Among NAFLD patients without known diabetes or high fasting plasma glucose at or above 7.0 mmol/I, 21% had undiagnosed diabetes and 29% had impaired glucose tolerance. In this population, post-challenge hyperglycemia was associated with NASH and liver fibrosis. Oral glucose tolerance test should be considered in the evaluation of NAFLD patients. / NAFLD is closely related to metabolic syndrome. Using the ethnic-specific IDF criteria, 70% of NAFLD patients had metabolic syndrome, compared to 7% of the general population. A significant proportion of NAFLD patients had body mass index between 23 and 25 kg/m2. The diagnosis of NAFLD may predate the development of different components of metabolic syndrome. / NAFLD patients have lower serum adiponectin level than healthy controls. NASH patients have higher serum tumor necrosis factor alpha (TNF-alpha) level than those with simple steatosis. The differences in adipokines remained significant after adjustment for traditional metabolic risk factors. These suggest that abnormalities in adipokines may be involved in the pathogenesis of NAFLD and NASH. On the other hand, genetic polymorphisms of the adiponectin and TNF-alpha genes are not associated with histological severity. / Since advanced fibrosis is less common in Chinese NAFLD patients, performing liver biopsies on every NAFLD patient for disease staging does not appear to be essential or cost-effective. Recently, the NAFLD fibrosis score is developed using 6 clinical parameters including age, BMI, impaired fasting glucose or diabetes, AST/ALT ratio, platelet count and albumin. The score had high negative predictive value of 91% in excluding advanced fibrosis in Chinese NAFLD patients. It may also reduce the burden of liver biopsies in the majority of cases. / Through a series of clinical studies in Chinese nonalcoholic fatty liver disease (NAFLD) patients in Hong Kong, we demonstrated that nonalcoholic steatohepatitis (NASH) and advanced fibrosis do occur in Chinese, with around 10% of NAFLD patients having advanced fibrosis, and 80% having necroinflammation. Importantly, up to half of the patients had progression of liver fibrosis upon long-term follow-up. / by Wong Wai Sun. / Adviser: Lik Yuen Henry Chan. / Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: . / Thesis submitted in: May 2008. / Thesis (M.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 219-263). / 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. / School code: 1307.

published_or_final_version / Public Health / Master / Master of Public Health

在世界範圍內，肝細胞癌(HCC)是其中一種惡性程度很高並且預後很差的疾病。和其他的癌症一樣，肝癌是一种基因疾病，基因異常的積累在肝癌的生成中扮演著重要的角色。近年來，第二代測序技術（NGS）的迅速發展顯現了前所未見的能力揭示癌癥基因組中分子的複雜性，這為癌癥的生物學，診斷和药物治療提供了一個嶄新的思路。 / 非酒精脂肪性肝炎（NASH）是一種與代謝有關的疾病，在發達國家地區例如美國，歐洲，日本和加拿大，這是其中一種越來越常見的HCC 的病因。在本論文的第一部份，三個NASH 相關的HCC 以及其配對的血DNA 進行了全基因組測序（WGS）。另外還有一個來源于這三個NASH 相關的HCC其中之一的細胞株也進行了全基因組測序。在全部樣品中，測序深度範圍介乎29.1X 到102.2X，序列覆蓋度均大於99%。結果顯示發現的新的單核苷酸變異（SNVs）數量介乎于6,898 至17,129，平均值是11,133。根據這些找到的SNV，隨機選出56 個體細胞SNV 進行Sanger 測序，其中92.9%可以被確認。基因突變譜顯示有頻繁的A:T>G:C 和C：G>A:T 體細胞突變，而C：G>T:A 則在CpG 位點頻繁出現。在眾多的非同義體細胞突變基因中，我們選擇了CTNNB1，PNLIP 和MLL2 這三個基因進行進一步研究，此三個基因都在多於一個病例中發現有突變。在額外的一組44 對NASH 相關HCC及50 對HBV 相關的HCC 的癌變組織和其臨近非腫瘤組織中，我們進一步對這三個基因和TP53 的編碼區域進行了測序，而TP53 是在HBV 相關HCC中被報導有高頻率突變的。在NASH 相關的HCC 中，這些基因都只在腫瘤組織中發現重複出現的錯義突變，在臨近的非腫瘤肝組織沒有發現有突變。在NASH 相關的HCC 中，CTNNB1 的突變率（36.4%）明顯高於在HBV相關的HCC 中的突變率（12.0%，P=0.007）。PNLIP 和MLL2 的突變只在NASH相關的HCC 中發現，其突變率分別為12.1%和7.1%，而在HBV 相關的HCC中，則沒有發現突變。然而，TP53 的突變率在NASH 相關的HCC 及HBV相關的HCC 中差別不明顯（P>0.1）。在功能性研究的實驗中，我们发现在HKCI-10（有PNLIP 突變D396N）細胞株中，PNILP 的活性比在正常肝細胞L02 細胞株（野生型PNLIP）中要低。在永生性肝細胞L02 細胞株中，CTNNB1的突變引起了TOPFLASH 活性的提高以及增加了細胞群落形成的能力。HKCI-10 是一條我們實驗室建立的NASH 相關的肝細胞癌細胞株，在HKCI-10細胞株中，抑制CTNNB1 表達引起了細胞生長和增殖的減少。另外，在DEN誘導肝癌的有代謝失衡的db/db 轉基因鼠中，發現了一個CTNNB1 的突變T41A。據報導，CTNNB1 發生的致癌突變會引起蛋白的穩定並且因此激活經典的Wnt/β-catenin 信號通路，從而引致特定基因的轉錄。對於CTNNB1中最常見的突變S45P(在發現的CTNNB1 突變中占31.3%)，我們做了ChIP-array 實驗，結果顯示，在HKCI-10（CTNNB1 有S45P 突變）中，CTNNB1比在Hep3B 中（野生型CTNNB1）有著更緊密的啟動子結合能力（P<0.001）。Gene ontology 分析結果表明，被S45P 富集的生物過程涉及有RNA 代謝調節，轉錄因子活性和凋亡。MYC，E2F1 和ZFX 被ChIP-PCR 證實是與CTNNB1突變子S45P 有著更緊密結合能力的轉錄因子。 / 在本論文的第二部份，爲了進一步闡明致癌性的CTNNB1 突變S45P在HCC 中的角色，我們研究了一個此前未在HCC 報導過的基因ZFX。ZFX是一個在脊椎動物中高度保守，屬於Zfy 家族的zinc finger 蛋白。有報導指出ZFX 對於胚胎和造血幹細胞的自我更新有著重要的作用。另外亦有文獻報導ZFX 在一系列人類癌癥病例中有過度表達，例如食道癌，胃癌，前列腺癌和神經膠質瘤，並且顯現出致癌基因的特性。在本研究中，qRT-PCR結果提示ZFX 在HCC 腫瘤中的表達明顯高於正常肝組織。ZFX 的表達在51.8%的HCC 腫瘤中明顯高於其配對的鄰近非腫瘤肝組織。功能性研究表明，在MTT 實驗中，細胞的生存力在ZFX 缺失的穩定克隆中明顯減弱（P<0.0001）。在細胞群落形成實驗中，ZFX 缺失的穩定克隆顯示出明顯減弱的群落生長能力（P<0.0001）。在單細胞克隆生成實驗中，ZFX 缺失的HCC穩定克隆展示出數量更少，體積更小的細胞群落。另外，ZFX 基因抑制或者cisplatin 單獨處理均顯示細胞生存力的抑制，其抑制效率分別是24.0%和30.9%。當ZFX 基因抑制合併cisplatin 處理時，細胞生存力的抑制效率顯著地提高至65.2%，這個結果提示ZFX 基因抑制和cisplatin 處理兩者有協同增效作用（P<0.0001）。ZFX 基因的缺失會引起兩個為人熟知的胚胎幹細胞（ESCs）標誌物SOX-2 和NANOG 表達的明顯降低。 / 綜上所述，通過進行全基因測序，本論文的研究結果為NASH 相關的HCC 分子層面上的異常提供了一個高解析度的視覺角度。在NASH 相關的HCC 中，一些可能對於肝細胞癌變有重要作用並且重複出現突變的基因被確定，例如CTNNB1 和PNLIP。CTNNB1 的突變體S45P 的其中一個下游目標基因ZFX，在HCC 中被證實有幹細胞和腫瘤啟動細胞特性。闡明在HCC發展過程中的分子改變以及機制，對於為肝癌病人探索新的治療手段有著重要意義。 / Hepatocellular Carcinoma (HCC) is one of the most malignant diseases worldwide with poor prognosis. Like other human cancers, HCC is a genetic disease, where accumulation of genetic aberration plays important role in the liver carcinogenesis. The rapid advances in Next Generation Sequencing (NGS) technology in recent years have allowed unprecedented ability to unravel the molecular complexity of the cancer genome, providing new insights into the cancer biology, diagnosis and therapeutic drug development. / Non-alcoholic steatohepatitis (NASH), which is related to metabolic disorder, is an increasing common etiological factor of HCC, especially in developed countries such as United States, Europe, Japan and Canada. In first part of this thesis, Whole Genome Sequencing (WGS) was performed on three NASH-associated HCCs and their matched lymphocytic DNA. A cell line developed from one of the three NASH-associated HCCs was also subjected to WGS. The sequencing depth ranged from 29.1X to 102.2X with the coverage >99% shown in all samples. Novel SNVs identified ranged from 6,898 to 17,129 with an average of 11,133. Based on the SNVs found, the validation rate was 92.9% in 56 randomly selected somatic SNVs by Sanger sequencing. Mutational spectrum showed frequent somatic substitution of A:T>G:C and C:G>A:T while C:G>T:A transition exhibited a predominant somatic mutation rate in CpG sites. Among the non-synonymous somatic mutated genes, we selected CTNNB1, PNLIP and MLL2 which were mutated in more than one tumor for further study. In additional cohort of 44 NASH-associated and 50 HBV-associated HCC tumors and adjacent non-tumoral tissues, further sequencing all the coding regions of these three genes and TP53, which has been reported to be highly mutated in HBV-associated HCCs, were carried out. In NASH-associated HCCs, all genes harboured recurrent missense mutations exclusive in tumor but none in adjacent ,non-tumoral liver. The prevalence of CTNNB1 mutations was significantly higher in NASH-associated HCCs (36.4%) when compared to HBV-associated HCCs (12.0%, P=0.007). Mutations of PNLIP and MLL2 were detected only in NASH-associated HCCs with rates of 12.1% and 7.1%, respectively, but none in HBV-associated HCCs. The mutation rate of TP53, however, did not differ much between NASH-associated and HBV-associated HCCs (P>0.1). In functional study, for PNLIP, a loss-of-function in PNLIP activity was found in HKCI-10 harbouring D396N mutation as compared to normal liver cell, L02 with wild-type PNLIP. We demonstrated that CTNNB1 mutants conferred elevated TOPFLASH activity and enhanced colony growth in an immortalized hepatocyte cell line L02. Knockdown of CTNNB1 in HKCI-10, which is a NASH-associated HCC in-house cell line, resulted in inhibition of cell growth and proliferation. Also, a CTNNB1 mutation (T41A) was found in DEN-induced liver cancer of db/db transgenic mouse with metabolic disorder. Since oncogenic mutations of CTNNB1 were reported to be contributed to the stabilization of the protein and hence activate the canonical Wnt/β-catenin signaling pathway, inducing transcription of specific gene sets. We performed ChIP-array focusing on the most common CTNNB1 S45P mutation (accounted for 31.3% of all detected CTNNB1 mutants) in NASH-associated HCCs, the result showed more intense promoter binding affinities in HKCI-10 with CTNNB1 S45P mutation than Hep3B with wild-type CTNNB1 (P<0.001). Gene ontology analysis revealed that S45P mutant enriched the process including RNA metabolic regulation, transcription factor activities and apoptosis. Furthermore, we found that CTNNB1 S45P mutant showed more profound binding affinity to the promoter regions of transcription factors MYC, E2F1 and ZFX by ChIP-PCR. / In the second part of the thesis, we aimed to further understand the role of oncogenic CTNNB1 S45P mutant in HCC. Zinc finger protein X-linked (ZFX) gene which is previously undescribed in HCC was studied. ZFX is a zinc finger protein of Zfy family that is highly conserved among vertebrates. It has been reported that ZFX is required for self-renewal of embryonic and hematopoietic stem cells. Also, ZFX is suggested to be overexpressed in a number of human cancers such as esophageal carcinoma, gastric cancer, prostate cancer and glioma, conferring oncogenic characteristics. In this study, qRT-PCR analysis showed a significant higher ZFX expression in HCC tumors compared to normal livers. 51.8% of HCC tumors showed significant up-regulations of ZFX when compared to paired adjacent non-tumoral livers. Functional studies showed significant reduction in in-vitro cell proliferation in HCC by MTT assay (P<0.0001) and colony formation ability by colony formation assay (P<0.0001) in ZFX-deficient stable clones. In single-cell clonogenic assay, ZFX-depleted HCC exhibited fewer and smaller colonies. In addition, while ZFX knockdown or cisplatin treatment alone showed an inhibitory effect on cell viability by 24.0% and 30.9%, respectively, the reduction efficiency of cell viability increased dramatically to 65.2% when combine ZFX-knockdown and cisplatin treatment, indicating a synergistic effect between them (P<0.0001). Also, significant reduced expressions of SOX-2 and NANOG, both well-known embryonic stem cells (ESCs) markers, were observed as a result of ZFX depletion. / In summary, findings from this thesis provided high-resolution insight into the molecular aberrations in NASH-associated HCCs by performing WGS. Recurrent mutated genes which may be of great importance in hepatocellular carcinogenesis induced by NASH were identified, such as CTNNB1 and PNLIP. One of the S45P CTNNB1 downstream targets ZFX was demonstrated to confer stemness and tumor-initiating cells (TICs) characteristics in HCC. The understanding of molecular changes and mechanisms underlying HCC development will thus facilitate the exploration of new therapeutic options in patients with this deadly disease. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Jiawei. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 166-190). / Abstracts also in Chinese.

Thesis (Ph. D.)--George Mason University, 2007. / Title from PDF t.p. (viewed Jan. 18, 2008). Thesis director: Ancha Baranova Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biosciences. Vita: p. 213 Includes bibliographical references (p. 188-212). Also available in print.

Background and objectives: Nonalcoholic steatohepatitis (NASH), which is characterized by concurrent existence of hepatic steatosis and predominantly lobular necroinflammation, represents the more advanced stage in the spectrum of nonalcoholic fatty liver disease (NAFLD). NASH exhibits dramatically increased risk of progression to end-stage liver diseases than simple steatosis. Therefore, the progression of hepatic steatosis to steatohepatitis is the crucial step in the development of obesity-related NASH. Toll like receptor 4 (TLR4), a master regulator of innate immunity, is the principal receptor for endotoxin, which is a central mediator of liver inflammation associated with both alcoholic and nonalcoholic liver disease. However, due to a lack of suitable animal models which fully recapitulate the natural history of obesity-induced NASH, the precise pathophysiological function of TLR4 signaling in the development of this disease remains poorly understood. The objective of this study is to investigate the role of TLR4 in mediating inflammatory responses in obesity-induced NASH using both in vivo and ex vivo approaches, and to unveil cellular and molecular mechanisms responsible for TLR4 actions. Key findings: 1. To address the role of TLR4 in the pathogenesis of NASH, we crossed ApoEdeficient mice (ApoE-/-) with TLR4 mutant mice (TLR4-/-) to generate ApoE-/- /TLR4 wild type mice (ApoE-/-/TLR4-WT) and ApoE-/-/TLR4-/- mice. Noticeably, when fed with high fat high cholesterol (HFHC) diet, ApoE-/-/TLR4-WT mice developed the typical pathology of NASH (hepatic steatosis, lobular inflammation, and hepatocyte ballooning) in the context of obesity and metabolic syndrome, suggesting HFHC-fed ApoE-/- mice as a suitable animal model for NASH. 2. TLR4 inactivation protected ApoE-/- mice against HFHC diet-induced liver injury, as indicated by a significant improvement in liver histology, a a marked reduction in serum ALT activity, a dramatic repression of inflammatory infiltrates, as well as an obvious decrease in hepatic production of pro-inflammatory cytokines. 3. In ApoE-/-/TLR4-WT mice, TLR4 expression was selectively elevated in Kupffer cells in response to HFHC diet feeding. 4. The activation of XBP1, a transcription factor involved in endoplasmic reticulum stress, was markedly elevated in liver of ApoE-/-/TLR4-WT mice fed with HFHC diet, whereas this change was abrogated in HFHC diet-fed ApoE-/-/TLR4-/- mice. 5. In rat primary Kupffer cells, treatment with anti-oxidants blocked endotoxininduced activation of XBP1 and NF-κB, leading to decreased cytokine production. In addition, siRNA-mediated knockdown of XBP1 inhibited NF-κB activation and cytokine production resulted from the treatment with the TLR4 agonist LPS. 6. In ApoE-/-/TLR4-WT mice, adenovirus-mediated expression of dominant negative XBP1 had no obvious effect on HFHC diet-induced hepatic steatosis and ROS production, but markedly decreased lobular inflammation, NF-κB activation, cytokine production in the liver and significantly reduced serum levels of ALT. Conclusions: These findings support the role of TLR4 in Kupffer cells as a key player in mediating the progression of simple steatosis to NASH, by inducing ROS-dependent activation of XBP1. In light of the obligatory role of XBP1 in TLR4-induced liver inflammation and injury, therapeutic interventions that inhibit TLR4/XBP1 activation may represent a promising strategy for treatment of NASH. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Non-alcoholic fatty liver disease is affecting an increasing population worldwide. NAFLD is closely associated with obesity and diabetes. Research has shown that the expression of some important hepatic transporters and enzymes are altered under inflammatory conditions. We examined the effect of NAFLD on the gene expression of several hepatic transporters and enzymes, as well as the impact of exercise in attenuating the effect of NAFLD. We have demonstrated that the mRNA expression of several hepatic transporters and enzymes, as well as FXR were significantly downregulated in liver of rats treated with a HFHCD. We concluded that HFHCD-induced hepatic steatosis, together with the reduced expression of FXR, contributed to the downregulation of expression of hepatic transporters and enzymes. The mRNA expression of TNF-α and IL-1β were unaffected. Interestingly, exercise was found to improve the expression levels of some transporters and enzymes.

Non-alcoholic fatty liver disease (NAFLD) includes simple hepatic steatosis (SS) and non-alcoholic steatohepatitis (NASH). NAFLD is tightly linked to obesity and is thought to be secondary to various noxious signals, some of which may originate from the intestinal microbiota (IM). Despite a growing body of evidence supporting a link between obesity and altered IM, there are no studies assessing the IM of patients with NAFLD. In this cross-sectional study we aimed at comparing fecal levels of total bacteria, Bacteroidetes, C. coccoides, C. leptum, Bifidobacteria, E. coli, and Archaea between healthy controls (HC) and patients with SS or NASH. We found higher C. coccoides levels in NASH compared to SS and lower percentage Bacteroidetes in NASH compared to SS and HC. Controlling for body mass index and fat intake we found an association between presence of NASH and percentage Bacteroidetes. The latter inversely correlated with insulin resistance.

Non-alcoholic fatty liver disease is affecting an increasing population worldwide. NAFLD is closely associated with obesity and diabetes. Research has shown that the expression of some important hepatic transporters and enzymes are altered under inflammatory conditions. We examined the effect of NAFLD on the gene expression of several hepatic transporters and enzymes, as well as the impact of exercise in attenuating the effect of NAFLD. We have demonstrated that the mRNA expression of several hepatic transporters and enzymes, as well as FXR were significantly downregulated in liver of rats treated with a HFHCD. We concluded that HFHCD-induced hepatic steatosis, together with the reduced expression of FXR, contributed to the downregulation of expression of hepatic transporters and enzymes. The mRNA expression of TNF-α and IL-1β were unaffected. Interestingly, exercise was found to improve the expression levels of some transporters and enzymes.

No description available.