Orientador: Márjorie de Assis Golim / Coorientador: Paulo Eduardo de Abreu Machado / Banca: Giovanni Faria Silva / Banca: Paulo Inácio da Costa / Resumo: O vírus da hepatite C crônica (VHC) é causa de doença hepática crônica afetando cerca de 3% da população mundial, aproximadamente 170 milhões de pessoas. Investigações sobre a resposta imune em indivíduos infectados com HCV é justificável pelo fato desta constituir doença infecciosa cujas alterações imunológicas têm relação direta com o desenvolvimento e a manutenção da infecção. Este projeto visou avaliar aspectos que compõem a resposta imune de pacientes com hepatite C crônica com diferentes graus de lesão hepática. Foram incluídos no estudo pacientes VHC+ (n=83), genótipo 1, pré-tratamento, os quais foram estratificados conforme o grau de fibrose hepática determinada por biópsia (classificação METAVIR), compondo: G1 (n = 15) pacientes nos estágios F0 (nenhuma fibrose) + F1 (fibrose em expansão dos espaços porta); G2 (n = 21) pacientes no estágio F2 (poucas fontes de fibrose nos espaços porta); G3 (n = 15) pacientes em estágio F3 (início da formação de nódulos); G4 (n = 32) pacientes em estágio F4 (fibrose severa); G5 (n = 16) doadores de sangue voluntários (grupo controle). Foram avaliadas características imunofenotípicas de subpopulações de leucócitos do sangue periférico (células NK totais, NKdim, NKbright, células dendríticas mielóides e plasmocitóides, monócitos clássicos e pro-inflamatórios, linfócitos T CD4+/CD8+), níveis de citocinas e quimiocinas plasmáticas (TGF-β, TNF-α, CCL2, CCL3, CCL4, CCL5, CXCL8, CXCL9, CXCL10), analisando a relação destes com a progressão da fibrose. Todas as frequências das subpopulações leucocitárias avaliadas estão alteradas em portadores de hepatite C crônica comparados com indivíduos saudáveis. Além disso, há correlação entre o grau de fibrose e a quantidade de linfócitos T CD4, T CD8, monócitos pro-inflamatórios e células NKbright circulantes. Níveis aumentados de TNF-α foram observados ... / Abstract: The chronic hepatitis C virus (HCV) is the cause of chronic liver disease affecting about 3% of world population, approximately 170 million people. Investigations of the immune response in individuals infected with HCV is justifiable because this form infectious disease whose immunological changes are directly related to the development and maintenance of infection. This project aimed to evaluate aspects that make up the immune chronic hepatitis C patients with different degrees of liver damage response. The study included patients HCV+ (n = 83), genotype 1, pretreatment, which were classified according to the rate of hepatic fibrosis determined by biopsy (METAVIR classification), composed of: G1 (n = 15) patients in stages F0 (no fibrosis) + F1 (fibrosis expansion of port spaces), G2 (n = 21) patients in the F2 stage (few sources of fibrosis in portal ), G3 ( n = 15 ) patients with stage F3 (early nodule formation), G4 (n = 32) patients with stage F4 (severe fibrosis), G5 (n = 16) volunteer blood donors (control group). Immunophenotypic characteristics of subpopulations of peripheral blood leukocytes (total NK cells, NKdim , NKbright , myeloid dendritic cells and plasmacytoid , classical monocytes, pro- inflammatory CD4 + / CD8 + cells), cytokine levels and plasma chemokines (were evaluated TGF- β , TNF - α , CCL2 , CCL3 , CCL4 , CCL5 , CXCL8 , CXCL9, CXCL10), analyzing their relationship with the progression of fibrosis. All frequencies of evaluated leukocyte subpopulations are altered in patients with chronic hepatitis C compared with healthy subjects. In addition, no correlation between the degree of fibrosis and the amount of CD4, CD8, pro-inflammatory monocytes and circulating NKbright cells. Increased levels of TNF-α were observed in cirrhotic patients (G4) and TGF - β in HCV + groups with moderate hepatic fibrosis (G2 and G3). Plasma chemokine CXCL9/MIG, CXCL10/IP-10, CXCL8/IL-8, CCL2/MPC-1, ... / Mestre

The incidence of Type 2 Diabetes Mellittus (T2DM) is increasing world wide. In Africa the limited access to health care and the insidious course of the disease lead to more severe illness and diabetic complications. There is a need to find alternative approaches to treatment and prevention that address the problems and needs of Africa. Sutherlandia frutescens (S.frutescens) is a traditional herbal plant with known anti-diabetic properties, the precise mechanism of action of S.frutescens is not known. In order to develop new approaches for treatment and prevention of T2DM the pathophysiology of T2DM must be understood. T2DM is the final outcome of a multi-organ disease characterized by early defects in muscle, adipocytes, hepatocytes and pancreatic β-cells. In this study the role of the liver was investigated because of its central role in glucose and lipid metabolism. It is hard to differentiate between all the influences in an in vivo model, so the aim of this study was to develop an in vitro model of T2DM in Chang liver cells and to determine if S.frutescens can reverse the state of insulin resistance in this model. Different culture media conditions were screened to identify a method that can be used as the T2DM model in Chang liver cells. Serum free medium (MCBD-201) supplemented with human diabetic serum, (2.5%-10%), high insulin concentrations (0.1μM-1μM), high fructose concentrations (1-10mM). and a combination of high insulin and high fructose was used for this screening. Chang liver cells cultured in MCBD-201 medium supplemented with 1mM fructose and 0.1μM insulin showed reduced glucose uptake and increased lipid accumulation. The effect of two S.frutescens extracts, two anti-diabetic drugs, metformin and ciglitazone, and a hypolipidemic drug ciprofibrate were determined and shown to increase glucose uptake and reduce lipid accumulation. It was postulated that exposing the cells to excess nutrients in the form of high fructose would stimulate the cells to become adipogenic and accumulate lipids, which would interfere with the glucose uptake and induce insulin resistance. Gene expression of PPARγ, PPARα, and SREBP-1 transcription factors regulating lipid metabolism was determined in Chang liver cells cultured in insulin resistance inducing medium over a 48 hour time course. The expression of PPARγ, known to stimulate adipogenesis was increased after 6, 24 and 48 hours of exposure (P(H1)<0.0001). The expression of PPARα, known to stimulate β-oxidation expression, was significantly decreased after 24 hours of exposure (P(H1)<0.0001). The presence of the plant extracts in the insulin resistance inducing media protect against this increase in adipogenesis and decrease in β-oxidation after 48 hours of exposure by increasing PPARα expression and decreasing PPARγ expression. A PCR Array was performed which identified 32 more potential molecular targets of S.frutescens. Five of the 32 targets identified with the PCR Array were validated using qRT-PCR. These genes play a role in lipid and glucose metabolism and protection against oxidative stress and inflammation. In summary a cellular model of insulin resistace in hepatocytes has been established and the capacity of S.frutescens to reverse this process has been demonstrated by acting as a dual PPARγ/α agonist. New genes have been identified in the development of insulin resistance and as targets of S.frutescens.

The burden of liver diseases is increasing worldwide, accounting for two million deaths annually. In the past decade, tremendous progress has been made in the basic and translational research of liver tissue engineering, which seeks to build physiologically relevant liver models to better understand liver diseases, accelerate drug development, and advance regenerative medicine. Liver microtissues are small, three-dimensional (3D) hepatocyte cultures that recapitulate liver physiology and have been used in many biomedical applications. However, sourcing of high-quality human hepatocytes for microtissue fabrication poses a significant challenge. Since the inception of induced pluripotent stem cell (iPSC) technology, iPSC-derived hepatocyte-like cells (HLCs) have demonstrated significant improvement over other hepatocyte cell sources in many studies. Despite their promising potential, HLCs face certain challenges: they resemble fetal hepatocytes rather than adult hepatocytes; they undergo dedifferentiation quickly after reaching maturity; they are produced on a small scale; and they exhibit large donor-to-donor and batch-to-batch variability. This doctoral thesis focuses on engineering patient-specific liver microtissues with prolonged phenotypic maintenance and disease modeling potential. Chapter 1 provides a review of recent advances, challenges, and future directions in liver microtissue research. 3D microtissues can be generated by scaffold-free assembly or scaffold-assisted methods using macroencapsulation, droplet microfluidics, and bioprinting. Optimization of the hepatic microenvironment entails incorporating the appropriate cell composition for enhanced cell-cell interactions and niche-specific signals, and creating scaffolds with desired chemical, mechanical and physical properties. Perfusion-based culture systems such as bioreactors and microfluidic systems are used to achieve efficient exchange of nutrients and soluble factors in the microtissues. Chapter 2 describes our efforts in optimizing methods of generating human HLCs from the peripheral blood of selected donors. Peripheral blood mononuclear cells (PBMCs) were first reprogrammed to iPSCs using Sendai viruses carrying the four Yamanaka factors. We developed an optimized protocol for hepatocyte differentiation from iPSCs, and obtained HLCs that exhibited hepatocyte-specific phenotypes and functions that were comparable to other reports. We then demonstrated the one-step generation of homogeneous, microencapsulated liver microtissues in Chapter 3. Droplet microfluidics was used to produce double emulsion droplets that served as individual microenvironments where HLCs were encapsulated in methylated collagen and alginate. The cells self-assembled in <16 hours through dynamic interactions with methylated collagen, and individual spheroids were encapsulated in polymerized alginate gel to prevent cell fusion and attachment. HLC spheroids remained viable and functional for >24 days, whereas 2D HLCs underwent dedifferentiation within 7 days of reaching maturity. The spheroids showed further maturation compared to the 2D HLCs at peak maturity. Co-culture of HLCs with human endothelial cells was also investigated in the 3D system, but no improvement was observed over monoculture spheroids with our current methods. To our knowledge, this is the first study to utilize droplet microfluidics to generate homogeneous, compartmentalized droplets that serve as optimized 3D microenvironments for HLC aggregation and maturation. It demonstrated the potential of using high-throughput droplet microfluidics to produce and encapsulate mature, functional human HLCs for long-term applications. In Chapter 4, we developed a TM6SF2 knockout and overexpression model in iPSCs to investigate its molecular function and potential role in nonalcoholic fatty liver disease (NAFLD). Transmembrane 6 superfamily member 2 (TM6SF2) is a protein of unknown function, and analysis from our model suggested that TM6SF2 dysregulation has a biphasic response. Our data showed that both knockout and overexpression can result in the upregulation of cholesterol biosynthesis and a defect in the proper processing of lipid droplets. Additionally, high expression of the TM6SF2 rs58542926 variant has an increased risk for cholesterol upregulation, compared to the major allele. Future works will focus on generating liver microtissues from the TM6SF2 knockout and transgene-expressing cells using droplet microfluidics, and validating our hypotheses with established biochemical and functional assays.

The natural history of hepatitis C virus infection differs between women and men. Women demonstrate a slow rate of disease progression until menopause. Older women are more likely to develop fibrosis and are less responsive than younger women to pegylated interferon and ribavirin. Women of childbearing age have higher rates of sustained virologic response, but current therapies are contraindicated during pregnancy. Vertical transmission of hepatitis C virus occurs, but data supporting recommendations for prevention of mother-to-infant transmission are limited.

The natural history of hepatitis C virus infection differs between women and men. Women demonstrate a slow rate of disease progression until menopause. Older women are more likely to develop fibrosis and are less responsive than younger women to pegylated interferon and ribavirin. Women of childbearing age have higher rates of sustained virologic response, but current therapies are contraindicated during pregnancy. Vertical transmission of hepatitis C virus occurs, but data supporting recommendations for prevention of mother-to-infant transmission are limited.

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A nonsynonymous, loss of function variant (rs58542926, E167K) located in the gene encoding TM6SF2 was identified in multiple genetic association studies as significantly correlating with increased risk for non-alcoholic fatty liver disease (NAFLD) and decreased risk for hyperlipidemia. Given the pivotal role that lipoproteins play at the juncture of these two conditions, researchers hypothesize that the ER-membrane spanning TM6SF2 protein regulates the degree of lipidation of VLDL particles synthesized in the liver. However, not all published data supports this theory and contradictions regarding many aspects of the mechanistic function of TM6SF2 remain. The inconsistencies observed in the literature are in part due to drawbacks of the models used to study TM6SF2 activity; thus, there is an obvious need for an improved hepatocyte model to better understand how TM6SF2 impacts lipid metabolism.To address this need, we present an optimized protocol for the differentiation of inducible pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs), created in collaboration with the Leong Lab. We provide extensive validation of HLC maturity and hepatic functionality, including prolonged albumin secretion, evidence of membrane polarity, and cytochrome P450 induction. We also demonstrate that HLCs express proteins essential for lipoprotein metabolism, secrete authentic VLDL particles, and respond to metabolic perturbations, supporting their value for modeling hepatosteatosis and VLDL metabolism in vitro. To investigate the effect of TM6SF2 variant expression on hepatic lipid metabolism, we produced HLCs derived from 4 homozygous TM6SF2-carrier individuals (KK) and 4 age- and sex-matched unaffected siblings (EE). We describe the variability in differentiation efficiency that we observed in our sibling-matched HLC model and present the gene editing strategy we developed using CRISPR/Cas9 technology and transgene-induced expression to create isogenic iPSCs differing only in their TM6SF2 genotype [EE, KK, or knockout (KO)]. After extensive confirmation of successful gene editing, we explore the effect of TM6SF2 on lipid metabolism in the edited iPSCs. RNA-sequencing and qPCR validation reveal that the Sterol Regulatory Element Binding Protein 2 (SREBP2)-mediated transcriptional program regulating cholesterol synthesis is significantly increased in TM6SF2 KO iPSCs. However, lipidomics analysis and de novo lipogenesis functional assays show that free cholesterol (FC) levels are unchanged. In TM6SF2 KO iPSCs, we further show a reduction in the activities of Acyl-Coenzyme A: Cholesterol Acyltransferase 1 (ACAT1) and Phosphatidylserine Synthase 1 (PSS1), two enzymes that display optimal function when specifically localized to cholesterol enriched ER lipid raft-like domains. Our findings suggest that TM6SF2 may impact cholesterol localization within ER subdomains, which regulate expression levels of cholesterol synthesis genes and activities of ER lipid-raft associated enzymes. In summary, we present here methodological approaches for generating multiple cell culture models in which to investigate the function of TM6SF2, as well as novel evidence supporting a role for TM6SF2 in iPSC cholesterol metabolism.

Finally, the hepatoprotection of the 50%EtWZ was evaluated using rat model. The results indicated that the 50%EtWZ possessed potent hepatoprotective activities. The protective effect of the extract against hepatotoxicity induced by long-term treatment with ethanol might be attributed to its inhibitory action on oxidative stress. Although multiple factors could be involved in the inhibition of oxidative injury in the liver, the inhibition of CYP2E1 pathway and the enhanced GSH-related antioxidant capacity might be responsible for the protective effect. In addition, the 50%EtWZ also produced anti-inflammatory effect partly by interfering Toll-Like-Receptor-4 (TLR-4)-mediated signal pathway and reducing the production of Tumor Necrosis Factor-alpha (INF-alpha) in Kupffer cells during long-term ethanol exposure. / First, in order to determine which kind of extract possesses the strongest hepatoprotective effect on ethanol-induced cytotoxicity, various extracts were screened for cytochrome P450 2E1 isoenzyme (CYP2E1) inhibitory activity using the fluorogenic CYP2E1 substrate and HepG2 cells overexpressing human CYP2E1. The results showed that all extracts (aqueous, 50% ethanol, and 90% ethanol) of WZ produced inhibitory effect on CYP2E1. The 50% ethanol extract of WZ (50%EtWZ) displayed a stronger CYP2E1 inhibition than the aqueous and 90% ethanol extracts. The aqueous extract and 50%EtWZ showed protective effect against ethanol-induced cytotoxicity at concentrations equivalent to 100 and 1000 mug raw herb/ml. At the same concentration of 100 1.1g/ml, the 50%EtWZ exhibited a more potent protective effect. Higher degree of cytotoxicity was found in the 90% ethanol extract of WZ. Thus, 50%EtWZ was chosen for further study. / In summary, all data suggest that the inhibition of CYP2E1 pathway and the inhibition of oxidative stress by the 50%EtWZ, together with the anti-inflammatory effect on Kupffer cells, may contribute to its hepatoprotection against chronic ethanol-induced liver injury. / Second, the chemical components of the 50%EtWZ were analyzed by chromatographic fingerprints. The fingerprint revealed six hepatoprotective compounds including schisandrin B, schisandrin, deoxyschisandrin, betaine, hyperin, and quercitrin in the formula. / Third, the protective mechanism of the 50%EtWZ was investigated in E47 cells model. The 50%EtWZ protected against CYP2E1-dependent toxicity and oxidative stress induced by ethanol. The mechanism of protection involved the decrease of reactive oxygen species production and the inhibition of lipid peroxidation. The hepataprotection was associated with the maintenance of mitochondrial GSH. Pre-treating E47 cells with the 50%EtWZ significantly inhibited the expression of CYP2E1. Therefore, the protective effect of the 50%EtWZ was most likely attributed to its antioxidant activities and the inhibition of CYP2E1. In addition, the 50%EtWZ prevented ethanol-induced apoptosis and protected against oxidative damage to mitochondria which are critical for maintenance of cell viability. / Wu-Zi-Yan-Zong-Wan (WZ), a traditional medicinal formula, is used for treatment of male sexual dysfunctions. In this study, the hepatoprotection afforded by Wu-Zi-Yan-Zong-Wan treatment and its biochemical mechanism involved against chronic alcohol-induced injury were investigated. / Chen, Mengli. / "May 2008." / Adviser: Che Chun Tao. / Source: Dissertation Abstracts International, Volume: 70-03, Section: B, page: 1609. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (p. 157-179). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.