosis, among others. The abnormalities in the metabolism of fatty acids that frequently occur in obesity, contribute to the dysfunction of a number of organs and systems. In the liver, the simple accumulation of fat in the parenchyma cells leads to steatosis which is somewhat benign and reversible. However, as far as the condition is maintained, and depending on some yet to be known genetic factors, an inflammatory response can be installed leading to steatohepatitis, fibrosis and cirrhosis, a terminal liver disease. During the installation of steatohepatitis, a number of proinflammatory cytokines are expressed, contributing to the progression of the liver damage. However, some recent studies have shown that interleukin-10 (IL- 10), an anti-inflammatory cytokine, is produced in the liver and plays a role in the negative modulation of the inflammatory response. The objective of the present study was to evaluate the roles of IL-10 as an endogenous factor controlling inflammatory and metabolic parameters in diet induced hepatic steatosis. IL-10 was inhibited in vivo by two distinct methods, a neutralizing antibody and an antisense oligonucleotide. The inhibition of IL-10 in diet induced steatohepatitis increased proinflammatory cytokine expression and worsened a number of metabolic parameters, including insulin signal transduction. Thus, IL-10 is an endogenous factor that modulates inflammatory response in diet-induced hepatic disease. / Doutorado / Ciencias Basicas / Doutor em Clínica Médica

L’huile de palme est l’huile végétale la plus consommée au monde. Du fait de sa teneur élevée en acides gras saturés (AGS), notamment en acide palmitique, cette huile est considérée par certains auteurs comme potentiellement nocive pour la santé. Cette étude avait pour objectif de comparer les effets de l’huile de palme (rouge ou oléine), à l’huile d’olive (réputée bonne pour la santé) et aux saindoux (riche en AGS), sur la santé. Pour réaliser cette étude, 40 rats mâles Wistar ont été répartis en 5 groupes de 8 rats chacun : 1 groupe contrôle et 4 groupes nourris par des régimes obésogènes contenant respectivement de l’huile de palme rouge, de l’oléine de palme, de l’huile d’olive ou du saindoux. Après 12 semaines de régime, les rats ont été sacrifiés et les tissus prélevés. Les examens réalisés sur les tissus ont montré que l’huile de palme (rouge ou oléine) induit un statut antioxydant et un profil lipidique superposables à ceux de l’huile d’olive. Tous les régimes obésogènes ont favorisé la prise de poids, l’altération de la fonction mitochondriale et la perturbation du métabolisme glucidique par l’induction d’une insulino-résistance. Il ressort de cette étude que l’huile d’olive est plus délétère pour le foie que l’huile de palme (rouge ou oléine) et le saindoux. Hormis l’huile de palme rouge, l’oléine de palme, l’huile d’olive et le saindoux influencent négativement les tissus adipeux. Les études menées sur l’aorte ont montré que les effets vasculaires de l’huile de palme sont moins délétères pour l’aorte que le saindoux et l’huile d’olive.Les résultats de cette étude indiquent que globalement, l’huile de palme (rouge ou oléine) n’a pas d’effets délétères supérieurs à ceux de l’huile d’olive concernant les organes qui ont été étudiés / Palm oil is the most consumed vegetable oil in the world. Because of its high content of saturated fatty acids (SFA), particularly palmitic acid, this oil is considered by some authors as potentially harmful to health. The aim of this study was to compare the effects of palm oil (red or olein), olive oil (considered good for health) and lard (rich in SFA), on health. To do this, 40 male Wistar rats were divided into 5 groups of 8 rats each: 1 control group et 4 groups fed by high fat diet (HFD) containing respectively red palm oil, palm olein, olive oil or lard. After 12 weeks of diet, the rats were sacrificed and the tissues removed. Tissue tests have shown that palm oil (red or olein) induces an antioxidant status and a lipid profile superimposed on those of olive oil. All HFD contributed to weight gain, impaired mitochondrial function, and disturbance of carbohydrate metabolism by the induction of insulin resistance. The study shows that olive oil is more deleterious to the liver than palm oil (red or olein) and lard. Apart from red palm oil, palm olein, olive oil and lard negatively influence adipose tissue. Studies on the aorta have shown that the vascular effects of palm oil are less deleterious to the aorta than lard and olive oil.Overall, the results of this study show that harmfull effects of palm oil (red or olein) were not worse than that of olive oil on organ that were analyzed

Motivées par l'observation mondiale de l'augmentation de la morbidité et de la mortalité associées à des pathologies liées à l'obésité, dont la stéatose, les études pré-cliniques et cliniques s'intéressent à la recherche de nouveaux biomarqueurs pour le diagnostic de la stéatose. Actuellement, la stéatose est diagnostiquée et gradée par des analyses histologiques à partir d'une biopsie du foie. Dans l'intérêt du patient, et afin de permettre un suivi de la stéatose lors d'un régime ou d'un traitement, il est apparu important de se tourner vers des modalités de diagnostic moins invasives. Dans ce cadre, la spectroscopie par résonance magnétique (SRM), non-invasive et non-ionisante, est une méthode de choix pour le diagnostic de la stéatose par la mesure de la fraction lipidique hépatique. De plus, à partir des informations observables sur un spectre de SRM acquis au niveau hépatique, il est possible d'envisager une quantification de la composition en acides gras (AG) des lipides hépatiques, potentiel biomarqueur pour le suivi d'une stéatose. Les travaux de cette thèse ont été réalisés à partir d'objets-tests, et dans le cadre d'études pré-cliniques (4,7 T) et cliniques (3,0 T). Une étude du protocole d'acquisition de spectres de SRM pour la quantification de la composition en AG des lipides a été réalisée, avec notamment un questionnement quant à la nécessité de l'utilisation d'un module de suppression du signal de l'eau. Un état de l'art des algorithmes de quantification de la composition en AG des lipides a été effectué, et des tests de validations de ces algorithmes ont été réalisés afin de déterminer le plus approprié à la problématique hépatique, dans nos conditions expérimentales. Enfin, toujours dans l'objectif de déterminer des nouveaux biomarqueurs de la stéatose, une méthode de mesure par SRM in vivo du T1 de l'eau et de la résonance majeure des lipides hépatiques (LOREEDE pour LOngitudinal RElaxation time Evaluation from Dynamic Equilibrium) a été développée, et validée au cours d'une étude préliminaire sur des objets-tests et in vivo sur modèles murins. / In recent years, there has been an unprecedented increase in the morbidity and mortality associated with diseases such as the steatosis, linked to obesity. In this context, pre-clinical and clinical studies are of interest in the search for new biomarkers allowing the diagnosis of steatosis. Currently, steatosis is diagnosed and graded by histological analyzes from a liver biopsy. On the other hand, it is advantageous to use non-invasive diagnostic modalities, especially in longitudinal studies. In this context, magnetic resonance spectroscopy (MRS), as a non-invasive and non-ionizing approach, is an attractive alternative method for the diagnosis of steatosis by measuring the hepatic fat fraction. Moreover, from the MRS spectrum acquired in the liver, it is possible to quantify the fatty acids (FA) composition of the hepatic lipids, which could be a potential biomarker for the follow-up of steatosis. The work of this thesis has been performed in vitro and in vivo, in the context of pre-clinical (4.7 T) and clinical (3.0 T) studies. An investigation of the optimal MRS acquisition protocol for the quantification of FA was carried out, with particular attention to the role of the water signal suppression module. Different quantification algorithms of the lipid composition were studied and validation of these algorithms was carried out in vitro and in vivo. Finally, still with the objective of determining new biomarkers of steatosis, a method (LOREEDE: LOngitudinal RElaxation time Evaluation from Dynamic Equilibrium) for the measurement in vivo of the T1 of the water resonance and the major lipid resonance, by MRS, was developped and validated in a preliminary study.

La chromatine est connue pour son rôle dans le maintien de l'identité cellulaire. Des perturbations dans la dynamique de la chromatine sont des événements courants dans les cancers. La structure de la chromatine et sa dynamique sont fortement dépendante des protéines HP1, connues pour être impliquées dans l’extinction de l’hétérochromatine, mais également dans la régulation de l'expression des gènes, la réplication et la réparation des dommages de l'ADN. Afin de mieux caractériser les fonctions d’HP1 chez les mammifères, nous avons étudié les conséquences de l'inactivation de leurs gènes chez la souris. De façon inattendue, nous démontrons que l'inactivation d’HP1a ou d’HP1g conduit à une prédisposition élevée des souris à développer des tumeurs spécifiquement dans le foie. Par conséquent, nous avons établi des modèles murins permettant l'inactivation simultanée d’HP1a/HP1b et HP1a/HP1g spécifiquement dans les hépatocytes. Ces modèles ont montré une augmentation significative de l'incidence du développement des tumeurs dans le foie, ce qui montre que les protéines HP1 sont des suppresseurs spécifiques de tumeurs hépatiques. L'analyse histologique de foies HP1abliverKO a montré des défauts qui ressemblent à ceux observés dans une pathologie connue du foie humain, la stéatohépatite non alcoolique. Afin de caractériser les mécanismes moléculaires sous-jacents ces fonctions des HP1, nous avons analysé le transcriptome de foies de souris âgées de 5 semaines. Ces analyses ont révélé que les gènes sur-exprimés en réponse à l’absence d’HP1ag ou HP1ab sont fortement enrichis en gènes codant pour des membres de la famille de répresseurs de transcription KRAB-ZFP. Ce résultat est intéressant car il est connu que ces répresseurs sont régulés par le corépresseur TRIM28 qui a besoin d’interagir avec HP1 pour remplir ses fonctions. Cela suggère donc une boucle d'autorégulation entre HP1, TRIM28 et KRAB-ZFP. En utilisant des souris exprimant une protéine TRIM28 qui est incapable d'interagir avec HP1 spécifiquement dans les hépatocytes, nous avons démontré que la perturbation de l'interaction entre TRIM28 et HP1 conduit au développement spontané de tumeurs dans le foie et conduit également à une surexpression des mêmes KRAB-ZFP que ceux dérégulée chez les souris HP1abliverKO et HP1agliverKO. L’immunoprécipitation de la chromatine (ChIP) a mis en évidence que TRIM28 et HP1 sont recrutés de façon interdépendante dans les régions 5 'et/ou 3' des gènes de KRAB-ZFP afin de réguler leur expression. Nous avons également observé la dérégulation de certains gènes liés au cancer, comme Tert, Nox4, AR, GPC3 et Arid1a. Ces modifications sont dépendantes de l’isotope d’HP1 inactivé, ce qui reflète les différents mécanismes moléculaires de l’oncogenèse. Afin d'élucider l'impact possible d’HP1 sur l'organisation générale du noyau, j'ai effectué une analyse par immunofluorescence sur cryosections du foie. Nos données suggèrent que les caractéristiques hétérochromatiques constitutives (H3K9me3) sont remplacées par des caractéristiques hétérochromatiques facultatives (H3K27me3) en l'absence de HP1ag et que les foyers péricentriques hétérochromatiques ont une légère tendance à être délocalisés. Enfin, pour mieux comprendre les profils chromosomiques dans la tumeur du foie HP1-dépendante, nous avons effectué une hybridation génomique comparative dans les foies tumoraux. Comme prévu, plusieurs événements de gain et de perte dans les variations du nombre de copies (CNV) dans certaines régions subchromosomales ont été observés, en particulier pour les chromosomes 4, où certains membres de KRAB-ZFP sont touchés. En résumé, nos résultats montrent que les protéines HP1 sont des suppresseurs de tumeur spécifique du foie. Ces données suggèrent également que la fonction principale d’HP1 au sein du foie est de réguler l'activité de TRIM28 et ainsi réguler l'expression et l'activité de répression des KRAB-ZFP et, finalement, l'homéostasie du foie. / Chromatin is known for its essential role in establishment and maintenance of cellular identity. Accordingly, disturbances in chromatin’s dynamics are common events in cancers. Chromatin structure and dynamics is highly dependent upon HP1, small non-histone chromosomal proteins that are known to be involved in heterochromatin silencing but also in gene expression regulation, DNA replication and DNA damage repair. To better characterize HP1 functions in mammals, we have studied the consequences of the inactivation of the corresponding genes in mice. Unexpectedly, we demonstrated that inactivation of either HP1a or HP1g lead to a high predisposition of mice to develop tumors specifically within liver. Hence, we established mice models allowing simultaneous inactivation of HP1a/HP1b and HP1a/HP1g specifically within hepatocytes. These models (HP1abliverKO and HP1agliverKO) displayed a significant increased incidence of tumor development within liver, demonstrating that HP1 are liver specific tumor suppressors. Histological analysis of HP1abliverKO livers showed defects that resembled those observed in a human liver pathology known as nonalcoholic steatohepatitis (NASH) characterized by an increase of steatosis, followed by an increased inflammation and the development of fibrosis that finally leads to tumors in old animals. In the case of HP1agliverKO mice, even though inflammation and tumor development were observed, this was not linked with steatosis, strongly suggesting that the underlying mechanisms are specific of each HP1 isoform. In order to reveal molecular mechanisms, we did expression analysis in the liver of 5 weeks old mice, which revealed a strong enrichment of genes encoding for members of the KRAB-ZFP of transcriptional repressors family within genes regulated by HP1ag or HP1ab. This result is of particular interest since it is known that these repressors are regulated by the corepressor TRIM28 which has been shown to require its interaction with HP1 to fulfill its functions suggesting a loop of auto-regulation between HP1, TRIM28 and KRAB-ZFP. Using mice expressing a TRIM28 protein unable to interact with HP1 specifically within hepatocytes, we demonstrated here that the disruption of the interaction between TRIM28 and HP1 lead to spontaneous development of tumors within liver and to over-expression of the same KRAB-ZFP as those deregulated in HP1abliverKO and HP1agliverKO mice. Chromatin immunoprecipitation (ChIP) pinpointed that TRIM28 and HP1 are inter-dependently recruited to the 5’ and/or 3’ ends of KRAB-ZFP genes to regulate their expression. We also observed deregulation of some cancer related genes, such as Tert (Telomerase reverse transcriptase), Nox4 (NADPH oxidase 4), AR (Androgen receptor), GPC3 (Glypican3), Arid1a (AT-Rich Interaction Domain 1A), and interestingly these alterations are depended upon the inactivated HP1 isotype, reflecting distinct molecular oncogenesis. In order to elucidate the possible impact of HP1 on global organization of the nucleus, I performed immunofluorescence analysis in the liver cryosections of 5 weeks old mice. Our data suggest that constitutive heterochromatic features (H3K9me3) are replaced by facultative heterochromatic features (H3K27me3) in absence of HP1ag and that heterochromatic pericentric foci tend to slightly be delocalized. Finally, to better understand the chromosomal rearrangements profile in HP1-dependent liver tumor, we performed Comparative genomic hybridization (CGH) in old tumoral liver. As anticipated, multiple events of gain and loss in copy number variations (CNV) in subchromosomal regions were observed, especially for chromosomes 4, where some KRAB-ZFP members are affected. Altogether, our data demonstrated that HP1 are liver-specific tumor suppressor. They also suggest that HP1 main function within liver is to regulate TRIM28 activity and thereby regulate the expression and repression activity of KRAB-ZFP and ultimately liver homeostasis.

We tested the hypothesis that triacylglycerol (TAG) accumulation in the liver induced by short-term high-fat diet (HFD) in rats leads to the dysregulation of endogenous TAG degradation via lysosomal pathway and is causally linked with the development of hepatic insulin resistance. Lysosomal lipase (LAL) is stored in qualitatively different depots (light and dense lysosomes). In contrast to dense lysosomal fraction, LAL associated with light lysosomes exhibits high activity on intracellular TAG and prandial- or diet-dependent regulation. On standard diet, LAL activity was up-regulated in starved and down-regulated in fed animals. In the HFD group, we demonstrated elevated LAL activity, increased TAG content, enhanced production of diacylglycerol and the abolishment of prandial-dependent LAL regulation in light lysosomal fraction. The impairment of insulin signalling and increased activation of PKCε was found in liver of HFD-fed animals. Lipolysis of intracellular TAG, mediated by LAL, is increased in steatosis probably due to the enhanced formation of phagolysosomes. Consequent overproduction of diacylglycerol may represent the causal link between HFD-induced hepatic TAG accumulation and hepatic insulin resistance via PKCε activation.

No description available.