Focal liver lesions are commonly encountered in clinical practice. To be able to differentiate potentially life-threatening lesions from clinically insignificant lesions, and to be able to treat them effectively are the two basic problems of a clinician who comes across such lesions. Percutaneous intervention of the liver with a needle enables a clinician to solve the above two problems in a minimally invasive manner. To date, there is a diversity of needle-related percutaneous interventional procedures that are applicable to the clinical management of patients with liver lesions, such as biopsy of focal lesions, drainage of abscesses, and ablation of tumors. Despite a reasonable safety and efficacy associated with these procedures, there are always grounds of further improvement in techniques and technology of needle-related percutaneous procedures to achieve an even better outcome. It was hypothesized that the application of needle-related interventional radiology to clinical management of focal liver lesions could be facilitated and extended with advancement and refinement in needle-related techniques and technology. This thesis was based on a series of nine studies that aimed to explore the potential of needle-related percutaneous interventions in the clinical management of focal liver lesions and to study the effect of the introduction of innovations in needle-related techniques and technology on such clinical applications. It was concluded that the hypothesis was confirmed. / Yu Chun Ho. / "April 2006." / Adviser: Anil Ahuja. / Source: Dissertation Abstracts International, Volume: 68-08, Section: B, page: 5176. / Thesis (M.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 219-235). / 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.

To Kin Wah. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 293-345). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

丹參為雙子葉植物唇形科鼠尾草族植物的乾燥根及根莖。在中國，丹參為廣泛用於治療心血管疾病的藥用植物，而在西方，丹參也常作為一種輔助性藥物。《中國藥典2010版》收錄了35個以上含有丹參的複方或者方劑。在這些複方中，採用了富含丹酚酸和丹參酮的丹參水提物、乙醇提取物或兩者的混合物。丹參提取物具有較強的抗氧化作用，被認為在化學預防和化療的輔助治療中有一定用途。作為主要的丹參水溶性成分，熱敏感的丹酚酸在提取與加熱過程中可能會降解為其他丹酚酸。丹參水提取物的化學組成可能會在不同熱水提取溫度下有所不同，進而影響其藥理活性。在本研究中，通過加熱回流提取和在不同溫度下的微波提取（MAE-W）獲得了6種丹參水提取物，並對這些提取物進行化學成分和藥理分析，考察它們的抗氧化、抗凋亡和血管舒張作用。在這些提取物中，第三輪的微波提取物（100 oC）含有最多的丹酚酸和丹參酮，在1，1-二苯基-2-三硝基苯肼（DPPH）法和鐵還原/抗氧化能力（FRAP）法中具有最強的抗氧化活性，在2,2'-偶氮二(2-脒基丙烷)二鹽酸鹽（AAPH）誘導人血紅細胞的溶血實驗和過氧化氫誘導大鼠心肌細胞H9c2凋亡實驗中還顯示了最強的抑制作用，對大鼠腦基底動脈有最強的鬆弛效應。這些丹參水提取物的抗氧化作用與它們的血管舒張效應呈一定的線性關係（回歸係數r = 0.895 - 0.977）。通過多元線性回歸分析發現，丹參素可以作為丹參水提物的抗氧化和血管舒張功能的顯著性標記物，而丹參酮IIA則是抑制過氧化氫誘導大鼠心肌H9c2細胞凋亡的標記物。 / 作為丹參中主要的脂溶性成分，丹參酮在不同的腫瘤細胞系和荷瘤小鼠模型中展示了抗癌潛力。這些丹參酮的抗癌機制包括細胞週期阻滯，觸發半胱天冬酶（Caspase）依賴的內源性和外源性的凋亡途徑和絲裂原激活的蛋白激酶（MAPK）信號通路等。丹參新酮（miltirone）是從丹參中分離得到的松香烷型二萜醌類化合物，具有多種的藥理活性，如抗氧化，抗焦慮和抗腫瘤等。本研究評估了丹參新酮在人肝癌HepG2細胞系和P-糖蛋白（P-gp）過表達的阿霉素耐藥HepG2細胞系（R-HepG2）中的凋亡作用及其機制。丹參新酮在HepG2細胞中顯示了細胞毒性（EC₅₀值為7.06 微摩），而丹參新酮在抑制HepG2和R-HepG2細胞增殖中的濃度依賴性沒有顯著性差異。丹參新酮（1.56 - 6.25微摩）與阿霉素（DOX）對R-HepG2細胞的增殖具有協同效應，在達到50的生長抑制時，它們的聯合用藥指數為0.3至0.5。流式細胞術分析表明，丹參新酮降低了R-HepG2細胞中P-gp介導的阿霉素外排，分子對接研究表明該效果是通過抑制P-gp的藥物結合位點。在非壞死濃度（25微摩或以下），丹參新酮在HepG2和R-HepG2細胞中活化了Caspase依賴的凋亡途徑，誘導產生活性氧（ROS）和氧化應激，且觸發ROS介導的包括p38 MAPK，應激活化蛋白激酶/c-Jun氨基末端激酶（SAPK / JNK）以及細胞外調節激酶1和2在內的MAPK信號通路。綜上所述，在R-HepG2中丹參新酮是P-gp和細胞增殖的雙重抑制劑，顯示了其在治療肝癌（HCC）的潛力。 / 為了增加藥物開發的成功率，在藥物發現的早期階段應考察新化學實體（NCEs）的蛋白結合率，清除率，藥動學參數，以及藥物代謝相互作用等體內代謝參數。以往的研究已經顯示了從丹參中分離得到的四種主要丹參酮對人和大鼠的細胞色素P450酶介導的探針底物的代謝具有不同程度的抑制作用，需要注意丹參和其他藥物間的相互作用。本研究的另一目的是在人類肝微粒體中探討丹參新酮與探針底物間的細胞色素P450酶介導的代謝相關的相互作用。人肝微粒體孵育實驗結果表明丹參新酮對CYP1A2（IC₅₀值為 1.73微摩）和CYP2C9（IC₅₀值為8.61微摩）有中等強度的抑制，對CYP2D6（IC₅₀值為30.20微摩）和CYP3A4（IC₅₀值為33.88微摩）有弱的抑制。酶動力學和分子對接研究的結果進一步表明，丹參新酮為CYP1A2（Ki值為3.17微摩）的中等強度混合型抑制劑，是CYP2C9（Ki值為1.48微摩）的中等強度競爭型抑制劑，也是CYP2D6（Ki值為24.25微摩）和CYP3A4（Ki值為35.09微摩）的弱的混合型抑制劑。這些結果表明，應考慮丹參新酮與CYP1A2和CYP2C9代謝的藥物間的相互作用，但是可認為其與CYP2D6及CYP3A4代謝的藥物間幾乎不存在相互作用。 / 總之，本研究考察了不同提取方法對丹參提取物成份及其藥效的影響，確定了不同用途的丹參提取物的質控標記物。本研究還考察了丹參新酮體外抗肝癌的能力及其藥物代謝相互作用為基礎的類藥性,為其進一步的體內試驗提供了依據。 / Danshen, the dried root and rhizome of Salvia miltiorrhiza Bg. (Fam. Labiatae), is a widely used medicinal plant for the treatment of cardiovascular diseases in China and also a complementary medicine in the West. Danshen is indexed in the Pharmacopoeia of People’s Republic of China (2010 Edition), with more than 35 formulations and concoctions containing Danshen water-extracts, ethanolic extracts or their combination which are rich in phenolic acids and tanshinones with various contents. Danshen extracts have been considered for the use as an adjunct in chemoprevention and chemotherapy due to their strong antioxidant effects. Phenolic acids, the major water-soluble components in Danshen, are thermosensitive and may degrade to other phenolic acids during extractions upon heating. The chemical profiles of Danshen water-extracts may vary with different heat water extraction at different temperatures, affecting the composition and bioactivity of the extracts obtained. In this study, six water-extracts of Danshen obtained from heat reflux water extraction and microwave-assisted extraction with water (MAE-W) at different temperatures were prepared for evaluation of their composition and pharmacological effects such as antioxidant, anti-apoptosis and vascular relaxation. Among these extracts obtained, the third-round MAE-W (100 °C) product, which was the last round product obtained by extracting the same crude material three times, had the highest contents of phenolic acids and tanshinones, with the strongest antioxidant activity estimated by 2, 2-diphenyl-1-(2, 4, 6-trinitrophenyl) hydrazyl (DPPH) assay and ferric reducing / antioxidant potential (FRAP) assay. This extract also possessed the strongest inhibitory effects on 2, 2'-azobis-2-amidino-propane (AAPH)-induced haemolysis in human red blood cells, hydrogen peroxide-induced apoptosis in rat heart H9c2 cells and the highest relaxation effects on rat basilar artery. The antioxidant effects of Danshen water-extracts linearly correlated to their relaxation effects (r = 0.895 to 0.977). Through multiple linear regression analysis, danshensu was found to be the most significant marker in the antioxidant and vasodilation effects of Danshen water-extract, while tanshinone IIA as the marker on hydrogen peroxide-induced apoptosis in rat heart H9c2 cells. Danshensu is, therefore, a useful marker for the quality control of Danshen water-extracts in antioxidant and vasodilation, while tanshinone IIA for anti-apoptotic potential of water-extracts. / Tanshinones, the major lipid-soluble components isolated from Danshen, have been reported for their anti-cancer potential in various cell lines and tumor-bearing mice models. Their anti-cancer mechanisms are also well-studied, mainly through cell cycle arrest, caspase-dependent apoptotic pathways and mitogen activated protein kinase (MAPK) signaling pathways. Miltirone, another abietane type-diterpene quinone isolated from Danshen, has been reported for its anti-oxidative, anxiolytic and anti-cancer effects. This study evaluated the apoptotic effect of miltirone and the underlying mechanisms in a human hepatoma HepG2 cell line and its p-glycoprotein (P-gp)-overexpressed doxorubicin-resistant counterpart (R-HepG2). Miltirone showed similar cytotoxicity in HepG2 (EC₅₀ = 7.06 μM) and R-HepG2 (EC₅₀ = 12.0 μM), demonstrated synergistic effects (1.56 - 6.25 μM) with doxorubicin (DOX) on the growth inhibition of R-HepG2 (synergism: 0.3 < CI < 0.5 at 50 % inhibition). Flow cytometric analysis showed that miltirone decreased P-gp-mediated DOX efflux in R-HepG2, and molecular docking studies illustrated that this effect was through inhibition on the active site of P-gp. At non-necrotic concentrations (25 μM or below), miltirone activated caspase-dependent apoptotic pathways, and induced the generation of reactive oxygen species (ROS) and oxidative stress which triggered ROS-mediated MAPK signaling pathways, including p38 MAPK, stress-activated protein kinase / c-Jun N-terminal kinase (SAPK/JNK) and extracellular regulated kinase 1/2, in both HepG2 and R-HepG2 cells. It is therefore concluded that miltirone is a dual inhibitor on P-gp and cell proliferation in R-HepG2 cells, with potential for the treatment of human hepatocellular carcinoma (HCC). / In order to improve the successful rates in drug development, the in vivo metabolic parameters of new chemical entities (NCEs), such as protein bindings, clearance rate, pharmacokinetic parameters and metabolism-based drug-drug interactions, should be considered at the early stage of drug discovery. Previous studies have shown that major tanshinones isolated from Danshen inhibited the metabolism of model probe substrates of human and rat CYP450 enzymes, with potential in causing herb-drug interactions. The aim of this study was to study the effect of miltirone on the metabolism of model probe substrates of CYP1A2, 2C9, 2D6 and 3A4 in pooled human liver microsomes. Miltirone showed moderate inhibition on CYP1A2 (IC₅₀ = 1.73 μM) and CYP2C9 (IC₅₀ = 8.61 μM), and weak inhibition on CYP2D6 (IC₅₀ = 30.20 μM) and CYP3A4 (IC₅₀ = 33.88 μM). Enzyme kinetic studies showed that miltirone competitively inhibited CYP2C9 (Ki = 1.48 μM), and displayed mixed type inhibitions on CYP1A2, CYP2D6 and CYP3A4 with Ki values of 3.17 μM, 24.25 μM and 35.09 μM, respectively. Molecular docking study further confirmed the ligand-binding conformations of miltirone in the active sites of human CYP450 isoforms. These findings suggested that miltirone may have potential drug-drug interactions with CYP1A2- and CYP2C9-metabolized drugs, and to a lesser extent with CYP2D6- and CYP3A4-metabolized drugs. / In conclusion, this study investigated the effects of Danshen water-extracts produced by different extraction methods on the chemical compositions and pharmacological activities, and consequently confirmed the biomarkers for the quality control of Danshen water-extracts for different medicinal uses. This study also demonstrated the anti-cancer potential of miltirone for HCC in vitro and the metabolism-based drug-drug interactions for its drug-likeness, which may provide useful and promising data for in vivo anti-cancer study of miltirone and further pre-clinical studies. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhou, Xuelin / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 195-224). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 論文摘要 --- p.v / Publications based on the work in this thesis --- p.viii / Acknowledgements --- p.x / Abbreviations --- p.xii / Table of Contents --- p.xv / Chapter Chapter 1 --- General introduction --- p.1 / Chapter 1.1 --- Reactive oxygen species and carcinogenesis --- p.1 / Chapter 1.2 --- Reactive oxygen species and tumor progression & metastasis --- p.2 / Chapter 1.3 --- Antioxidant enzymes in chemoprevention and chemotherapy --- p.3 / Chapter 1.3.1 --- Glutathione and Glutathione reductase --- p.5 / Chapter 1.3.2 --- Glutathione Peroxidase --- p.5 / Chapter 1.3.3 --- Glutathione S-transferases --- p.6 / Chapter 1.3.4 --- NAD(P)H: quinone reductase 1 --- p.7 / Chapter 1.3.5 --- Heme oxygenase-1 --- p.8 / Chapter 1.3.6 --- Thioredoxin reductase --- p.9 / Chapter 1.3.7 --- Superoxide Dismutase --- p.10 / Chapter 1.3.8 --- Catalase --- p.11 / Chapter 1.4 --- Medicinal uses of Danshen --- p.12 / Chapter 1.5 --- Analysis of Danshen and its components --- p.14 / Chapter 1.6 --- Antioxidant effects of Danshen extract and its bioactive compounds in chemoprevention and chemotherapy-related disease --- p.19 / Chapter 1.7 --- Anti-cancer effects of tanshinones isolated from Danshen --- p.21 / Chapter 1.7.1 --- Tanshinone IIA --- p.22 / Chapter 1.7.2 --- Tanshinone I --- p.26 / Chapter 1.7.3 --- Cryptotanshinone --- p.27 / Chapter 1.7.4 --- Dihydrotanshinone --- p.27 / Chapter 1.8 --- Metabolism / disposition of Danshen and its major active ingredients --- p.28 / Chapter 1.9 --- Herb-drug interactions with Danshen --- p.31 / Chapter 1.10 --- Effects of Danshen (and its major active ingredients) on model probe substrates of CYP isoforms --- p.33 / Chapter 1.11 --- CYPs induction by Danshen and its active components --- p.38 / Chapter 1.12 --- Effects of Danshen / active ingredients on drug transporter proteins --- p.40 / Chapter 1.13 --- CYP450 inhibition screening for new chemical entity --- p.42 / Chapter 1.14 --- Molecular docking analysis --- p.44 / Chapter 1.15 --- The Aim of this study --- p.45 / Chapter Chapter 2 --- Quantitative and qualitative studies to evaluate the efficiency of different heat water-extractions --- p.48 / Chapter 2.1 --- Introduction --- p.48 / Chapter 2.2 --- Materials and methods --- p.51 / Chapter 2.2.1 --- Materials and apparatus --- p.51 / Chapter 2.2.2 --- Extraction procedures --- p.51 / Chapter 2.2.3 --- HPLC analysis --- p.54 / Chapter 2.2.4 --- DPPH assay and FRAP assay --- p.54 / Chapter 2.2.5 --- Inhibition of 2,2'-azobis-2-amidinopropane (AAPH)-induced haemolysis in human red blood cells --- p.55 / Chapter 2.2.6 --- Protective effects on hydrogen peroxide-induced apoptosis in rat heart H9c2 cells --- p.56 / Chapter 2.2.7 --- Vasodilation effects on rat basilar artery --- p.57 / Chapter 2.2.8 --- Statistical analysis --- p.58 / Chapter 2.3 --- Results and Discussion --- p.59 / Chapter 2.3.1 --- Chemical profiles analyzed by HPLC analysis --- p.59 / Chapter 2.3.2 --- DPPH assay and FRAP assay --- p.63 / Chapter 2.3.3 --- Inhibition of AAPH-induced haemolysis --- p.65 / Chapter 2.3.4 --- Protective effects on hydrogen peroxide-induced apoptosis --- p.69 / Chapter 2.3.5 --- Vasodilation effects on rat basilar artery --- p.71 / Chapter 2.3.6 --- Multiple linear regression analysis --- p.76 / Chapter Chapter 3 --- Effects of miltirone on cell proliferation in a hepatoma HepG2 cell line and its doxorubicin-resistant counterpart --- p.83 / Chapter 3.1 --- Introduction --- p.83 / Chapter 3.2 --- Materials and Methods --- p.87 / Chapter 3.2.1 --- Chemicals --- p.87 / Chapter 3.2.2 --- Cell culture --- p.87 / Chapter 3.2.3 --- Cell viability test --- p.88 / Chapter 3.2.4 --- Drug-efflux study by flow cytometry --- p.89 / Chapter 3.2.5 --- Molecular docking study and Ligand-based prediction --- p.90 / Chapter 3.2.6 --- Measurement of ROS generation by confocal microscopy and flow cytometry --- p.91 / Chapter 3.2.7 --- GSH and GSSG determination for oxidative stress --- p.93 / Chapter 3.2.8 --- Apoptosis-related proteins expression detected by Western blotting analysis --- p.94 / Chapter 3.2.9 --- Data analysis --- p.96 / Chapter 3.3 --- Results --- p.97 / Chapter 3.3.1 --- Cytotoxicity in hepatoma cells --- p.97 / Chapter 3.3.2 --- Drug-efflux study by flow cytometry --- p.104 / Chapter 3.3.3 --- Molecular docking study and Ligand-based prediction --- p.108 / Chapter 3.3.4 --- ROS generation --- p.113 / Chapter 3.3.5 --- Determination of GSH/GSSG ratio --- p.117 / Chapter 3.3.6 --- Caspase-dependent apoptosis. --- p.121 / Chapter 3.3.7 --- Phosphorylation of MAPKs --- p.126 / Chapter 3.4 --- Discussion --- p.134 / Chapter Chapter 4 --- Enzyme kinetic and molecular docking studies of miltirone on major human cytochrome P450 isozymes inhibitions --- p.139 / Chapter 4.1 --- Introduction --- p.139 / Chapter 4.2 --- Material and Methods --- p.141 / Chapter 4.2.1 --- Materials and Reagents --- p.141 / Chapter 4.2.2 --- Incubation conditions --- p.142 / Chapter 4.2.3 --- Samples preparation --- p.143 / Chapter 4.2.4 --- HPLC analysis --- p.143 / Chapter 4.2.5 --- CYP inhibition and enzymatic kinetic study --- p.144 / Chapter 4.2.6 --- Molecular docking analysis --- p.145 / Chapter 4.2.7 --- Data analysis --- p.146 / Chapter 4.3 --- Results --- p.148 / Chapter 4.3.1 --- CYP inhibition and enzymatic kinetic study --- p.148 / Chapter 4.3.2 --- Molecular docking study of miltirone --- p.167 / Chapter 4.4 --- Discussions --- p.184 / Chapter Chapter 5 --- General discussion --- p.188 / References --- p.195

MicroRNA（miRNAs）是一類細小的非編碼RNA（ncRNA），能透過轉錄後機制調節靶標基因的表達。miRNA的發現，不僅提出一個嶄新的基因調節機制，更強調了小ncRNA於不同的生理和發展過程中的重要性。最近的研究更進一步展示了miRNA失調與癌症發展之間的因果關係。 / 我們此前曾利用陣列分析，發現miRNA在肝細胞癌（HCC）中的失調模式，揭示了miR-145在HCC的普遍下調。在本論文的第一部分，定量逆轉錄聚合酶鏈反應（qRT-PCR）進一步證實了miR-145在50的肝細胞癌患者（n=80）的腫瘤中出現表達下調，而且miR-145的表達下調更與較短的无病生存期相關。其中一個低內源性miR-145的肝癌腫瘤樣本被建立為細胞株─HKCI-C2。此體外模型保持低miR-145水平，並於恢復miR-145表達後，抑製細胞存活和增殖。多個計算機演算法均預測了miR-145可針對胰島素樣生長因子（IGF）信號通路中的多個基因，包括胰島素受體底物（IRS1）-1，IRS2和胰島素樣生長因子1受體。這些假定目標的蛋白表達亦被miR-145下調。熒光素酶檢測進一步驗證了miR-145和IRS1/IRS2 3'-非編碼區的直接目標關聯。隨後的分析也確定miR-145能下調 IGF信號通路下游的信號傳導，即活性β-catenin水平。 / 最近出現的深度測序技術，為研究miRNome提供了一個前所未有的平台，以識別已知和新的miRNA。此外，現代生物信息學技術可同時對不同類型的小ncRNA，如PIWI-interacting RNA（piRNAs）進行分析。在本論文的第二部分中，我們利用Illumina大規模並行測序對兩個肝癌細胞株（HKCI-4和HKCI-8）和正常肝細胞株（MIHA）的小RNA轉錄組進行研究。生物信息學和生物功能分析揭示一種新型piRNA（取名為piR-Hep1）在肝腫瘤發生中的重要角色。在73例肝癌中，qRT-PCR結果顯示piR-Hep1在47的肝癌組織出現上調。PiR-Hep1的沉默能抑制肝癌細胞存活、遷移和侵襲，同時亦減少了Akt的磷酸化。在miRNA的分析中，miR-1323被發現在肝癌組織中大量表達，並與肝硬化背景下產生的肝腫瘤相關。此外，miR-1323出現過表達的肝硬化肝癌患者的無病和整體存活率亦較差（P<0.009）。 / 總觀來說，本論文首次發現miR-145可同時抑制引致肝癌的IGF信號通路中的多個傳導因子，亦突出了piR-Hep1的功能重要性和miR-1323在肝癌患者中的預後意義。此外，本研究表明，傳統的陣列分析和新一代的測序技術均能發現重要的miRNA。新一代測序技術對轉錄組的全面分析，將對研究各種不同類型的ncRNAs在肝癌發生發展過程中的參與提供新的思路。 / MicroRNAs (miRNAs) are a class of small non-coding RNAs (ncRNA) that post-transcriptionally regulate gene expression. The discovery of miRNAs not only puts forth an alternate gene regulatory mechanism, but also underscores the importance of small ncRNAs as pivotal regulators of diverse physiological and developmental processes. Recent studies have emphasized a causal link between miRNA deregulation and cancer development. / Our group has previously reported on dysregulated miRNA pattern in hepatocellular carcinoma (HCC) by array-based profiling, which revealed common downregulation of miR-145. In the first part of this thesis, quantitative reverse transcription polymerase chain reaction (qRT-PCR) corroborated reduced miR-145 expression in 50% of tumors in a cohort of 80 HCC patients, which also correlated reduced miR-145 expression with shorter disease-free survival of patients. One HCC tumor analyzed with low endogenous miR-145 was propagated as cell line. This in vitro model HKCI-C2 maintained low miR-145 level and upon restoration of miR-145 expression, a consistent inhibitory effect on cell viability and proliferation was readily observed. Multiple in silico algorithms predicted that miR-145 could target a number of genes along the insulin-like growth factor (IGF) signaling, including insulin receptor substrate (IRS1)-1, IRS2 and insulin-like growth factor 1 receptor. Protein expression of these putative targets was concordantly downregulated in the presence of miR-145. Luciferase reporter assay further verified direct target association of miR-145 to specific sites of IRS1 and IRS2 3’-untranslated regions. Subsequent analysis also affirmed the modulation of IGF signaling cascade by miR-145 as evident by reduction of the downstream mediator, namely, the active β-catenin level. / The recent advent of deep sequencing has provided an unprecedented platform to study the miRNome, in which both known and novel miRNAs can be identified. Moreover, bioinformatics advances have enabled different types of small ncRNAs, e.g. piwi-interacting RNAs (piRNAs), to be analyzed simultaneously. In the second part of this thesis, small RNA transcriptomes of two HCC cell lines (HKCI-4 and HKCI-8) and an immortalized hepatocyte line (MIHA) were examined using Illumina massively parallel sequencing. Combined bioinformatic and biological analyses revealed the involvement of a novel piRNA, designated as piR-Hep1, in liver tumorigenesis. piR-Hep1 was found to be up-regulated in 47% of HCC in a cohort of 73 HCC patients by qRT-PCR. Silencing of piR-Hep1 inhibited cell viability, motility and invasiveness with a concomitant reduction of Akt phosphorylation. In the analysis of miRNA, miR-1323 was found to be abundantly expressed in HCC and distinctly associated with tumors arising from a cirrhotic background. Furthermore, miR-1323 overexpression in cirrhotic-HCC correlated with poorer disease-free and overall survivals of patients (P<0.009). / Taken together, results from this thesis showed for the first time the pleiotropic effect of miR-145 on targeting multiple components of the oncogenic IGF signaling pathway in HCC. In addition, the functional importance of piR-Hep1 and the prognostic significance of miR-1323 in HCC were highlighted. Studies conducted demonstrated that important miRNAs can be discovered by both traditional array-based profiling and next-generation sequencing. Moreover, comprehensive definition of transcriptome by next-generation sequencing unveils virtually all types of ncRNAs and provides new insight into the liver carcinogenic events. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Law, Tak Yin. / "December 2012." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 180-200). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstracts also in Chinese. / Acknowledgements --- p.i / Publications --- p.ii / Abstract --- p.iii / 摘要 --- p.vi / Contents --- p.viii / List of Figures --- p.xiii / List of Tables --- p.xv / Abbreviations --- p.xvi / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Hepatocellular Carcinoma - One of the world’s most deadly killers --- p.2 / Chapter 1.2 --- MicroRNAs - a tiny molecule with enormous impacts --- p.10 / Chapter 1.2.1 --- Discovery of miRNAs --- p.11 / Chapter 1.2.2 --- Biogenesis and actions of miRNA --- p.13 / Chapter 1.3 --- MiRNAs and cancer --- p.16 / Chapter 1.4 --- Involvements of miRNAs in HCC etiological factors --- p.18 / Chapter 1.4.1 --- Viral hepatitis infection --- p.19 / Chapter 1.4.2 --- Chronic heavy alcohol consumption --- p.26 / Chapter 1.4.3 --- Dietary aflatoxin exposure --- p.28 / Chapter 1.4.4 --- Male gender --- p.31 / Chapter 1.4.5 --- Obesity --- p.33 / Chapter 1.5 --- Regulation of cancer-associated signaling network by microRNAs --- p.34 / Chapter 1.5.1 --- Apoptotic pathway --- p.37 / Chapter 1.5.1.1 --- Intrinsic pathway --- p.38 / Chapter 1.5.1.2 --- Extrinsic pathway --- p.39 / Chapter 1.5.2 --- Cell cycle regulators --- p.41 / Chapter 1.5.2.1 --- G₁/S transition --- p.42 / Chapter 1.5.2.2 --- G₂/M transition --- p.43 / Chapter 1.5.3 --- Receptor tyrosine kinase-mediated pathways --- p.45 / Chapter 1.5.3.1 --- c-MET-activated signaling --- p.45 / Chapter 1.5.3.2 --- PI3K-Akt --- p.47 / Chapter 1.5.3.3 --- RAS-RAF-MEK-ERK cascade --- p.48 / Chapter 1.5.4 --- TGF-ß signaling pathways --- p.50 / Chapter 1.5.5 --- Metastatic pathways --- p.52 / Chapter 1.5.5.1 --- MiRNAs with metastatic suppressing effects --- p.52 / Chapter 1.5.5.2 --- MiRNAs with metastatic promoting effects --- p.53 / Chapter 1.6 --- Clinical potentials of microRNAs - a killer or a cure? --- p.56 / Chapter 1.6.1 --- MiRNAs involvements in HCC risk prediction --- p.57 / Chapter 1.6.2 --- MiRNAs as diagnostic biomarkers --- p.59 / Chapter 1.6.3 --- MiRNAs as prognostic biomarkers --- p.60 / Chapter 1.6.4 --- Effects of miRNAs on responses to therapy --- p.61 / Chapter 1.7 --- Non-coding RNAs --- p.62 / Chapter 1.8 --- Aims of study --- p.63 / Chapter 2 --- Materials and Methods --- p.65 / Chapter 2.1 --- Quantitative reverse transcription polymerase chain reaction (qRT-PCR) --- p.66 / Chapter 2.1.1 --- Materials --- p.66 / Chapter 2.1.1.1 --- Total RNA extraction --- p.66 / Chapter 2.1.1.2 --- DNase treatment --- p.66 / Chapter 2.1.1.3 --- Reverse transcription --- p.66 / Chapter 2.1.1.4 --- Quantitative polymerase chain reaction --- p.66 / Chapter 2.1.2 --- Methods --- p.67 / Chapter 2.1.2.1 --- Total RNA extraction --- p.67 / Chapter 2.1.2.2 --- DNase treatment --- p.68 / Chapter 2.1.2.3 --- Reverse transcription --- p.69 / Chapter 2.1.2.4 --- Quantitative polymerase chain reaction --- p.69 / Chapter 2.2 --- Transfection --- p.70 / Chapter 2.2.1 --- Materials --- p.70 / Chapter 2.2.2 --- Methods --- p.70 / Chapter 2.2.2.1 --- Evaluation of HCC cells transfection efficiency --- p.70 / Chapter 2.2.2.2 --- Transfection --- p.71 / Chapter 2.3 --- In vitro functional assay --- p.72 / Chapter 2.3.1 --- Materials --- p.72 / Chapter 2.3.1.1 --- Cell viability assay --- p.72 / Chapter 2.3.1.2 --- Colony formation assay --- p.72 / Chapter 2.3.1.3 --- Cell cycle analysis --- p.72 / Chapter 2.3.1.4 --- Apoptosis assay --- p.72 / Chapter 2.3.1.5 --- Cell motility and invasion assay --- p.73 / Chapter 2.3.2 --- Methods --- p.73 / Chapter 2.3.2.1 --- Cell viability assay --- p.73 / Chapter 2.3.2.2 --- Colony formation assay --- p.74 / Chapter 2.3.2.3 --- Cell cycle analysis --- p.75 / Chapter 2.3.2.4 --- Apoptosis assay --- p.75 / Chapter 2.3.2.5 --- Cell motility and invasion assay --- p.76 / Chapter 2.4 --- Luciferase reporter assay --- p.78 / Chapter 2.4.1 --- Materials --- p.78 / Chapter 2.4.1.1 --- Cloning --- p.78 / Chapter 2.4.1.2 --- Cycle sequencing --- p.78 / Chapter 2.4.1.3 --- Luciferase reporter assay --- p.79 / Chapter 2.4.2 --- Methods --- p.79 / Chapter 2.4.1.1 --- Cloning --- p.79 / Chapter 2.4.2.2 --- Cycle sequencing --- p.81 / Chapter 2.4.2.3 --- Luciferase reporter assay --- p.82 / Chapter 2.5 --- Western blot --- p.84 / Chapter 2.5.1 --- Materials --- p.84 / Chapter 2.5.2 --- Methods --- p.85 / Chapter 2.5.2.1 --- Cell harvesting and protein quantitation --- p.86 / Chapter 2.5.2.2 --- Western blotting --- p.86 / Chapter 2.6 --- Small RNA Sequencing --- p.88 / Chapter 2.6.1 --- Materials --- p.88 / Chapter 2.6.2 --- Methods --- p.88 / Chapter 2.6.2.1 --- Sample preparation --- p.88 / Chapter 2.6.2.2 --- Cluster generation by bridge amplification --- p.88 / Chapter 2.6.2.3 --- Sequencing by synthesis --- p.89 / Chapter 2.7 --- Northern blot analysis --- p.94 / Chapter 2.7.1 --- Materials --- p.94 / Chapter 2.7.2 --- Methods --- p.94 / Chapter 2.7.2.1 --- Polyacrylamide gel electrophoresis (PAGE) --- p.94 / Chapter 2.7.2.2 --- Probe preparation --- p.95 / Chapter 2.7.2.3 --- Hybridization, stringency washes and signal detection --- p.95 / Chapter 3 --- Conventional miRNA profiling reveals miR-145 as a tumor suppressor in HCC --- p.97 / Chapter 3.1 --- Introduction --- p.98 / Chapter 3.2 --- Materials and Methods --- p.102 / Chapter 3.2.1 --- Patients --- p.102 / Chapter 3.2.2 --- qRT-PCR --- p.104 / Chapter 3.2.3 --- Cell line --- p.105 / Chapter 3.2.4 --- Transfection --- p.106 / Chapter 3.2.5 --- In vitro functional assay --- p.107 / Chapter 3.2.5.1 --- Cell viability assay --- p.107 / Chapter 3.2.5.2 --- Colony formation assay --- p.107 / Chapter 3.2.5.3 --- Flow cytometry assay --- p.107 / Chapter 3.2.6 --- miRNA target prediction --- p.109 / Chapter 3.2.7 --- Luciferase reporter assay --- p.110 / Chapter 3.2.8 --- Western blot --- p.112 / Chapter 3.2.9 --- Immunohistochemistry --- p.113 / Chapter 3.2.10 --- Statistical analysis --- p.114 / Chapter 3.3 --- Results --- p.115 / Chapter 3.3.1 --- Down-regulation of miR-145 in primary HCC --- p.115 / Chapter 3.3.2 --- Re-expression of miR-145 induced G₂-M arrest and apoptosis --- p.119 / Chapter 3.3.3 --- IRS1, IRS2 and IGF1R expressions --- p.124 / Chapter 3.3.4 --- miR-145 targeted both IRS1 and IRS2 and elicited IGF signaling --- p.126 / Chapter 3.4 --- Discussion --- p.131 / Chapter 4 --- Small RNA Deep sequencing reveals novel non-coding RNAs in HCC --- p.134 / Chapter 4.1 --- Introduction --- p.135 / Chapter 4.2 --- Materials and Methods --- p.136 / Chapter 4.2.1 --- Cell lines --- p.136 / Chapter 4.2.2 --- Patients --- p.137 / Chapter 4.2.3 --- Small RNA Sequencing --- p.139 / Chapter 4.2.4 --- Bioinformatics analysis --- p.140 / Chapter 4.2.4.1 --- Sequence mapping and ncRNA identification --- p.140 / Chapter 4.2.4.2 --- Putative miRNA prediction --- p.140 / Chapter 4.2.4.3 --- Putative piRNA identification --- p.140 / Chapter 4.2.4.4 --- Differentially-expressed ncRNAs identification --- p.141 / Chapter 4.2.5 --- qRT-PCR --- p.142 / Chapter 4.2.6 --- Northern blot analysis --- p.143 / Chapter 4.2.7 --- Transfection --- p.144 / Chapter 4.2.8 --- In vitro functional assays --- p.145 / Chapter 4.2.8.1 --- Cell viability assay --- p.145 / Chapter 4.2.8.2 --- Cell motility and invasion assay --- p.145 / Chapter 4.2.9 --- Western blot analysis --- p.147 / Chapter 4.2.10 --- Statistical analysis --- p.148 / Chapter 4.3 --- Results --- p.149 / Chapter 4.3.1 --- Small RNA Sequencing --- p.149 / Chapter 4.3.2 --- Up-regulation of putative piR-Hep1 in HCC --- p.155 / Chapter 4.3.3 --- piR-Hep1 silencing reduced cell viability and invasiveness --- p.159 / Chapter 4.3.4 --- Novel miR-1323 overexpression in HCC --- p.162 / Chapter 4.4 --- Discussion --- p.171 / Chapter 5 --- Concluding remarks and future perspectives --- p.175 / Chapter 6 --- References --- p.179

Yun Jing-ping. / "March 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 185-226). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

雖然錯誤調控微小核糖核酸 (miRNA) 引起肝細胞癌 (HCC) 發生發展的生物途徑得到了廣泛的研究，但是對於上游的調控機制卻知之甚少。以往的研究表明，組蛋白甲基化轉移酶 (EZH2) 介導的組蛋白3上27位賴氨酸三甲基化（H3K27me3）是一類通過沉默腫瘤抑制基因而誘發癌症的機制，並且與脫氧核糖核酸 (DNA) 啟動子甲基化機制獨立存在。另一方面，基因抑制也與 H3K27和DNA甲基化相關聯。盡管如此，miRNA沉默機制，特別是在肝癌中，仍然是知之甚少。 / 在這項研究中，我們使用整合全基因組定位和表達分析方法，以探討在肝癌細胞中miRNA表達的表觀遺傳和轉錄控制。通過染色質免疫沉澱偶聯人類啟動子芯片的方法，我們發現在Hep3B和HKCI - 8肝癌細胞中分別有8.4和12.2%的審問miRNA有豐富的H3K27me3。另一方面，在甲基結合域捕捉偶聯芯片實驗中，我們發現在Hep3B和HKCI-8肝癌細胞中分別有15.5和14.7% 的miRNA出現DNA超甲基化。與以往的蛋白質編碼基因結果相同，大多數 H3K27me3豐富的miRNA沒有被檢測到DNA超甲基化，並且反之亦然。 敲除EZH2基因引起H3K27me3水平廣泛下降,並且恢復 H3K27me3抑制的 miRNA表達，而DNA去甲基化劑5-氮雜 -2'-脫氧胞苷 (5-aza-dC) 卻不能重新啟動他們的表達，進一步顯示EZH2基因介導的H3K27me3引發miRNA沉默的機制是獨立存在的。然而，一些過往研究證明有腫瘤抑制功能的miRNA，包括 miR-9-1，miR-9-2和 miR-9-3 被發現同時被 H3K27me3和DNA甲基化調節。我們進一步發現，在肝瘤細胞中，miR-9 特異性調控致癌性的基因結合核因 (NF-κB) 信號通路，並且與配對的非腫瘤肝組織相比，miR-9 的表達在大約一半的原發性肝癌腫瘤（五十九分之三十零）中顯著被壓抑。 / 為了調查在H3K27me3介導的miRNA基因沉默中參與的轉錄因子，我們應用轉錄因子結合位點分析的方法檢查H3K27me3結合蛋白編碼和miRNA基因的調控區域。在包括miR-9亞型的miRNA中，滎陽 1（YY1）的結合位點在這些調控區域中反覆出現並有很高的代表性。定量芯片聯合聚合酶鏈反應結果顯示，在Hep3B細胞中，敲除YY1不僅大大降低了自身的結合力，同時在 miR-9-1，miR-9-2和 miR-9-3 的啟動子中，EZH2基因和H3K27me3結合也大大降低了。尤其重要的是，敲除YY1可以顯著地重新激活他們的表達，表明在肝癌細胞中YY1在EZH2基因介導的的miR-9 表觀遺傳沉默中發揮重要作用。功能研究證明，下調YY1能夠抑制肝癌細胞的增殖，增加細胞凋亡和減少體內的腫瘤生長。定量實時聚合酶鏈反應進一步證實在miR-9 被下調的子集肝癌腫瘤中，有超過85的樣本顯示YY1和EZH2基因同時過量表達，表明我們的研究結果具有臨床相關性。 / 總之，我們完整的分析表明miRNA的調控在肝癌上的獨特表觀遺傳模式。 H3K27me3介導的miRNA沉默可由擁有致癌功能的YY1誘發，它亦可能代表一個可能公認的肝癌癌基因。綜合表觀遺傳和miRNA表達的轉錄控制的結果，能夠提高我們對肝癌發生發展的認識和闡明利用表觀遺傳方法針對性治療肝癌的新的發展方向。 / Although the biological pathways by which mis-regulated microRNAs (miRNAs) contribute to the development of hepatocellular carcinoma (HCC) have been extensively investigated, little is known about the upstream regulatory mechanisms. Previous studies demonstrated that EZH2-mediated histone H3 lysine 27 trimethylation (H3K27me3) is a mechanism of tumor-suppressor gene silencing in cancer that is potentially independent of promoter DNA methylation. On the other hand, gene repression can be associated with both H3K27 and DNA methylation. However, the mechanisms underlying miRNA silencing, particularly in HCC, are poorly understood. / In this study, we used an integrated genome-wide location and expression analysis to investigate the epigenetic and transcriptional controls of miRNA expression in HCC cells. Chromatin immunoprecipitation (ChIP) coupled with human promoter microarrays revealed that 8.4 and 12.2% of the interrogated miRNA were enriched with H3K27me3 in Hep3B and HKCI-8 HCC cells, respectively. On the other hand, Methyl-binding domain capture coupled with microarray (MethylCap-chip) uncovered that 15.5 and 14.7% of miRNA were hypermethylated in Hep3B and HKCI-8 HCC cells, respectively. Consistent with previous observation on protein-coding genes, most of the miRNAs enriched with H3K27me3 had no detectable DNA hypermethylation and vice versa. Knockdown of EZH2 decreased global H3K27me3 level and restored expression of the H3K27me3-targeted miRNAs while the DNA demethylating agent 5-aza-2’-deoxycytidine (5-aza-dC) did not reactivate their expression, further suggesting the independence of EZH2-mediated H3K27me3 in miRNA silencing. Nevertheless, a few miRNAs reported to exhibit tumor-suppressive functions including miR-9-1, miR-9-2 and miR-9-3 were found to be regulated by both H3K27me3 and DNA methylation. We further found that miR-9 targeted the oncogenic NF-κB signaling pathway in HCC cells and were significantly repressed in approximately half of the primary HCC tumors (30/59) compared to the paired non-tumor liver tissues. / To investigate the involvement of transcription factors in H3K27me3-mediated gene silencing of miRNAs, the regulatory regions of H3K27me3-bound protein-coding and miRNA genes were submitted to transcription factor binding site analysis. The binding sites for Ying Yang 1 (YY1) were recurrently over-represented in these loci including the miR-9 isoforms. Quantitative ChIP-PCR demonstrated that knockdown of YY1 in Hep3B cells not only significantly reduced its own binding, but also the EZH2 and H3K27me3 promoter occupancy at miR-9-1, miR-9-2 and miR-9-3. Importantly, their expression levels were significantly reactivated by YY1 knockdown, suggesting that YY1 plays part in the EZH2-mediated epigenetic silencing of miR-9 in HCC cells. Functionally, down-regulation of YY1 was shown to inhibit HCC cell proliferation, increase cell apoptosis and reduce tumor growth in vivo. Quantitative RT-PCR further demonstrated that YY1 and EZH2 were concordantly over-expressed in over 85% of the same subset of HCC tumors that exhibited miR-9 down-regulation, demonstrating the clinical relevance of our findings. / In conclusion, our integrated analysis demonstrated differential epigenetic patterns of miRNA regulation in HCC. H3K27me3-mediated silencing of miRNAs may be initiated by YY1, which possesses oncogenic functions and may represent a putative HCC oncogene. The findings of combinatorial epigenetic and transcriptional control of miRNA expression enhance our understanding of hepatocarcinogenesis and shed light on the development of novel epigenetic targeted therapy of HCC. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Tsang, Pui Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 111-121). / Abstracts also in Chinese. / Abstract (English version) --- p.i / Abstract (Chinese version) --- p.iv / Acknowledgements --- p.vi / table of contents --- p.vii / List of tables --- p.x / List of Figures --- p.xi / Abbreviations --- p.xiii / Chapter CHAPTER 1 --- INTRODUCTION4 / Chapter 1.1 --- Hepatocellular carcinoma --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.1 / Chapter 1.1.2 --- Etiology --- p.2 / Chapter 1.2 --- Epigenetic mechanisms --- p.3 / Chapter 1.2.1 --- Epigenetic silencing by DNA hypermethylation --- p.3 / Chapter 1.2.2 --- Epigenetic silencing by Polycomb group protein --- p.5 / Chapter 1.2.3 --- Interplay between H3K27me3 and DNA hypermethylation --- p.7 / Chapter 1.3 --- microRNA --- p.10 / Chapter 1.3.1 --- Transcriptional gene silencing by miRNA --- p.11 / Chapter 1.3.2 --- miRNA and cancers --- p.12 / Chapter 1.3.3 --- miRNA and liver cancer --- p.13 / Chapter 1.4 --- Signal transduction pathway and cancers --- p.14 / Chapter 1.5 --- Aims of study --- p.15 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Cell lines --- p.16 / Chapter 2.2 --- Clinical samples --- p.16 / Chapter 2.3 --- Plasmid DNA transfection --- p.16 / Chapter 2.4 --- Small interfering RNA transfection --- p.17 / Chapter 2.5 --- Extraction of total RNA --- p.19 / Chapter 2.6 --- Western blot analysis --- p.19 / Chapter 2.7 --- Quantitative RT-PCR --- p.20 / Chapter 2.8 --- miRNA Real Time RT-PCR --- p.22 / Chapter 2.9 --- ChIP-chip assay --- p.24 / Chapter 2.10 --- MethylCap-chip --- p.27 / Chapter 2.11 --- miRNA microarray --- p.28 / Chapter 2.12 --- ChIP Assay and Quantitative ChIP-PCR Assay --- p.28 / Chapter 2.13 --- Colony formation assay --- p.33 / Chapter 2.14 --- Cell proliferation assay --- p.33 / Chapter 2.15 --- Annexin V apoptosis assay --- p.34 / Chapter 2.16 --- Cancer 10-pathway reporter array --- p.34 / Chapter 2.16.1 --- Transfection of siEZH2 with 5-aza-dC treatment --- p.34 / Chapter 2.16.2 --- Transfection of siYY1 and pcDNA3-miR9 plasmid --- p.35 / Chapter 2.16.3 --- Luciferase reporter array --- p.35 / Chapter 2.17 --- Animal Studies --- p.36 / Chapter 2.18 --- Statistical Analysis --- p.36 / Chapter CHAPTER 3 --- Results / Chapter 3.1 --- Occupancy of miRNA genes by epigenetic marks in HCC cells --- p.37 / Chapter 3.1.1 --- Identification of H3K27me3-occupied miRNAs --- p.37 / Chapter 3.1.2 --- Identification of DNA methylation-occupied miRNAs --- p.41 / Chapter 3.1.3 --- Relationship between H3K27me3 and DNA methylation occupancy of miRNAs in HCC cells --- p.45 / Chapter 3.2 --- Regulation of miRNA expression by H3K27me3 and DNA methylation in HCC cells --- p.51 / Chapter 3.3 --- Epigenetic regulation and molecular function of miR-9 in HCC cells --- p.56 / Chapter 3.3.1 --- Confirmation of H3K27me3 and DNA methylation occupancy in miR-9 genes --- p.59 / Chapter 3.3.2 --- Synergistic reactivation of miR-9 upon removal of epigenetic marks --- p.62 / Chapter 3.3.3 --- Effect of miR-9 re-expression on NFKB1 (p50) expression and NF-κB signaling in HCC cells --- p.66 / Chapter 3.4 --- Role of the transcription factor YY1 in the epigenetic regulation of miR-9 --- p.72 / Chapter 3.4.1 --- Identification of transcription factors involved in the regulation of H3K27me3-bound genes --- p.72 / Chapter 3.4.2 --- Occupancy of YY1 on miR-9 in HCC cells --- p.75 / Chapter 3.4.3 --- Effects of YY1 on EZH2/H3K27me3 occupancy and expression of miR-9 --- p.78 / Chapter 3.4.4 --- Effects of YY1 on p50/p65 expression and NF-κB signaling in HCC cells --- p.81 / Chapter 3.5 --- Functional significance of YY1 in HCC --- p.84 / Chapter 3.5.1 --- Effect of YY1 on HCC cell growth --- p.84 / Chapter 3.5.2 --- Effect of YY1 on HCC cell apoptosis --- p.87 / Chapter 3.5.3 --- Effect of YY1 on HCC cell growth in vivo --- p.90 / Chapter 3.5.4 --- Expressions of YY1, EZH2 and miR-9 on clinical HCC samples --- p.92 / Chapter CHAPTER 4 --- DISCUSSION / Chapter 4.1 --- Independence of EHZ2-mediated H3K27me3 and DNA methylation --- p.97 / Chapter 4.2 --- Concordant H3K27 and DNA methylation-mediated silencing of miR-9 --- p.101 / Chapter 4.3 --- Ectopic expression of miR-9 inhibits NF-kB signaling in HCC cells --- p.102 / Chapter 4.4 --- YY1 is involved in the regulation of H3K27me3-bound genes --- p.103 / Chapter 4.5 --- Knockdown of YY1 inhibits NF-kB signaling in HCC --- p.105 / Chapter 4.6 --- Clinical relevance and therapeutic significance of miR-9 silencing by YY1-mediated recruitment of EZH2 --- p.106 / Chapter 4.7 --- Limitations and future studies --- p.109 / REFERENCES --- p.111 / PUBLICATION --- p.122

O câncer colorretal (CCR) é a neoplasia mais prevalente no mundo e, cerca de 60% apresentarão metástases hepáticas, representando uma importante causa de mortalidade. Aproximadamente 35% dos pacientes apresentam metástases hepáticas no momento do diagnóstico do tumor primário ou desenvolverão metástases durante o primeiro ano após o tratamento da neoplasia colorretal, conhecidas como metástases sincrônicas. Inúmeros trabalhos têm demonstrado que as metástases sincrônicas representam importante fator prognóstico negativo na evolução destes pacientes, representando um grupo de pacientes que necessita uma abordagem multidisciplinar mais agressiva. O objetivo deste trabalho foi de avaliar os resultados do tratamento cirúrgico das metástases sincrônicas de CCR e determinar os possíveis fatores que pudessem interferir no prognóstico de sobrevida livre de doença e sobrevida atuarial. Entre maio de 1996 e dezembro de 2007, 59 pacientes submetidos à ressecção hepática por metástases sincrônicas foram avaliados retrospectivamente através de análise uni e multivariada. A mortalidade pós-operatória foi de 3,38%, e a morbidade pós-operatória de 30,50%. A sobrevida estimada em 5 anos foi de 38,45% e a sobrevida livre de doença no mesmo período foi de 23,96. O valor do antígeno carcinoembrionário igual ou superior a 50 ng/ml e o número de metástases hepáticas maior que três lesões representaram fatores prognósticos limitados da sobrevida livre de doença, porém sem interferir na sobrevida atual. Pacientes com metástases no fígado e com doença extra-hepática, selecionados para a ressecção, não apresentaram sobrevida livre de doença acima de 20 meses, porém sem impacto na sobrevida a longo prazo. Nenhum dos fatores prognósticos estudados interferiu na sobrevida atual tardia. Entretanto,não foi observada sobrevivência além de 40 meses em pacientes com mais de três metástases hepáticas. A ressecção de metástases sincrônicas de câncer colorretal pode propiciar sobrevida tardia em mais de um terço dos pacientes. O valor do CEA e do número de metástases representaram fatores prognósticos limitantes da sobrevida livre de doença / Colorectal cancer is the world´s most prevalent digestive neoplasia and about 60% of the patients will present liver metastases, representing an important cause of mortality. About 35% of the patients present hepatic metastases at the diagnosis of the colorectal tumor or will develop metastases during the first year after the treatment of the primary tumor, known as synchronous metastases. Innumerable studies have shown that synchronous metastases represent a negative prognostic factor in the evolution of these patients, representing a group of patients that need an aggressive multidisciplinary approach. The purpose of this study was to evaluate results of the surgical treatment of colorectal cancer synchronous metastases and to determine possible factors that might interfere in the prognosis of disease-free and actuarial survival. Between May 1996 and December 2007, 59 patients submitted to liver resection for synchronous metastases were retrospectively evaluated through univariate and multivariate analysis. Postoperative morbidity and mortality were 30.5% and 3.38%, respectively. Cumulative survival estimated in 5 years was 38.45% and disease-free survival in the same period was 23.96%. Levels of carcino-embrionary antigen (CEA) higher than 50 ng/ml and the number of hepatic metastases higher than three lesions represented negative prognostic factors limiting disease-free survival; however, with no impact on cumulative survival. Patients with liver metastases and extrahepatic disease selected for resection didnt present a disease-free survival above 20 months, yet without impact in global survival. None of the prognostic factors studied interfered in long term actuarial survival, however survival beyond 40 months in patients with more than three hepatic metastases was not observed. Resection of synchronous metastases of colorectal cancer may provide late survival in more than one third of the patients. CEA values and the number of metastases represented prognostic factors with negative impact on disease-free survival

Meng Jie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references. / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Table of Contents --- p.vi / Abbreviation --- p.x / List of Figures --- p.xi / List of Tables --- p.xiii / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Black Mustard and Sinigrin --- p.2 / Chapter 1.2 --- Hepatocellular Carcinoma --- p.5 / Chapter 1.2.1 --- Different Stages of HCC --- p.6 / Chapter 1.2.2 --- Risk Factors --- p.8 / Chapter 1.2.3 --- Treatments of HCC --- p.10 / Chapter 1.3 --- Biomarkers Used to Evaluate Effects of Sinigrin on HCC --- p.12 / Chapter 1.3.1 --- AST & ALT --- p.12 / Chapter 1.3.2 --- Glutathione S Transferase -p (GST-p) --- p.13 / Chapter 1.4 --- Tumor Suppressor Genes and Oncogenes --- p.14 / Chapter 1.4.1 --- "p53, the Tumor Suppressor Gene" --- p.15 / Chapter 1.4.2 --- p53-dependent pathway --- p.15 / Chapter 1.4.2.1 --- Mdm2 --- p.16 / Chapter 1.4.2.2 --- Bax and Bcl-2 --- p.17 / Chapter 1.4.2.3 --- PCNA and p21wAF1/CIP1 --- p.18 / Chapter 1.5 --- Aim of the Project --- p.19 / Chapter Chapter 2: --- Materials and Methods --- p.20 / Chapter 2.1 --- In vitro Studies --- p.21 / Chapter 2.1.1 --- Neutral Red Assay --- p.21 / Chapter 2.1.1.1 --- Chemicals and Reagents --- p.21 / Chapter 2.1.1.2 --- Liver Cells --- p.23 / Chapter 2.1.1.3 --- Neutral Red Assay --- p.24 / Chapter 2.1.2 --- Flow Cytometery --- p.24 / Chapter 2.1.2.1 --- Chemicals and Reagents --- p.25 / Chapter 2.1.2.2 --- Flow Cytometery Analysis --- p.25 / Chapter 2.1.3 --- DNA Fragmentation --- p.26 / Chapter 2.1.3.1 --- Chemicals and Reagents --- p.26 / Chapter 2.1.3.2 --- DNA Extraction --- p.28 / Chapter 2.1.3.3 --- DNA Agarose Gel Electrophoresis --- p.29 / Chapter 2.1.4 --- cDNA Microarray --- p.29 / Chapter 2.1.4.1 --- Chemicals and Reagents --- p.30 / Chapter 2.1.4.2 --- RNA Extraction --- p.33 / Chapter 2.1.4.3 --- RNA Quantity and Quality Control --- p.34 / Chapter 2.1.4.4 --- RT-PCR --- p.35 / Chapter 2.1.4.5 --- cRNA Convention and Purification --- p.36 / Chapter 2.1.4.6 --- Hybridization --- p.37 / Chapter 2.1.4.7 --- Washing and Detection --- p.37 / Chapter 2.1.4.8 --- Data Analysis --- p.38 / Chapter 2.2 --- In vivo Studies --- p.39 / Chapter 2.2.1 --- Animal Treatment --- p.39 / Chapter 2.2.1.1 --- Chemicals and Reagents --- p.39 / Chapter 2.2.1.2 --- Chemical Carcinogens --- p.40 / Chapter 2.2.1.3 --- Promotion Stage --- p.41 / Chapter 2.2.1.4 --- Progression Stage --- p.44 / Chapter 2.2.2 --- Measurement of Serum ALT and AST Activities --- p.46 / Chapter 2.2.2.1 --- Chemicals and Reagents --- p.46 / Chapter 2.2.2.2 --- Activity Assay --- p.46 / Chapter 2.2.3 --- Histological Analysis --- p.47 / Chapter 2.2.3.1 --- Chemicals and Reagents --- p.47 / Chapter 2.2.3.2 --- Preparation of Slides --- p.49 / Chapter 2.2.3.3 --- H&E Staining --- p.49 / Chapter 2.2.3.4 --- GST-p Immuno-staining --- p.50 / Chapter 2.2.4 --- Semi-Quantitative RT-PCR Analysis of mRNA Expression --- p.53 / Chapter 2.2.4.1 --- Chemicals and Reagents --- p.53 / Chapter 2.2.4.2 --- Extraction of total RNA from rat liver --- p.53 / Chapter 2.2.4.3 --- Quantity and Quality Control of RNA --- p.53 / Chapter 2.2.4.4 --- RT-PCR (Reverse Transcription) --- p.54 / Chapter 2.2.4.5 --- PCR --- p.54 / Chapter 2.2.4.6 --- DNA gel electrophoresis --- p.55 / Chapter 2.2.4.7 --- Data Analysis --- p.56 / Chapter 2.2.5 --- Western Blot Analysis for Biomarkers --- p.56 / Chapter 2.2.5.1 --- Chemicals and Reagents --- p.56 / Chapter 2.2.5.2 --- Extraction of the Cytosol Protein --- p.60 / Chapter 2.2.5.3 --- Extraction of the Nuclear protein --- p.61 / Chapter 2.2.5.4 --- SDS Gel Electrophoresis --- p.61 / Chapter 2.2.5.5 --- Western Blot --- p.62 / Chapter 2.2.5.6 --- Interaction with Antibodies --- p.63 / Chapter 2.2.5.7 --- ECL Detection --- p.63 / Chapter 2.2.5.8 --- Data Analysis --- p.64 / Chapter Chapter 3: --- Results --- p.65 / Chapter 3.1 --- In vitro Studies --- p.66 / Chapter 3.1.1 --- Cell Viability test and IC50 --- p.66 / Chapter 3.1.2 --- Cell Cycle Analysis --- p.68 / Chapter 3.1.3 --- DNA Fragmentation --- p.71 / Chapter 3.1.4 --- Effects of Sinigrin on Gene Expression --- p.73 / Chapter 3.2 --- In vivo Studies --- p.77 / Chapter 3.2.1 --- Effects of Sinigrin on HCC Development (Promotion stage) in Rats --- p.77 / Chapter 3.2.1.1 --- Direct Observation --- p.77 / Chapter 3.2.1.2 --- Relative Liver / Body Weight Ratio --- p.79 / Chapter 3.2.1.3 --- AST/ALT Assay --- p.81 / Chapter 3.2.1.4 --- Basic Structure of Hepatocytes --- p.83 / Chapter 3.2.1.5 --- GST-p Foci Area --- p.85 / Chapter 3.2.1.6 --- mRNA Expression of p53 and Mdm2 --- p.88 / Chapter 3.2.1.7 --- Protein Expression of Biomarkers --- p.90 / Chapter 3.2.2 --- Effects of Sinigrin on HCC Development (Progression stage) in Rats --- p.97 / Chapter 3.2.2.1 --- Direct Observation --- p.97 / Chapter 3.2.2.2 --- Relative Liver / Body Weight Ratio --- p.99 / Chapter 3.2.2.3 --- AST/ALT Assay --- p.101 / Chapter 3.2.2.4 --- Basic Structure of Hepatocytes --- p.103 / Chapter 3.2.2.5 --- GST-p Foci Area --- p.105 / Chapter 3.2.2.6 --- mRNA Expression of p53 and Mdm2 --- p.108 / Chapter 3.2.2.7 --- Protein Expression of Biomarkers --- p.110 / Chapter Chapter 4: --- Discussion --- p.116 / Chapter 4.1 --- Protective and Therapeutic Benefits of Sinigrin --- p.117 / Chapter 4.1.1 --- Effects of SIN on Cancer and Normal Cells --- p.117 / Chapter 4.1.2 --- Effective Tumor Induction by DEN-CC14 Treatment --- p.118 / Chapter 4.1.3 --- Protective Effect of SIN in the Promotion Stage of HCC --- p.118 / Chapter 4.1.4 --- Therapeutic Effect of SIN in the Progression Stage of HCC --- p.119 / Chapter 4.2 --- Biological Activities of SIN --- p.121 / Chapter 4.3 --- Summary --- p.134 / References --- p.xiv

Leung, Ching Hei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 101-110). / Abstracts in English and Chinese. / Acknowledgements --- p.i / 摘要（Abstract in Chinese) --- p.iii / Abstract --- p.v / List of Abbreviations --- p.vii / List of Figures and Tables --- p.ix / Table of Content --- p.xi / Chapter CHAPTER 1 --- Introduction / Chapter 1.1 --- History and development of photodynamic therapy --- p.1 / Chapter 1.2 --- Basic principle of photodynamic therapy: the beauty of the treatment --- p.3 / Chapter 1.3 --- "Photosensitizers: From discovery, synthesis to modifications" --- p.6 / Chapter 1.4 --- Enhancement of selective retention of PS in cancerous tissue --- p.10 / Chapter 1.5 --- Development of silicon (IV) phthalocyanine derivatives --- p.14 / Chapter 1.6 --- Death mechanisms in photodynamic therapy --- p.17 / Chapter 1.7 --- Objectives of the present study --- p.18 / Chapter CHAPTER 2 --- Materials and Methods / Chapter 2.1 --- Synthesis of BAM-SiPc --- p.20 / Chapter 2.2 --- Preparation of BAM-SiPc solution for photodynamic treatment --- p.20 / Chapter 2.3 --- Cell line and culture conditions --- p.21 / Chapter 2.4 --- Animal tumor model --- p.23 / Chapter 2.5 --- PDT laser source --- p.23 / Chapter 2.6 --- In vitro photodynamic activity assay --- p.23 / Chapter 2.6.1 --- Preparation of cells for photodynamic treatment / Chapter 2.6.2 --- In vitro photodynamic treatment / Chapter 2.6.3 --- Cell viability assay / Chapter 2.7 --- "Determination of reactive oxygen species production by 2',7'- dichlorofluorescein diacetate (DCFDA) assay" --- p.28 / Chapter 2.8 --- Analysis of cell cycle arrest --- p.28 / Chapter 2.9 --- Biodistribution of BAM-SiPc --- p.29 / Chapter 2.10 --- In vivo photodynamic treatment --- p.30 / Chapter 2.11 --- Assay for plasma enzyme activities --- p.30 / Chapter 2.12 --- Determination of cellular uptake of BAM-SiPc --- p.31 / Chapter 2.13 --- Metabolism of BAM-SiPc --- p.31 / Chapter 2.14 --- Histochemical staining --- p.32 / Chapter 2.14.1 --- Preparation of paraffin-embedded tissue section / Chapter 2.14.2 --- Haematoxylin and Eosin (H & E) staining / Chapter 2.14.3 --- Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay / Chapter 2.15 --- Conjugation of BAM-SiPc with LDL --- p.34 / Chapter 2.15.1 --- Analysis of the phototoxicity and cellular uptake of BAM- SiPc in the presence of LDL / Chapter 2.15.2 --- Gel filtration analysis of the mixture of LDL and BAM- SiPc / Chapter 2.16 --- Statistical analysis --- p.35 / Chapter CHAPTER 3 --- Results / Chapter 3.1 --- In vitro photodynamic activity assays --- p.36 / Chapter 3.2 --- Tissue distribution of BAM-SiPc in HepG2- bearing nude mice --- p.39 / Chapter 3.3 --- Anti-tumor activities of in vivo PDT with BAM-SiPc --- p.42 / Chapter 3.3.1 --- In vivo effect of PDT treatment with BAM-SiPc on HepG2 and HT29 tumor growth / Chapter 3.3.2 --- Dosage effect on anti-tumor activities by BAM-SiPc mediated PDT / Chapter 3.4 --- Analysis of intrinsic toxicity induced by BAM-SiPc mediated PDT --- p.48 / Chapter 3.4.1 --- H & E staining of liver sections of nude mice after in vivo PDT / Chapter 3.4.2 --- Plasma enzyme activity assays of PDT treated mice / Chapter 3.5 --- BAM-SiPc metabolism in in vitro culture cells and liver homogenate --- p.53 / Chapter 3.5.1 --- Cellular uptake of BAM-SiPc / Chapter 3.5.2 --- BAM-SiPc metabolism in cultured normal liver cells and cancer cells / Chapter 3.5.3 --- BAM-SiPc metabolism by mice liver homogenate / Chapter 3.6 --- Death mechanism induced by BAM-SiPc mediated PDT --- p.62 / Chapter 3.6.1 --- Events related to cell death induced by in vitro BAM-SiPc mediated PDT / Chapter 3.6.2 --- Death mechanism exerted by in vivo BAM-SiPc mediated PDT / Chapter 3.7 --- Effect on phototoxicity of BAM-SiPc in the presence of LDL --- p.70 / Chapter 3.7.1 --- Effect on phototoxicity of BAM-SiPc after mixing BAM- SiPc with LDL / Chapter 3.7.2 --- Gel filtration for analysis of the LDL-BAM-SiPc mixture / Chapter CHAPTER 4 --- Discussion / Chapter 4.1 --- Anti-cancer effect of BAM-SiPc on different cancer cell lines --- p.76 / Chapter 4.2 --- Tissue distribution of BAM-SiPc in HepG2 bearing nude mice --- p.77 / Chapter 4.3 --- In vivo effect of BAM-SiPc mediated PDT on HepG2 and HT29 tumor growth --- p.80 / Chapter 4.4 --- Analysis of the safety of using BAM-SiPc as a potential agent in PDT --- p.83 / Chapter 4.5 --- Metabolism of BAM-SiPc --- p.84 / Chapter 4.6 --- Mechanism of the apoptosis triggered by BAM-SiPc mediated PDT --- p.88 / Chapter 4.7 --- Death mechanism induced by in vivo PDT with BAM-SiPc --- p.93 / Chapter 4.8 --- Phototoxicity of BAM-SiPc in the presence of LDL --- p.94 / Chapter CHAPTER 5 --- Conclusion and Future perspective / Chapter 5.1 --- Conclusion --- p.97 / Chapter 5.2 --- Future perspective --- p.98 / References

Lau Ven Gie Vengie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 113-121). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.ii / 論文摘要 --- p.iv / TABLE OF CONTENTS --- p.vi / LIST OF FIGURES --- p.ix / LIST OF TABLES --- p.x / LIST OF ABBREVIATIONS --- p.xi / INTRODUCTION --- p.1 / Chapter I --- Hepatocellular Carcinoma --- p.1 / Risk factors --- p.1 / Stages in chemical carcinogenesis --- p.2 / Initiation --- p.2 / Promotion --- p.3 / Progression --- p.5 / Treatment of hepatocarcinoma --- p.6 / Chemotherapy ´ؤ hepatic arterial infusion (HAI) --- p.6 / Trans-arterial chemoembolization (TACE) --- p.7 / Radiofrequency ablation (RFA) --- p.8 / Percutaneous ethanol injection (PEI) --- p.9 / Liver resection --- p.9 / Liver transplantation --- p.10 / Chapter II --- Molecular Mechanisms: Oncogenes and Tumor-suppressor genes --- p.11 / Cell cycle control --- p.12 / p53 mutation in HCC --- p.13 / Normal functions of p53 and its target genes --- p.13 / p21(Wafl/Cipl/Sdil) --- p.13 / PCNA --- p.14 / Bcl-2 and Bax: the Bcl-2 family --- p.14 / Mdm2 --- p.17 / Chapter III --- Evaluation of the effects of hepatocarcinogenesis --- p.19 / GST-Pi --- p.19 / AST & ALT --- p.19 / Chapter IV --- Traditional Chinese Medicine (TCM) --- p.21 / Andrographis Paniculata --- p.21 / Pharmacological properties of andrographolide --- p.23 / Chapter V --- Aim of the project --- p.26 / MATERIALS AND METHODS --- p.27 / Chapter 1 --- Effects of andrographolide on cell viability and cell cycle --- p.27 / Chapter 1.1 --- Materials and solutions --- p.27 / Chapter 1.2 --- Preparation of solutions --- p.28 / Chapter 1.3 --- Procedures --- p.29 / Chapter 1.3.1 --- Seeding cells into culture flask --- p.29 / Chapter 1.3.2 --- Subculturing technique --- p.30 / Chapter 1.3.3 --- Neutral red assay --- p.30 / Chapter 1.3.4 --- DNA purification of HepG2 cells --- p.31 / Chapter 1.3.5 --- DNA gel electrophoresis --- p.32 / Chapter 1.3.6 --- Flow cytometry --- p.32 / Chapter 2 --- Effects of andrographolide on gene expressions --- p.33 / Chapter 2.1 --- Materials and solutions --- p.33 / Chapter 2.2 --- Preparation of solutions --- p.34 / Chapter 2.3 --- Procedures --- p.35 / Chapter 2.3.1 --- Cell treatments --- p.35 / Chapter 2.3.2 --- mRNA extraction from cell --- p.35 / Chapter 2.3.3 --- Determination of total RNA yield and quality yield --- p.36 / Chapter 2.3.4 --- RNA formaldehyde agarose gel electrophoresis --- p.36 / Chapter 2.3.5 --- cDNA synthesis --- p.37 / Chapter 2.3.6 --- "cRNA synthesis, labeling and amplification" --- p.39 / Chapter 2.3.7 --- cRNA purification --- p.40 / Chapter 2.3.8 --- Oligo GEArray hybridization --- p.41 / Chapter 2.3.9 --- Chemiluminescent detection --- p.43 / Chapter 2.3.10 --- Data analysis --- p.44 / Chapter 3 --- Effects of andrographolide on hepatocarcinogenesis in rats --- p.45 / Chapter 3.1 --- Materials and solutions --- p.45 / Chapter 3.2 --- Preparation of solutions --- p.46 / Chapter 3.3 --- Procedures --- p.47 / Chapter 3.3.1 --- Animal treatment --- p.47 / Chapter 3.3.2 --- Promotion (Experiment 1) --- p.48 / Chapter 3.3.3 --- Progression (Experiment 2) --- p.49 / Chapter 3.3.4 --- Extraction of blood serum --- p.52 / Chapter 3.3.5 --- Measurement of absorbance --- p.52 / Chapter 3.3.6 --- Tissue processing --- p.53 / Chapter 3.3.7 --- Hematoxylin and Eosin (H&E) Staining --- p.53 / Chapter 3.3.8 --- Immunohistochemical staining of GST-P --- p.54 / Chapter 3.3.9 --- Examination of liver sections --- p.55 / Chapter 4 --- "Effects of andrographolide on the expressions of Mdm2, p53, PCNA, Bax, Bcl-2 & p21" --- p.56 / Chapter 4.1 --- Materials and solutions --- p.56 / Chapter 4.2 --- Preparation of solutions --- p.57 / Chapter 4.3 --- Procedures --- p.59 / Chapter 4.3.1 --- Total mRNA extraction from liver --- p.59 / Chapter 4.3.2 --- Reverse transcription of mRNA to cDNA --- p.59 / Chapter 4.3.3 --- Protocol for polymerase chain reaction (PCR) --- p.60 / Chapter 4.3.4 --- DNA gel electrophoresis --- p.61 / Chapter 4.3.5 --- Nuclear protein extraction --- p.61 / Chapter 4.3.6 --- Cytosolic protein extraction --- p.62 / Chapter 4.3.7 --- Determination of protein concentration --- p.62 / Chapter 4.3.8 --- Immunoprecipitation of p53 from liver nuclear protein --- p.62 / Chapter 4.3.9 --- Protein gel electrophoresis by SDS-PAGE --- p.63 / Chapter 4.3.10 --- Western blotting --- p.64 / RESULTS --- p.66 / Chapter 1 --- Effects of andrographolide on cell viability and cell cycle --- p.66 / Chapter 2 --- Effects of andrographolide on gene expressions --- p.76 / Chapter 3 --- Effects of andrographolide on hepatocarcinogenesis in rats --- p.79 / Chapter 4 --- "Effects of andrographolide on the expressions of Mdm2, p53, PCNA, Bax, Bcl-2 & p21" --- p.91 / DISCUSSION --- p.102 / CONCLUSION --- p.111 / REFERENCES --- p.113