A utilização de fatores hepatotróficos (FH) tem trazido importantes avanços no tratamento de algumas doenças hepáticas. A avaliação dos efeitos dessas substâncias pode ser feita com o uso de modelos in vivo, como a regeneração hepática após a hepatectomia parcial ou in vitro, como a cultura de hepatócitos, células estreladas ou outros tipos celulares do fígado. O modelo de cultura demonstra ser útil por possibilitar a análise individualizada de determinadas substâncias ou soluções diretamente nas células-alvo, facilitando o delineamento de seu mecanismo de ação. Dessa forma, o presente trabalho estudou in vitro os efeitos da administração de fatores hepatotróficos (FH) em hepatócitos isolados de fígados de ratos, avaliando seu metabolismo e proliferação celular. Trinta ratos Wistar fêmeas foram utilizados, o fígado retirado e, por digestão enzimática in situ, suas células foram dissociadas e os hepatócitos separados em gradiente de Percoll 45%; cultivados em meio DMEM/F12, tratadas com diferentes concentrações dos FH (1X, 5X e 10X) e analisadas em intervalos de 24, 48 e 72 horas. A viabilidade e a proliferação celular foram avaliadas pelo método colorimétrico MTT, a toxicidade pelo iodeto de propídeo, o potencial elétrico da membrana mitocondrial pela Rodamina 123 e a expressão da citoqueratina 8, 18 e desmina como marcadores do citoesqueleto. O metabolismo hepático foi avaliado pela depuração do verde de indocianina (VIC), pela quantificação de colágeno tipo I e pela formação de radicais lipídicos poliinsaturados peroxidados. A técnica utilizada para a obtenção de hepatócitos mostrou-se eficaz com viabilidade superior a 90%, sem apresentar toxicidade, com manutenção do potencial mitocondrial e com marcação positiva para citoqueratinas 8, 18 e desmina. A adição dos FH aumentou a proliferação dos hepatócitos nos três períodos analisados em relação ao grupo controle. Os FH não demonstraram toxicidade em cultura primária de hepatócitos em nenhuma das concentrações avaliadas ao longo de todos os períodos experimentais. A adição dos FH na concentração de 10X evitou a formação de radicais livres, protegendo os hepatócitos da lipoperoxidação. Por outro lado, a avaliação funcional das culturas primárias pelo teste VIC mostrou-se eficaz na determinação do metabolismo dos hepatócitos, como a manutenção dos níveis das transaminases e amilase. Os hepatócitos mantidos em cultura primária após a adição dos FH em todos os períodos analisados aumentaram a produção de colágeno, e também foram capazes de modificar a distribuição da população de células nas fases quiescentes, aumentando sua capacidade de síntese. A adição dos FH nas concentrações estudadas nas culturas de hepatócitos mostrou-se eficaz na manutenção dessas células, mantendo-se funcionalmente ativos, com a expressão dos marcadores de seu metabolismo e diferenciação. / The use of hepatrotophic hic factors (HF) has provided important advances in the treatment of several hepatic disorders. The evaluation of this treatment may be carried out by in vivo models, as experiments on hepatic regeneration after partial hepatectomy; or in vitro models as culture of hepatocytes, stellate cells or any other hepatic cell. This last model allows an individual analysis of the studied substances on their target cells, providing the possibility of further investigation on the mechanisms involved. Therefore, the present study evaluated the in vitro effects of the hepatotrophic factors administration on the metabolism and proliferation of cultured hepatocytes from normal rat liver. Liver from 30 Wistar rats were used, and after in situ enzimatic dissociation, hepatocytes were collected and separated by 45% Percoll density gradient, cultivated in DMEN/F12 media supplemented with different HF concentrations (1×, 5× and 10×) and finally analyzed at 24, 48 and 72hs intervals. Cell viability and proliferation were assessed by MTT colorimetric assay, cell toxicity by propidium iodide, mitochondrial membrane electrical potential by 123 rodamine test and cytokeratin 8, 18 and desmin as cytoskeleton markers. Hepatic metabolism was evaluated by infusion of indocianine green (ICG), by type I collagen quantification and by peroxided polyunsaturated lipid radicals production. The techniques used in order to collect hepatocytes proved to be efficient, with a viability higher than 90%, no toxicity, and the maintenance of the mitochondrial membrane potential and positive labeling for cytokeratins (CK) 8, 18 and desmin. The HF addition increased the proliferation of hepatocytes in the three periods analyzed, when compared to the control group. The HF did not show any toxicity in primary hepatocytes culture regardless of the concentration evaluated along the experimental periods. The HF addition impaired the production of free radicals, protecting the hepatocytes from the lipid peroxidation. On the other hand, the functional evaluation of primary hepatocytes cultures using the ICG test proved to be efficient in the determination of the hepatocytes metabolism, as the maintenance of the transaminase and amylase levels. The hepatocytes kept in primary culture after HF addition in all the analyzed periods increased the collagen production and were also able to shift the distribution of quiescent cells population of, thus increasing their synthesis capacity. The HF addition in the studied concentrations in hepatocytes cultures proved to be efficient in the maintenance of these cells, that remain functionally active and expressing their metabolism and differentiation characteristic markers.

As neoplasias de glândula salivar (NGS) são tumores raros que despertam interesse por sua diversidade histopatológica e comportamento clínico. A compreensão da patobiologia assim como, dos mecanismos envolvidos no comportamento invasivo destas lesões é necessária para melhor entender a biologia das NGS e posteriormente delinear novas estratégias terapêuticas. A presente tese foi dividida em dois artigos. O objetivo do primeiro estudo foi descrever os dados demográficos, clinicopatológicos e de prognóstico das NGS diagnosticados em um centro de atenção terciário. Para tal, foi realizada uma análise retrospectiva utilizando os dados de arquivos e de prontuários. Foram identificados 109 casos de NGS cuja média de idade dos pacientes foi de 46.47 anos e a relação homens:mulheres foi de 0.94:1. As glândulas salivares maiores foram mais acometidas (75.2%) e os tumores benignos os mais prevalentes (75.2%) sendo o adenoma pleomórfico o tumor benigno mais comum e o carcinoma adenóide cístico o principal maligno. O objetivo do segundo estudo foi analizar o padrão de expressão da via de sinalização do HGF/c-Me/PI3K em NGS e correlacionar com o perfi proliferativo e desfechos clínicos das lesões. Foram construídos microarranjos de tecido (TMAs) de 93 casos de NGs e as lâminas foram submetidas a análise imunoistoquímica para HGF, p-Met, p-Akt e Ki67. Foi observada maior expressão de HGF nos tumores benignos (p=0.04), enquanto que as protínas p-Met (p=0.03), p-Akt (p=0.00) e Ki-67 (p=0.00) foram mais expressas nos tumores malignos. Nas neoplasias malignas houve maior ativação da via HGF observada pela maior expressão do seu receptor fosforilado (p-Met) bem como, maior ativação da via do PI3k pela fosforilação de Akt (p-Akt) resultando em um maior perfil proliferativo. Pode-se concluir que a via de sinalização do HGF/c-Met/PI3k parece estar ativa nas NGS regulando a proliferação especialmente nas neoplasias malignas. / Salivary gland tumors (SGT) are rare yet interesting neoplasms due to their histopatological diversity and clinical behavior. Understanding the pathobiology as well as the mechanisms involved in the invasive behavior of these lesions is needed to better comprehend the biology of SGT and further delineate new therapeutic strategies. This thesis was divided in two papers. The aim of the first study was to describe the demographic, clincopathological and prognostic data of SGT diagnosed in a tertiary care center. For this purpose, a retrospective analysis using data from the archives and records was performed. One hundred and nine cases of SGT were identified. The patients mean age was 46.47 years and the male:female ratio was 0.91:1. The major salivary glands were the most affected (75.2%) and the benign SGT were more prevalent (78%) being pleomorphic adenoma the most common benign tumor and adenoid cystic carcinoma the most common malignant tumor. The objective of the second study was to analyze the expression pattern of HGF/c-Met/PI3K signaling pathway in SGT and correlate the findings with the proliferative profile and clinical outcomes of cases. Tissue microarrays (TMAs) of 93 cases of SGT were constructed; the slides were submitted to immunohistochemical analysis for HGF, p-Met, p-Akt and Ki-67. Increased expression of HGF was observed in benign tumors (p = 0.04), while p-Met (P = 0.03), p-Akt (p = 0:00) and Ki-67 (p = 0:00) were most expressed in malignant tumors. In salivary glands carcinomas there was a higher activation of the HGF pathway observed by the higher expression of its phosporylated receptor (p-Met) as well as the higher activation of PI3k pathway through Akt (p-Akt) phosphorilation, resulting in a higher proliferative profile. It can be concluded that HGF/c-Met/PI3K signaling pathway appears to be active in SGT regulating the proliferation specially in malignant tumors.

Avaliaram-se os efeitos da administração do extrato seco de maracujá (Passiflora incarnata), veiculado na dieta, sobre a morfologia dos hepatócitos de juvenis de tilápias do Nilo (Oreochromis niloticus). Peixes isolados (86,50 &plusmn; 10,17 g) receberam durante 28 dias ração comercial extrusada (32% PB - 2% biomassa) contendo o extrato diluído em alginato de sódio nas doses 0 (controle), 50, 100 e 200 mg/Kg, (n = 6 peixes/tratamento), registrando-se diariamente o consumo. No início e ao final do experimento cada indivíduo foi exposto (30 minutos) à reflexão da própria imagem em espelho (presença virtual de um coespecífico - estresse social), sendo a seguir anestesiado (2-fenoxietanol 0,5 mL/L) para realização de biometria e coleta de sangue (veia caudal) para determinação dos níveis plasmáticos de glicose e cortisol. Após 28 dias todos os animais foram sacrificados para remoção do fígado e obtenção de fragmentos utilizados na contagem de células e avaliação da morfometria do citoplasma dos hepatócitos (H/E), observando-se também as reservas de glicogênio hepático (PAS). Visando a comparação dos efeitos do estresse social natural com aquele empregado no experimento, seis peixes provenientes de um grupo de 30 indivíduos foram também sacrificados e utilizados como referência (valores basais) para avaliação dos parâmetros histológicos. Os dados foram submetidos a ANOVA, utilizando-se o proc mixed SAS 8.0 (p<0,05) para os parâmetros consumo de alimento, ganho em peso e bioquímica sangüínea (cortisol e glicose) e GLM SAS 8.0 (p<0,01) para a contagem de células e morfometria dos hepatócitos. Verificou-se que os pesos e os comprimentos iniciais não diferiram, que o consumo de alimento não foi alterado pela adição do extrato e que todos os peixes, independentemente do tratamento, cresceram significativamente. Os níveis de cortisol e de glicose também não diferiram inicialmente entre os grupos e não foram alterados pela presença do agente ou pela adição do extrato. Porém, observou-se um aumento significativo da glicose e redução dos níveis de cortisol em todos os peixes. A adição do extrato nas diferentes doses provocou aumento crescente e significativo da área citoplasmática e redução do número de células em todos os animais, com destaque para a dose 100 mg/Kg. O mesmo não ocorreu nos peixes do grupo controle, cujas áreas citoplasmáticas foram significativamente menores, em decorrência de um menor acúmulo de glicogênio hepático. Embora os efeitos do agente estressor empregado não tenham sido detectados pela análise dos parâmetros bioquímicos sangüíneos, verificou-se que este procedimento provocou alterações metabólicas que contribuíram para a depleção dos estoques de glicogênio no fígado. Este efeito parece ter sido revertido nos peixes que receberam a dieta contendo o extrato de maracujá que, por meio de mecanismos não elucidados neste trabalho, contribuiu para a manutenção ou aumento dos estoques de glicogênio hepático. Concluiu-se que o extrato de maracujá veiculado na dieta, na dose de 100 mg/Kg, protege juvenis de tilápia da depleção dos estoques de glicogênio hepático causada pelo estresse social. / Effects of Passion fruit (Passiflora incarnata) dry extract administration on hepatocyte morphometry of juveniles Nile tilapias (Oreochromis niloticus) were investigated. Male isolated fish (86,50 &plusmn; 10,17 g) were daily fed (28 days) with extruded commercial ration (32% crude protein - 2% biomass) containing the extract diluted in sodium alginate in graded doses 0 (control), 50, 100 and 200 mg/Kg (n= 6 animals/treatment), registering daily consumption. At the start and after 28 days of experiment each fish was displayed to the reflection of own mirror image (30 minutes) in order to simulate the virtual presence of a coespecific (social stress). Then the animals were anesthetized (2-fenoxietanol) for biometric measures and blood collection (caudal vein) for determination of the plasmatic levels of glucose and cortisol. After 28 days the animals were sacrificed for removal of liver fragments samples used to histological examination (cytoplasmic area and number of cells - HE stained; hepatic glycogen supplies - PAS stained). Aiming the comparison of the natural social stress with that utilized in this experiment, six fish from a group of 30 individuals were also sacrificed and used as reference (basal values) for the histological parameters evaluation. Food consumption, weight gain and sanguine biochemist parameters (cortisol and glucose) were submitted to ANOVA, SAS 8.0 proc mixed (p<0,05) and data of cells counting and hepatocyte morfometry to the same test, using SAS 8.0 GLM (p<0,01). The initial weights and lengths were similar, the food consumption was not modified by the addition of the extract and all the fish grew significantly during the experiment. Initial levels of cortisol and glucose were also similar between groups and did not modify by the stressor agent or treatments with the extract. However, a significant increase of glucose and reduction of cortisol levels were observed for all the fish. The addition of different extract doses provoked significant and noticeable increase of the cytoplasmic area and reduction of the cells number, mainly for 100 mg/Kg dose. In the control group cytoplasmatic area was significantly minor due to lesser hepatic glycogen accumulation. The stressor agent did not affect sanguine biochemists parameters but seems to lead metabolic alterations that had collaborated with the depletion of liver glycogen supplies. This effect seems to have been reverted in the fish that received the diet contend Passion fruit extract that, by means of a mechanism not elucidated in this experiment, contributed for the maintenance or increase of hepatic glycogen supplies. It was conclude that Passion fruit extract diet inclusion at 100 mg/Kg dose protects the tilapia against the hepatic glycogen depletion caused by social stress.

Orientador: Mario José Abdalla Saad / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T17:03:51Z (GMT). No. of bitstreams: 1 Araujo_TiagoGomes_D.pdf: 9136785 bytes, checksum: 25c68d3dd56714d164986b415f98070d (MD5) Previous issue date: 2012 / Resumo: A resistência à insulina está presente na obesidade e na diabetes tipo 2, e está associada à hiperplasia das ilhotas pancreáticas e hiperinsulinemia como resposta compensatória, entretanto, as forças motrizes por trás desse mecanismo compensatório não são totalmente compreendidos. Dados anteriores sugeriram o envolvimento de um fator circulante desconhecido que na resistência à insulina atua como um fator de crescimento das células ?. Neste contexto, procurando por candidatos a serem este fator circulante, percebemos que o fator de crescimento de hepatócitos (HGF) é um forte candidato a ser este elo entre a resistência à insulina e o aumento da massa de ilhotas / hiperinsulinemia. Nossa abordagem teve como objetivo mostrar uma possível relação de causa-efeito entre o aumento dos níveis circulantes de HGF e a hiperplasia da ilhota / hiperinsulinemia compensatória, assim mostrando a força da associação. Ainda, se esta associação, apresenta ou não uma resposta dose-dependente, temporalidade, consistência, plausibilidade e reversibilidade. Nesse sentido, os nossos dados mostraram: a) uma correlação forte e consistente entre o HGF e o mecanismo de compensação em três modelos animais de resistência à insulina; b) o HGF aumenta a massa de célula ? de uma forma dose-dependente; c) o bloqueio do HGF interrompe os mecanismos de compensação; d) o aumento nos níveis de HGF precede a resposta compensatória associada com a resistência à insulina, indicando que estes eventos ocorrem em um modo sequencial. Além disso, o bloqueio do receptor de HGF (Met) piorou a já prejudicada sinalização da insulina no fígado de ratos obesos induzidos por dieta. Em geral, os nossos dados indicam que o HGF é um fator de crescimento que desempenha um papel fundamental no aumento da massa de ilhotas e hiperinsulinemia em ratos obesos induzidos por dieta, e sugerem um efeito protetor da interação HGF-Met na sinalização de insulina no fígado / Abstract: Insulin resistance is present in obesity and in type 2 diabetes, and is associated with islet cell hyperplasia and hyperinsulinemia but the driving forces behind this compensatory mechanism are incompletely understood. Previous data have suggested the involvement of an unknown circulating insulin resistance-related ?-cell growth-factor. In this context, looking for candidates to be a circulating factor, we realized that hepatocyte growth factor (HGF) is a strong candidate as a link between insulin resistance and increased mass of islets/hyperinsulinemia. Our approach aimed to show a possible cause-effect relationship between increase in circulating HGF levels and compensatory islet hyperplasia/hyperinsulinemia by showing the strength of the association, whether or not is a dose-dependent response, the temporality, consistency, plausibility and reversibility of the association. In this regard, our data showed: a) a strong and consistent correlation between HGF and the compensatory mechanism in three animal models of insulin resistance; b) HGF increases ?-cell mass in a dose-dependent manner; c) blocking HGF shuts down the compensatory mechanisms; d) an increase in HGF levels seems to precede the compensatory response associated with insulin resistance, indicating that these events occur in a sequential mode. Additionally, blockages of HGF receptor (Met) worsen the impaired insulin-induced insulin signaling in liver of diet-induced obesity rats. Overall, our data indicate that HGF is a growth factor playing a key role in islet mass increase and hyperinsulinemia in diet-induced obesity rats, and suggest a protective effect of the HGF-Met axis on insulin signaling in the liver / Doutorado / Medicina Experimental / Doutor em Ciências

In pharmaceutical development cell cultures are used as in vitro models to evaluate the function of drug candidates. In such research it is vital to have models that resemble the in vivo environment to get reliable results. In 3D models with hydrogels ECM like scaffolds are supporting the cells in a more in vivo like environment than flat 2D cultures. In this project PEG-peptide based hydrogels with cell binding RGD incorporated on one PEG-peptide type has been evaluated for culturing of HepG2 cells. Structure and viscoelastic properties were evaluated with techniques like circular dichroism spectroscopy, dynamic light scattering and rheology. Sterilisation impact was also evaluated for PEG-peptides. For cell culturing, observations in light microscope and evaluation with Live/Dead assay and albumin assay were performed. A few companies were interviewed regarding 3D culturing and interest in mechanically tuneable hydrogels. The HepG2 cells grows and forms spherical clusters in the 3D environment with hydrogels, percentage of RGD seems to not impact cell adhesion, growth or albumin secretion. UV irradiation was the most suitable sterilisation method for gel components. The most rigid gel combination formed had storage modulus of around 230 Pa. Mechanically tuneable hydrogels is interesting for the industry. The PEG-peptide based gels are suitable tor growing cells but too soft to closely resemble the in vivo rigidity of hepatocytes.

Abstract The carbonic anhydrases (CAs) catalyse the hydration of CO2 to bicarbonate at physiological pH. This chemical interconversion is crucial since HCO3- is the substrate for several biosynthetic reactions. Carbonic anhydrases are involved in many physiological processes connected with respiration and transport of CO2/bicarbonate between metabolising tissues and the lungs, pH homeostasis and electrolyte secretion in a variety of tissues/organs. The present work was undertaken to study the distribution and expression of CA isoenzymes in the normal and neoplastic gastrointestinal tissues. The expression of CA I, II, IX and XII in the human intestine and colorectal tumours was investigated by immunohistochemistry and western blotting. In the present study, immunohistochemical methods were also used to examine the location of CA IX and XII in the human pancreas and pancreatic tumours. The expression of CA XIV in the murine liver and intestine was studied using immunostaining and northern blotting. The present results suggest that transmembrane CA XII is absent from the small intestine, but is expressed in all segments of the normal large intestine. The positive signal for CA XII was confined to the basolateral plasma membranes of the epithelial cells of the surface epithelial cuff. In tumours, the signal for CA XII became stronger in the deep part of the lesion. The intensity of the immunostaining for CA I and II was clearly found to decrease in benign lesions and became very weak in malignant colorectal tumours. The reciprocal pattern of expression observed for membrane-associated (CA IX and XII) and cytoplasmic (CA I and II) isoenzymes in intestinal samples suggests that CA IX and XII may be functionally involved in tumour progression to malignancy and/or in invasion. CA I and II, which are thought to play important physiological roles in the normal colorectal mucosa, may not be required for growth of colorectal cancers and their expression consistently diminishes with progression to malignancy. In the human pancreas CA IX and XII appeared to be sporadically expressed in the basolateral plasma membrane of the normal acinar and ductal epithelium. The increased expression of CA IX in hyperplastic ductal epithelium may contribute to the pancreatic tumourigenesis. CA XIV was expressed in the hepatocyte plasma membrane and its localization on both apical and basolateral membrane domains suggests an important role for this isoenzyme in the regulation of ion and pH homeostasis in the liver.

Le foie a des capacités de régénérations importantes qui lui permettent de reconstituer progressivement sa masse cellulaire suite à une agression. L’induction d’une régénération hépatique est une approche qui a été utilisée dans de nombreux modèles animaux afin de favoriser la prise de greffe hépatocytaire pour le traitement des maladies métaboliques héréditaires hépatiques (MMHH). Par ailleurs, les capacités de régénération du foie sont utilisées en pratique clinique courante dans le cadre de la chirurgie hépatique afin de préparer le foie à une hépatectomie majeure. Les principaux objectifs de ce travail ont été d’étudier des moyens peu invasifs pour induire une importante régénération hépatique dans deux buts précis : i) favoriser la transplantation d’hépatocytes thérapeutiques pour le traitement des MMHH ; ii) élargir les possibilités de prise en charge des patients nécessitant une hépatectomie.Dans un premier temps, nous avons évalué l’effet d’une embolisation portale partielle (EPP) au cours de la transplantation d’hépatocytes dans un des modèles animaux de l’hypercholestérolémie familiale de type IIA, le lapin Watanabe. Dans un deuxième temps, nous avons mis au point chez le rat, une technique d’EPP résorbable répétée visant à entrainer un stimulus répété de régénération hépatocytaire afin d’envisager par la suite l’utilisation de cette technique avant transplantation d’hépatocytes. En parallèle de ces travaux fondamentaux, nous avons évalué en approche clinique l’intérêt de l’EPP résorbable avant hépatectomie majeure. Nos travaux dans le modèle du lapin Watanabe ont montré la faisabilité du protocole, une correction phénotypique in vitro, une amélioration de la prise de greffe hépatocytaire et une expression prolongée du transgène. Notre équipe a développé chez le rat un stimulus additionnel de prolifération hépatocytaire qui permet une augmentation du poids et du volume du foie non embolisé en comparaison à une seule EPP résorbable. Enfin, dans une série rétrospective préliminaire, la technique d’EPP résorbable a été utilisée avant hépatectomie majeure. L’approche a été bien tolérée chez tous les patients et a permis de systématiquement envisager la chirurgie.L’EPP résorbable est une technique peu invasive capable d’induire une régénérative hépatique efficace. Cette approche pourrait permettre notamment d’augmenter les capacités de proliférations hépatiques par la répétition du stimulus d’embolisation. A terme, l’EPP résorbable répétée pourrait permettre de modeler à la demande l’organisation du volume hépatique favorisant ainsi l’hypertrophie de tel ou tel secteur en fonction des besoins. / The liver has an important regenerative capacity allowing progressive reconstitution of the hepatic volume after an aggression. The induction of liver regeneration was used in different animal models in order to increase engraftment during hepatocyte transplantation for the treatment of inherited metabolic liver diseases (IMLD). Furthermore, liver regenerative capacities are used in routine clinical practice before liver surgery in order to prepare the liver for major hepatectomy.The main objective of this doctoral thesis was to evaluate minimally invasive approaches inducing substantial liver regeneration, focusing in two specific aims: i) increasing the engraftment of therapeutic cells for the treatment of IMLD; ii) expanding the therapeutic options for patients requiring an hepatectomy In a first study, we evaluated the impact of a partial portal vein embolization (PVE) during hepatocyte transplantation in the animal model of familial hypercholesterolemia type IIA, the Watanabe rabbit. In a second investigation, we developed an approach of repeated reversible PVE in a rat model to further boost liver hypertrophy, planning to apply this approach in hepatocyte transplantation. In parallel, we evaluated the clinical interest of reversible PVE before major hepatectomy.Our results of PVE during hepatocyte transplantation in the Watanabe rabbit model demonstrated the feasibility of the procedure, in vivo phenotypic correction, increase of liver cell engraftment and stable transgene expression. Our team developed in the rat an additional stimulus of hepatocyte proliferation allowing increase of non-embolized liver lobe weight and volume in comparison to a single reversible PVE. Finally, the reversible PVE approach was evaluated before major hepatectomy in a preliminary retrospective series of 20 patients. The procedure was well tolerated and allowed to plan surgery in all patients.Reversible PVE is a minimally invasive technique allowing to successfully induce liver regeneration. This approach could increase hepatocyte proliferation capacity by using an additional stimulus of repeated embolization. In the future, reversible PVE may allow on demand modeling of liver volumes organization by supporting the hypertrophy of a specific liver lobe when required.

PCSK9 est un régulateur clé du métabolisme du cholestérol par le foie à travers la dégradation lysosomiale du récepteur aux LDL (low-density lipoprotein). Alors que les mutations gain de fonction (GOF) de PCSK9 induisent une hypercholestérolémie autosomique dominante, les mutations pertes de fonctions (LOF) entraînent un taux spontanément bas de LDL-cholestérol, ainsi qu’un protection cardiovasculaire. Du fait des limitations inhérentes aux modèles d’études, tels que les lignées cellulaires transfectées ou des animaux transgéniques, les fonctions de PCSK9 restent encore mal connues. Ainsi, nous avons utiliser des cellules souches pluripotentes induites (hiPSC) spécifiques de patients pour les différencier en hépatocytes et modéliser la physiopathologie liée aux mutations de PCSK9 GOF-S127R et LOF-R104C/V114A. Nous avons démontré que les hépatocytes obtenus récapitulaient la physiopathologie liés aux mutations de PCSK9. De plus, les cellules portant la mutation S127R ont montré une importante réponse au traitement par les statines, qui est corrélée à la réponse clinique des patients portant cette même mutation. Enfin, notre étude nous a permis de mettre à jour une fonction inattendue de PCSK9 dans les hiPSC et pendant leur différenciation. Elle montre que PCSK9 affecterait la prolifération des hiPSC ainsi qu’une voie de signalisation clé du développement régulée par NODAL. Cette régulation se ferait à travers une interaction directe entre PCSK9 et DACT2, un régulateur intracellulaire de la voie de signalisation de NODAL. En conclusion, les hiPSC s’avèrent être un modèle cellulaire translationnel pertinent pour mettre à jour de nouvelles fonctions hépatiques de PCSK9. / PCSK9 has been identified as a key regulator of cholesterol metabolism by the liver through inducing lysosomal degradation of the low-density lipoprotein receptor (LDLR). While PCSK9 gain-of-function (GOF) mutations induced autosomal dominant hypercholesterolemia and increased cardiovascular risk, loss-of-function (LOF) mutations are associated with low LDL-cholesterol levels and cardiovascular protection. Due to limitations inherent to current models including animal and human cells lines transfected with DNA constructs or transgenic animal models, PCSK9 functions are not fully understood. Therefore, we took advantage of patient related somatic cells reprogramming intoinduced pluripotent stem cells (hiPSC) to generate hepatocyte-like cells (HLC) and model the pathophysiology of PCSK9 mutations in dyslipidemia through focusing on two intracellular mutation forms; GOF (S127R) and LOF (R104C/V114A). We showed that HLC could recapitulate the key pathophysiological features of PCSK9 mutations. Moreover, HLC with the S127R mutation displayed an increased uptake of LDL upon statin treatment, which was correlated with the original patient clinical response. In parallel, this model enabled us to unravel a new unexpected role of PCSK9 in hiPSC and during differentiation. PCSK9 was found to affect the proliferation of hiPSC and regulate a key developmental signaling pathway mediated by NODAL. This regulation might occur by a direct interaction between PCSK9 and DACT2, an intracellular attenuator of NODAL signaling pathway. In conclusion, hiPSC provide a pertinent translational model to decipher PCSK9 hepatic functions and a novel cellular environment to highlight new functions.

Ebola virus (EBOV) infection causes a severe disease in humans and leads to widespread liver necrosis, a dysregulated cytokine response, and coagulopathy. However, little is known about the specific liver response to EBOV infection in humans. Here we present the utilization of an induced pluripotent stem cell (iPSC)-derived hepatocyte platform to define the host response to EBOV infection. We demonstrate that iPSC-derived hepatocytes are a suitable platform for investigating innate immune responses to viral infections. We compared the host response to EBOV infection in iPSC-derived hepatocytes, immortalized hepatocytes, and primary human hepatocytes and identified minimal transcriptomic changes 1 day post infection (dpi). Between 2-3 dpi, EBOV infection led to a significant upregulation of interferon-beta (IFN-β) and select interferon-stimulated genes (ISGs) in iPSC-derived hepatocytes. In addition, the acute phase response and coagulation cascade was downregulated in these hepatocytes, mimicking known liver dysfunction in EBOV disease. Using fluorescent in situ hybridization (RNA-FISH), we showed at single cell resolution that EBOV-infected iPSC-derived hepatocytes express IFN-β, indicating that infected cells mount an antiviral response to EBOV infection. This platform can be utilized to investigate therapeutic targets in human hepatocytes that may attenuate EBOV infection in patients. In addition, we present in this dissertation the development of a minigenome system for the filovirus Lloviu virus (LLOV). LLOV is closely related to EBOV and is known to circulate in bats throughout Europe. The complete sequence of LLOV has yet to be resolved, and therefore investigation of LLOV biology is limited. As part of this work, we established a functional LLOV minigenome system based on sequence complementation of other filoviruses. We demonstrate that the LLOV replication and transcription strategy is generally more similar to ebolaviruses than marburgviruses. We show that a single nucleotide at the 3ꞌ end of the LLOV genome determines specificity of the LLOV polymerase complex. This minigenome system can now be used to elucidate replication and transcription mechanisms employed by this novel filovirus. / 2023-02-19T00:00:00Z

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23368号 / 医博第4737号 / 新制||医||1051(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 妹尾 浩, 教授 稲垣 暢也 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM