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A conexina 32 (Cx32) é uma estrutura protéica que constitui os canais que promovem as comunicações intercelulares via junções comunicantes (GJIC), permitindo difusão de pequenas moléculas citoplasmáticas de uma célula à outra. Este trabalho objetivou os estudos destas estruturas devido a sua importância em processos hepáticos, mais especificamente, a fibrose hepática. O presente estudo foi realizado através da administração oral da droga hepatotoxica dimetilnitrosamina (DMN) em ratas Wistar duas vezes por semana em dias consecutivos no prazo de cinco semanas. A necropsia destes animais foi realizada após cinco semanas da última administração da droga e revelou um quadro de fibrose hepática, em contra partida aos resultados obtidos em um grupo controle com a mesma quantidade de animais. O material fibrótico foi submetido à análise imunohistoquímica que revelou uma presença preferencial de Cx32 dispersa no citoplasma, o que pode levar à hipótese de problemas no mecanismo de transporte citoplasmático destas estruturas, em contrapartida ao material pertencente ao grupo controle que evidenciou a presença das Cx32 na membrana plasmática formando placas juncionais. Quando submetido à análises moleculares o fígado fibrótico revelou uma diminuição da expressão gênica embora o produto protéico deste material quando comparado ao grupo controle não tenha se mostrado diminuído. / The connexin 32 (Cx32) is a proteic structure that constitute the channels that promote the cell communication by means of the gap junction (GJIC), allowing the diffusion of short cytoplasmic molecules from a cell to another. This work aimed to study these structures due to their importance in the hepatic metabolic processes. The hepatic fibrosis was triggered by the oral administration of dimethylnitrosamine (DMN) in the female rat Wistars twice a week in consecutive days during five weeks. The necropsy of these animals was carried out after the last drug administration. They presented a hepatic fibrosis state. The fibrotic material was submitted to the imunohistochemical analysis, which showed a preferencial presence of Cx32 in the cytoplasm, whereas in the control group the Cx32 was located at the membranes, in the junctional plaques. The molecular analysis showed a decrease of the genic expresson of the fibrotic material, however the proteic product wasn? t reduced in comparison with the control group as it was shown by western blot. We concluded that the fibrotic state introduced a disturbance in the intracellular distribution and genic expression of the connexin 32.

A conexina 32 (Cx32) é uma estrutura protéica que constitui os canais que promovem as comunicações intercelulares via junções comunicantes (GJIC), permitindo difusão de pequenas moléculas citoplasmáticas de uma célula à outra. Este trabalho objetivou os estudos destas estruturas devido a sua importância em processos hepáticos, mais especificamente, a fibrose hepática. O presente estudo foi realizado através da administração oral da droga hepatotoxica dimetilnitrosamina (DMN) em ratas Wistar duas vezes por semana em dias consecutivos no prazo de cinco semanas. A necropsia destes animais foi realizada após cinco semanas da última administração da droga e revelou um quadro de fibrose hepática, em contra partida aos resultados obtidos em um grupo controle com a mesma quantidade de animais. O material fibrótico foi submetido à análise imunohistoquímica que revelou uma presença preferencial de Cx32 dispersa no citoplasma, o que pode levar à hipótese de problemas no mecanismo de transporte citoplasmático destas estruturas, em contrapartida ao material pertencente ao grupo controle que evidenciou a presença das Cx32 na membrana plasmática formando placas juncionais. Quando submetido à análises moleculares o fígado fibrótico revelou uma diminuição da expressão gênica embora o produto protéico deste material quando comparado ao grupo controle não tenha se mostrado diminuído. / The connexin 32 (Cx32) is a proteic structure that constitute the channels that promote the cell communication by means of the gap junction (GJIC), allowing the diffusion of short cytoplasmic molecules from a cell to another. This work aimed to study these structures due to their importance in the hepatic metabolic processes. The hepatic fibrosis was triggered by the oral administration of dimethylnitrosamine (DMN) in the female rat Wistars twice a week in consecutive days during five weeks. The necropsy of these animals was carried out after the last drug administration. They presented a hepatic fibrosis state. The fibrotic material was submitted to the imunohistochemical analysis, which showed a preferencial presence of Cx32 in the cytoplasm, whereas in the control group the Cx32 was located at the membranes, in the junctional plaques. The molecular analysis showed a decrease of the genic expresson of the fibrotic material, however the proteic product wasn? t reduced in comparison with the control group as it was shown by western blot. We concluded that the fibrotic state introduced a disturbance in the intracellular distribution and genic expression of the connexin 32.

Orientador: Rejane Maria Tommasini Grotto / Resumo: A progressão da fibrose hepática somada à infecção pelo vírus da hepatite C (VHC) tem sido associada à resposta imunológica permanente. O estudo no repertório de anticorpos Anti-VHC na progressão da fibrose hepática foi explorado através de ferramentas de sequenciamento em larga escala (NGS) possibilitando uma análise de repertórios altamente variáveis como as porções variáveis VH (cadeia pesada) e Vk (cadeia leve) das imunoglobulinas, e a determinação de famílias e subfamílias dos genes V-D-J associadas à resposta humoral encontrada nas diferentes fases da doença proporcionam uma ferramenta importante no entendimento da resposta imune frente à infecção viral pelo VHC. As porções VH e Vk das imunoglobulinas foram obtidas a partir da amplificação de RNA de sangue de paciente VHC positivos e com diferentes graus de fibrose, e sequenciadas na plataforma Illumina® Miseq, fornecendo uma grande variabilidade de sequências que foram pré-processadas por ferramentas de bioinformática e analisadas em dois bancos de anticorpos diferentes: IgBlast (NCBI) e IMGT® quanto às famílias e subfamílias mais expressas. A expressão restrita de algumas famílias e subfamílias: IGHV1, IGHV3, IGHV4 e subfamílias já descritas em vários estudos associados ao VHC corrobora com nossos achados de que existe uma tendência do uso de algumas subfamílias como: IGHV1-2, IGHV1-8, IGVH1-69, IGHV3-11, IGHV3-21, IGHV3-23, IGVH3-30, IGHV4-4, IGHV4-34 IGHV4-39 na cadeia pesada; assim como IGkV3-15 e IGkV3-20 na cadei... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The progression of hepatic fibrosis in the hepatitis C virus (HCV) infection has been linked with permanent immune response. The study in the repertoire of Anti-HCV antibodies in the progression of hepatic fibrosis was analysed for large-scale sequencing (NGS) tools enabling a highly variable analysis such as the VH (heavy chain) and Vk (light chain) portions of immunoglobulins and the determination of families and subfamilies of the V-D-J genes in the humoral response found in the different phases of the disease, a great tool in the understanding of the immune response to HCV viral infection. The VH and V portions of the immunoglobulins were obtained from the amplification of HCV positive HCV patient blood with different degrees of fibrosis and sequenced on the Illumina® Miseq platform, providing a large sequence variability that was preprocessed by tools of bioinformatics and analyzed in two different antibody banks: IgBlast (NCBI) and IMGT® for the most expressed families and subfamilies. The restricted expression of some families: IGHV1, IGHV3, IGHV4 and subfamilies already described in several HCV-related studies confirm our findings that there is a tendency to use some subfamilies, such as: IGHV1-2, IGHV1-8, IGVH1- 69, IGHV3-11, IGHV3-21, IGHV3-23, IGVH3-30, IGHV4-4, IGHV4-34 IGHV4-39 in the heavy chain; as well as IGkV3-15 and IGkV3-20 in the light chain, but the subfamilies: IGHV1-8, IGHV3-11, IGHV4-39, IGkV1-5, IGkV1-12, IGkV1-39 were also among the most expressed, i... (Complete abstract click electronic access below) / Mestre

Obesity and insulin resistance are associated with a constellation of features including hypertension, dyslipidaemia, type 2 diabetes, and premature cardiovascular disease, collectively termed the metabolic syndrome. Non-alcoholic fatty liver disease (NAFLD) represents the hepatic component of this syndrome, incorporating a spectrum of liver disease with increasing morbidity and mortality, from simple steatosis, to non-alcoholic steatohepatitis (or NASH), fibrosis, cirrhosis and ultimately hepatocellular carcinoma. However, factors influencing this progression are incompletely understood. In this thesis I sought to investigate pathways which promote hepatic inflammation and fibrosis by studying two contrasting dietary models of NAFLD in mice in which the risk of hepatic inflammation, insulin resistance and fibrosis differ; namely the methionine and choline deficient diet (MCDD) which induces steatohepatitis, hepatic insulin resistance, and weight loss, and the choline deficient diet (CDD) which may be protected from insulin resistance, and leads to steatosis without inflammation or weight loss. I investigated the possible molecular mechanisms underlying these differences, and whether they influenced progression to hepatic fibrosis induced by carbon tetrachloride (CCl4).

Chronic liver disease is a worldwide health problem, and hepatic fibrosis (HF) is one of the hallmarks of the disease. Pathology diagnosis of HF is based on textural change in the liver as a lobular collagen network that develops within portal triads. The scale of collagen lobules is characteristically on order of 1mm, which close to the resolution limit of in vivo Gd-enhanced MRI. In this work the methods to collect training and testing images for a Hotelling observer are covered. An observer based on local texture analysis is trained and tested using wet-tissue phantoms. The technique is used to optimize the MRI sequence based on task performance. The final method developed is a two stage model observer to classify fibrotic and healthy tissue in both phantoms and in vivo MRI images. The first stage observer tests for the presence of local texture. Test statistics from the first observer are used to train the second stage observer to globally sample the local observer results. A decision of the disease class is made for an entire MRI image slice using test statistics collected from the second observer. The techniques are tested on wet-tissue phantoms and in vivo clinical patient data.

La bilharziose reste un problème de santé majeur. L'équipe du Pr Dessein a montré que les infections élevées étaient déterminées par un locus majeur en 5q31 et que des polymorphismes dans un gène à ce locus,IL13, aggravent l'infection. Notre premier objectif était d'évaluer si des variants d'autres gènes de la voie de l'IL13 intervenaient dans le contrôle de l'infection. Nous avons observé une association entre le SNP rs324013, dans le promoteur de STAT6,et les niveaux d'infection à S. haematobium. Ce polymorphisme a un effet additif avec le polymorphisme IL13rs1800925. Ce SN modifie la fixation de facteurs nucléaires au niveau du promoteur de STAT6. L'équipe du Pr Dessein avait également montré que les fibres hépatiques avancées et sévères étaient déterminées par un autre locus majeur localisé en 6q23. Notre deuxième objectif fut d'évaluer dans le laboratoire du Pr Dessein et en étroite collaboration avec le laboratoire du Pr Li(Yueyang Institute of Parasitic disease)deux gènes candidats(IFNGR1 et CTGF) situés dans cette région chromosomique. Nous avons observé une association entre les deux polyporphismes(rs17066192 er rs673156)localisés dans le promoteur du gène. Nous avons observé une association entre les deux polymorphismes(rs17066192 et rs673156)localisés dans le promoteur du gène IFNGR1 et la fibrose hépatique: le génotype rs673156A/A et rs17066192C/C sont associés à un risque 7.3 fois et 1.5 fois plus élevé, respectivement, de fibrose avancée. Nous avons également montré que les variants rs9402373 et rs12526196 du gène CTGF sont indépendamment associés à la fibrose chez les fermiers et pêcheurs chinois infectés par S.japonicum. Sur la population chinoise d'étude, les risques relatifs associés aux polymorphismes rs9402373 et rs12526196 sont de 2.8 et 3 / Schistosomiasis remains one of the world’s most prevalent diseases. It comprises a group of chronic diseases caused by helminths of the Schistosoma genus. Schistosoma haematobium causes obstructive nephropathy that can be aggravated by urinary bacterial infections. S.japonicum and S.mansoni cause hepatic fibrosis associated with portal blood hypertension, which can be lethal. In previous studies, our laboratory had shown that worm burden in S.haematobium infections were aggravated by IL13 variants and that severe hepatic fibrosis (HF) was controlled by gene(s) located on 6q23. The present study is to further evaluate other IL-13 pathway genes (STAT6) in the control of infection in Malian farmers and to test candidate genes in the 6q23 region in hepatic fibrosis (HF) in S.japonicum infected Chinese fishermen and farmers. First we have developped an improved FTA® technology technique to perform SNP genotyping. This technique allows us to use saliva samples for genotyping SNPs. Subsequently, this improved FTA® technology was used in our study on HF.Our work on a Malian sample infected with S. haematobium indicated that a polymorphism (rs324013) in the promoter of STAT6 gene was associated with the control of S. haematobium infection levels and has an additive effect with IL13rs1800925, a polymorphism previously associated with infection in this same population. Both SNPs modify the binding of nuclear factors to the promoter regions of their respective genes. Thus, both SNPs may play a crucial role in controlling S. haematobium infection levels. In order to study HF in S.japonicum infections, we have participated actively in the study that recruited of a large sample of Chinese fishermen and farmers who had been exposed to the infection for most of their life. HF was evaluated by ultrasound and covariates that could affect HF were evaluated by interviews. Then, we tested two genes (IFNGR1, CTGF) of the 6q23 region that were good candidates for the control of HF on these samples. Both genes encode molecules that were shown in animal and human studies to have strong effect on extracellular matrix proteins deposition and turnover. We found that two polymorphisms (rs17066192 and rs673156) in IFNGR1 promoter were associated with HF: the rs673156A/A genotype was associated with a 7.3-fold increased risk of advanced HF; and rs17066192C/C genotype with a 1.5-fold increased risk of HF. These results must now be confirmed in another population sample. We also found that variants of CTGF rs9402373 and rs12526196 were independently associated with HF in Chinese fishermen and farmers, in Sudanese, and in Brazilians infected with either S. japonicum or S. mansoni. Our results provide additional evidence for a protective role of IL-13 in schistosome infections, and they also demonstrate that TGFβ / CTGF pathway plays a key role in HF and should be targeted by chemotherapy. Ongoing studies evaluate whether CTGF variants could be used in the prognosis of the HF caused by schistosomes and also by other infectious agents.

Chronic liver disease is a worldwide health problem, and hepatic fibrosis (HF) is one of the hallmarks of the disease. The current reference standard for diagnosing HF is biopsy followed by pathologist examination; however, this is limited by sampling error and carries a risk of complications. Pathology diagnosis of HF is based on textural change in the liver as a lobular collagen network that develops within portal triads. The scale of collagen lobules is characteristically in the order of 1 to 5 mm, which approximates the resolution limit of in vivo gadolinium-enhanced magnetic resonance imaging in the delayed phase. We use MRI of formalin-fixed human ex vivo liver samples as phantoms that mimic the textural contrast of in vivo Gd-MRI. We have developed a local texture analysis that is applied to phantom images, and the results are used to train model observers to detect HF. The performance of the observer is assessed with the area-under-the-receiver-operator-characteristic curve (AUROC) as the figure-of-merit. To optimize the MRI pulse sequence, phantoms were scanned with multiple times at a range of flip angles. The flip angle that was associated with the highest AUROC was chosen as optimal for the task of detecting HF. (C) The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

Les bilharzioses hépatospléniques sont des maladies parasitaires provoquées essentiellement par Schistosoma japonicum et Schistosoma mansoni. Ces infections provoquent une fibrose hépatique sévère chez 5 à 20% des sujets infectées vivant en zone endémique. Plusieurs études ont démontré que le développement de cette fibrose était régulé par des cytokines et chemokines et que la susceptibilité génétique à cette maladie dépendait chez l'homme d'un locus majeur sur le chromosome humain 6 en position q23. L'IL-22 est une cytokine produite essentiellement par les lymphocytes T auxiliaires et les cellules NK et qui a été impliqué dans la protection du foie et de l'intestin chez la souris. Le gène IL22RA2 codant pour le récepteur inhibiteur soluble de l'IL-22 (IL-22BP) est localisé dans la région de susceptibilité à la FH (6q23). Nous avons donc étudié l'implication de l'IL-22 dans la pathologie bilharzienne par une approche immunogénétique. Notre étude démontre que la production d'IL-22 est augmentée en réponse aux œufs de S. japonicum et aux antigènes d'œufs de S. mansoni dans des cultures de cellules mononuclées du sang périphérique d'individus chinois et brésiliens vivant dans des zones endémiques. Le traitement par le Praziquantel, qui favorise l'élimination des parasites et la réversion de la fibrose, est associé à une augmentation des taux d'IL-22 en culture. Nous avons observé que dans le sang des patients chinois, l'IL-22 est produit majoritairement par les lymphocytes T CD4+ et des cellules CD3-CD4- ne produisant pas d'IL-17A. Les taux d'IL-22 en culture et la proportion des cellules CD3-CD4-IL22+ sont inversement corrélés à la FH. / Hepatosplenic schistosomiasis is a parasitic disease caused primarily by Schistosoma japonicum and Schistosoma mansoni. These infections cause severe hepatic fibrosis (HF) in 5-20% of infected subjects living in endemic areas. Several studies have shown that the development of this fibrosis was regulated by cytokines and chemokines. Our laboratory has shown that genetic susceptibility to HF map to a major locus on human chromosome 6 at position q23. IL-22 is a cytokine produced primarily by T cells and NK cells and has been involved in protecting the liver and intestine in mice. The gene IL22RA2, encoding the soluble inhibitor receptor of IL-22 (IL-22BP), is located in the region of susceptibility to HF (6q23). We therefore examined the involvement of IL-22 in schistosomiasis pathology by an immunogenetic approach. Our study shows that the production of IL-22 is increased in response to eggs of S. japonicum and egg antigens of S. mansoni in cultures of peripheral blood mononuclear cells from Chinese and Brazilian subjects living in endemic areas. Treatment with praziquantel, which helps eliminate parasites and reversion of HF, is associated with increased levels of IL-22 in culture. We observed that in the blood of Chinese patients, IL-22 is produced mainly by CD4+ T cells and CD3-CD4-cells that do not produce IL-17A. The levels of IL-22 in culture and the proportion of CD3+CD4-IL22+ are inversely correlated with HF. These observations suggest that IL-22 may play a protective role in HF. To confirm this implication, we performed association studies between SNPs located in IL22 and IL22RA2 genes and HF.