A cirrose hepática é uma doença crônica e irreversível com modificações histológicas caracterizadas por nódulos de regeneração e desarranjo de hepatócitos, com aumento difuso de tecido conjuntivo e perda de função. As células-tronco têm sido amplamente estudadas para o tratamento de injúrias hepáticas. Fibrose e cirrose foram induzidas em ratos Wistar, através da aplicação de tioacetamida (TAA) intraperitoneal a 200 mg/kg por 8 ou 14 semanas, seguido de terapia celular com aplicação intravenosa única de 1x106 células-tronco de membrana amniótica de ratas. Amostras de sangue foram coletadas para análises bioquímicas, e tecido hepático para histologia e imunohistoquímica. A histopatologia para colorações de hematoxilina-eosina para graduação e estadiamento da lesão hepática foi realizada, com observação de alterações celulares; picrosírius para quantificação do colágeno parenquimal; ácido periódico-Schiff para mucopolissacarídeos neutros e glicogênio; e alcian blue para mucopolissacarídeos ácidos. Imunohistoquímica foi realizada para marcação de PCNA, colágenos tipo 1 e 3, e α-SMA. O cultivo celular apresentou ótimo crescimento, sendo transduzido com GFP e negativo para micoplasma. Os animais induzidos experimentalmente mostraram baixo ganho de peso durante o experimento, com notável recuperação nas duas primeiras semanas após término da indução. A mortalidade experimental foi de 10 %. A indução da fibrose e cirrose hepática obtiveram sucesso no período de 8 e 14 semanas, respectivamente Análises macroscópicas mostraram melhor aspecto de fígados cirróticos após terapia celular. Não houve diferenças estatísticas após a terapia celular para as análises bioquímicas, graduação, estadiamento, alterações celulares, proliferação de ductos, mucopolissacarídeos ácidos e neutros e imunohistoquímicas para colágeno 1 e 3, PCNA e α- SMA. Houve, porém, uma tendência a melhora na graduação e estadiamento da fibrose e cirrose com redução da atividade necroinflamatória; redução na proliferação de ductos; menor deposição de mucopolissacarídeos ácidos e neutros; diminuição das alterações celulares com indicativos de menor grau de lesão tecidual e maior grau de regeneração; diminuição da expressão de α-SMA e colágenos 1 e 3; e aumento do PCNA. O colágeno intralobular teve diminuição após terapia celular em fígados fibróticos. Em animais induzidos a cirrose, houve diminuição de ambos colágenos interlobular e intralobular. Conclui-se que houve uma tendência de melhora dos animais após tratamento com células-tronco da membrana amniótica de ratas, o que nos encoraja a aplicação de protocolos de terapia celular. Apesar dos resultados promissores, um maior número de animais precisa ser testado para que se tenham mais dados, que possam dar subsídios para o uso destas células na prática clínica para o tratamento da cirrose hepática / Liver cirrhosis is a chronic and irreversible disease with histological changes characterized by regenerative nodules and disarray of hepatocytes with diffuse increase in connective tissue and loss of function. Stem cells have been extensively studied for the treatment of liver injury. Fibrosis and cirrhosis was induced in Wistar rats by application of thioacetamide (TAA) intraperitoneally at 200 mg/kg for 8 or 14 weeks, followed by cell therapy with a single intravenous administration of 1x106 stem cells from amniotic membrane of rats. Blood samples were collected for biochemical analyzes, and liver tissue for histology and immunohistochemistry. Histopathology by hematoxylin-eosin for grading and staging of hepatic injury with observation of cellular changes was performed; picrosirius for quantification of parenchymal collagen; periodic acid-Schiff for neutral mucopolysaccharides and glycogen; and alcian blue for acid mucopolysaccharides. Immunohistochemistry was performed for PCNA, type 1 and 3 collagens, and α-SMA. Cell culture showed optimal growth, being transduced with GFP and negative for mycoplasma. Animals induced to liver disease showed low weight gain during the experiment, with remarkable recovery in the first two weeks after completion of induction. Experimental mortality was 10%. Induction of fibrosis and cirrhosis was successful after the period of 8 and 14 weeks, respectively. Macroscopic analysis showed improvement in the aspect of cirrhotic livers after cell therapy. There were no statistical differences after cell therapy for biochemical analyzes, grading, staging, cellular changes, proliferation of ducts, acidic and neutral mucopolysaccharides and immunohistochemistry for collagen 1 and 3, PCNA and α-SMA. There was, however, a tendency on improvement in grading and staging of fibrosis and cirrhosis with reduced necroinflammatory activity; reduction in the proliferation of ducts; lower deposition of acidic and neutral mucopolysaccharides; decrease in cellular changes indicating a lower degree of tissue damage and a greater degree of regeneration; decreasing on the expression of α-SMA and type 1 and 3 collagens; and increased PCNA. Intralobular collagen decreased after cell therapy in fibrotic livers. In cirrhotic animals, there was a decrease of both interlobular and intralobular collagen. We conclude that was a trend to improvement of animals after treatment with stem cells derived from amniotic membrane of rats, which encourages the application of cell therapy protocols. Despite the promising results, a larger number of animals must be tested, so we can have more data to make allowances for the use of these cells in clinical practice for the treatment of liver cirrhosis

A infecção crônica pelo vírus da hepatite C (HCV) é importante fator de risco para o desenvolvimento de cirrose hepática e de carcinoma hepatocelular. A evolução para formas mais graves está relacionada a fatores ligados ao vírus, ao hospedeiro e à resposta imune. O objetivo deste estudo foi avaliar a associação entre os polimorfismos do gene HLA-E, a expressão da molécula HLA-E e a gravidade da doença hepática pelo HCV. Foram incluídos 112 pacientes com hepatite C crônica e avaliados parâmetros clínicos, bioquímicos e histológicos (esteatose, atividade inflamatória e fibrose hepática). A variabilidade do gene HLA-E foi avaliada por sequenciamento de Sanger, e a expressão hepática da molécula, por imunoistoquímica. Para comparação da expressão hepática da molécula HLA-E e da variabilidade do gene HLA-E, foram usados dois grupos controles de indivíduos sem hepatopatia da mesma região geográfica. A imunoistoquímica para HLA-E identificou expressão da molécula nos hepatócitos e nas células de Kupffer. A expressão de HLAE em hepatócitos e células de Kupffer foi encontrada em 56,3% e 43,8% dos pacientes com HCV e em 20% e 10% nos controles (P = 0,008 e 0,02), respectivamente. Foi identificado que o percentual de pacientes do sexo masculino, com expressão moderada de HLA-E em células de Kupffer, foi maior em relação aos pacientes do sexo feminino (22,8% x 7,3%; P = 0,03). As amostras de fígado classificadas como esteatose, atividade necroinflamatória e fibrose graves apresentaram maior grau de expressão de HLA-E em células de Kupffer e hepatócitos, com associação linear significativa. Na análise multivariada, as variáveis que influenciaram significativamente a gravidade da doença foram a expressão da molécula HLA-E nos hepatócitos, a idade avançada e o índice de massa corporal maior que 25. Foram identificados 14 haplótipos diferentes do gene HLA-E, quatro deles ainda não descritos na literatura. A frequência do alelo HLA-E*01:01:01:03 foi menor no grupo de pacientes, quando comparada ao controle (P = 0,0001). O alelo HLA-E*01:03:05 associou-se a maior probabilidade (OR = 4,69) de expressão da molécula HLA-E, na célula de Kupffer (P = 0,046). O genótipo TT do polimorfismo +424 T/C (rs1059510) associou-se a menor probabilidade (OR = 0,06) de expressão da molécula HLA-E, na célula de Kupffer, em relação à ausência de expressão (P=0,009), a menor probabilidade (OR = 0,22) de atividade inflamatória moderada/grave em relação à leve (P = 0,047) e esteve associado a menor probabilidade (OR = 0,17) de fibrose hepática moderada/grave em relação à fibrose leve (P = 0,049). Os resultados do presente estudo sugerem que a pesquisa de fatores imunogenéticos, como a expressão hepática da molécula HLA-E e a identificação da variabilidade genética do HLA-E, pode ter aplicabilidade no manejo clínico dos pacientes, uma vez que auxilia na discriminação daqueles com maior risco de atingir formas avançadas da hepatite C crônica. / Chronic hepatitis C is an important risk factor for the development of cirrhosis and hepatocellular carcinoma. The severity of liver disease can be influenced by factors related to the virus, the host and the immune response. The aim of this study was to evaluate the association between HLA-E gene polymorphisms, HLA-E molecule expression and HCV liver disease severity. We included 112 patients with chronic hepatitis C and evaluated clinical, biochemical and histological parameters (steatosis, inflammatory activity and liver fibrosis). The variability of the HLA-E gene was assessed by Sanger sequencing and liver HLA-E expression by immunohistochemistry. Two control groups of individuals without hepatopathy from the same geographical region were used to compare the HLA-E expression and the gene variability. Immunohistochemistry for HLA-E showed positivity in hepatocyte and Kupffer cell. HLA-E positivity in hepatocytes and Kupffer cells were found in 56.3% and 43.8% of HCV patients and in 20% and 10% in the controls (P = 0.008 and 0.02), respectively. We found that the percentage of male patients with moderate HLA-E expression in Kupffer cells was higher than in females (22.8% vs. 7.3%, P = 0.03). The liver samples classified as severe fibrosis, necroinflammatory activity and steatosis presented greater expression of HLA-E on Kupffer cells and hepatocytes. There was a positive linear association between HLA-E expression and severity of liver damage (P<0.05). In the multivariate analysis, the variables that significantly influenced the severity of the disease were HLA-E molecule expression in hepatocytes, advanced age and body mass index greater than 25. Fourteen different HLA-E haplotypes were identified, four of them not yet described in the literature. The frequency of the HLA-E * 01: 01: 01: 03 allele was lower in the group of patients than in the control group (P = 0.0001). The HLA-E * 01: 03: 05 allele was associated with increased likelihood (OR = 4.69) of HLA-E expression in the Kupffer cell (P = 0.046). The TT genotype of the +424 T / C polymorphism (rs1059510) was associated with a lower probability (OR = 0.06) of HLA-E expression in the Kupffer cell in relation to the absence of its expression (P = 0.009), was associated with a lower probability (OR=0,22) of moderate/severe necroinflammatory activity in relation to the mild inflammatory activity (P=0,047) and was associated with a lower probability (OR = 0.17) of moderate / severe hepatic fibrosis in relation to mild fibrosis (P = 0.049). The results of the present study suggest that the study for immunogenic factors such as HLA-E liver expression and the identification of certain polymorphisms and alleles of the HLA-E gene may have applicability in the clinical management of patients since it aids in discrimination of those at greatest risk of reaching advanced forms of chronic hepatitis C.

INTRODUÇÃO: Pacientes com coinfecção HIV e hepatite C, em geral, apresentam rápida progressão da fibrose hepática. No entanto, a maior parte dos estudos, que avaliam essa questão, caracteriza a progressão da fibrose de forma indireta, utilizando para isso uma única biópsia hepática e tendo como base de cálculo o tempo estimado de infecção pelo vírus da hepatite C. OBJETIVO: Os objetivos do presente trabalho são: 1-Estimar a taxa de progressão da fibrose hepática de forma direta, através da análise de biópsias pareadas em pacientes coinfectados HIVhepatite C, não submetidos a tratamento prévio para hepatite C; 2- Avaliar a possível associação dessa progressão a determinadas variáveis clínicas. MÉTODOS: Trinta pacientes coinfectados com HIV e hepatite C, sem antecedente de tratamento prévio da hepatite C, submetidos a duas biópsias hepáticas pareadas foram avaliados. Calculou-se a taxa de progressão de fibrose direta e foram feitas determinações de progressão, estabilização e regressão da fibrose. Procedeu-se então à análise estatística, testando-se a associação entre progressão de fibrose e algumas variáveis clínicas e demográficas. RESULTADOS: A taxa de progressão média foi de 0,13 UF/ano, com 36,7% dos pacientes configurando-se como progressores. Em análise univariada, a progressão de fibrose hepática esteve associada a níveis de alanina aminotransferase (p<0,001) e aspartato aminotransferase (p<0,0340) acima de 3 vezes o limite superior da normalidade à época da primeira biópsia. Verificou-se também associação entre níveis de alanina aminotransferase (p=0,049) e aspartato aminotransferase (p=0,013) acima da normalidade e atividade necroinflamatória à primeira biópsia. CONCLUSÕES: Elevações de alanina aminotransferase e aspartato aminotransferase parecem estar associadas a uma maior atividade necroinflamatória em pacientes co-infectados pelo HIV e hepatite C e também a uma progressão mais acelerada da fibrose hepática nesse grupo de pacientes / INTRODUCTION: HIV and hepatitis C virus co-infected patients usually exhibit rapid liver fibrosis progression. However, most studies evaluating this issue indirectly characterize fibrosis progression via single liver biopsy, further using this as basis for calculating the estimated duration of hepatitis C infection. OBJECTIVE: Objectives of this study include: 1- Estimate the annual rate of direct liver fibrosis progression, using analyses of paired biopsy samples from HIVhepatitis C co-infected patients without prior treatment for hepatitis C; 2- Assess the possible association of fibrosis progression with certain clinical variables. METHODS: We evaluated 30 HIV-hepatitis C co-infected patients, with no history of prior treatment for hepatitis C, who underwent two paired liver biopsies. We calculated the annual rate of direct fibrosis progression and determined fibrosis progression, stabilization, and regression. We then performed statistical analysis, testing the association between fibrosis progression and several clinical and demographic variables. RESULTS: The average annual progression rate was 0.13 FU/year, with 36.7% of patients defined as progressors. In univariate analysis, liver fibrosis progression was associated with alanine aminotransferase (p<0.001) and aspartate aminotransferase (p<0.0340) levels over three times the upper limit of normal present at first biopsy. There was also an association between above-normal alanine aminotransferase (p=0.049) and aspartate aminotransferase (p=0.013) levels and necroinflammatory activity at first biopsy. CONCLUSION: Elevated alanine aminotransferase and aspartate aminotransferase levels appear to be associated with higher necroinflammatory activity and more accelerated liver fibrosis progression among HIV-hepatitis C co-infected patients

During embryonic development, generation of functional organs is dependent on proper interactions between different cell types. Elucidation of the mechanisms operating during organ formation might provide insights into the origin of many pathological disorders in the adult. Gene inactivation studies in mice have provided invaluable tool to study the function of genes critical for morphogenesis of distinct organs. A LIM-homeodomain transcription factor Lhx2 has previously been reported to play a role in fetal liver development and hematopoiesis, as its inactivation leads to lethal anemia due to underdeveloped liver. This thesis focuses on the function of Lhx2 in the development of these two organ systems. Reciprocal signaling between ventral foregut endoderm and mesenchyme of the septum transversum regulates the liver formation, expansion and differentiation. A fully formed liver is composed of endoderm-derived hepatocytes and cholangiocytes and a variety of mesenchyme-derived cell types, such as endothelial cells and hepatic stellate cells. In early stages of liver development Lhx2 is expressed in the liver-associated septum transversum mesenchyme, a part of which becomes integrated into the liver organ and develops into hepatic stellate cells. Functional Lhx2 expression in the hepatic mesenchyme is necessary for normal liver outgrowth and differentiation. Loss of Lhx2 from developing hepatic stellate cells leads to their activation and excessive deposition of collagen fibres, resulting in hepatic fibrosis and severely distorted liver architecture. Transfection of Lhx2 to human stellate cell line downregulates genes associated with stellate cell activation and fibrogenesis. Thus, Lhx2 is the first gene identified to negatively regulate events leading to hepatic fibrosis. Elucidation of the molecular mechanisms involved in this process might therefore be instrumental for the development of novel therapies useful in treatment of this disorder. Fetal liver is also a major site of hematopoiesis in the embryo and provides physiological conditions necessary for the efficient expansion of hematopoietic stem cells (HSCs). The hematopoietic defect observed in Lhx2-deficient embryos is cell-nonautonomous, indicating that Lhx2 might control secreted factors involved in the self-renewal of HSCs. This putative second role of Lhx2 has been investigated by analyzing the mechanism whereby Lhx2 expression generates in vitro self-renewing HSC-like cell lines. Interestingly, in agreement with the cell nonautonomous phenotype of the lethal anemia in Lhx2-/- embryos, the mechanism of self-renewal is dependent on Lhx2 expression and occurs via secreted factor(s). Identification of these factor(s) might potentially allow ex vivo expansion of HSCs for therapeutic purposes. The Lhx2-immortalized HSC-like cell lines share many basic features with HSCs and self-renew in vitro in presence of Steel factor (SF). SF/c-Kit signaling mediates a wide variety of biological activities in cells at many different levels in the hematopoietic hierarchy. We used the HSC-like cell lines as an in vitro model system to compare signal transduction pathways from c-Kit receptor in stem cells versus differentiated hematopoietic cells. HSCs require PI-3K dependent activation of Raf1-Mek-Erk cascade for their survival and self-renewal in response to SF, whereas activation of Erk is PI-3K independent in committed myeloid and mast cells. Thus, the mode of SF/c-Kit signaling is dependent on the differentiation status of the cells.

<p>Tissue stiffness can be an indicator of various types of ailments. However, no standard diagnostic imaging modality has the capability to depict the stiffness of tissue. To overcome this deficiency, various elasticity imaging methods have been proposed over the past 20 years. A promising technique for elasticity imaging is acoustic radiation force impulse (ARFI) based shear wave imaging. Spatially localized acoustic radiation force excitation is applied impulsively to generate shear waves in tissue and its stiffness is quantified by measuring the shear wave speed (SWS).</p><p>The aim of this thesis is to contribute to both the clinical application of ARFI shear wave imaging and its technical development using the latest advancements in ultrasound imaging capabilities.</p><p>To achieve the first of these two goals, a pilot imaging study was conducted to evaluate the suitability of ARFI shear wave imaging for the assessment of liver fibrosis using a rodent model of the disease. The stiffness of severely fibrotic rat livers were found to be significantly higher than healthy livers. In addition, liver stiffness was correlated with fibrosis as quantified using collagen content.</p><p>Based on these findings, an imaging study was conducted on patients undergoing liver biopsy at the Duke University Medical Center. A robust SWS estimation algorithm was implemented to deal with noisy patient shear wave data using the random sample consensus (RANSAC) approach. RANSAC estimated liver stiffness was found to be higher in severely fibrotic and cirrhotic livers, suggesting that ARFI shear wave imaging may potentially be useful for the staging of severe</p><p>fibrosis in humans.</p><p>To achieve the second aim of this thesis, a system capable of monitoring ARFI induced shear wave propagation in 3D was implemented using a 2D matrix array transducer. This capability was previously unavailable with conventional 1D arrays. This system was used to study the precision of time-of-flight (TOF) based SWS estimation. It was found that by placing tracking beam locations at the edges of the SWS measurement region of interest using the 2D matrix array, TOF SWS precision could be improved in a homogeneous medium.</p><p>The 3D shear wave imaging system was also used to measure the SWS in muscle, which does not conform to the isotropic mechanical behavior usually assumed for tissue, due to the parallel arrangement of muscle fibers. It is shown that the SWS along and across the fibers, as well as the 3D fiber orientation can be estimated from a single 3D shear wave data-set. In addition, these measurements can be made independent of the probe orientation relative to the fibers. This suggests that 3D shear wave imaging can be useful for characterizing anisotropic mechanical properties of tissue.</p> / Dissertation

INTRODUÇÃO: Pacientes com coinfecção HIV e hepatite C, em geral, apresentam rápida progressão da fibrose hepática. No entanto, a maior parte dos estudos, que avaliam essa questão, caracteriza a progressão da fibrose de forma indireta, utilizando para isso uma única biópsia hepática e tendo como base de cálculo o tempo estimado de infecção pelo vírus da hepatite C. OBJETIVO: Os objetivos do presente trabalho são: 1-Estimar a taxa de progressão da fibrose hepática de forma direta, através da análise de biópsias pareadas em pacientes coinfectados HIVhepatite C, não submetidos a tratamento prévio para hepatite C; 2- Avaliar a possível associação dessa progressão a determinadas variáveis clínicas. MÉTODOS: Trinta pacientes coinfectados com HIV e hepatite C, sem antecedente de tratamento prévio da hepatite C, submetidos a duas biópsias hepáticas pareadas foram avaliados. Calculou-se a taxa de progressão de fibrose direta e foram feitas determinações de progressão, estabilização e regressão da fibrose. Procedeu-se então à análise estatística, testando-se a associação entre progressão de fibrose e algumas variáveis clínicas e demográficas. RESULTADOS: A taxa de progressão média foi de 0,13 UF/ano, com 36,7% dos pacientes configurando-se como progressores. Em análise univariada, a progressão de fibrose hepática esteve associada a níveis de alanina aminotransferase (p<0,001) e aspartato aminotransferase (p<0,0340) acima de 3 vezes o limite superior da normalidade à época da primeira biópsia. Verificou-se também associação entre níveis de alanina aminotransferase (p=0,049) e aspartato aminotransferase (p=0,013) acima da normalidade e atividade necroinflamatória à primeira biópsia. CONCLUSÕES: Elevações de alanina aminotransferase e aspartato aminotransferase parecem estar associadas a uma maior atividade necroinflamatória em pacientes co-infectados pelo HIV e hepatite C e também a uma progressão mais acelerada da fibrose hepática nesse grupo de pacientes / INTRODUCTION: HIV and hepatitis C virus co-infected patients usually exhibit rapid liver fibrosis progression. However, most studies evaluating this issue indirectly characterize fibrosis progression via single liver biopsy, further using this as basis for calculating the estimated duration of hepatitis C infection. OBJECTIVE: Objectives of this study include: 1- Estimate the annual rate of direct liver fibrosis progression, using analyses of paired biopsy samples from HIVhepatitis C co-infected patients without prior treatment for hepatitis C; 2- Assess the possible association of fibrosis progression with certain clinical variables. METHODS: We evaluated 30 HIV-hepatitis C co-infected patients, with no history of prior treatment for hepatitis C, who underwent two paired liver biopsies. We calculated the annual rate of direct fibrosis progression and determined fibrosis progression, stabilization, and regression. We then performed statistical analysis, testing the association between fibrosis progression and several clinical and demographic variables. RESULTS: The average annual progression rate was 0.13 FU/year, with 36.7% of patients defined as progressors. In univariate analysis, liver fibrosis progression was associated with alanine aminotransferase (p<0.001) and aspartate aminotransferase (p<0.0340) levels over three times the upper limit of normal present at first biopsy. There was also an association between above-normal alanine aminotransferase (p=0.049) and aspartate aminotransferase (p=0.013) levels and necroinflammatory activity at first biopsy. CONCLUSION: Elevated alanine aminotransferase and aspartate aminotransferase levels appear to be associated with higher necroinflammatory activity and more accelerated liver fibrosis progression among HIV-hepatitis C co-infected patients

A cirrose hepática é uma doença crônica e irreversível com modificações histológicas caracterizadas por nódulos de regeneração e desarranjo de hepatócitos, com aumento difuso de tecido conjuntivo e perda de função. As células-tronco têm sido amplamente estudadas para o tratamento de injúrias hepáticas. Fibrose e cirrose foram induzidas em ratos Wistar, através da aplicação de tioacetamida (TAA) intraperitoneal a 200 mg/kg por 8 ou 14 semanas, seguido de terapia celular com aplicação intravenosa única de 1x106 células-tronco de membrana amniótica de ratas. Amostras de sangue foram coletadas para análises bioquímicas, e tecido hepático para histologia e imunohistoquímica. A histopatologia para colorações de hematoxilina-eosina para graduação e estadiamento da lesão hepática foi realizada, com observação de alterações celulares; picrosírius para quantificação do colágeno parenquimal; ácido periódico-Schiff para mucopolissacarídeos neutros e glicogênio; e alcian blue para mucopolissacarídeos ácidos. Imunohistoquímica foi realizada para marcação de PCNA, colágenos tipo 1 e 3, e &alpha;-SMA. O cultivo celular apresentou ótimo crescimento, sendo transduzido com GFP e negativo para micoplasma. Os animais induzidos experimentalmente mostraram baixo ganho de peso durante o experimento, com notável recuperação nas duas primeiras semanas após término da indução. A mortalidade experimental foi de 10 %. A indução da fibrose e cirrose hepática obtiveram sucesso no período de 8 e 14 semanas, respectivamente Análises macroscópicas mostraram melhor aspecto de fígados cirróticos após terapia celular. Não houve diferenças estatísticas após a terapia celular para as análises bioquímicas, graduação, estadiamento, alterações celulares, proliferação de ductos, mucopolissacarídeos ácidos e neutros e imunohistoquímicas para colágeno 1 e 3, PCNA e &alpha;- SMA. Houve, porém, uma tendência a melhora na graduação e estadiamento da fibrose e cirrose com redução da atividade necroinflamatória; redução na proliferação de ductos; menor deposição de mucopolissacarídeos ácidos e neutros; diminuição das alterações celulares com indicativos de menor grau de lesão tecidual e maior grau de regeneração; diminuição da expressão de &alpha;-SMA e colágenos 1 e 3; e aumento do PCNA. O colágeno intralobular teve diminuição após terapia celular em fígados fibróticos. Em animais induzidos a cirrose, houve diminuição de ambos colágenos interlobular e intralobular. Conclui-se que houve uma tendência de melhora dos animais após tratamento com células-tronco da membrana amniótica de ratas, o que nos encoraja a aplicação de protocolos de terapia celular. Apesar dos resultados promissores, um maior número de animais precisa ser testado para que se tenham mais dados, que possam dar subsídios para o uso destas células na prática clínica para o tratamento da cirrose hepática / Liver cirrhosis is a chronic and irreversible disease with histological changes characterized by regenerative nodules and disarray of hepatocytes with diffuse increase in connective tissue and loss of function. Stem cells have been extensively studied for the treatment of liver injury. Fibrosis and cirrhosis was induced in Wistar rats by application of thioacetamide (TAA) intraperitoneally at 200 mg/kg for 8 or 14 weeks, followed by cell therapy with a single intravenous administration of 1x106 stem cells from amniotic membrane of rats. Blood samples were collected for biochemical analyzes, and liver tissue for histology and immunohistochemistry. Histopathology by hematoxylin-eosin for grading and staging of hepatic injury with observation of cellular changes was performed; picrosirius for quantification of parenchymal collagen; periodic acid-Schiff for neutral mucopolysaccharides and glycogen; and alcian blue for acid mucopolysaccharides. Immunohistochemistry was performed for PCNA, type 1 and 3 collagens, and &alpha;-SMA. Cell culture showed optimal growth, being transduced with GFP and negative for mycoplasma. Animals induced to liver disease showed low weight gain during the experiment, with remarkable recovery in the first two weeks after completion of induction. Experimental mortality was 10%. Induction of fibrosis and cirrhosis was successful after the period of 8 and 14 weeks, respectively. Macroscopic analysis showed improvement in the aspect of cirrhotic livers after cell therapy. There were no statistical differences after cell therapy for biochemical analyzes, grading, staging, cellular changes, proliferation of ducts, acidic and neutral mucopolysaccharides and immunohistochemistry for collagen 1 and 3, PCNA and &alpha;-SMA. There was, however, a tendency on improvement in grading and staging of fibrosis and cirrhosis with reduced necroinflammatory activity; reduction in the proliferation of ducts; lower deposition of acidic and neutral mucopolysaccharides; decrease in cellular changes indicating a lower degree of tissue damage and a greater degree of regeneration; decreasing on the expression of &alpha;-SMA and type 1 and 3 collagens; and increased PCNA. Intralobular collagen decreased after cell therapy in fibrotic livers. In cirrhotic animals, there was a decrease of both interlobular and intralobular collagen. We conclude that was a trend to improvement of animals after treatment with stem cells derived from amniotic membrane of rats, which encourages the application of cell therapy protocols. Despite the promising results, a larger number of animals must be tested, so we can have more data to make allowances for the use of these cells in clinical practice for the treatment of liver cirrhosis

L’actine est impliquée dans de nombreuses fonctions cellulaires physiologiques et pathologiques. Au cours de ma thèse j'ai analysé le rôle de l'actine i) lors de l’invasion tumorale et ii) dans la formation des fenêtres des cellules endothéliales sinusoïdales hépatiques. i) Les cellules tumorales forment des structures d’actine permettant la dégradation de la matrice extracellulaire (MEC) nommés invadosomes. Mes travaux ont permis de démontrer que la RhoGTPase Cdc42 régule la formation de la structure d’actine qu’est l’invadosome, tandis que la protéine d’échafaudage Tks5 est requise pour l’activité de dégradation aboutissant à un invadosome fonctionnel. Ces deux molécules constituent la signature moléculaire minimale des invadosomes. Nous avons établi que le collagène de type I qui est surexprimé dans le microenvironnement tumoral induit la formation d’invadosomes linéaires (Lis). Nous avons identifié le récepteur à domaine discoïdine 1 (DDR1) comme spécifiquement responsable de la formation des Lis. Son interaction avec le collagène fibrillaire permet le recrutement du facteur d’échange des RhoGTPases, Tuba et l’activation de la Cdc42 conduisant à la formation d’un Li. DDR1 est impliqué dans l’invasion tumorale et sa surexpression est de mauvais pronostic dans plusieurs cancers comme le poumon ou encore le sein. Le récepteur DDR1 est également impliqué dans la cohésion cellulaire au cours de la migration collective des cellules tumorales. Nous avons démontré que dans un contexte riche en collagène de type I, DDR1 a une double localisation et donc différents rôles associés dans la migration collective. D’une part un rôle de cohésion cellulaire et d’autre part un rôle dans la dégradation de la MEC. Nous tentons de démontrer que ces différentes fonctions impliquent différentes isoformes de DDR1. Nous souhaitons par la suite déterminer les mécanismes moléculaires qui régulent l’expression, la localisation et la signalisation associées aux différentes isoformes de DDR1. ii) Dans un contexte physiologique, les capillaires sanguins du foie présentent des pores transcellulaires ou fenêtres, qui permettent les échanges bidirectionnels entre le sang et les hépatocytes pour assurer la fonction de filtration de cet organe. Au cours du processus de fibrose ces fenêtres sont perdues, réduisant les échanges. Nous avons démontré le caractère réversible de la perte des fenêtres mais aussi que l’actine n’était pas impliquée dans la formation de ces structures. Nous avons développé une méthode de visualisation en microscopie haute résolution STED de ces structures, permettant pour la première fois une analyse sur cellule vivante. Par une approche de spectrométrie de masse couplée à notre nouvelle méthode d’observation en STED, nous voulons valider la co-localisation des fenêtres avec des marqueurs potentiels identifiés. / Actin is involved in many physiological and pathological cellular functions. In my thesis I analyzed the role of actin i) during tumor invasion and ii) in the formation of fenestrae in liver sinusoidal endothelial cells. i) Tumor cells form actin-based structures, invadosomes, involved in extracellular matrix (ECM) degradation. My work has demonstrated that the RhoGTPase Cdc42 regulates the formation of invadosomes, while the Tks5 scaffold protein is required for the matrix degradation activity. These two molecules form a minimum molecular signature for invadosomes. We found that type I collagen, which is overexpressed in the tumor microenvironment, induces the formation of linear invadosomes (Lis). We have identified the discoidin receptor 1 (DDR1) to be specifically responsible for Lis formation. Its interaction with the fibrillar collagen allows the recruitment of GEF Tuba and the activation of Cdc42 RhoGTPase leading to Li formation. DDR1 is implicated in tumor invasion and its overexpression is a poor prognosis in many cancers like lung or breast. The DDR1 receptor is also involved in cell cohesion in the collective migration of tumor cells. We have demonstrated that in a context rich in type I collagen, DDR1 has a dual location and therefore possesses different roles in the collective migration of tumor cells: a role in cell cohesion and a role in ECM degradation. We are analyzing the role of the different DDR1 isoforms in this process. We wish subsequently to determine the molecular mechanisms that regulate the expression, localization and signaling associated with these different isoforms. ii) In a physiological context, the liver capillaries have transcellular pores that allow bidirectional exchanges between the blood and the hepatocytes to ensure the proper filtering function of that organ. During the fibrosis process, these pores are lost thus decreasing the exchanges. We have demonstrated that the loss of these “fenestrae” is reversible and also that actin does not play a role in their formation. We have developed a novel method to analyze these structures in living cells using high resolution STED microscopy. Now, by using mass spectrometry approach coupled to our new observation methods in STED, we want to validate the fenestrae co-localisation with potential markers identified.

1 Abstract Liver fibrosis is a condition described by extensive accumulation of scar tissue in the liver. With further progression, it leads to cirrhosis or even to hepatocellular carcinoma. Liver fibrosis accompanies every chronic liver disease and its prevalence in adult European population is estimated to be around 4%. During my dissertation work, I studied the function of three members of Metzincin family of metalloproteinases - ADAM17, ADAM10 and MMP-19, in liver fibrosis and liver regeneration using mouse genetic models. ADAM17 and ADAM10 are important regulators of signalling pathways which are involved in immune response as well as differentiation. Both proteases are able to cleave ectodomains of their substrates from cell membrane, affecting bioavailability of ligands and functionality of receptors. Several of their substrates are involved in liver pathologies. MMP-19 on the other hand, is a metalloprotease mainly involved in extracellular matrix cleavage, important process in fibrosis development, as well as resolution of fibrosis. Our results demonstrate that ablation of ADAM10 results in increased susceptibility to liver fibrosis in mice, both spontaneous and toxin induced. ADAM10 deficiency affected biliary epithelium, as we detected higher markers of biliary damage in serum of ADAM10 deficient...

Hepatitis C Virus (HCV) is a small RNA virus that progresses to chronicity in 50-80% of infected individuals. Direct-acting antivirals (DAAs) are revolutionary treatments for HCV with 90-98% cure rates. However, over time, chronic HCV infections can result in advanced liver disease, including cirrhosis. Patients with advanced fibrosis experience a poor response to vaccination, recurrent infections and increased risk for hepatocellular carcinoma (HCC). These outcomes are, in part, a consequence of immune dysfunction. Increased inhibitory receptor and Galectin-9 (GAL-9) expression is a possible mechanism promoting lymphocyte dysfunction. In this study, blood samples were collected from chronic HCV patients with different degrees of liver fibrosis. I conducted a 13-parameter flow stain on the peripheral blood mononuclear cells (PBMC) of these patients. Next, I measured the expression of inhibitory receptors (PD-1, CTLA-4, LAG-3, TIGIT and TIM-3) and GAL-9 on bulk T cell and NK cells of 15 chronic HCV patients with no to moderate fibrosis (F0-F2) and 15 with advanced fibrosis (F3-F4). To analyze receptor co-expression, I employed t-distributed stochastic neighbor embedding (t-SNE) analysis to dimensionally reduce the multi-parametric data. Notably, I found that F3-F4 patients had higher frequencies of >3 inhibitory receptor co-expression on NK cells. Moreover, t-SNE analysis of bulk T cells revealed that F3-F4 patients manifest a higher frequency of cells in the clusters with CD25+TIGITmed-hi CD4+ T cells and PD-1medLAG-3med-hiGAL-9med-hi CD4+ T cells. t-SNE analysis of NK cells also showed that F3-F4 patients manifest a higher frequency of cells in the cluster with CD25+TIGITmed-hiTIM-3med-hi CD56Dim NK cells and CCR7+ PD-1medLAG-3med-hiGAL-9med-hi CD56Dim NK cells. Lastly, the frequency of cells in these clusters was found to positively correlate with patient’s extent of liver damage. In conclusion, I identified phenotypes of immune dysregulation that could explain the increased susceptibility to infection and HCC in chronic HCV patients with advanced fibrosis. These phenotypes could identify targets for combinatorial checkpoint blockade therapy to potentially improve immune function in these patients.