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Etude de la réponse humorale neutralisante contre le Virus de l’Hépatite C / Study of the neutralizing antibody response against the hepatitis C virusNdongo Thiam, Ndiémé 11 February 2010 (has links)
Le virus de l’hépatite C (HCV) est l’agent responsable de l’hépatite C, maladie qui touche environ 3% de lapopulation mondiale. Une des caractéristiques de cette infection est son évolution dans 60 à 90% des casvers des formes chroniques avec des complications sévères telles que la cirrhose et le carcinomehépatocellulaire. Un des handicaps majeurs de la recherche sur le HCV est l’absence de systèmes decultures in vitro efficaces et de modèles animaux adaptés car le HCV n’infecte que l’homme et le chimpanzé.l’anticorps D32.10. Pour cela, nous avons développé un test de cellbindinget nous avons montré quel’interaction des particules virales sériques (HCVsp) radiomarquées à l’Iode 125 avec les celluleshépatocytaires (Huh‐7 et HepaRG) est spécifique et saturable impliquant des sites de haute et faible affinité.De plus, l’anticorps D32.10 est capable d’inhiber spécifiquement et efficacement les interactions de hauteaffinité entre les HCVsp et les cellules HepaRG avec une IC50 ≤ 0,5 μg/ml. Nous avons mis en évidence quel’inhibition est plus efficace lorsque nous utilisons sélectivement une population de particules HCVenveloppées exprimant fortement E1E2. Récemment, nous avons développé un système d’infection originaldes cellules HepaRG qui sont des cellules progénitrices du foie par les HCVsp et avons montré quel’infection, la réplication et la propagation dépendent de l’état de prolifération/différenciation de cescellules. Nous avons aussi démontré que les particules virales produites dans ce système contiennent del’ARN viral, expriment les protéines d’enveloppe E1E2 et sont infectieuses. Des études préliminairesmontrent que l’anticorps D32.10 inhibe fortement l’infection (95% à 80% aux jours 14 et 21 aprèsinfection) vraisemblablement au niveau des étapes précoces du cycle viral.Dans un second temps, nous avons recherché la prévalence des anticorps de même spécificité que le D32.10(anti‐E1E2A,B) dans différents groupes de patients HCV positifs afin de déterminer leur significationbiologique. Par un test ELISA utilisant les peptides biotinylés E1, E2A et E2B dans la phase de capture, nousavons démontré que la réponse anticorps anti‐E1E2A,B était présente dans 90% des cas chez les patientsqui guérissent spontanément avec des titres élevées (≥ 1/1000). Cette réponse humorale est absente ourare (< 10%) chez les patients porteurs chroniques non traités ou non répondeurs aux traitementsantiviraux. Une étude longitudinale a été réalisée chez des patients non répondeurs ou répondeursdéveloppant une réponse virologique soutenue à une bithérapie standard, interféron pégylé plus ribavirine.L’analyse statistique des résultats a montré que les anticorps anti‐E1E2A,B pouvaient être prédictifs de laréponse au traitement avec une spécificité et une valeur prédictive positive de 100%.La convergence des résultats in vitro et in vivo supporte un rôle neutralisant de l’anticorps monoclonalD32.10, permettant d’envisager son utilisation en immunothérapie. / Hepatitis C Virus (HCV) is the major etiological agent of liver disease in the world with approximately180 million people who are seropositive. The majority (60‐90%) of infected individuals progressesto chronic hepatitis that increases their risk for developing cirrhosis and hepatocellular carcinoma.One of the major limitations of HCV research is the lack of efficient in vitro culture systems andappropriateanimal models. vitro direct cell‐binding assay and an infection system of the human HepaRG cell line were developedby using HCVsp. The HepaRG cells possess potent ability to acquire a mature hepatocyte phenotype.The E1E2‐specific mAb D32.10 was shown to inhibit efficiently and specifically high affinityinteractionsthrough glycosaminoglycans and the CD81 tetraspanin between HCVsp and HepaRGcells with an IC50 = 0.5 μg/ml. This inhibition was more efficient when E1E2‐positive envelopedHCVsp were used selectively for binding studies (IC50 < 0.5 μg/ml). Establishment of infection,replication and propagation of HCVsp were shown to depend on the proliferation/differentiationstage of HepaRG cells. Persistent HCV infection in HepaRG cells could be obtained with production ofE1E2/RNA(+) infectious HCV particles. Preliminary data showed a complete early inhibitory effect ofthe D32.10 mAb on virion RNA production in HepaRG culture supernatants (95% at D14 and 80% atD21 post‐infection).Furthermore, the detection of the anti‐E1E2/D32.10‐binding peptide antibodies during natural HCVinfection demonstrated significant prevalence (90%) of these antibodies: (1) in patients whorecovered spontaneously from HCV infection with high titers compared to patients with chronichepatitis C, and (2) in patients who are complete responders compared to non responders toantivirals. Kinetic analyses revealed that the anti‐E1E2/D32.10‐like humoral response appeared veryearly with high titers (≥ 1/1000) and was associated with complete virus eradication. The positiveand negative predictive values (ROC curve analysis) for achieving or not a sustained viral response toantiviral therapy are 100% and 86%, respectively, reflecting diagnostic accuracy. The anti‐E1E2/D32.10‐binding peptide antibodies may thus predict the outcome of HCV infection andrepresent a new relevant pronostic marker in serum for the HCV diagnosis.Convergence of in vitro and in vivo data strongly support the neutralizing activity of the D32.10 mAb,and thus immunotherapeutic potential of this unique anti‐E1E2 D32.10 mAb.
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Caractérisation par microscopie électronique des étapes précoces de l'entrée du virus de l'hépatite C dans les hépatocytes / Unraveling the details of the entry of hepatitis C virus into hepatocytic cells by electron microscopy imagingPerrault, Marie 22 November 2010 (has links)
L'infection par le virus de l'hépatite C (HCV) reste aujourd'hui une cause majeure d'hépatite chronique, de cirrhose du foie et de carcinome hépatocellulaire. L'attachement cellulaire et l'entrée de HCV sont médiés par les protéines d'enveloppe E1 et E2. De nouveaux récepteurs ont été récemment identifiés mais l'entrée du virus dans les hépatocytes reste énigmatique et n'a jamais été visualisée. Nous avons tout d'abord caractérisé le modèle des pseudo-particules HCV (HCVpp) encryomicroscopie électronique en transmission (cryo-MET). Ce sont des particules sphériques de 100 nm de diamètre portant à leur surface E1 et E2. Nous avons ensuite visualisé l'entrée des HCVpp dans les hépatocytes en MET conventionnelle en utilisant des lignées d'hépatome et des hépatocytes primaires humains(PHH). Ces derniers maintiennent leur polarité en culture comme en témoigne la persistance de canalicules biliaires, tels que dans les hépatocytes natifs. Après synchronisation à 4°C avec les cellules, les HCVpp sont retrouvées liées aux prolongements cellulaires via des 'piliers', et sont ensuite internalisées à 37°C par endocytose dépendante de la clathrine. Ces 'piliers', actuellement en cours d'identification par immunomarquages, sont internalisés avec les HCVpp dans les hépatocytes au sein de vésicules de clathrine ; ce suivi est effectué par des approches de congélation haute pression et de tomographie électronique. Enfin, les évènements d'endocytose des HCVpp dans les PHH se sont avérés rares, avec une cinétique ralentie comparée aux lignées cellulaires. Ces études en MET soulignent l'importance d'utiliser un modèle cellulaire physiologique polarisé pour l'étude du mécanisme d'entrée de HCV. / Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Receptor recognition, cell binding and membrane fusion rely on HCV envelope proteins E1 and E2. New receptors were recently discovered; however HCV entry into hepatocytes remains largely unknown and has not yet been visualized. At first, we characterized HCV pseudoparticles (HCVpp) by cryo-transmission electron microscopy (cryo-TEM). They appeared as regular spherical structures of ca. 100-nm, with E1 and E2 at their surface. By conventional TEM, we then visualized HCVpp entry into hepatocytes, using hepatoma cells and primary human hepatocytes (PHH) as a more physiological cell model.PHH maintain their polarity in culture as attested by TEM observation of persistent bile canaliculi. At 4°C, viral particles were primarily found attached to microvilli at the cell surface via molecular bridges and, after warming to 37°C, they were internalized by endocytosis in clathrin-coated pits and vesicles. Using freeze substitution and electron tomographyapproaches, these bridges were found intimately surrounding HCVpp inside the clathrincoated vesicles, suggesting a concomitant internalization. The nature of these bridges is currently under investigation by immunogoldlabeling approaches. Finally, we reproducibly observed less HCVpp internalization events in PHH compared to hepatoma cells, and the kinetics of these events seemed delayed, probably due to PHH polarity. To conclude, ourTEM approach proved powerful to visualize HCV entry, and highlights the importance of studying a physiological cell model to understand HCV entry mechanism.
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PGC-1 alfa como regulador inflamatório na esteato-hepatite não-alcoólica / PGC-1 alpha in regulating inflammatory in nonalcoholic steatohepatitisBarroso, Wermerson Assunção 22 March 2016 (has links)
A doença hepática gordurosa não-alcoólica (NAFLD, do inglês) é a manifestação clínica hepática da síndrome metabólica, cuja incidência aumenta consideravelmente em todo o mundo. A NAFLD pode progredir para um estado de esteatohepatite não-alcoólica (NASH, do inglês), caracterizado por inflamação hepatocelular, com ou sem fibrose. Dados na literatura mostram que o coativador-1 alfa do receptor ativado por proliferadores de peroxissoma gama (PGC-1alfa), além de estar envolvido em diversos processos metabólicos, representa uma estratégia terapêutica promissora na modulação da inflamação. Neste projeto investigamos as alterações inflamatórias no fígado induzida por dieta hiperlipídica e o papel do PGC-1alfa nesse processo. Camundongos C57black/6 receberam dieta hiperlipídica contendo 30% de gordura por 10 semanas. O peso dos animais foi avaliado semanalmente. Após a eutanásia, o tecido adiposo intra-abdominal (retroperitoneal e periepididimal) foi coletado e pesado. Analisamos o perfil glicêmico e lipídico sérico e expressão de genes envolvidos no metabolismo glicêmico e lipídico. Avaliou-se também o aspecto histológico e a inflamação do tecido hepático por quantificação das citocinas IL-6, TNF-alfa e IL-1beta. A dieta rica em gordura conduziu a um aumento dos depósitos de gordura intra-abdominal, hiperglicemia e hiperlipidemia. Os animais também apresentavam esteatohepatite, com aumento de citocinas pró-inflamatórias e diminuição na expressão de PGC-1alfa no tecido hepático. O envolvimento do PGC-1alfa na produção de mediadores inflamatórios por hepatócitos foi avaliado em células HepG2 utilizando RNA de interferência (RNAi). O knockdown da expressão de PGC-1alfa causou aumento na expressão e liberação de IL-6 em hepatócitos via aumento na fosforilação de IkBalfa e consequente ativação do NFkB. Portanto, nossos dados mostram que o PGC-1alfa inibe a produção de mediadores inflamatórios (IL-6) em hepatócitos, e fornecem novas evidências das conexões existentes entre as vias metabólicas e imunes / Nonalcoholic fatty liver disease (NAFLD) is the liver clinical manifestation of the metabolic syndrome, whose incidence increases considerably around the world. NAFLD may progress to a state of non-alcoholic steatohepatitis, characterized by hepatocellular inflammation, with or without fibrosis. Data in the literature show that the peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1alfa), a protein involved in various metabolic processes, represents a promising therapeutic strategy in the modulation of inflammation. In this project, we investigate the inflammatory changes in the liver induced by high fat diet and the role of PGC-1alfa in this process. C57black/6 mice were fed a high fat diet containing 30% fat for 10 weeks. The weight of the animals was measured weekly. After euthanasia, the intra-abdominal adipose tissue (retroperitoneal and periepididymal) was collected and weighed. We have analyzed the glycemic and serum lipid profile and expression of genes involved in glucose and lipid metabolism. We also assessed liver morphology by HE staining and inflammation by quantification of the cytokines IL-6, TNF-alfa and IL-1beta. The high fat diet led to an increase in intra-abdominal fat deposits, hyperglycemia and hyperlipidemia. Animals also presented steatohepatitis, an increased proinflammatory cytokines and decreased PGC-1alfa expression in the hepatic tissue. The involvement of PGC-1alfa on inflammatory mediators production by hepatocytes was evaluated in HepG2 cells using RNA interference (RNAi). The knockdown of PGC-1alfa expression caused an increase in IL6 expression and release by hepatocytes via the increase in the IkBalfa phosphorylation and subsequent activation of NFkB. Therefore, our data show that PGC-1alfa inhibits the production of inflammatory mediators (IL-6) in hepatocytes and provide further evidence of the connections between the metabolic and immune pathways
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Hépatocytes matures dérivés de cellules souches in vitro : améliorer la différenciation des cellules souches pluripotentes induites humaines en copiant l’organogénèse hépatiqueM'Callum, Marie-Agnès 04 1900 (has links)
No description available.
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PGC-1 alfa como regulador inflamatório na esteato-hepatite não-alcoólica / PGC-1 alpha in regulating inflammatory in nonalcoholic steatohepatitisWermerson Assunção Barroso 22 March 2016 (has links)
A doença hepática gordurosa não-alcoólica (NAFLD, do inglês) é a manifestação clínica hepática da síndrome metabólica, cuja incidência aumenta consideravelmente em todo o mundo. A NAFLD pode progredir para um estado de esteatohepatite não-alcoólica (NASH, do inglês), caracterizado por inflamação hepatocelular, com ou sem fibrose. Dados na literatura mostram que o coativador-1 alfa do receptor ativado por proliferadores de peroxissoma gama (PGC-1alfa), além de estar envolvido em diversos processos metabólicos, representa uma estratégia terapêutica promissora na modulação da inflamação. Neste projeto investigamos as alterações inflamatórias no fígado induzida por dieta hiperlipídica e o papel do PGC-1alfa nesse processo. Camundongos C57black/6 receberam dieta hiperlipídica contendo 30% de gordura por 10 semanas. O peso dos animais foi avaliado semanalmente. Após a eutanásia, o tecido adiposo intra-abdominal (retroperitoneal e periepididimal) foi coletado e pesado. Analisamos o perfil glicêmico e lipídico sérico e expressão de genes envolvidos no metabolismo glicêmico e lipídico. Avaliou-se também o aspecto histológico e a inflamação do tecido hepático por quantificação das citocinas IL-6, TNF-alfa e IL-1beta. A dieta rica em gordura conduziu a um aumento dos depósitos de gordura intra-abdominal, hiperglicemia e hiperlipidemia. Os animais também apresentavam esteatohepatite, com aumento de citocinas pró-inflamatórias e diminuição na expressão de PGC-1alfa no tecido hepático. O envolvimento do PGC-1alfa na produção de mediadores inflamatórios por hepatócitos foi avaliado em células HepG2 utilizando RNA de interferência (RNAi). O knockdown da expressão de PGC-1alfa causou aumento na expressão e liberação de IL-6 em hepatócitos via aumento na fosforilação de IkBalfa e consequente ativação do NFkB. Portanto, nossos dados mostram que o PGC-1alfa inibe a produção de mediadores inflamatórios (IL-6) em hepatócitos, e fornecem novas evidências das conexões existentes entre as vias metabólicas e imunes / Nonalcoholic fatty liver disease (NAFLD) is the liver clinical manifestation of the metabolic syndrome, whose incidence increases considerably around the world. NAFLD may progress to a state of non-alcoholic steatohepatitis, characterized by hepatocellular inflammation, with or without fibrosis. Data in the literature show that the peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1alfa), a protein involved in various metabolic processes, represents a promising therapeutic strategy in the modulation of inflammation. In this project, we investigate the inflammatory changes in the liver induced by high fat diet and the role of PGC-1alfa in this process. C57black/6 mice were fed a high fat diet containing 30% fat for 10 weeks. The weight of the animals was measured weekly. After euthanasia, the intra-abdominal adipose tissue (retroperitoneal and periepididymal) was collected and weighed. We have analyzed the glycemic and serum lipid profile and expression of genes involved in glucose and lipid metabolism. We also assessed liver morphology by HE staining and inflammation by quantification of the cytokines IL-6, TNF-alfa and IL-1beta. The high fat diet led to an increase in intra-abdominal fat deposits, hyperglycemia and hyperlipidemia. Animals also presented steatohepatitis, an increased proinflammatory cytokines and decreased PGC-1alfa expression in the hepatic tissue. The involvement of PGC-1alfa on inflammatory mediators production by hepatocytes was evaluated in HepG2 cells using RNA interference (RNAi). The knockdown of PGC-1alfa expression caused an increase in IL6 expression and release by hepatocytes via the increase in the IkBalfa phosphorylation and subsequent activation of NFkB. Therefore, our data show that PGC-1alfa inhibits the production of inflammatory mediators (IL-6) in hepatocytes and provide further evidence of the connections between the metabolic and immune pathways
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MicroRNA profiling of human hepatocytes induced by HBx in hepatocarcinogenesis.January 2009 (has links)
Yip, Wing Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 100-119). / Abstract also in Chinese. / Abstract (English version) --- p.i / Abstract (Chinese version) --- p.iii / Acknowledgments --- p.v / Table of Contents --- p.vii / List of Tables --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xiii / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Hepatocellular Carcinoma --- p.1 / Chapter 1.1.1 --- Epidermiology --- p.1 / Chapter 1.1.2 --- Etiology --- p.1 / Chapter 1.2 --- Hepatitis B Virus --- p.3 / Chapter 1.2.1 --- The Epidermiology of Hepatitis B Virus Infection --- p.3 / Chapter 1.2.2 --- The Morphology and Genome of Hepatitis B Virus --- p.4 / Chapter 1.2.3 --- HBV Genotypes and Their Significance --- p.8 / Chapter 1.3 --- Hepatitis B Virus X Protein --- p.9 / Chapter 1.3.1 --- HBx Alters Various Signal Transduction Pathways --- p.10 / Chapter 1.3.2 --- HBx Interacts with Various Transcription Factors and Co-activators --- p.12 / Chapter 1.3.3 --- HBx Induces Epigenetic Alterations --- p.14 / Chapter 1.3.4 --- Identification of COOH-terminal Truncated HBx in Liver Tumors --- p.15 / Chapter 1.4 --- MicroRNAs --- p.17 / Chapter 1.4.1 --- Transcriptional Regulation and Biogenesis of MicroRNAs --- p.18 / Chapter 1.4.2 --- MicroRNAs and Cancer --- p.21 / Chapter 1.4.3 --- MicroRNAs and HCC --- p.25 / Chapter 1.5 --- Hypothesis and Aims of the Study --- p.29 / Chapter CHAPTER 2 --- MATERIALS and METHODS --- p.30 / Chapter 2.1 --- Patients --- p.30 / Chapter 2.2 --- Cell Lines --- p.30 / Chapter 2.3 --- Cloning of Various HBx Constructs --- p.32 / Chapter 2.3.1 --- PCR Amplification of HBx Fragments --- p.32 / Chapter 2.3.2 --- Cloning of HBx Fragments into TA-vectos --- p.33 / Chapter 2.3.3 --- Heat Shock Transformation --- p.33 / Chapter 2.3.4 --- Sub-cloning of HBx Fragments into Lentiviral Vectors --- p.34 / Chapter 2.4 --- Generation of Lentivirus --- p.37 / Chapter 2.4.1 --- Lentivirus Infection --- p.37 / Chapter 2.5 --- RNA Extraction --- p.38 / Chapter 2.6 --- Western Blot Analysis --- p.39 / Chapter 2.7 --- MiRNA Microarray --- p.40 / Chapter 2.7.1 --- Cyanine3-pCp Labeling of RNA Samples --- p.40 / Chapter 2.7.2 --- Sample Hybridization --- p.41 / Chapter 2.7.3 --- Microarray Wash --- p.41 / Chapter 2.7.4 --- Array Slide Scanning and Processing --- p.41 / Chapter 2.8 --- Detection of HBx Gene Deletion by PCR --- p.43 / Chapter 2.9 --- Immunohistochemistry --- p.44 / Chapter 2.10 --- Quantitative Real-time PCR --- p.45 / Chapter 2.11 --- Proliferation Assay --- p.47 / Chapter 2.12 --- Cell Cycle Analysis --- p.48 / Chapter 2.13 --- Annexin V Apoptosis Assay --- p.49 / Chapter 2.14 --- Colony Formation Assay --- p.50 / Chapter 2.15 --- Statistical Analysis --- p.51 / Chapter CHAPTER 3 --- RESULTS --- p.52 / Chapter 3.1 --- Detection of Full-length and COOH-terminal Truncated HBx in HCC Tissues --- p.52 / Chapter 3.2 --- Confirmation of HBx Expression in HCC Tissues --- p.55 / Chapter 3.3 --- Comparison of HBx from Different HBV Genotypes for Study --- p.61 / Chapter 3.4 --- Functional Characterization of COOH-tterminal Truncated HBx --- p.64 / Chapter 3.4.1 --- Selection of COOH-terminal Truncated HBx --- p.64 / Chapter 3.4.2 --- Generation of Various HBx-expressing Hepatocyte Cell Lines --- p.66 / Chapter 3.4.3 --- Effect of Full-length and COOH-terminal Truncated HBx on Cell Proliferation --- p.69 / Chapter 3.4.4 --- Effect of Full-length and COOH-terminal Truncated HBx Cell Cycle --- p.34 / Chapter 3.4.5 --- Effect of Full-length and COOH-terminal Truncated HBx on Apoptosis --- p.45 / Chapter 3.5 --- MicroRNA Profiling of Various HBx-expressing Hepatocyte Cell Lines --- p.76 / Chapter 3.5.1 --- Identification of Deregulated MicroRNAs by Microarray --- p.76 / Chapter 3.5.2 --- Validation of Deregulated MicroRNAs by Real-time PCR Analysis --- p.80 / Chapter 3.5.3 --- Confirmation of Deregulated MiRNAs in HCC and Adjacent Non-tumor Tissues --- p.84 / Chapter 3.5.4 --- Potential Downstream Targets of the HBx-deregulated MiRNAs --- p.87 / Chapter CHAPTER 4 --- DISCUSSION --- p.91 / Chapter 4.1 --- The Impact of COOH-terminal Truncated HBx in HCC --- p.91 / Chapter 4.2 --- The Biological Significance of COOH-terminal Truncated HBx Induced MiRNAs --- p.94 / Chapter 4.3 --- Limitations of the Present Study --- p.97 / Chapter 4.4 --- Future Studies --- p.98 / Chapter CHAPTER 5 --- CONCLUSION --- p.99 / REFERENCES --- p.100
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LKB1, gardien de la prolifération hépatocytaire et de l’intégrité génomique / LKB1, gatekeeper of hepatocyte proliferation and genomic integrityMaillet, Vanessa 28 November 2017 (has links)
La Liver Kinase B1 (LKB1) est une protéine pléiotrope, impliquée dans divers processus biologiques. Dans le foie, LKB1 est notamment connue pour être un régulateur clé du métabolisme et de la polarité cellulaire. Au cours de notre étude, nous avons investigué l’implication de LKB1 dans le contrôle de la prolifération des hépatocytes au cours du processus de régénération hépatique physiologique (hépatectomie partielle des 2/3). Nous avons démontré que la perte de Lkb1, spécifiquement dans les hépatocytes, favorise la récupération de la masse hépatique après hépatectomie partielle, en induisant une augmentation drastique de la réponse proliférative hépatocytaire, indépendamment de la balance métabolique/énergétique. Ainsi, LKB1 agit comme un senseur négatif de la prolifération et régule la transition G0/G1, en particulier en contrôlant la signalisation de l’EGFR (Epidermal Growth Factor Receptor). Par ailleurs, plus tard pendant la régénération, LKB1 garantit également l’intégrité mitotique. En effet, la suppression de Lkb1 entraîne des altérations majeures de la formation du fuseau mitotique. Nos résultats établissent également que LKB1 contrôle la polarité de la division cellulaire, indépendamment de l'activité de l’AMPK (AMP-activated protein kinase), une cible clé de LKB1. Par conséquent, la perte de LKB1 conduit à une altération majeure du profil de ploïdie, au stade tardif du processus de régénération. L’ensemble de notre étude souligne le double rôle de LKB1, au cours de la régénération hépatique, en tant que gardien de la prolifération hépatocytaire et de l'intégrité génomique. / Liver Kinase B1 (LKB1) is involved in pleiotropic biological processes and known to be a key regulator of hepatic metabolism and polarity. Here, we investigated the contribution of LKB1 in hepatocyte proliferation and liver regeneration process. We demonstrated that loss of hepatic Lkb1 promotes liver mass recovery, through an increase of hepatocytes proliferation, independently on metabolic/energetic balance. LKB1 regulates G0/G1 progression, specifically by controlling Epidermal Growth Factor Receptor (EGFR) signaling. In addition, later during regeneration, LKB1 controls mitotic fidelity. Deletion of Lkb1 results in major alterations of mitotic spindle formation, along the polarity axis, independently of AMP- activated protein kinase (AMPK) activity, a key target of LKB1. Consequently, LKB1 deficiency leads to an alteration of ploidy profile, at late stage of regenerative process. Overall our study highlights the dual role of LKB1, during liver regeneration, as a guardian of hepatocyte proliferation and genomic integrity.
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Hinweise auf Reduktion von Steatosis hepatis durch Metformin in vitroSchramm, Stefanie 04 January 2013 (has links) (PDF)
Die Arbeit beschäftigt sich mit dem Problem der Fettlebererkrankung. In der Einleitung wird auf die aktuelle Relevanz der Gesundheitsstörung und Therapiemöglichkeiten eingegangen, insbesondere durch das, in der Therapie des Diabetes mellitus Typ 2 gebräuchliche Biguanid Metformin. Der Bezug zu molekularbiologischen Signalwegen wird hergestellt und verschiedene in vitro Modellsysteme werden vorgestellt.
Anschließend wird auf die Herkunft und genetische Besonderheiten der verwendeten primären Maushepatozyten und Hepatomzellen eingegangen, bevor die angewandten Methoden vorgestellt werden. Zum Einsatz kam in dieser Arbeit vor allem die Lipidmessung mittels Fettrot, um das Ausmaß an Steatosis quantifizierbar zu machen.
Im Ergebnisteil folgen zuerst Versuche zur Zytotoxizität der einzelnen Chemikalien und deren Einfluss auf intrazelluläre Energieniveaus, bevor der Einfluss auf die hepatozellulären Fetteinlagerungen im Detail untersucht wird. Unterstützt werden die Ergebnisse durch mikroskopische Bilder der Hepatozyten, welche die beschriebenen Effekte verdeutlichen.
Insgesamt konnten folgende Thesen aufgestellt werden:
• Zwischen primären Hepatozyten von Wildtyp- und Knockout-Mäusen, bestehen nach 24 stündiger Kultivierung Unterschiede bezüglich des intrazellulären Lipidgehaltes, welche sich nach 72 stündiger Kultivierungszeit nivellieren.
• Metformin- und Fructoseinkubation senken den intrazellulären ATP-Gehalt, gleichzeitige Anwesenheit von Metformin und Glucose vermindern den Effekt.
• Durch 72-stündige Inkubation der primären Hepatozyten und Behandlung mit Metformin konnte der intrazelluläre Lipidgehalt um circa 40% gesenkt werden.
• Durch 72-stündige Inkubation der primären Hepatozyten mit Glucose konnte der intrazelluläre Lipidgehalt um circa 100% gesteigert werden.
• Bei humanen Hepatomzellen (HuH7) konnte kein Metformin- und kein Glucoseeffekt beobachtet werden.
• Der LXR-Agonist TO901317 wirkt auf den intrazellulären Lipidgehalt Metformin entgegen.
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The study of glucose-6-phosphate dehydrogenase (G6PD) gene regulation in HepG2 cells by glucose induction and the study of G6PD mRNA localization by fluorescent in situ hybridization (FISH)Griffith, Brian Nelson. January 2002 (has links)
Thesis (M.S.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains viii, 100 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 80-96).
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Pro- and anti-apoptotic roles of map kinase signaling in liver exposed to alcoholLee, Youn Ju, January 2003 (has links)
Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 172-205). Also available on the Internet.
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