The liver regulates inflammation during sepsis, and most liver functions are carried out by hepatocytes. Bacterial lipids, including lipopolysaccharide (LPS) and lipoteichoic acid (LTA), can be cleared by hepatocytes, but the underlying mechanisms are uncertain. Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates uptake of LPS by hepatocytes, but it is unknown whether LTA uptake is similarly regulated. Therefore, our objectives were to characterize the PCSK9-regulated pathway of bacterial lipid uptake by hepatocytes by identifying whether low-density lipoprotein (LDL) receptor (LDLR) and LDLR-related protein 1 (LRP1) are the target receptors, and by determining which lipoproteins are involved. To study this pathway, we assessed the uptake of fluorescently-labeled LPS or LTA by human HepG2 hepatocytes using flow cytometry. We pre-treated HepG2 cells with PCSK9, alone or in combination with anti-LDLR or anti-LRP1 antibodies, in order to identify the PCSK9-regulated receptors that are involved, and utilized media containing normal serum or lipoprotein-deficient serum to investigate the lipoprotein- dependence of this pathway. We also determined the roles of LDL and HDL in bacterial lipid uptake through a series of add-back experiments to lipoprotein-deficient serum, and blocked LDLR to confirm that LDLR mediates LDL-dependent uptake. The HepG2 cell response to variable degrees of bacterial lipid uptake was also assessed in a subset of experiments by measuring several cytokines and extracellular alanine aminotransferase (ALT) activity in the cell culture supernatant. We found that PCSK9 regulates LDLR-mediated uptake of both LPS and LTA through an LDL-dependent mechanism, while LRP1 is not involved. Increased bacterial lipid uptake did not result in any hepatocellular injury or cytokine production, as measured by ALT activity and interleukin (IL)-6, IL-8, IL-10, and IL-17 concentrations. In conclusion, we completed our objective of characterizing the PCSK9-regulated pathway of bacterial lipid uptake, and provide supporting evidence for targeting PCSK9 as a novel therapeutic avenue in sepsis. / Thesis / Master of Science (MSc) / Bacterial compounds stimulate inflammation that can be overwhelming during sepsis. Understanding the processes behind uptake and clearance of these compounds may lead to better sepsis treatments. Therefore, our goal was to understand how uptake of two bacterial compounds, lipopolysaccharide and lipoteichoic acid, occurs by liver cells called hepatocytes. Hepatocytes are naturally equipped to clear foreign compounds, so understanding their role in clearing bacterial compounds is important. Another goal was to identify the role of the protein PCSK9 in this uptake process, as treatments targeting PCSK9 could be applied to sepsis once we understand its role in this disease. Our research demonstrates the negative role of PCSK9 in regulating uptake of lipopolysaccharide and lipoteichoic acid through a lipoprotein receptor called LDLR, and identifies the role of lipoproteins in this process. These findings further our understanding of the hepatocyte response to bacterial compounds in relation to sepsis, and identify PCSK9 as a potential target for new sepsis therapies.

The overall objective of this research was to understand the mechanims by which growth hormone (GH) regulates insulin-like growth factor-I (IGF-I) gene expression in liver. Previous studies have suggested that GH regulation of IGF-I gene expression in liver is mediated by binding of the transcription factor signal transducer and activator of transcription (STAT) 5 to four binding sites located distantly from the IGF-I promoter. The first specific objective of this research was to determine whether additional STAT5 binding sites were involved in GH stimulation of IGF-I gene expression in liver. Sequence analysis of 170 kb of mouse genomic DNA revealed nineteen consensus STAT5 binding sequences corresponding to fourteen ~200 bp chromosomal regions that were conserved in the corresponding human DNA sequence. Eight of these chromosomal regions were able to mediate STAT5 activation of reporter gene expression in cotransfection experiments. Two of these chromosomal regions corresponded to those previously identified. Gel-shift assays indicated that the eight new STAT5 binding sites and three of the four previously identified STAT5 binding sites could bind GH-activated STAT5 from mouse liver. Together, these results suggest that GH stimulation of IGF-I gene transcription in the mouse liver may be mediated by at least eleven STAT5 binding sites located distantly from the IGF-I promoter. In a previous study, I found that liver expression of liver-enriched transcription factor hepatocyte nuclear factor 3γ (HNF-3γ) was increased by GH in cattle. Therefore, the second specific objective of this research was to determine how GH stimulates HNF-3γ gene expression and whether the increased HNF-3γ mediates GH stimulation of IGF-I gene expression in bovine liver. Sequence analysis of the bovine HNF-3γ promoter revealed the presence of two putative binding sites for STAT5. The proximal putative STAT5 binding site appears to be conserved in other mammals. Chromatin immunoprecipitation (ChIP) assays demonstrated that GH increased the binding of STAT5 to the HNF-3γ promoter in bovine liver and that this binding was associated with increased HNF-3γ expression. Gel-shift assays demonstrated that the proximal STAT5 binding site in the HNF-3γ promoter could bind GH-activated STAT5 from bovine liver. Cotransfection analyses showed that the proximal STAT5 binding site was necessary for the HNF-3γ promoter to be activated by GH. The promoter of the bovine IGF-I gene contains three putative HNF-3 binding sites that seem to be evolutionarily conserved. ChIP assays indicated that GH stimulated the binding of HNF-3γ to the IGF-I promoter in bovine liver. Gel-shift assays showed that one of the putative HNF-3 binding sites could bind HNF-3γ protein from bovine liver. Co-transfection analyses demonstrated that this HNF-3 binding site was necessary for HNF-3γ activation of reporter gene expression from the IGF-I promoter. In summary, the results of this dissertation research suggest that GH-activated STAT5 directly stimulates IGF-I gene transcription in liver by binding to at least eleven distantly located STAT5 binding sites in the IGF-I locus and indirectly stimulates IGF-I gene transcription by enhancing HNF-3γ gene expression in the liver. / Ph. D.

La maladie de Wilson est une maladie héréditaire due à un déficit du transporteur du cuivre, l’ATP7B. Cette maladie se présente sous forme d’insuffisance hépatique aiguë ou chronique, pour lesquels le traitement médical actuel consiste en l’administration d’agents chélateurs, ce qui ne résulte cependant pas en une guérison complète de la maladie. La transplantation orthotopique du foie est le seul traitement définitif actuellement, avec tous les désavantages qu’elle comporte. Un traitement alternatif à cette option est donc souhaitable. Cette étude porte sur la faisabilité de la transplantation d’hépatocytes chez le modèle animal de la maladie de Wilson, le rat Long Evans Cinnamon (LEC), avec pour buts d’en déterminer la sécurité et l’efficacité tant sur le plan clinique (amélioration de la survie, prévention de l’hépatite) que pathologique. Douze rats LEC ont reçu une injection intrasplénique de 2,6 x 105 – 3,6 x 107 hépatocytes prélevés chez des rats donneurs de souche LE. Ils ont été suivis durant 6 mois puis sacrifiés. Ils ont ensuite été comparés à un groupe contrôle de douze autres rats LEC. Aucune différence significative n’a été notée au niveau du poids, du bilan hépatique et des concentrations de cuivre biliaire et hépatique. Cependant, une amélioration de l’activité oxydase de la céruloplasmine post-transplantation a été démontrée chez le groupe de rats transplantés (49,6 ± 31,5 versus 8,9 ± 11,7). Les rats transplantés ont aussi eu une amélioration sur tous les critères histologiques étudiés. Enfin, l’ARNm de l’atp7b a été retrouvé chez 58% des rats transplantés avec un taux d’expression de 11,9% ± 13,6 par rapport à un rat LE normal. L’immunohistochimie a quant à elle démontré la présence de l’atp7b chez tous les rats transplantés. Les résultats obtenus sont considérés favorables à ce traitement alternatif, et indiquent que la transplantation d’hépatocytes est une technique sécuritaire qui peut contribuer à renverser le processus pathologique en cours dans la maladie de Wilson. / Wilson’s disease (WD) is a hereditary metabolic disease caused by a deficiency of copper-transporting ATP7B, resulting in copper accumulating to toxic levels in the liver. Its manifestations range from acute or chronic hepatic insufficiency to fulminant liver failure. The mainstay of therapy is the use of chelating agents. However selected patients may also require orthotopic liver transplantation (OTL), an invasive and complex procedure with life-long implications. Hepatocyte transplantation is an appealing alternative to OLT. Its safety and efficacy were evaluated in the animal model of WD, the Long Evans Cinnamon (LEC) rat. Twelve LEC rats received an intrasplenic injection of 2,6 x 105 – 3,6 x 107 hepatocytes obtained from LE donor rats. They were followed for 6 months before sacrifice. They were then compared to a control group of twelve rats. No difference was found when comparing their weights, biochemical parameters such as liver function tests and bilirubin, as well as their biliary and hepatic copper concentrations. However, the ceruloplasmin oxydase activity was improved in the transplanted rats (49,6 ± 31,5 versus 8,9 ± 11,7). After sacrifice, histologic evaluation and demonstration of atp7b mRNA in the recipient liver were performed. There was evidence of histological improvement and atp7b mRNA was found in 58% of transplanted rats with an expression of 11,9% ± 13,6 when compared to a normal LE rat. Evidence of successful engraftment of the transplanted cells was found in every transplanted rat using the technique of immunohistochemistry. These encouraging results are in accordance with previous studies on hepatocyte transplantation in the LEC rat. Its application to the human clinical setting is the next step, as it has already been tried in other metabolic liver diseases.

Le récepteur à activité tyrosine kinase MET et son ligand le facteur de croissance des hepatocytes (Hepatocyte Growth Factor/Scattor Factor (HGF/SF)) sont essentiels pour la migration, la morphogenèse et la survie des cellules épithéliales. En plus de son implication et son importance physiologiques, la dérégulation de la signalisation de MET favorise la progression et l’invasion tumorales dans plusieurs types de cancers. Au sein des tumeurs, l’hypoxie est également un phénomène crucial qui induit une réponse adaptative menant à l’invasion, la métastase cancéreuses et la résistance aux traitements.Nous avons démontré que dans des conditions hypoxiques, la phosphorylation de MET induite par sa liaison au ligand, des mutations activatrices ou sa surexpression, est diminuée de manière importante in vitro et in vivo dans des modèles de tumeurs expérimentales chez la souris. Cette baisse de phosphorylation est très rapide et est réversible quand les cellules sont replacées en normoxie. Alors que la phosphorylation de GAB1, principal partenaire de MET, est également diminuée en hypoxie, l’activation des voies de signalisation en aval AKT et ERK n’est pas affectée et reste bien dépendante de l’activité du récepteur et du recrutement de GAB1. De la même façon, l’HGF/SF induit des réponses de motilité, de dispersion, de morphogenèse et de survie similaires en normoxie et en hypoxie. De manière intéressante, le traitement par deux inhibiteurs de tyrosine kinase (ITK) ciblant MET (PHA-665752 et SU11274) est moins efficace en hypoxie pour inhiber les voies de signalisation AKT et ERK ainsi les réponses cellulaires induites par MET. Comme pour la phosphorylation de MET, la résistance à ces ITK est un phénomène réversible. Ainsi, alors que l’hypoxie n’affecte pas les voies de signalisation en aval ni les effets biologiques, elle diminue la sensibilité de MET aux ITK induisant donc une résistance immédiate. L’ensemble de ces données pourrait fournir de nouvelles perspectives dans l’utilisation des thérapies ciblant MET dans les tumeurs solides. / The receptor tyrosine kinase MET and its ligand the Hepatocyte Growth Factor/Scattor Factor (HGF/SF) are essential for migration, morphogenesis and survival of epithelial cells. Beside its physiological involvement, deregulation of MET signaling has been shown to promote tumor progression and invasion in many cancers. Inside the tumors, hypoxia is also a crucial phenomenon promoting an adaptive response able to induce invasion, metastasis and resistance to treatment.We show that under hypoxia, MET phosphorylation induced by ligand-stimulation, activating mutation or overexpression, is drastically decreased both in cell culture and in experimental tumors. This decrease in MET phosphorylation occurs within minutes and is reversible when cells are returned to normoxia. While phosphorylation of the proximal signaling adaptor GAB1 is also decreased in hypoxia, activation of the downstream kinases ERK and AKT is not affected, but is still dependent on MET receptor activity. Consistently, several cellular responses induced by HGF/SF, including motility, morphogenesis or survival, are still efficiently induced. Interestingly, treatment with two tyrosine kinase inhibitors targeting MET (PHA-665752 and SU11274) are less efficient to inhibit the downstream kinases ERK and AKT and cellular responses induced by MET in hypoxia compared to normoxia. Similarly to MET phosphorylation, this resistance to TKI is a reversible phenomenon. Therefore, while hypoxia does not affect downstream signaling and cellular responses, it decreases MET sensitivity to TKIs targeting the receptor thus providing an immediate resistance. This may provide new insights in the use of MET targeted therapies in solid tumors.

A matriz extracelular serve como depósito para fatores biologicamente ativos, como fatores de crescimento e proteases, os quais influenciam no comportamento das células tumorais. A ADAMTS-1 (uma desintegrina e metaloproteinase com motivos trombospondina) é um membro da família de metaloproteases ADAMTSs. Neste trabalho, avaliamos o papel da ADAMTS-1 na regulação das atividades estimuladas pelo HGF ou TGF-β1, sobre as células de fibrossarcoma (HT1080). A superexpressão de ADAMTS-1 afetou a proliferação e a velocidade de migração das células HT1080 estimuladas por HGF, mas não por TGF-β1. Demonstramos que a superexpressão da ADAMTS-1 diminuiu a fosforilação do receptor c-Met e das vias downstream ERK1/2 e FAK. Adicionalmente, na presença do HGF, a superexpressão de ADAMTS-1 perturbou a formação de fibrossarcosferas in vitro e microtumores in vivo. Esses microtumores e células individuais apresentaram características morfológicas de lesões menos invasivas. Nossos dados sugerem que a ADAMTS-1 regula as atividades estimuladas pelo HGF no fibrossarcoma. / The extracellular matrix serves as a reservoir for biologically active factors, such as growth factors and proteases that influence the tumor cell behavior. ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin motifs) is a member of the ADAMTS family of metalloproteases. Here, we addressed the role played by ADAMTS-1 regulating HGF and TGF-β1 activities in fibrosarcoma cell line (HT1080). ADAMTS-1 overexpression affected the proliferation and migration velocity of HT1080 cells, after stimulation with HGF. However, ADAMTS-1 overexpression failed to affect TGF-β1 activity. We showed that ADAMTS-1 overexpression decreased the phosphorylation of c-Met receptor and downstream signaling pathways ERK1/2 and FAK. Additionally, in presence of HGF, ADAMTS-1 overexpression disrupted the formation of fibrosarcospheres in vitro and microtumors in vivo. These microtumors and individual cells presented characteristics of low invasive tumor cells (rounded morphology). Our results suggest that ADAMTS-1 is involved in regulating HGF-related functions on fibrosarcoma cells.

A interação entre células tumorais e células estromais tem um papel central na progressão neoplásica. As células estromais mesenquimais multipotentes (MSCs) podem se integrar ao microambiente tumoral onde modulam o crescimento dos tumores por meio de distintos mecanismos. Entretanto, pouco se sabe sobre o papel das MSCs na metástase, a principal causa de morte em pacientes com câncer. Utilizando um modelo de melanoma murino ortotópico, nós demonstramos que MSCs obtidas da medula óssea de camundongos (MO-MSCs) ocupam o nicho perivascular nos tumores primários e aumentam 2,5 vezes a incidência de micrometástases pulmonares quando co-infundidas com células de melanoma B16. Observamos ainda que o meio condicionado das MO-MSCs não altera o potencial de colonização pulmonar das células B16 infundidas sistemicamente. Isto indica que as MO-MSCs modulam as fases iniciais da cascata metastática, durante a qual ocorrem os processos de invasão e intravasão nos vasos sangüíneos. Em correlação com estes efeitos pró-metastáticos, o secretoma das MO-MSCs induziu a transição epitélio-mesenquimal (EMT) nas células de melanoma in vitro. Após cultivo em meio condicionado das MO-MSCs, as células B16 adquiriram uma morfologia evidentemente fibroblástica. Ao mesmo tempo, houve o rearranjo dos filamentos de actina e o aumento da expressão de marcadores mesenquimais como fibronectina, vimentina, FSP1, N-caderina e ZEB2, acompanhado da repressão transcricional de E-caderina. A ativação da EMT pelo secretoma das MO-MSCs resultou na aquisição de propriedades metastáticas nas células de melanoma. Após cultivo em meio condicionado de MO-MSCs, as células B16 tiveram seu potencial de ancoragem à fibronectina reduzido, ao passo que houve o aumento na mobilidade e no potencial de invasão em matrizes tridimensionais. Utilizando inibidores competitivos de ATP contra o receptor tirosina-cinase Met, demonstramos que a aquisição de todas as propriedades metastáticas avaliadas e a ativação da EMT nas células de melanoma é mediada pela ativação da via HGF/Met. Estes dados destacam o papel das MOMSCs no microambiente tumoral como fonte perivascular de moléculas indutoras da EMT, cuja ativação leva a aquisição de traços metastáticos nas células de melanoma. Além disso, a inibição da via HGF/Met pode neutralizar os efeitos das MO-MSCs sobre as células tumorais, contribuindo para a repressão de propriedades fundamentais que sustentam a progressão e a disseminação neoplásica. Estas informações são importantes para o desenvolvimento seguro das MO-MSCs como ferramenta terapêutica e demonstram a importância da sinalização entre MSCs e células tumorais na disseminação metastática. Mais especificamente, estas observações reforçam a inibição da via HGF/Met como uma abordagem promissora para o tratamento da metástase. / The crosstalk between tumor cells and stromal cells can profoundly impact tumor progression. Multipotent mesenchymal stromal cells (MSCs) have been reported to integrate the tumor microenvironment where they are described to modulate tumor growth by distinct mechanisms. However, little is known about the impact of MSCs on metastasis, the main cause of death in patients with cancer. Using an orthotopic mouse melanoma model, we showed that mouse bone marrow-derived MSCs (BMMSCs) occupy the perivascular niche within primary tumors and increased by 2.5-fold the incidence of lung micrometastases after co-infusion with B16 melanoma cells. Also, MO-MSCs conditioned medium did not affect the lung colonization ability of systemically infused B16 cells. This indicates that MO-MSCs induces the initial steps of the metastatic cascade, during which the invasion and intravasion occurs. Correlating with these metastatic effects, the BM-MSCs\' secretome activated the epithelial-to-mesenchymal transition (EMT) in B16 cells in vitro. After culture in BMMSCs\' conditioned medium, B16 cells acquired an evident fibroblastic morphology. Simultaneously, we observed the rearrangement of actin filaments and the upregulation of mesenchymal markers such as fibronectin, vimentin, FSP1, Ncadherin and ZEB2. In agreement with the loss of epithelial phenotype, BM-MSCs\' secretome also suppressed E-cadherin expression in B16 cells. The activation of EMT by BM-MSCs leaded to the acquisition of metastatic traits in melanoma cells. After culture in BM-MSCs\' conditioned medium, B16 cells displayed reduced anchorage to fibronectin and increased motility and invasiveness in threedimensional matrix plugs. Inhibition of Met receptor with competitive ATP inhibitors demonstrated that the induction of EMT and the resultant acquisition of metastatic traits are driven by activation of HGF/Met signaling pathway. Taken together, these evidences highlight the role of BM-MSCs as a perivascular source of EMT-inductive signals, whose activation leads to acquisition of metastatic traits in melanoma cells. Furthermore, inhibition of HGF/Met signaling pathway can neutralize the effects of BM-MSCs on tumor cells, thereby allowing the repression of fundamental properties which support tumor progression and metastasis. This information is useful to safely develop BM-MSCs as therapeutic tool and demonstrate the relevance of the signaling between MSCs and tumor cells during metastasis. More specifically, it reinforces that inhibition of Met signaling can be a promissory approach for the treatment of metastasis.

Volumetric muscle loss (VML) typically results from traumatic incidents; such as those presented from combat missions, where soft-tissue extremity injuries account for approximately 63% of diagnoses. These injuries lead to a devastating loss of function due to the complete destruction of large amounts of tissue and its native basement membrane, removing important biochemical cues such as hepatocyte growth factor (HGF), which initiates endogenous muscle regeneration by recruiting progenitor cells. Clinical strategies to treat these injuries consist of autologous tissue transfer techniques, requiring large amounts of healthy donor tissue and extensive surgical procedures that can result in donor site morbidity and limited functional recovery. As such, there is a clinical need for an off-the-shelf, bioactive scaffold that directs patient’s cells to align and differentiate into muscle tissue in situ. In this thesis, we developed fibrin microthreads, scaffolds composed of aligned fibrin material that direct cell alignment along the longitudinal axis of the microthread structure, with specific structural and biochemical properties to recreate structural cues lost in VML injuries. We hypothesized that fibrin microthreads with an increased resistance to proteolytic degradation and loaded with HGF would enhance the functional, mechanical regeneration of skeletal muscle tissue in a VML injury. We developed a crosslinking strategy to increase fibrin microthread resistance to enzymatic degradation, and increased their tensile strength and stiffness two- to three-fold. This crosslinking strategy enhanced the adsorption of HGF, facilitated its rapid release from microthreads for 2 to 3 days, and increased the chemotactic response of myoblasts twofold in 2D and 3D assays. Finally, we implanted HGF-loaded, crosslinked (EDCn-HGF) microthreads into a mouse model of VML to evaluate tissue regeneration and functional recovery. Fourteen days post-injury, we observed more muscle ingrowth along EDCn-HGF microthreads than untreated controls, suggesting that released HGF recruited additional progenitor cells to the injury site. Sixty days post-injury, EDCn-HGF microthreads guided mature, organized muscle to replace the microthreads in the wound site. Further, EDCn-HGF microthreads restored the contractile mechanical strength of the tissue to pre-injured values. In summary, we designed fibrin microthreads that recapitulate regenerative cues lost in VML injuries and enhance the functional regeneration of skeletal muscle.

Introdução: O fígado é um órgão chave na manutenção da homeostasia corpórea e o transplante hepático ainda continua sendo o padrão-ouro no tratamento da insuficiência hepática aguda. A falta de doadores tem favorecido o desenvolvimento da terapia celular. Células derivadas de medula óssea podem se diferenciar em células tipo-hepatócitos em menos de 24 horas e a comunicação através de vesículas extracelulares (VEs) é um dos mecanismos propostos para explicar essa capacidade. Muitos estudos têm demonstrado que as células-tronco mesenquimais (CTMs) da medula tem alta plasticidade para se diferenciar em hepatócitos, mas os protocolos normalmente utilizados levam entre 7 e 28 dias. Objetivo: Analisar a capacidade de diferenciação das CTMs da medula em se tornar uma célula tipo-hepatócito através do mecanismo de comunicação celular por VEs em cultura (6 e 24 horas). Materiais e métodos: Para avaliar o efeito de hepatócitos primários isolados de ratos saudáveis e lesados com CCl4 na diferenciação das CTMs foi utilizado um sistema de co-cultivo com insertos que não permitem o contato entre as células colocando as CTMs na câmera superior e os hepatócitos na câmera inferior do sistema. Meio condicionado de hepatócitos com lesão foi utilizado para avaliar a capacidade das CTMs de capturar VEs e se diferenciar em célula-tipo hepatócito. Os marcadores de célula tipo-hepatócito avaliados foram expressão gênica (alfa fetoproteina, albumina e citoqueratina-18), armazenamento de glicogênio e liberação de ureia. Para rastrear VEs, hepatócitos de ratos lesados foram marcados com PKH-26. As VEs foram obtidas por ultracentrifugação do sobrenadante e analisadas por citometria de fluxo. Hepatócitos e CTMs também foram analisados por citometria de fluxo na busca de marcação positiva. Resultados: CTMs co-cultivadas durante 6 e 24 horas com hepatócitos não apresentaram expressão de genes hepáticos, mesmo quando expostas a um ambiente de lesão. Os ensaios funcionais confirmaram a falta de sinais de diferenciação, sendo que não foi observado armazenamento de glicogênio nem liberação de ureia nas CTMs. Um achado interessante foi que ao analisar o sobrenadante da câmera superior do sistema de co-cultivo, não foram achadas VEs marcadas com PKH-26 nem CTMs com rastros do marcador. Por outro lado, os experimentos utilizando meio condicionado mostraram que as CTMs têm capacidade de capturar VEs. A citometria de fluxo mostrou que às 6 horas e 24 horas respetivamente 2,28% e 3,97% das CTMs eram positivas para o marcador PKH-26. Quando analisadas no microscópio de fluorescência, foram vistos pontos vermelhos nas CTMs alguns dos quais parecem carregar a proteína albumina. Porém a expressão gênica e analise de ureia não se adequaram a um perfil de célula tipo-hepatócito. Conclusões: O sistema de co-cultivo não foi adequado para permitir a transferência e comunicação através de VEs entre hepatócitos e CTMs sendo que as VEs não conseguem atingir a câmara superior. Os experimentos com meio condicionado sugerem que as CTMs têm capacidade de capturar VEs derivadas de hepatócitos, porém a captação não conduz ao desenvolvimento de um perfil de célula tipo-hepatócito em 6 e 24 horas. São necessários mais estudos para esclarecer a dinâmica de transferência das VEs e suas consequências em longo prazo. / Introduction: Liver is a key organ for corporeal homeostasis maintenance and whole organ replacement still remains the gold standard procedure to treat acute liver failure. Shortage of liver donor has promoted the increase on cell-therapy research. Bone marrow (BM) derived cell have shown potential for differentiation into hepatocyte-like cells in a short time and extracellular vesicles communication (EVs) is one of the proposed mechanisms. Plasticity of bone marrow mesenchymal stem cells (BM-MSCs) is extensively supported by scientific literature but protocols applied to differentiation usually take from 7 to 28 days. Objective: To analyze in vitro differentiation potential of BM-MSCs into hepatocyte-like cells through EVs transfer mechanism in 6 and 24 hours. Materials and Methods: Co-culture system with cell-impermeable inserts and conditioned medium experiments were used to explore the effects of healthy and CCl4-injured hepatocytes, over BM-MSCs differentiation. Assessment of hepatocyte-like cell profile on BM-MSCs was revealed by gene expression (alpha fetoprotein, albumin and cytokeratin-18), glycogen storage and urea release. Hepatocytes from CCl4-injured rats were labeled by PKH-26 to track EVs. Ultracentrifugation was used to isolate EVs from supernatant medium of the two chamber of the co-culture system. PKH-26 positive EVs and PKH-26 positive cells were revealed by flow cytometry analysis and fluorescent microscopy. BM-MSCs cultured with conditioned medium were stained with ALB-FITC antibody. Results: Co-cultured BM-MSCs for 6 and 24 hours, showed no expression of hepatocyte-like genes, even after exposure to damaged microenvironment. Functional assays confirm the lack of differentiation signs there were no glycogen storage or urea release. Interestingly, EVs traffic analysis revealed no PKH-26 positive EVs at the upper chamber of co-culture system and no positive BM-MSCs were found either. On the other hand, conditioned medium experiment showed that BM-MSCs could uptake EVs. Flow cytometry analysis showed positive PKH-26 BM-MSCs at 6 (2.28%) and 24 (3.97%) hours. Flourescence microscopy revealed red points into BM-MSCs and immunofluorescence suggest that some EVs contain albumin. Gene expression and urea assay of BM-MSCs were not in accordance with a hepatocyte-like profile. Conclusions: Co-culture system, by using cell-impermeable membrane, was not adequate to promote EVs transfer between hepatocyte and BM-MSCs since EVs do not pass from the lower to the upper chamber. Conditioned medium experiments can suggest that BM-MSCs could uptake hepatocyte-derived EVs but this not drive to a hepatocyte-like profile in a short period of time. More studies will be necessary to clarify the dynamic of EVs transfer and their long time effects.

Les formes sévères de l’infection par les virus de la fièvre jaune (YFV) et de la dengue (DENV) sont caractérisées par une atteinte du foie, plus sévère lors d’une infection YFV. L’objectif de cette thèse est de comparer les infections de YFV et DENV dans un modèle d’hépatocytes humains dérivés de cellules souches (iHeps) afin d’identifier des facteurs à l’origine de cette différence de pathogenèse. Dans un premier temps, nous avons comparé le tropisme de YFV aux 4 sérotypes de DENV dans notre modèle hépatique établi en monocouche cellulaire. Nous avons observé une faible propagation de DENV dans les iHeps par rapport YFV. Les mêmes observations ont été faites dans des hépatocytes primaires. L’utilisation de souches chimériques 17D/DENV a permis de mettre en évidence que cette faible propagation serait liée à une faible efficacité d’entrée de DENV dans les hépatocytes. Nous avons également étudié l’infection dans des sphéroïdes iHeps, métaboliquement plus actifs que les iHeps 2D. Une infection productive a été observée uniquement avec YFV. Ce résultat pourrait s’expliquer par la faible accessibilité des cellules à l’intérieur des sphéroïdes. Dans un 2ème temps, nous avons étudié les réponses cellulaires induites dans les iHeps 2D après infection par les différents virus en utilisant une approche RNAseq. Les résultats préliminaires suggèrent un lien entre le taux de réplication et le nombre de gènes activés. La réponse interféron est plus précocement détectée dans le cas de YFV, mais l’infection par DENV induit un plus grand nombre de gènes. De plus, DENV-1 et DENV-4 induisent une augmentation d’expression de certains gènes impliqués dans la présentation d’antigène comme HLA-E et TAP-2, alors que YFV diminue l’expression de certains gènes de chimiokines et molécules d’adhésion. L’analyse préliminaire des voies liées au métabolisme hépatique révèle une inhibition de la voie de la coagulation dans le cas de l’infection par YFV, qui n’est pas observée lors de l’infection par DENV. Des observations similaires ont été décrites in vivo, au niveau protéique, confirmant la pertinence du modèle iHeps / Severe forms of infection with yellow fever virus (YFV) and dengue virus (DENV) are characterized by liver damage, with more severe symptoms observed during YFV infection. The aim of this thesis is to compare YFV and DENV infections in a model of human hepatocytes derived from stem cells (iHeps) in order to identify factors that could explain their difference in pathogenesis.First, we compared YFV tropism to the four DENV serotypes in 2D iHeps. We observed a low spread of DENV compared to YFV in both iHeps and primary hepatocytes. By using chimeric 17D/DENV strains, we demonstrate that this low propagation is linked to a low DENV entry efficiency in hepatocytes. We also studied infection in iHeps spheroids, metabolically closer to primary cells than 2D iHeps. A productive infection was observed with YFV only. The low accessibility of cells inside the spheroids could explained this result. Second, we studied cellular responses induced following infection by different viruses in 2D iHeps using an RNAseq approach. Preliminary results suggest a link between replication rate and the number of activated genes. The interferon response is detected earlier following YFV infection, but DENV induces a greater number of genes implicated in this pathway. Moreover, DENV-1 and DENV-4 up-regulate some genes involved in antigen presentation such as HLA-E and TAP-2, while YFV down-regulates genes encoding chemokines and adhesion molecules. Preliminary analysis of hepatic metabolism pathways reveals inhibition of the coagulation pathway induced by YFV infection, which is not observed during DENV infection. Similar observations have been described in vivo, at the protein level, confirming the relevance of the iHeps model

La phénylcétonurie (PCU est une maladie métabolique génétique causée par une déficience d'activité phénylalanine hydroxylase (PAH). Une hypomyélinisation du cerveau a été documentée chez les patients non traités, mais sa pathophysiologie reste floue. Nous avons investigué l'influence de la phénylalanine (Phe), phénylpyruvate (PP) et phénylacétate (PA) sur les oligodendrocytes. Nous avons premièrement montré dans un modèle murin de PCU que le nombre d'oligodendrocytes n'était pas différent dans le corps calleux entre animaux PCU et sains. Ensuite, en utilisant la technique des co-cultures myélinisantes nous avons pu déterminer que Phe, PP et PA n'ont pas d'effet direct sur la synthèse des gaines de myéline. Ces données indiquent que ces trois composés n'exercent probablement pas de rôle direct dans l'hypomyélinisation du système nerveux central constatée dans le cadre de la PCU. Ces données suggèrent donc des mécanismes d'action indirects. De plus, nous avons investigué la faisabilité d'un modèle de thérapie génique pour la PCU. Celui-ci implique la transduction ex vivo d'hépatocytes ou cellules souches mésenchymateuses par un vecteur lentiviral puis leur implantation dans le foie de l'organisme receveur. Phenylketonuria (PKU) is a metabolic genetic disease characterized by deficient phenylalanine hydroxylase (PAH) enzymatic activity. Brain hypomyelination has been reported in untreated patients, but its mechanism remains unclear. We therefore investigated the influence of phenylalanine (Phe), phenylpyruvate (PP), and phenylacetate (PA) on oligodendrocytes. We fisrt showed in a mouse model of PKU that the number of oligodendrocytes is not different in corpus callosum sections from adult mutants or from control brains. Then, using enriched oligodendroglial cultures, we detected no cytotoxic effect of high concentrations of Phe, PP, or PA. Finally, we analyzed the impact of Phe, PP, and PA on the myelination process in myelinating cocultures using both an in vitro index of myelination, based on activation of the myelin basic protein (MBP) promoter, and the direct quantification of myelin sheaths by both optical measurement and a bioinformatics method. None of these parameters was affected by the increased levels of Phe or its derivatives. Taken together, our data demonstrate that high levels of Phe, such as in PKU, are unlikely to directly induce brain hypomyelination, suggesting involvement of alternative mechanisms in this myelination defect. Moreover, we investigated the feasibility of a gene therapy for phenylketonuria. This project involved the ex vivo transduction of hepatocytes and mesenchymal stem cells with lentivirus vector and the engraftment of these cells in the liver's recipient.