A fosfoetanolamina sintética (FO-S), um fosfomonoéster, apresenta relevante atividade antitumoral. Contudo, a utilização de um carreador para encapsular a FO-S em lipossomas poderia favorecer a sua disponibilidade no microambiente tumoral, possibilitando o aumento da sua eficácia. Desta forma, o presente estudo avaliou a eficiência de encapsulamento da FO-S em lipossomas de DODAC e o seu potencial antitumoral. Os lipossomas foram preparados por ultrasonicação e caracterizados físicoquimicamente. A citotoxidade foi avaliada nas linhagens tumorais B16F10 (melanoma murino), Hepa1c1c7 (hepatocarcinoma murino) e Skmel-28 (melanoma humano) e nas células normais HUVEC, após o tratamento com diferentes concentrações dos lipossomas DODAC/FO-S, no tempo de 24 horas. A internalização dos lipossomas e o potencial elétrico mitocondrial foram analisados por microscopia confocal a laser. Adicionalmente, a expressão das proteínas caspases 3 e 8 ativas, receptor DR4, citocromo c, p53, p21, Bax, p27, CD44, CD90, Bcl-2 e ciclina D1 foi quantificada por citometria de fluxo. Para os estudos in vivo, os camundongos C57BL/6J portadores de hepatocarcinoma foram tratados com FO-S, DODAC/FO-S e DODAC, pelas vias intraperitoneal (IP) e intrahepática (IH), durante 20 dias. Os resultados demonstraram que os lipossomas apresentaram aspecto esférico e alta eficiência de encapsulação da FO-S, como também promoveram maior citotoxicidade nas linhagens tumorais estudadas, em comparação com FO-S. Além disto, nas células B16F10 e Hepa1c1c7, ocasionou parada nas fases S e G2/M do ciclo celular. A linhagem Hepa1c1c7 foi a mais sensível ao tratamento com os lipossomas DODAC/FO-S, os quais foram internalizados em até 6 horas e promoveram a diminuição de CD90, CD44, ciclina D1 e Bcl-2, o aumento de p53, p21, p27, Bax e caspases 8 e 3 ativas e a liberação do citocromo c. O aumento significativo das caspases 8 e 3 ativas, expressão do receptor DR4 e a liberação do citocromo c também ocorreu nas linhagens B16F10 e Skmel-28. Os resultados in vivo mostraram que os lipossomas DODAC/FO-S e a FO-S não induziram hepatotoxicidade, nefrotoxicidade e caquexia. Os lipossomas DODAC/FO-S não ocasionaram mielossupressão e hemólise, apresentando menor toxicidade em relação a FO-S, administrada pelas vias IP e IH. Além disto, os tratamentos com DODAC/FO-S (IH) e FO-S (IH e IP) foram efetivos em diminuir o número de células na fase S. Contudo, apenas os lipossomas DODAC/FO-S (IH) reduziram significamente os focos tumorais, aumentando as áreas de necrose, promovendo também o aumento da expressão gênica da p53, ciclina B1 e caspases 8 e 3. O conjunto dos resultados in vivo e in vitro demonstraram que a formulação lipossomal DODAC/FO-S foi capaz de maximizar os efeitos antitumorais da FO-S, ativando as vias intrínsecas e extrínsecas da apoptose / Synthetic phosphoethanolamine (PHO-S) - a phosphomonoester - has shown relevant anticancer effects. However, the utilization of a carrier to encapsulate the PHOS in liposomes can maximize its availability in the tumor microenvironment, allowing an increase in its effectiveness. Thus, the present study has evaluated efficiency of PHO-S encapsulation in DODAC liposomes and its antitumor potential. The liposomes were prepared by ultrasonication and physico-chemically characterized. The cytotoxic effects were evaluated on B16F10 cells (murine melanoma), Hepa1c1c7 cells (murine hepatocellular carcinoma), Skmel-28 (human melanoma) and in endothelial cells HUVEC, after treatment with DODAC/PHO-S liposomes at different concentrations for 24 hours. The internalization of the liposomes and mitochondrial electrical potential were analyzed by confocal laser microscopy. Additionally, the expression of active caspases 3 and 8, receptor DR4, cytochrome c, p53 p53, p21, Bax, p27, CD44, CD90, Bcl-2 and cyclin D1 proteins was quantified by flow cytometry. For in vivo studies, C57BL/6J mice with hepatocellular carcinoma were treated with PHO-S, DODAC/PHO-S and DODAC, by intraperitoneal (IP) and intratumoral (IT) routes for 20 days. The results demonstrated that liposomes presented spherical aspect and high PHO-S encapsulation efficiency, as also promoted high cytotoxic effect - compared with PHO-S. Furthermore, in B16F10 and Hepa1c1c7 cells, the liposomes induced S and G2/M cell cycle arrest. Hepa1c1c7 cells showed greater sensitivity to the DODAC/PHO-S formulation, which were internalized until 6 hours and promoted a decrease in the expression of CD90, CD44, cyclin D1 and Bcl-2, an increase of de p53, p21, p27, Bax and active caspases 8 and 3 and the liberation of cytochrome c. The significant increase in the expression of active caspases 3 and 8, DR4 receptor and liberation of cytochrome c also occurred in B16F10 and Skmel-28 cells. In vivo results showed that DODAC/PHO-S liposomes and PHO-S did not induce nephrotoxicity, hepatotoxicity and cachexia. DODAC/PHO-S liposomes did not cause myelosuppression and hemolysis, presenting lower toxicity in relation to PHO-S - when administered by IP and IT routes. Moreover, treatment with DODAC/PHO-S (IT) and PHO-S (IT and IP) effectively decreased the number of cells in S phase. However, only DODAC/PHO-S liposomes significantly reduced the number of tumor foci, increasing area of necrosis, and also promoting an increase in gene expression of p53, cyclin B1 and caspases 8 and 3. The set of in vitro and in vivo results demonstrated that DODAC/PHO-S liposomal formulation was capable of maximizing the PHO-S antitumor effects, activating the intrinsic and extrinsic pathways of the apoptosis

Orientadora: Profa. Dra. Ana Carolina Santos de Souza Galvão / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2018. / O hepatorcarcinoma é um dos tipos tumorais mais comuns e está entre as três malignidades que mais causam óbito. Embora grandes avanços terapêuticos tenham sido realizados nos últimos anos, ainda se faz grande a necessidade de desenvolvimento de novas terapias mais efetivas na eliminação desta doença e menos severas quanto aos efeitos adversos resultantes. Diante deste cenário, neste trabalho a metformina foi avaliada quanto à sua capacidade citotóxica, antiproliferativa e antimetastática sobre células de carcinoma hepático humano - HepG2. Desta forma, objetivou-se no presente trabalho avaliar as alterações na viabilidade, proliferação e potencial metastático de HepG2 após o tratamento com metformina, A viabilidade celular foi verificada através do teste de redução do MTT e incorporação do vermelho neutro. A progressão do ciclo celular foi avaliada por citometria de fluxo e a análise de marcadores de morte celular foi realizada por meio de avaliação de expressão/atividade das moléculas sinalizadoras caspase 3, PARP e PARP clivada através de Western blotting. Variações no potencial metastático foram verificadas através de estudo das atividades de adesão, via teste de redução do MTT, migração celular, por Scratch Assay, migração e invasão por ensaios em placas do tipo transwell e determinação da atividade das metaloproteinases MMP2 e MMP9, por Zimografia em gelatina. Os resultados indicam que a metformina atua de forma citotóxica e citostática sobre HepG2 de modo concentração e tempo-dependente, e sugerem também a ocorrência de morte celular envolvendo ativação da caspase 3. Adicionalmente, o tratamento com metformina reduziu as atividades de migração e invasão celular, redução esta provavelmente associada à diminuição nas atividades de MMP2 e MMP9, ao mesmo tempo em que aumentou a atividade de adesão celular. Conclui-se, assim, que a ação antitumoral da metformina está associada com a indução de efeito citotóxico e citostático em HepG2, e que o euglicemiante parece reduzir o potencial metastático dessas células. Estes achados indicam que, futuramente, a metformina poderá embasar uma nova estratégia terapêutica e preventiva contra o hepatocarcinoma humano. / Hepatocellular carcinoma is one of the most common types of tumor and represents the third leading cause of cancer-related death. In despite to the major progresses made in recent years, there is still a great needed for the development of new therapies. More efficient treatments should eliminate the disease while triggers few smoother adverse effects as possible. Metformin is a traditional hypoglycemic prescribed for the treatment of type II diabetes. It has received considerable attention in the past years due to its potential anti-tumor activity. Indeed, previous studies involving this drug showed interesting results not only for the treatment itself, but also in preventing the cancer development. In this project, effects of metformin over human hepatic carcinoma cells (HepG2 lineage) were evaluated, assessing changes on cell proliferation, viability and metastatic potential. HepG2 cells were exposed to different metformin concentrations for 24 and 48 hours, and the cell viability/proliferation was determined by MTT assay. The metastatic potential analysis, adhesive and migratory abilities of HepG2 cells were evaluated by the determination of metalloproteinases (MMP-2 and -9) activities by zymography, cell motility by scratch assay and cell adhesion by MTT, respectively. Analysis of cell death markers was performed by the evaluation of caspase 3, PARP and cleaved-PARP expression by Western blotting. Results suggested that metformin promotes the reduction of cell viability in a concentration and time-dependent manner. HepG2 cells had their viability reduced through the cytotoxic and cystostatic action of metformin. Regulation over caspase 3 activation and cleavage of its substrate, PARP, indicated occurrence of programmed cell death by apoptosis. Metformin increased the adhesion simultaneously to the reduction of HepG2 migration and invasion abilities. Finally, it seems that the drug was able to modulate MMP-2 and -9 activities. Thus, confirming literature data, metformin seems able to decrease the metastatic potential of HepG2 cells. These findings indicate that, in the future, metformin may support a new therapeutic and preventive strategy against human hepatocarcinoma.

O câncer é um dos principais problemas de saúde pública no mundo. Dentre as neoplasias primárias que acometem o fígado, o carcinoma hepatocelular (HCC) é a mais frequente. Diversos fatores de risco predispõem ao HCC, entre eles a doença hepática gordurosa não alcóolica (NAFLD). Segundo estudos prévios do grupo, a &#946-ionona (BI), apresenta potencial quimiopreventivo na hepatocarcinogênese, promovendo redução de lesões preneoplásicas (LPN). Assim pretendeu-se investigar se a NAFLD potencializaria o desenvolvimento de LPN em ratos Wistar submetidos ao modelo do hepatócito resistente (RH) na etapa de iniciação/promoção inicial da hepatocarcinogênese e se a BI apresenta efeito quimiopreventivo nesse contexto. Para tanto, os animais foram distribuídos em 4 grupos experimentais: grupo não-tratado (NT), grupo submetido ao RH (RH), grupo submetido ao RH e ao modelo de NAFLD, que consiste na administração de emulsão hiperlipídica, (AS) e grupo AS tratado com BI (AS + BI). Em uma primeira instância, 5 animais pertencentes aos grupos NT, AS,AS + BI foram eutanasiados após 6 semanas de administração de emulsão hiperlipídica, antes da aplicação do modelo RH, para se confirmar o desenvolvimento da NAFLD. Foi possível observar que a administração de emulsão hiperlipídica durante 6 semanas foi suficiente para o desenvolvimento de esteatose hepática. Após as 6 semanas foi introduzido o modelo do RH concomitante à administração da emulsão hipercalipídica até o fim do experimento na 13a semana . Após 13 semanas o grupo AS apresentou maiores (p<0,05) valores de focos de inflamação, hepatócitos balonizados e grau de esteatose hepática em relação ao grupo AS+BI, assim como maiores (p<0,05) níveis séricos de triacilgliceróis, colesterol, HDL, LDL, VLDL, e maior (p<0,05) valor de MDA em relação ao grupo RH embora sem diferença estatística. O grupo AS também apresentou maior (p<0,05) incidência, número total e multiplicidade de nódulos, além de maior (p<0,05) número e tamanho de LPN persistentes (pLPN) e índice de proliferação quando comparado aos grupos RH e AS+BI. O grupo AS + BI, por sua vez, demonstrou menores (p<0,05) valores de escore de células ovais e menores valores de comprimentos de cometa e danos no DNA quando comparado ao grupo AS, embora sem diferença estatista para este último parâmetro. Em relação à expressão gênica, o grupo AS apresentou menores (p<0,05) valores de expressão do gene Hmgcr em relação ao grupo RH e maiores (p<0,05) valores dos genes Insig 1 e Thy 1 quando comparados ao grupo AS+BI. Portanto, no contexto de esteatose hepática associada ao modelo do RH, a administração de BI durante a etapa de iniciação/promoção em ratos Wistar resultou em atividade quimiopreventiva que se deu pela diminuição de pLNP, redução da proliferação celular e do número de células ovais, consideradas potenciais alvos para o desenvolvimento da hepatocarcinogênese, entretanto os genes analisados parecem não serem modulados pela BI. / Cancer is a major public health problem in the world. Among the primary neoplasms affecting the liver, hepatocellular carcinoma (HCC) is the most frequent. Several risk factors predispose to HCC, including the nonalcoholic fatty liver disease (NAFLD). According to previous studies of the group, the &#946-ionone (BI), has potential chemopreventive in hepatocarcinogenesis, promoting reduction of preneoplastic lesions (LPN). Thus we investigated whether NAFLD would increase the development of LPN in Wistar rats resistant hepatocyte model (RH) at the stage of initiation / promotion of hepatocarcinogenesis and if BI has chemopreventive effect in this context. Therefore, the animals were divided into 4 groups: non-treated group (NT), the group submitted to HR (HR), the group submitted to HR and NAFLD model, consisting of the fatty emulsion administration (AS) and AS group treated with BI (AS + BI). In a first point, 5 animals belonging to the groups NT, AS, AS + BI were euthanized after 6 weeks of administration of fat emulsion prior to application of the HR model, to confirm the development of NAFLD. It was observed that the administration of fatty emulsion for 6 weeks was sufficient to the development of hepatic steatosis. After 6 weeks it was introduced into the model HR the concomitant administration of fatty emulsion until the end of the experiment at 13 weeks. In the endpoint, the AS group had higher (p <0.05) of serum triacylglycerols, cholesterol, HDL, LDL, VLDL although no statistical difference inrelation to RH group, and increased (p <0.05) amount of MDA in relation to the group RH. The AS group also had higher (p <0.05) incidence, multiplicity and total number of nodes and greater (p <0.05) number and size of persistent LPN (pLPN) and proliferation index when compared to HR groups and AS + BI. AS + BI group. It was observed in AS+BI group lower (p <0.05) cell oval score values compared to AS group. In addition the AS+BI group showed lower values of the comet length and DNA damage compared to the AS group, although no statistical differences. In relation to gene expression, the AS group showed lower(p <0.05) HMGCR gene expression values in relation to HR group and higher (p <0.05) expression of Insig genes 1 and Thy 1 compared to group AS + BI.Therefore, in the context of hepatic steatosis associated with HR model BI for administration to the stage of initiation / promotion in rats resulted in chemopreventive activity was due to decrease in area of pLNP, reducing cell proliferation, and the number of oval cells, as potential targets for the development of hepatocarcinogenesis, however the genes do not seem to be modulated analyzed by BI.

Mutação 249Ser no TP53 no Carcinoma Hepatocelular (CHC), frequente em países da África e Ásia, é uma evidência molecular de exposição à aflatoxina. O objetivo deste estudo é analisar a freqüência de 249Ser em 74 amostras de CHC no Brasil. A mutação foi analisada por RFLP e sequenciamento. A presença de vírus da hepatite B (VHB) foi analisada por PCR em tempo real. 249Ser foi encontrada em 13/74 (28%) e VHB em 13/74 (16%). A mutação foi encontrada em maior freqüência em tumores indiferenciados (OR = 2,415, IC = 1,001 - 5,824). O tamanho médio de tumores com 249Ser foi de 9,4 cm contra 5,5 cm de amostras sem a mutação (p=0,012). Não foi encontrada relação entre VHB e 249Ser. Os resultados indicam que 249Ser é um fator importante na carcinogênese do CHC no Brasil sendo associada à uma forma maior e menos diferenciada de tumor. / TP53 249Ser mutation has been proved a molecular evidence for aflatoxin-related Hepatocellular Carcinoma (HCC) and is frequent in Africa and Asia. The aim of our study was to analyze the frequency of 249Ser mutation in HCC from Brazil. We studied 74 samples of paraffin embedded HCC. 249Ser mutation was analyzed by RFLP and sequencing. Presence of HBV DNA was determined by Real-Time PCR. 249Ser was found in 21/74 (28%) samples while HBV DNA was found in 13/74 (16%). Poorly differentiated HCC was more likely to have 249Ser mutation (OR = 2.415, IC = 1.001 - 5.824). The mean tumor size of 249Ser HCC was 9.4 cm versus 5.5cm on wild type (p=0.012). HBV DNA was not related with 249Ser. Results indicate that 249Ser is an important factor of HCC carcinogenesis in Brazil and is associated with large and poorly differentiated tumors.

Introdução: A injeção percutânea de etanol (PEI) é um método de ablação local considerado curativo para carcinomas hepatocelulares (CHC) pequenos. Não há dados sobre PEI para tratamento do CHC na América Latina. Pacientes e métodos: Incluídos 100 pacientes cirróticos submetidos à PEI como terapia primária para o CHC no Serviço de Gastroenterologia do HCFMUSP entre setembro de 1997 e dezembro de 2005. Avaliados resposta ao tratamento e taxa de sobrevida. Resultados: Resposta completa ao tratamento ocorreu em 41% dos casos. A sobrevida foi de 83% em 1 ano, 49% em 3 anos e 29% em 5 anos, mas no grupo com tumores de até 2 cm, sem invasão vascular e com resposta completa ao tratamento, atingiu 89%, 70% e 70%, respectivamente. Conclusão: PEI apresentou boa sobrevida em pacientes com tumores 2 cm, sem invasão vascular e resposta completa ao tratamento. / Introduction: Percutaneous ethanol injection (PEI) is a curative local ablative method for treatment of small hepatocellular carcinoma (HCC). There is no data about PEI for HCC treatment in Latin America. Patients and Methods: A total of 100 consecutive cirrhotic patients were enrolled. All of them had been submitted to PEI as first-line therapy for the treatment of HCC at the Service of Gastroenterology of HCFMUSP in the period of September 1997 to December 2005. Response to treatment and survival rates were assessed. Results: Complete response to treatment was obtained in 41% of the patients. Survival rates were 83% in 1 year, 49% in 3 years and 29% in 5 years, but in patients with tumors up to 2 cm, no vascular invasion and complete response to treatment, were 89%, 70% and 70%, respectively. Conclusion: PEI is associated with long-term survival in patients with tumors 2 cm, absence of vascular invasion and complete response to treatment

Avec près de 350 millions de personnes chroniquement infectées, et malgré l’existence de vaccins efficaces, le virus de l’Hépatite B (VHB) reste un problème majeur de santé publique. Parmi les protéines virales, la protéine régulatrice HBx possèdent des activités qui pourraient être particulièrement impliquées dans le développement de CHC. Au cours de ce travail, nous nous sommes intéressés aux différences biologiques entre la protéine HBx issue d’une région non tumorale (HBx-NT) et la protéine HBx issue d’une région tumorale (HBx-T) d’un même patient. En particulier, nous nous sommes intéressés à la régénération hépatique après hépatectomie partielle et à la carcinogenèse hépatique dans un modèle murin transgénique. Nous avons démontré l’absence d’impact de la forme tronquée de la protéine HBx sur la régénération hépatique. Nous avons démontré que la protéine HBx entière avait la capacité d’activer la sécrétion d’IL-6 dans la phase d’initiation de la régénération hépatique, conduisant à l’hyperactivation de STAT3, l’accumulation de SOCS3 et la diminution de phosphorylation de ERK. Au final, la protéine HBx entière induit un retard de régénération hépatique. Nous avons démontré une cinétique d’apparition de tumeurs plus rapide chez les souris HBx-T que chez les souris HBx-NT après injection d’un carcinogène chimique. Nous avons aussi pu observer que les deux formes HBx-T et HBx-NT sensibilisaient les hépatocytes à l’apoptose, au cours d’un dommage hépatique aigue, et que cette sensibilisation à l’apoptose pouvait en partie rendre compte de l’effet co-carcinogène observé chez les souris HBx-T et HBx-NT. L’ensemble de mes résultats a permis de mieux comprendre les mécanismes par lesquels la protéine HBx participe au développement de CHC. / Hepatitis B virus (HBV) is a worldwide health issue, as it is estimated that 350 millions people are chronically infected. Among the viral proteins, HBx is thought to be involved in hepatocellular carcinoma (HCC) development. In this work, we were interested in biological differences between HBx sequence from non tumoral region (HBx-NT) compared to HBx from tumoral region (HBx-T) from a single patient. In particular, we studied liver regeneration after partial hepatectomy et hepatocarcinogenesis in a transgenic mice model. We demonstrated that HBx-T did not modulate liver regenereation. We also showed that HBx-NT induced IL-6 overexpression during priming phase of liver regeneration, and that IL-6 overexpression was involved in STAT3 hyperactivation, SOCS3 accumulation and inhibition of ERK. Overall, HBx-NT induced IL-6 overexpression was responsible for a delay in liver regeneration. Moreover, we showed that HBx-T induced a faster development of hepatic tumor after DEN initiation, compared to HBx-NT. Both HBx forms were involved in an apoptosis sensibilization during acute liver injury, that could be involved in co-carcinogenic effect of HBx-T and HBx-NT. Overall, my results participate to the comprehension of HBx impact on liver carcinogenesis

Hepatitis B is one of the most common infectious diseases, which leads to public health problems in the world, especially in Asian counties. In recent years, extensive human genetic association studies have been carried out to identify susceptible genes and genetic polymorphisms to understand the genetic contributions to the disease progression of HBV infection. HLA-DQ gene variations have been reported to be associated with HBV infection/clearance, disease progression and the development of hepatitis B-related complications, including liver cirrhosis (LC) and hepatocellular carcinoma (HCC). However, the results are either inconclusive or controversial. Therefore, to derive a more precise estimation of the association, a meta-analysis was performed. Our data revealed that the HLA-DQ alleles rs2856718-G, rs7453920-A and rs9275319-G were significantly associated with decreased risk of HBV infection and HBV natural clearance. Logistic regression analyses showed that HLA-DQ alleles rs9275572-A significantly increased HBV infection clearance, and decreased HBV natural clearance. However, rs2856718-G and rs9275572-A were not associated with development of cirrhosis. The HLA-DQ polymorphisms (rs2856718 and rs9275572) were associated with a decreased HBV-related HCC risk in all genetic models, but rs9272105-A increased the risk of HBV-related HCC. In addition, no significant association was observed between HLA-DQ rs9275319-G polymorphism and HBVrelated HCC. These stratified analyses were limited due to relatively modest size of correlational studies. In future, further investigation on a large population and different ethnicities are warranted. Our findings contribute to the personalized care and prognosis in hepatitis B.

Le virus de l'hépatite C (HCV) est responsable de maladies chroniques du foie et l'une des principales causes de développement du carcinome hépatocellulaire (HCC). Cependant, les mécanismes moléculaires qui permettent le développement d’un HCC suite à une infection chronique par le HCV restent incompris. Les microARN (miR), de petits ARNs non codants qui régulent l'expression des gènes au niveau post-transcriptionnel, sont connus pour jouer un rôle important dans l'homéostasie cellulaire du foie. De plus en plus d’études suggèrent que l'infection par le HCV induit la modification de réseaux intracellulaires impliquant les miRs hépatiques contribuant au développement des lésions du foie, y compris le HCC. En utilisant des techniques d'analyse systématiques, nous avons identifié des miRs qui modulent le cycle viral du HCV mais également des miRs modulés lors de l'infection par le HCV. Cette analyse globale des interactions entre les miRs de l'hôte et le HCV améliore les connaissances actuelles sur les interactions entre le HCV et l’hôte qui contribuent vraisemblablement à la tumorigenèse dans le foie, et ouvre des perspectives pour de potentielles nouvelles approches pour prévenir et/ou traiter le HCC chez les patients infectés par le HCV. / Hepatitis C virus (HCV)-induced chronic liver disease is one of the leading causes of hepatocellular carcinoma (HCC). However, the molecular mechanisms that enable HCC development following chronic HCV infection remain poorly understood. MicroRNAs (miRs), small non coding RNAs that regulate gene expression at a post-transcriptional level have been reported to play an important role in cellular homeostasis within the liver. Increasing evidence suggests that HCV infection induces alteration of intrahepatic miR networks and that deregulation of miRs contributes to liver disease including HCC. Using high-throughput screening and RNA sequencing, we identified miRs that modulate the HCV life cycle and miRs that are modulated upon HCV infection. This comprehensive analysis of the HCV-host miR network improves the current knowledge of the HCV-host interactions that likely contribute to tumorigenesis in the liver and opens perspectives for novel potential approaches to prevent and/or treat HCC in HCV-infected patients.

La prévalence de l’obésité et du diabète de type 2 est en constante augmentation dans les pays industrialisés. Ces pathologies sont associées à des troubles de l’équilibre énergétique de l’organisme, ce qui se traduit au niveau hépatique par le développement de NAFLD (Non‐alcoholic Fatty Liver Disease). La NAFLD est engendrée par l’accumulation excessive de lipides dans les hépatocytes ou stéatose et est associée à la résistance à l’insuline. L’insulinorésistance hépatique est dite sélective car l’insuline n’est plus capable d’inhiber la production hépatique de glucose, contribuant à l’hyperglycémie, alors que la synthèse lipidique est exacerbée, induisant une stéatose hépatique. La stéatose peut évoluer en pathologies plus graves, telles que la fibrose, la cirrhose ou le cancer hépatocellulaire (CHC). L’objectif de ma thèse a été d’étudier les mécanismes moléculaires pouvant contribuer à la sélectivité de la résistance à l’insuline hépatique et à l’évolution de la stéatose simple vers le CHC. Dans un premier temps, nous avons étudié le rôle de l’adaptateur moléculaire Grb14, un inhibiteur de l’activité kinase du récepteur de l’insuline, dans la régulation du métabolisme hépatique chez la souris. L’invalidation de Grb14 dans le foie conduit d’une part à une activation de la signalisation de l’insuline et une amélioration de tolérance au glucose, et d’autre part à une diminution de la lipogenèse consécutive à l’inhibition du facteur lipogénique LXR via la voie Nrf2. L’invalidation de Grb14 dans le foie de souris obèses et diabétiques permet de ramener la glycémie et la stéatose hépatique à des valeurs similaires aux témoins. Ces données suggèrent que Grb14 est un nouvel acteur impliqué dans la sélectivité de la résistance à l’insuline du foie. La seconde étude a permis de montrer l’implication de la stéatose hépatique induite par un régime hypercalorique sur le développement de CHC. En effet, sur un fond génétique favorisant la carcinogenèse, un régime riche en graisse et en sucre contribuant à l’insulinorésistance hépatique accélère la cinétique d’apparition des tumeurs et augmente leur nombre. / The prevalence of metabolic diseases, including obesity and type 2 diabetes, are expanding in a worldwide epidemic way. These diseases are associated with metabolic disorders, resulting in the development of NAFLD (Non‐alcoholic Fatty Liver Disease) in the liver. NAFLD is generated by excessive accumulation of lipids in hepatocytes, and is associated with insulin resistance. In liver, insulin resistance leads to a blunted inhibitory action on hepatic glucose production, inducing hyperglycemia, whereas de novo lipogenesis, which is positively regulated by insulin, is paradoxically exacerbated, contributing to hepatic steatosis. Steatosis may also evolve into more serious diseases such as fibrosis, cirrhosis and hepatocellular carcinoma (HCC). The aim of my thesis was to study the molecular mechanisms that may contribute to the selectivity of hepatic insulin resistance and the development of HCC. Initially, we studied the role of the molecular adapter Grb14, an inhibitor of the insulin receptor kinase activity, in the regulation of hepatic metabolism in mice. Invalidation of Grb14 in the liver leads on the one hand to an activation of the insulin signaling and improved glucose tolerance, and on the other hand to a decrease of LXR activity resulting in lipogenesis inhibition. Invalidation of Grb14 in the liver of obese and diabetic mice restores blood glucose levels and hepatic steatosis similar to control values. These data suggest that Grb14 is a new player involved in the selectivity of the insulin resistance in the liver. The second study demonstrated the involvement of hepatic steatosis induced by a high‐calorie diet on the development of HCC. Indeed, on a genetic background favoring carcinogenesis, a diet rich in fat and sugar contributing to hepatic insulin resistance accelerates appearance of tumors and increases their number.

Philosophiae Doctor - PhD / To ameliorate Hepatitis C Virus (HCV) therapeutic and diagnostic challenges requires robust intervention strategies, including approaches that leverage the plethora of rich data published in biomedical literature to gain greater understanding of HCV pathobiological mechanisms. The multitudes of metadata originating from HCV clinical trials as well as low and high-throughput experiments embedded in text corpora can be mined as data sources for the implementation of HCV-specific resources. HCV-customized resources may support the generation of worthy and testable hypothesis and reveal potential research clues to augment the pursuit of efficient diagnostic biomarkers and therapeutic targets. This research thesis report the development of two freely available HCV-specific web-based resources: (i) Dragon Exploratory System on Hepatitis C Virus (DESHCV) accessible via http://apps.sanbi.ac.za/DESHCV/ or http://cbrc.kaust.edu.sa/deshcv/ and (ii) Hepatitis C Virus Protein Interaction Database (HCVpro) accessible via http://apps.sanbi.ac.za/hcvpro/ or http://cbrc.kaust.edu.sa/hcvpro/. DESHCV is a text mining system implemented using named concept recognition and cooccurrence based approaches to computationally analyze about 32, 000 HCV related abstracts obtained from PubMed. As part of DESHCV development, the pre-constructed dictionaries of the Dragon Exploratory System (DES) were enriched with HCV biomedical concepts, including HCV proteins, name variants and symbols to enable HCV knowledge specific exploration. The DESHCV query inputs consist of user-defined keywords, phrases and concepts. DESHCV is therefore an information extraction tool that enables users to computationally generate association between concepts and support the prediction of potential hypothesis with diagnostic and therapeutic relevance. Additionally, users can retrieve a list of abstracts containing tagged concepts that can be used to overcome the herculean task of manual biocuration. DESHCV has been used to simulate previously reported thalidomide-chronic hepatitis C hypothesis and also to model a potentially novel thalidomide-amantadine hypothesis. HCVpro is a relational knowledgebase dedicated to housing experimentally detected HCV-HCV and HCV-human protein interaction information obtained from other databases and curated from biomedical journal articles. Additionally, the database contains consolidated biological information consisting of hepatocellular carcinoma (HCC) related genes, comprehensive reviews on HCV biology and drug development, functional genomics and molecular biology data, and cross-referenced links to canonical pathways and other essential biomedical databases. Users can retrieve enriched information including interaction metadata from HCVpro by using protein identifiers, gene chromosomal locations, experiment types used in detecting the interactions, PubMed IDs of journal articles reporting the interactions, annotated protein interaction IDs from external databases, and via “string searches”. The utility of HCVpro has been demonstrated by harnessing integrated data to suggest putative baseline clues that seem to support current diagnostic exploratory efforts directed towards vimentin. Furthermore, eight genes comprising of ACLY, AZGP1, DDX3X, FGG, H19, SIAH1, SERPING1 and THBS1 have been recommended for possible investigation to evaluate their diagnostic potential. The data archived in HCVpro can be utilized to support protein-protein interaction network-based candidate HCC gene prioritization for possible validation by experimental biologists. / South Africa