NANOG is a transcription factor that functions as a central regulator of pluripotency and determines the cell fate in embryonic stem cells (ESCs). Dr Nateri’s laboratory and others have reported the expression of embryonic NANOG in a small subpopulation of cells in several human cancers including human colorectal cancer (CRC). NANOG positive CRC cells express the normal intestinal stem cell marker LGR5, as well as the induced pluripotent stem (iPS)-linked transcription factors SOX2, c-MYC, and high β-catenin. These cells strongly resemble a small subpopulation of poorly differentiated cancer stem cells (CSCs). Recent studies have reported that, in CSCs, the self-renewal and survival signals are dominant over the differentiation signals. This suggests that NANOG is an essential modulator of drug resistance complexity and tumour heterogeneity in cancer cells. Dedifferentiation is an established hallmark of carcinogenesis and is accompanied by key signalling pathways mediated in drug resistance, such as the mitogen-activated protein kinase (MAPK) and glycogen synthase kinase-3β (GSK-3β)/β-catenin pathways, via epithelial–mesenchymal transition (EMT). However, the gain of stemness and the concomitant loss of differentiation might affect and alter the signalling pathways exclusive to NANOG-expressing cells that are required for drug resistance, not fully studied. Therefore, we aimed to characterise the mechanism by which CSCs gain self-renewal ability and lose differentiation ability through NANOG. Hence, this study was initially focused on testing the role of NANOG in chemotherapy drug resistance in human CRC cells, using 5-fluorouracil (5-FU)–treated HCT116 cells stably overexpressing exogenous human NANOG (HCT116GFP/NANOG) and control GFP expressing cells (HCT116GFP). We show that NANOG overexpression promotes 5-FU resistance in HCT116GFP/NANOG cells versus HCT116GFP cells (Chapter 3). To define the possible downstream regulatory pathways directly or indirectly mediated by NANOG, we examined the MAPKs; JNK (Jun-N-terminal kinase), ERK1/2 (extracellular signal–regulated kinase) and Wnt/β-catenin signalling pathways through GSK-3β association with β-catenin in HCT116GFP/NANOG cells versus HCT116GFP cells, and if NANOG mediated in activation of the EMT (epithelial–mesenchymal transition). Our data show that overexpression of NANOG protein in the CRC cells mimics previously reported ESC differentiation mechanism mediated by induction of the phosphorylated ERK1/2 (p-ERK1/2) expression and phosphorylated GSK-3β (p-GSK-3β) at Ser9 (Chapter 3). Consequently, NANOG overexpression increases the β-catenin and represses the E-cadherin, while significantly increases the vimentin level, leading to EMT activation (Chapter 4). In this study, we have also demonstrated NANOG induction of the EMT signature, results increasing activity of symmetrical cell division, while reducing differentiation of HCT116 derived colonosphere formation (Chapter 5). Taken together, this study describes the mechanisms of NANOG induction of EMT and NANOG sustainment of CSC-like traits via activation of the ERK/GSK-3β/β-catenin pathways in CRC cells. These findings highlight the specific mechanism of action of the NANOG-CSC signalling pathways in human CRC tumour heterogeneity, in which it might eventually identify promising approaches to cancer treatment via selectively targeting of CSCs.

Cten (also known as Tensin4) is the fourth member of the Tensin gene family. It lacks the N terminal actin binding domain while retaining the C terminal SH2 and PTB domains. This helps to bind Cten to the intracytoplasmic tail of β1 integrin and puts it at the heart of focal adhesions. It is reported to be a tumour suppressor in kidney and prostate cancer where normal tissues show high expression. However in a number of tumours, including colorectal cancer, Cten has been labelled as an oncogene. Cten which normally is a cytoplasmic protein gives nuclear staining in colorectal metastatic deposits. It increases motility, invasion and colony formation in colorectal cancer cells. In this study we have tried toexplore the mechanism of functional activity and regulation of Cten. We looked at Cten in the nucleus in vitro and identified new downstream binding targets. In addition we investigated the role of the SH2 domain of Cten concentrating on its downstream signalling molecules and binding partners. Furthermore, we explored regulators of Cten. In this study we have forced nuclear localisation of Cten by tagging it with a nuclear localisation sequence (NLS) and found a significant increase in cell motility. In order to investigate the SH2 domain we used site directed mutagenesis to change potentially important amino acids namely Arginine at 474 to Alanine (R474A), which is important for binding tyrosine phosphorylated proteins. Moreover, we displayed that Cten underwent tyrosine phosphorylation and additionally changed three tyrosine residues i.e. Y449F, Y479F and Y530F via site directed mutagenesis. We found R474 and Y479 to be important in regulating cell motility and that known downstream targets such as ILK and FAK are dependent on an intact SH2 domain. Furthermore we have identified Cten to be physically bound to FAK in the cytoplasm and nucleus and new downstream targets identified such as Src and Paxillin. Regarding possible regulators of Cten, we found that Cten might be a possible substrate for calpain. Another regulator considered was CD24 due to its role in movement of integrins into lipid rafts and we found it was a positive regulator of Cten. In conclusion localisation of Cten into the nucleus causes an augmentation of its motility enhancing functions. Cten regulates cell motility via its SH2 domain. Arginine 474 and Tyrosine 479 are important for its function. Cten regulates levels of ILK, FAK, Src and Paxillin through its SH2 domain and binds to FAK in both cytoplasm and nucleus. Calpain and CD24 were found to possible regulators of Cten in colorectal cancer. Future studies are needed to define its role in signalling at focal adhesions and these studies should be validated in other cancer cell models as well to establish Cten as regulator of cell motility in cancer.

Cystic fibrosis (CF) is one of the commonest life-limiting genetic disorders in the Caucasian population. Management involves frequent administration of antibiotics including aminoglycosides. With improving survival, it is time to focus on various age-related and treatment-associated adverse influences. The objective of this research was to evaluate renal function in CF, determine the effects of cumulative antibiotic exposure and to identify ways to reduce associated comorbidity. A cross-sectional study showed that a small number of adults and children with CF had low glomerular filtration rate (GFR), and there was no association between GFR and cumulative antibiotic exposure. An above normal GFR was identified in one in four children with CF. Estimated GFR calculated by creatinine-based equations did not accurately predict the GFR measured by the gold standard 51Cr-EDTA (51chromium-ethylenediamine tetraacetic Acid). Pure tone audiograms identified a raised hearing threshold in one in four people with CF, which did not correlate with increasing aminoglycoside exposure. A randomised controlled study established that there is no difference in the pharmacokinetics of tobramycin when administered intravenously in the morning or evening. A Cochrane systematic review concluded that there was insufficient evidence to support a routine use of bronchoalveolar lavage in the management of pulmonary infections with Pseudomonas aeruginosa in children with CF below 5 years old. CF gene (Cystic Fibrosis Transmembrane Conductance Regulator, CFTR) is expressed in pig kidneys. Histological and molecular experiments established that there is no difference between the newborn pigs with genotypes CFTR -/- (knockout) and CFTR +/- (heterozygous) or CFTR +/+ (wild-type) pig kidneys in the renal morphology and in the expression of various renal endocytic receptor proteins. The vascular haemodynamic parameter, augmentation index ascertained in a small group of children with CF suggests a possibility that the vascular age may be advanced in people with CF right from their childhood. In summary, these studies have established a low prevalence of renal disease in CF and a lack of association between cumulative antibiotic exposure and GFR. Further research is needed to evaluate the natural history of high GFR in paediatric CF population. Kidneys from pig model of CF may provide an alternative model to investigate the renal disease in CF.

CD24 is a small (81 amino acids) GPI anchored protein which is involved in promoting cell motility and stemness and may be a part of the metastatic process. It is a heavily glycosylated molecule and contains numerous O-glycosylation sites together with two N-glycosylation sites. N-glycosylation is thought to be important in protein function, and therefore, the aim of this study is to (a) investigate the importance of N-glycosylation in the function of CD24, (b) identify other potentially functional sites in CD24 by deletion mapping, (c) define downstream targets of CD24, and (d) identify the extrinsic signals of which activate CD24. (a) Through site-directed mutagenesis, we changed the glycosylated residues N32 (ACC to CAA) and Q52 (AAT to CAG) in CD24. Mutating each of these sites individually, when compared to pCCD24WT (wild-type CD24), caused a partial reduction in ability to induce cell motility and cell invasion (cell motility p=0.0001 cell invasion p=0.0001) and, unexpectedly, resulted in significantly enhanced cell proliferation (p=0.0001). Mutation of both sites resulted in a near loss of motility induction and retained cell proliferation. (b) We mapped the functional sites of CD24 by deleting seven amino acid segments of the whole of the mature peptide. Apart from the N-glycosylation sites, no other functional domains were identified which altered cell motility or proliferation. (c) Previously, in our lab it has been shown that Cten is downstream motility-inducing target of CD24. We hypothesised that CD24 may signal through the Notch pathway since Notch1 has an important role in maintaining CSCs. Results showed that forced expression of CD24 upregulates Notch1 and Cten whilst knockdown of CD24 causes loss of Notch1 and Cten expression. However, forced expression of CD24 with simultaneous knockdown of Notch1 resulted in failure to induce Cten. (d) CD24 is reported to act as a ligand of P-selectin. We found that stimulating CD24 expressing cell lines induced with P-selectin induced cell motility (p=0.0011) and caused an increased in the protein expression of downstream targets of CD24. Stimulating cell lines expressing CD24 with mutant glycosylation sites resulted in a failure to induce motility or CD24 targets. We conclude, the removal of the N-glycosylation sites in CD24 resulted in a loss of cell migration and invasion, thereby suggesting the importance of these sites in mediating the migration and invasion functions of CD24. Unexpectedly, these mutations also appeared to stimulate cell proliferation, suggesting that wild type CD24 can functionally inhibit cell proliferation. Deletion mapping did not reveal any other functional sites on the mature CD24 suggesting that O-glycosylation is relatively affecting the glycosylation in the biology of CD24. Notch1 was to be an important downstream target of CD24 and a regulator of Cten. The binding of P-selectin with CD24 resulted in increased motility of CD24 which is also dependent on N-glycosylation.

In health, the gut provides a barrier between the external and internal environment. This selectively permeable barrier allows absorption of nutrients and water from the gastrointestinal contents, whilst preventing the transfer of noxious material such as bacteria. During episodes of inflammation, this barrier becomes compromised, allowing transfer of noxious material into the systemic circulation, leading to disease states such as inflammatory bowel disease and septic shock. There are no clinically available compounds to combat this increase in permeability directly. The endocannabinoid system is a group of endogenous lipid signalling molecules which activate membrane-bound receptors. Plant-derived and synthetic compounds also act at these receptors, generating a wide variety of secondary effects. The aims of this study were to identify compounds with action on the endocannabinoid system which could be used clinically to treat inflammation and hyperpermeability of the gastrointestinal tract, exploring mechanisms of action. Systematic review and meta-analysis of existing literature revealed 51 preclinical studies, and 2 clinical studies examining the effect of cannabinoid compounds. In preclinical studies, cannabinoid drugs reduced myeloperoxidase activity in the gastrointestinal mucosa within mouse and rodent models of colitis (standard mean difference -1.26, 95% confidence interval (CI)-1.54 to -0.97, I2=48.1%) and macroscopic disease activity scores (standard mean difference -1.36, 95% CI -1.62 to -1.09, I2=61%). Clinical trials found no overall benefit of cannabinoid drugs in Crohn’s disease (mean difference -74.97, 95% CI –229 to 0.79, I2=75%). Two compounds, cannabidiol and palmitoylethanolamide, possessing positive outcomes and preferable side effect profiles, were put forward for further study to examine potential clinical benefit. The mechanism of action of palmitoylethanolamide and cannabidiol were explored further by examination of their effects on the immune response, permeability of cultured cell monolayers, intracellular signalling pathways, expression of membrane-bound proteins governing permeability and receptors of the cannabinoid system. We found that these agents were anti-inflammatory in both cultured Caco-2 cells and explant human colonic tissue, prevented increases in permeability secondary to inflammation, and were likely to act through adenylyl cyclase, protein kinase A and extracellular signal-regulated kinases. The downstream effects of these compounds prevented down-regulation of the TRPV1 receptor, upregulation of aquaporin 3 expression, and prevention of downregulation of claudin-3. The effects of palmitoylethanolamide and cannabidiol were then examined on permeability in the human colon in vivo by means of a double blinded, randomised controlled trial. This study demonstrated that aspirin increased the permeability of the human gut, determined by increases in urinary concentrations of lactulose and D-mannitol, quantified by mass spectrometry. Groups receiving oral cannabidiol or palmitoylethanolamide demonstrated lower urinary concentrations of lactulose and D-mannitol, suggesting that these two drugs could be used clinically to prevent disease-induced hyperpermeability. In conclusion, cannabidiol and palmitoylethanolamide have shown consistent anti-inflammatory actions in colonic ex vivo and in vitro models, and also prevented increases in intestinal permeability in vitro and also in vivo in a randomised, double blind, placebo-controlled trial. Their clinical use in IBD should now be assessed in phase II clinical trials.

C-terminal tensin-like (Cten, also known as Tensin4) is the member of the tensin gene family. Cten functions as an oncogene in a variety of cancer types and its expression is commonly associated with poor prognosis and metastasis in colorectal cancer (CRC). Although several studies have shown that Cten has a critical role in the regulation of cell motility and invasion in different tumour tissues, the underlying signalling mechanisms have not been fully elucidated. This thesis investigated the biological activity of Cten in four different ways in order to further elucidate the mechanisms of Cten signalling in CRC cells. Potential downstream targets of Cten signalling involved in the regulation of epithelial-to-mesenchymal transition (EMT) induced cell motility i.e. Rho-associated protein kinase1 (ROCK1), Src and Snail were investigated. Cten expression was manipulated in different cell lines using multiple approaches including forced expression, gene knockdown and constitutive depletion (through Crispr/Cas9 gene deletion) to eliminate artefacts of methodology and cell line specific effects. Snail, Src and ROCK1 were identified as novel downstream targets of Cten signalling and additionally, Cten was shown to increase the stabilisation of both Src and Snail proteins. The functional relevance of Cten-Snail, Cten-Src and Cten-ROCK1 signalling was assessed, and the overall findings demonstrated that Cten could promote cell motility and colony formation directly through the positive regulation of the Src/ROCK1/Snail dependent axis. To gain a deeper insight into the mechanisms of Cten’s biological function, mutations, at two important residues (i.e. arginine 474 and tyrosine 479) in the Src homology 2 (SH2) domain of Cten were introduced into one construct (GFP-CtenR474A+Y479F) using site directed mutagenesis. These two residues in the SH2 domain of Cten were found to not only be important for interacting with Src, ROCK1, or Snail signalling, but also for regulating cell motility and colony formation efficiency. Numerous Cten regulatory factors have been identified, however, little is known about how Cten is activated and regulated in cancer cells. The relationship between transforming growth factor beta 1 (TGFβ1) and Cten was investigated and stimulation of cells with TGFβ1 or knockdown of TGFβ1 resulted in changes in Cten expression as well as its downstream targets of ROCK1, Src, Snail, and N-cadherin. Furthermore, this positive interaction between TGFβ1 and Cten was functionally relevant and caused changes in cell motility. and the nuclear translocation of ROCK1, Src, and Snail protein increased by TGFβ1 is probably mediated via upregulation of the Cten signalling pathway The biological function of Cten in the nucleus was further investigated and shown to increase nuclear localisation of Src, ROCK1, and Snail, further promoting the migratory capability and colony formation efficiency in CRC cells. Finally, Cten expression was shown to positively correlate with both ROCK1 and Src expression in a series of primary CRCs. This correlation was consistent with that observed following manipulation of Cten expression in CRC cell lines. In conclusion, this study has revealed a number of novel findings regarding the biological function of Cten signalling in CRC. However, further validation of the findings may enhance the understanding of the role of Cten in the invasion-metastasis cascade in the future.

Introduction: Oesophageal cancer is more common in men than women. Oestrogen, which mediates its effects via oestrogen receptors (ERs), may be responsible for the gender disparity. This thesis investigates the role of ERs in oesophageal cancer development and explores potential therapeutic possibilities. Methods: ERα and ERβ expression in oesophageal AC cell lines (OE19 and OE33) was knocked down using siRNA, and the effect of knockdown on the expression of proliferation-associated proteins (Ki67, PCNA, E-cadherin, Cyclin D) was assessed. The effect of the SERM, tamoxifen, on oesophageal cancer cell proliferation was investigated using proliferation assays and by evaluating the effect of tamoxifen on proliferation-associated proteins. Finally, a pilot study of tamoxifen in patients with oesophageal cancer was undertaken to assess feasibility of a clinical trial and to determine the short-term biological effect of tamoxifen on proliferation, assessed by a change in the immunohistochemical expression of Ki67 between paired biopsies. Results: ERα and ERβ are expressed at the mRNA (RT-PCR) and protein level (Western Blotting) in the OE19 and OE33 cell lines. ERβ mRNA knock down was achieved in the OE33 cell line (p = < 0.0001). However, reproducible significant ERβ protein knockdown was not demonstrated, and there was no change in the expression of proliferation-associated proteins. Treatment with tamoxifen significantly inhibited OE33 cell proliferation in a dose-dependent manner (p = < 0.0001). Interestingly, treatment with tamoxifen decreased the expression of E-cadherin, but failed to change the expression of the remaining proliferation-associated proteins. Eight patients (6 male with AC and 2 female with SCC) included in the pilot study completed a median on 30 days (range: 28 – 45 days) tamoxifen treatment; the mean Ki67 Labelling Index between paired biopsies increased by 0.625% (ns). Of the two women included, Ki67 expression decreased with tamoxifen treatment. A correlation was demonstrated between a reduction in Ki67 and mean ERβ expression (r= -0.2272, ns). Discussion: ERβ is the dominant ER subtype expressed in oesophageal cancer cell lines and human cancer tissue. Tamoxifen inhibits the proliferation of oesophageal cancer cell lines in-vitro. Further studies to define the role of the ERβ subtype in oesophageal cancer and a clinical trial of tamoxifen in patients with oesophageal cancer is needed.

The objective of this thesis was to develop new methodologies to assess upper gastro-intestinal function in health and disease. Several different technologies were studied in a range of upper gastro-intestinal diseases and adapted to try and provide more meaningful insights. The thesis has three main sections. In the first section, High Resolution Oesophageal Manometry (HRM) was used to assess unexplained upper gastro-intestinal symptoms in a group of patients referred to a tertiary centre. 46 patients were diagnosed with rumination syndrome following HRM. A retrospective review was completed of these patients case notes and HRM data. The predominant aim of this section was to identify if common mechanisms exist within rumination and its variations and to establish if the variety of presenting symptoms is due to different underlying problems or a common behavioural response to a variety of stimuli, with symptoms being dependent on the circumstance the behaviour exists in. This would support a generic biofeedback technique being useful regardless of presenting complaint. Comparing the variety of symptoms, exhibited behaviour and manometric findings, a new classification system for rumination was then developed; 1. Primary or “classical” rumination a. Increase in abdominal strain with corresponding rise in intra-gastric pressure and return of gastric contents to the mouth 2. Secondary or reflux-related rumination a. Reflux event causes the patient to respond with increase in intra-abdominal muscle strain and subsequent rumination 3. Supra-gastric belching independent of meals. a. Rise in intra-gastric pressure whilst a closed gastro-oesophageal junction, therefore producing rapid belching of air from the oesophagus without any return of gastric contents Generic biofeedback therapy was used (regardless of presenting symptoms) to control the abnormal behavioural response to symptoms. 20/46 patients reported full resolution of their symptoms and a further 13 / 46 reported improvement in their symptoms with this, while underlying mechanisms were targeted e.g. reflux with proton pump inhibitors, pain in functional dyspepsia. In the second main section of this thesis, gastro-oesophageal reflux disease (GORD) is considered. GORD is currently diagnosed by 24 hour pH studies. These are often difficult for patients to tolerate and require time off medication. A more attractive method would be for diagnosis to occur at the same time as gastroscopy. A novel instrument is the EndoFLIP® device. This measures cross-sectional area (CSA) and distensibility at the gastro-oesophageal junction (GOJ) via a long catheter with a balloon at the end that straddles the GOJ. It has been hypothesised that these measurements will be increased in those with GORD, as the GOJ is more distensible, allowing more retrograde movement of gastric contents. The aim of this section of the thesis was to establish if GOJ CSA and distensibility differentiate between healthy volunteers (HV) and GORD patients based on i) symptoms and ii) prolonged oesophageal acid exposure. 21 HV and 18 patients with GORD (based on symptoms) had EndoFLIP® measurements and wireless pH studies to assess this. 14% of HV and 50% GORD patients had pathological acid exposure. CSA and distensibility were both significantly higher in the HV’s compared to GORD patients. However, there was an inverse correlation between CSA and body mass index (BMI) which was significantly higher in the patient population. This may explain differences seen due to corresponding higher intra-abdominal pressure in those individuals with a high BMI, sub-sequentially affecting the CSA and distensibility. The complex structure of the GOJ and multiple factors involved in the pathogenesis of GORD present difficulties in using EndoFLIP® to diagnose GORD. It may find applications in other areas, such as serial measurements in single patients. In the final section of this thesis, gastric emptying is the focus and its pathogenesis in functional dyspepsia (FD). Current gastric emptying studies only find abnormalities in approximately 40% of patients with FD. Gamma scintigraphy is used in routine clinical practice for gastric emptying studies. Magnetic resonance imaging (MRI) is emerging as a modality in gastric emptying assessment and potentially provides additional information. This thesis hypothesised that standard gastric emptying studies may not be measuring the parameters reflective of underlying pathophysiology in FD. Also, most have a relatively small meal size that may be too small to trigger dysfunction. MRI may provide additional insights as can assess gastric contents and surrounding structure (unlike GS). To investigate these a 400ml test meal was utilised and gastric emptying parameters i) gastric contents volume at time 0 (GCV0, representative of early emptying), ii) gastric emptying rate at the time taken for half the meal volume to empty (GE rate @T50, representative of later emptying) and the more traditional measurement iii) time taken for half the gastric contents to empty (T50) in bopth GS and MRI studies. The hypothesis of this study is that early emptying is more rapid in FD due to impaired accommodation (therefore a lower GCV0) leading to a slower later emptying (therefore a lower GE rate @ T50). Following validation studies in a large healthy population (n=53), GS and magnetic resonance imaging (MRI) studies with a test meal of 400ml were used in 8 FD patients and 24 matched HV (from the pool of HV) . FD had a significantly lower BMI. Early emptying (represented by gastric contents volume after ingestion of meal (GCV0)) was significantly lower in GS for FD patients but higher in MRI. Time for half the meal to empty (T50) and gastric emptying rate at T50 (GE rate @T50) were similar. The difference between the two modalities was thought to be due to increased secretion production in the patients, which is measureable in MRI but not in GS. A further study with a solid component of 12 non-nutrient agar beads in addition to the liquid component was completed. 24 HV’s, 17 FD patients and 11 gastro-oesophageal reflux disease (GORD) patients were studied. FD patients and GORD patients had rapid early gastric emptying in comparison to HV in gamma scintigraphy (represented by GCV0) but higher GCV0 in MRI (significantly so between HV and GORD), suggesting increased secretion production is present in both conditions. These findings do support impaired fundal accommodation within the FD population but that other factors, such as secretion production and the rate of this in comparison to gastric emptying are important in the later stages of emptying. Further work is ongoing within the MRI department to quantify and measure the emptying of these secretions. This thesis explores how existing and new technologies can be applied to clinical conditions to identify possible pathophysiology and potential targets for treatment. Only by these ongoing efforts can we endeavour to improve the care we deliver to our patients.

Tese de mestrado integrado. Engenharia Electrotécnica e de Computadores (Major em Telecomunicações). Faculdade de Engenharia. Universidade do Porto. 2009

Pioglitazone (PGZ) is a peroxisome proliferator-activated receptor (PPAR)-γ agonist that improves peripheral insulin sensitivity and reduces hepatocellular injury/ inflammation in non-alcoholic steatohepatitis (NASH). However, the underlying hepatic mechanism of action is not clearly understood with PGZ treatment. Therefore, liver biopsies were used to study genes, protein and immunohistochemistry expression of hepatocyte and hepatic stellate cell markers. PGZ decreased both αSMA and PPAR β expression in stellate cell and PPAR α hepatocyte expression, hence inhibit both stellate cell activation and β-oxidation in hepatocytes. PGZ also inhibit cell proliferation by reducing both PCNA and Ki67 in the liver. Up regulation of PPAR β, PXR, LXRα, IkBα and TNFRSF1B were observed (using Taqman Low density array gene expression), which indicates that PGZ exerts an anti-inflammatory and anti-fibrotic effect. PPAR β, PGC1α, ACADVL and UCP2 up regulation lead to increase β-oxidation and reduced reactive oxygen species (ROS) production. LXRα, ChREBP and SREBP1C activation leads to lipogenesis in the liver was also observed in PGZ treatment group. In vitro study was also conducted in freshly isolated human hepatic stellate cells, where these cells were incubated in vehicle and 5μM of PGZ for 72 hours consecutively. After 24 hours, 15ng/ml of PDGF-BB was incubated for 48 consecutive hours, followed by cell proliferation and gene expression analysis. PGZ up regulate the adipogenic genes expression followed by reduction in stellate cells marker expression and cell proliferation induced by PDGF-BB. PPAR β elevation after PGZ treatment in human liver and HSCs culture are novel findings in this study, but the role of PPAR β is clearly unknown in the liver and stellate cells. Therefore, similar treatment was performed using PDGF-BB on freshly isolated human HSCs, followed by treatment with PPAR β agonist (GW0742). GW0742 restores the adipogenic genes expression maintaining the quiescent phenotype of the stellate cell in contrast to previous study where PPAR β has been reported to cause stellate cells activation and proliferation. Overall, PGZ improved injury and fibrosis, inhibiting cell proliferation, increased both lipogenesis and β-oxidation in NASH patients. PGZ also inhibit stellate cells activation and proliferation and up regulated the adipogenic genes.