Baicalein is known to be a potent antifungal agent and induces programmed cell death in Candida albicans. In the present study, we found that baicalein also inhibited the growth of C. krusei isolates. The minimal inhibitory concentrations of baicalein against eight C. krusei isolates were 1.35--2.70 microg/ml. One-hour exposure to baicalein elicited a consistent and moderate post-antifungal effect on the C. krusei isolates. Further flow cytometric study demonstrated a depolarization of mitochondrial membrane potential. However, both the levels of reactive oxygen species and DNA fragmentation were not significantly changed after baicalein treatment in C. krusei. It can be concluded that the antifungal activity of baicalein was mitochondria-dependent in both C. krusei and C. albicans, but the antifungal mechanism was different. Reactive oxygen species may not play a direct role and baicalein does not initiate programmed cell death or apoptosis in C. krusei. The structure-activity relationship study showed that the three hydroxyl groups in baicalein were essential for its antifungal potency. / Candidiasis has become a serious infection with very high mortality and morbidity in the world if not providing effective treatments. However, due to clinical limitation and resistance of the current antifungal agents, there is an urgent need to search for novel antifungals. In this study, after screening a compound library (n=400) for antifungal activity, three members (metergoline, purpurin and baicalein) were chosen for further study. Their antifungal characteristics and the antifungal mechanisms were investigated. / Metergoline, a serotonin receptor antagonist, was found to have potent antifungal activity against the intrinsically fluconazole-resistant human fungal pathogen Candida krusei. The minimal inhibitory concentration and minimal fungicidal concentration of metergoline against C. krusei were 4 microg/ml and 8 microg/ml respectively. Metergoline induced post-antifungal effect. Significant synergism was found in combination of metergoline with amphotericin B by a checkerboard assay, which may be due to the perturbation of cell permeability and increase in the intracellular accumulation of antifungal agents. Metergoline also inhibited extracellular phospholipase production in C. krusei. To gain insights into the mechanisms, intracellular changes that accompany apoptosis were examined by flow cytometry and spectrophotometry. The results showed an increase in the level of reactive oxygen species, depolarization of mitochondrial membrane potential, phosphatidylserine externalization, and positive terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labelling in the metergoline-treated C. krusei . Taken together, we conclude that metergoline may promote apoptosis in C. krusei through reactive oxygen species production and perturbation in mitochondrial homeostasis, implying its antifungal potential to treat candidiasis. / The antifungal activity of purpurin, a natural red anthraquinone pigment in madder root (Rubia tinctorum L.), was evaluated against Candida isolates by a broth microdilution assay. The minimal inhibitory concentrations of purpurin against Candida species isolates were 1.28--5.12 microg/ml. Mechanistic studies indicated that purpurin inhibited energy-dependent efflux pumps of Candida isolates. Furthermore, purpurin demonstrated a depolarization of mitochondrial membrane potential, suggesting a possible linkage of the antifungal mechanism of purpurin to Candida apoptosis. / Kang, Kai. / Adviser: Fong Wing Ping. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 98-123). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

TRAIL has been widely investigated as an anti-cancer agent due to its high efficacy in vitro and its safety to normal cells. However, TRAIL-based agents only showed modest effect in clinical studies because of TRAIL resistance. In addition to apoptosis, TRAIL has also been reported to promote pro-survival signalings, cell migration and metastasis. One of the current strategies in the development of TRAIL-based therapeutics focuses on the search of sensitizing agents that help overcome TRAIL resistance without increasing harm to normal cells. / This study reports a novel combination of TRAIL and 4-methylumbelliferone (4-MU) which can kill HeLa cells and HepG2 cells synergistically without cytotoxicity to Hs68 non-tumorigenic cells. This combination also effectively inhibited cancer cell proliferation and potentiated apoptosis by accumulation of tBid, down-regulation of anti-apoptotic proteins and inhibition of Akt. More importantly, 4-MU could suppress the recovery of HeLa cells from TRAIL-induced apoptosis, a process previously implicated to be associated with cancer relapse and tumor heterogeneity. This study has provided solid evidences substantiating further research on TRAIL-4-MU combination. / 腫瘤壞死因子相關凋亡誘導配體 (TRAIL) 在體外實驗中有良好抗癌作用，且不會傷害正常細胞，使之得到廣泛研究，成為近年熱門的新抗癌分子。然而TRAIL 在臨床實驗中並沒有顯著抗癌功效，一般認為人體腫瘤細胞對TRAIL 具有耐藥性。研究文獻亦指出，除了細胞凋亡外，TRAIL亦會誘發細胞存活機制、促進細胞移行及癌細胞轉移。目前，對於TRAIL相關藥品抗癌作用的研究有幾個大方向，其中之一就是尋找良好的增敏分子。良好的增敏分子應能夠增力癌細胞對TRAIL的敏感性，對抗癌細胞對TRAIL的耐藥性，同時不能殺傷正常細胞。 / 本研究揭示了一個全新的抗癌藥物聯合。當TRAIL聯合4-甲基伞形酮(4-MU)能產生協同作用，殺傷HeLa癌細胞和HepG2癌細胞而不會傷害Hs68正常細胞。此組合能有效抑制癌細胞生長，並透過增加tBid蛋白表達、減少抗凋亡蛋白表達及抑制Akt來促進細胞凋亡。更為重要的是，4-MU能抑制HeLa癌細胞自TRAIL誘導凋亡的恢復和逆轉。而癌細胞凋亡逆轉一般被視為與癌症復發及腫瘤多樣性有關。本研究提供了實質證據，支持對TRAIL-4-MU組合的後續研究。 / Wu, Hoi Yan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2015. / Includes bibliographical references (leaves 90-107). / Abstracts also in Chinese. / Title from PDF title page (viewed on 05, October, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Yang, Yaling. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 187-207). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Wong Sze Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 124-144). / Abstracts in English and Chinese. / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Cancer treatment and potential novel antitumor agents --- p.1 / Chapter 1.2 --- History of mushroom polysaccharides in medical uses --- p.1 / Chapter 1.3 --- Life cycle of mushroom --- p.3 / Chapter 1.4 --- Classification of antitumor mushroom polysaccharides --- p.5 / Chapter 1.4.1 --- (3-glucans --- p.5 / Chapter 1.4.2 --- Heteropolysaccharides --- p.7 / Chapter 1.4.3 --- Polysaccharide-protein complexes --- p.7 / Chapter 1.5 --- Structure-activity relationship of mushroom polysaccharides --- p.8 / Chapter 1.5.1 --- Lentinan as typical example --- p.9 / Chapter 1.5.2 --- Molecular weight --- p.10 / Chapter 1.5.3 --- Conformation --- p.10 / Chapter 1.5.4 --- Chemical modification --- p.11 / Chapter 1.5.5 --- Degree of branching --- p.13 / Chapter 1.6 --- Antitumor mushroom polysaccharides obtained from different developmental stages --- p.17 / Chapter 1.7 --- Mechanisms of in vitro antitumor activity of mushroom polysaccharides: cell cycle arrest and apoptotic induction --- p.20 / Chapter 1.7.1 --- Cell cycle regulation --- p.21 / Chapter 1.7.2 --- Induction of apoptosis --- p.24 / Chapter 1.8 --- The novel strategies for cancer treatment --- p.27 / Chapter 1.9 --- Literature Review on Pleurotus tuber-regium --- p.30 / Chapter 1.10 --- Objectives --- p.33 / Chapter Chapter 2 --- Materials and Methods --- p.35 / Chapter 2.1 --- Materials --- p.35 / Chapter 2.1.1 --- Assay kits --- p.35 / Chapter 2.1.2 --- Mushroom samples --- p.35 / Chapter 2.1.3 --- Cell lines and their subculture --- p.36 / Chapter 2.1.4 --- Antibodies --- p.37 / Chapter 2.2 --- Extraction of mushroom polysaccharides --- p.38 / Chapter 2.2.1 --- Hot-water extracts from mushroom fruiting body --- p.38 / Chapter 2.2.2 --- Hot-water extracts from mushroom mycelia --- p.38 / Chapter 2.2.3 --- Exo-polysaccharides from submerged fermentation medium --- p.39 / Chapter 2.3 --- Chemical and physio-chemical composition of PTR extracts --- p.41 / Chapter 2.3.1 --- Neutral monosaccharides --- p.41 / Chapter 2.3.1.1 --- Acid Depolymerization --- p.41 / Chapter 2.3.1.2 --- Neutral sugar derivatization --- p.42 / Chapter 2.3.1.3 --- Determination of neutral sugar composition by GC- --- p.43 / Chapter 2.3.2 --- Uronic acid (acidic monosaccharides) content --- p.45 / Chapter 2.3.3 --- Total carbohydrate content --- p.46 / Chapter 2.3.4 --- Protein content --- p.46 / Chapter 2.3.5 --- Molecular weight and the homogeneity --- p.47 / Chapter 2.4 --- In vitro growth inhibitory effects --- p.48 / Chapter 2.4.1 --- Trypan blue dye exclusion method --- p.48 / Chapter 2.4.2 --- "Colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay" --- p.49 / Chapter 2.5 --- In vitro cell proliferation assay --- p.50 / Chapter 2.6 --- Cell-cycle analysis --- p.51 / Chapter 2.7 --- Apoptotic determination --- p.52 / Chapter 2.8 --- Expression of proteins involved in apoptosis and cell-cycle --- p.52 / Chapter 2.8.1 --- Preparation of cell lysates --- p.53 / Chapter 2.8.2 --- Determination of protein concentrations --- p.53 / Chapter 2.8.3 --- Western blot --- p.54 / Chapter 2.9 --- Statistics --- p.57 / Chapter Chapter 3 --- Results and Discussion --- p.58 / Chapter 3.1 --- Yield of extract samples isolated from different developmental stages of PTR --- p.58 / Chapter 3.2 --- Chemical characteristics of hot-water extracts isolated from different stages of PTR --- p.60 / Chapter 3.2.1 --- The total carbohydrate and protein content of PTR extracts- --- p.60 / Chapter 3.2.2 --- The monosaccharide composition of PTR extracts --- p.62 / Chapter 3.3 --- Molecular weight distribution of PTR extracts --- p.64 / Chapter 3.4 --- Chemical characterization of PTR extracts --- p.69 / Chapter 3.5 --- Cytotoxic effect of PTR extracts on various cell line in vitro --- p.71 / Chapter 3.5.1 --- Effect of PTR extracts on HL-60 cell viability --- p.71 / Chapter 3.5.2 --- Effect of PTR extracts on K562 cell viability --- p.74 / Chapter 3.5.3 --- Effect of PTR extracts on MCF-7 cell proliferation --- p.76 / Chapter 3.5.4 --- Effect of PTR extracts on HepG2 cell proliferation --- p.76 / Chapter 3.5.5 --- Effect of PTR extracts on normal cell proliferation --- p.78 / Chapter 3.6 --- Effect of PTR extracts on the proliferation rate of various cell lines in vitro --- p.78 / Chapter 3.6.1 --- Effect of PTR extracts on HL-60 cell proliferation --- p.79 / Chapter 3.6.2 --- Effect of PTR extracts on K562 cell proliferation --- p.79 / Chapter 3.6.3 --- Effect of PTR extracts on MCF-7 cell proliferation --- p.80 / Chapter 3.6.4 --- Effect of PTR extracts on HepG2 cell proliferation --- p.80 / Chapter 3.6.5 --- Effect of PTR extracts on normal cell proliferation --- p.84 / Chapter 3.7 --- Summary of the cytotoxic and antiproliferative activities exhibited by PTR extracts --- p.84 / Chapter 3.8 --- Analysis of the effect of PTR extracts on the cell-cycle phases of HL-60 and K562 cells --- p.87 / Chapter 3.8.1 --- Effect of CEP on cell-cycle phases of HL-60 and K562 cells --- p.87 / Chapter 3.8.2 --- Effect of EDP on cell-cycle phases of HL-60 and K562 cells --- p.92 / Chapter 3.8.3 --- Effect of HWE1 on cell-cycle phases of HL-60 and K562 cells --- p.95 / Chapter 3.8.4 --- Effect of HWE2 on cell-cycle phases of HL-60 and K562 cells --- p.98 / Chapter 3.8.5 --- Effect of HWE3 on cell-cycle phases of HL-60 and K562 cells --- p.102 / Chapter 3.8.6 --- Summary --- p.105 / Chapter 3.9 --- The effect of PTR extracts on expression of cellular proteins involved in cell-cycle control and apoptotic pathway in HL-60 cells --- p.106 / Chapter 3.9.1 --- Expression of Bcl-2 and Bax proteins in HL-60 cells treated with PTR extracts --- p.106 / Chapter 3.9.2 --- Expression of cyclins and Cdks in HL-60 cells by PTR extracts --- p.115 / Chapter 3.9.3 --- The plausible antiproliferative mechanism(s) involved in PTR extracts on HL-60 cells --- p.117 / Chapter Chapter 4 --- Conclusions and Future works --- p.120 / Chapter 4.1 --- Conclusions --- p.120 / Chapter 4.2 --- Future works --- p.122 / References --- p.124 / Related Publications --- p.144

Li, Yuk Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 121-132). / Abstracts in English and Chinese. / ACKNOWLEDGMENTS --- p.i / ABSTRACT --- p.ii / LIST OF ABBREVATIONS --- p.vii / TABLE OF CONTENTS --- p.x / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Theories of aging --- p.2 / Chapter 1.2.1 --- Free radical theory of aging --- p.3 / Chapter 1.2.2 --- Free radicals reactions and their effects --- p.4 / Chapter 1.2.2.1 --- Lipid peroxidation --- p.6 / Chapter 1.2.2.2 --- Protein oxidation --- p.6 / Chapter 1.2.2.3 --- DNA oxidation --- p.8 / Chapter 1.2.2.4 --- Carbohydrate oxidation --- p.8 / Chapter 1.2.3 --- Antioxidant defense systems --- p.9 / Chapter 1.2.3.1 --- Superoxide dismutase (SOD) --- p.9 / Chapter 1.2.3.2 --- Catalase --- p.11 / Chapter 1.2.3.3 --- Glutathione peroxidase (GPx) and reductase --- p.11 / Chapter 1.3 --- Models on aging study --- p.12 / Chapter 1.3.1 --- Non-mammalian model systems --- p.12 / Chapter 1.3.1.1 --- History of Drosophila on longevity study --- p.14 / Chapter 1.3.1.2 --- Advantages of Drosophila model --- p.14 / Chapter 1.3.2 --- Mammalian model systems --- p.15 / Chapter 1.4 --- Antioxidant effects on aging process --- p.16 / Chapter 1.4.1 --- Antioxidant capacity of tea --- p.16 / Chapter 1.4.2 --- Antioxidant capacity of vegetables --- p.19 / Chapter 1.5 --- Dietary fat on longevity --- p.21 / Chapter Chapter 2 --- Anti-aging: Effects of Green Tea Catechins on Life Span and Antioxidant Enzymes of Drosophila melanogaster / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Gbjectives --- p.27 / Chapter 2.3 --- Materials and methods --- p.28 / Chapter 2.3.1 --- Materials --- p.28 / Chapter 2.3.2 --- Preparation of GTCs --- p.28 / Chapter 2.3.3 --- Feeding experiment --- p.30 / Chapter 2.3.3.1 --- Dose determination --- p.31 / Chapter 2.3.3.2 --- Paraquat treatment --- p.31 / Chapter 2.3.3.3 --- Hydrogen peroxide (H202) treatment --- p.33 / Chapter 2.3.3.4 --- Enzyme activity assay --- p.34 / Chapter 2.3.4 --- Enzyme analysis --- p.34 / Chapter 2.3.4.1 --- SOD activity --- p.34 / Chapter 2.3.4.2 --- Catalase activity --- p.35 / Chapter 2.3.4.3 --- Lipid hydorperoxies (LOOHs) formation --- p.36 / Chapter 2.3.4.4 --- Determination of protein concentration --- p.37 / Chapter 2.3.5 --- Semi-quantitative RT-PCR assay --- p.37 / Chapter 2.3.5.1 --- RNA isolation --- p.37 / Chapter 2.3.5.2 --- Primer sequences --- p.38 / Chapter 2.3.5.3 --- Reverse Transcriptase PCR --- p.40 / Chapter 2.3.6 --- Statistics --- p.40 / Chapter 2.4 --- Results --- p.42 / Chapter 2.4.1 --- Dose determination --- p.42 / Chapter 2.4.2 --- Paraquat treatment --- p.45 / Chapter 2.4.3 --- H202 treatment --- p.48 / Chapter 2.4.4 --- SOD activity --- p.51 / Chapter 2.4.5 --- Catalase activity --- p.51 / Chapter 2.4.6 --- LOOHs formation --- p.53 / Chapter 2.4.7 --- Semi-quantitative RT-PCR assay --- p.53 / Chapter 2.5 --- Discussion --- p.55 / Chapter Chapter 3 --- Anti-aging: Effects of Vegetable Extracts on Life Span and Antioxidant Enzymes of Drosophila melanogaster / Chapter 3.1 --- Introduction --- p.62 / Chapter 3.2 --- Objectives --- p.64 / Chapter 3.3 --- Materials and methods --- p.65 / Chapter 3.3.1 --- Materials --- p.65 / Chapter 3.3.2 --- Preparation of vegetables extracts --- p.65 / Chapter 3.3.3 --- Feeding experiment --- p.65 / Chapter 3.3.3.1 --- Dose determination --- p.66 / Chapter 3.3.3.2 --- Paraquat and hydrogen peroxide (H2O2) treatment --- p.67 / Chapter 3.3.4 --- Lipid hydroperoxide (LOOHs) formation --- p.67 / Chapter 3.3.5 --- Enzyme analysis --- p.67 / Chapter 3.3.6 --- Semi-quantitative RT-PCR assay --- p.67 / Chapter 3.3.7 --- Statistics --- p.68 / Chapter 3.4 --- Results --- p.69 / Chapter 3.4.1 --- Dose determination --- p.69 / Chapter 3.4.2 --- Antioxidant effect among different vegetables --- p.69 / Chapter 3.4.3 --- SOD activity --- p.76 / Chapter 3.4.4 --- Catalase activity --- p.76 / Chapter 3.4.5 --- LOOHs formation --- p.78 / Chapter 3.4.6 --- Semi-quantitative RT-PCR assay --- p.78 / Chapter 3.5 --- Discussion --- p.80 / Chapter Chapter 4 --- Anti-aging Activity of Greeen Tea Catechins and Broccoli Extracts in Drosophila melanogaster Fed a High Fat Diet / Chapter 4.1 --- Introduction --- p.85 / Chapter 4.2 --- Objectives --- p.87 / Chapter 4.3 --- Materials and methods --- p.88 / Chapter 4.3.1 --- Materials --- p.88 / Chapter 4.3.2 --- Preparation of free fatty acid --- p.88 / Chapter 4.3.3 --- Prepartion of GTCs and BEs --- p.89 / Chapter 4.3.4 --- Feeding experiment --- p.89 / Chapter 4.3.5 --- LOOHs formation --- p.90 / Chapter 4.3.6 --- Enzyme analysis --- p.91 / Chapter 4.3.7 --- Semi-quantitative RT-PCR assay --- p.91 / Chapter 4.3.8 --- Statistics --- p.91 / Chapter 4.4 --- Results --- p.92 / Chapter 4.4.1 --- Dose determination --- p.92 / Chapter 4.4.2 --- Effects of GTCs or BEs on life span with lard consumption --- p.95 / Chapter 4.4.3 --- LOOHs formation --- p.101 / Chapter 4.4.4 --- SOD activity --- p.103 / Chapter 4.4.5 --- Catalase activity --- p.107 / Chapter 4.4.6 --- Semi-quantitative RT-PCR assay --- p.110 / Chapter 4.5 --- Discussion --- p.112 / References --- p.121

肝癌是造成癌症相關死亡的主要原因之一。而常規化療受耐藥性的發展和各種副作用的限制。由於無毒性和鲜明的生物药物能力，從蘑菇提取的代謝物在癌症治療中獲得更多的注意和关注。我們以前的研究已經證明來自平菇香菇多醣蛋白複合物的抗癌作用。本研究的目的是探討一種含有多醣蛋白複合物的秀珍菇（PP）熱水提取物在肝癌細胞中抗癌活性的分子機制。 / 我們的研究結果表明，用PP处理过的肝癌細胞，不僅顯著的显示出降低的體外腫瘤細胞的增殖和侵襲，也增強化療藥物順鉑的藥物敏感性。無論是口服和腹腔注射都顯著抑制移植免疫BALB / c裸小鼠的腫瘤生長。同时，PP也能在體外和體內实验顯著抑制PI3K/Akt信號通路在肝癌細胞。有趣的是，当过表达AKT时，Myr-AKT，PP的這種抑制癌细胞生长的效果有减弱的趋势，同时也反映在PP对癌细胞侵襲抑制的作用上。印跡和酶聯免疫吸附試驗結果表明，在PP处理过的肝癌細胞中，血管內皮生長因子（VEGF）的表達和分泌減少了。此外， rhVEGF的加入减弱了 PP对PI3K/Akt通路和肝癌细胞表型的抑製作用。 / 我們的研究結果表明，PP能在體外和體內试验中抑制肝癌細胞增殖，侵襲和耐藥性，通过抑制分泌血管內皮生長因子誘導PI3K/Akt的信號通路。這項研究表明了PP的潛在治療肝癌的治療意義。 / Liver cancer or hepatocellular carcinoma is one of the leading causes of cancer-related deaths. Conventional chemotherapies are limited by the development of drug resistance and various side effects. Because of its non-toxicity and potent biopharmacological activity, metabolites derived from mushrooms have received more attention in cancer therapy. Our previous studies have demonstrated the anti-cancer effects of polysaccharide-protein complexes derived from the Pleurotus mushrooms. The aim of this study was to investigate the underlying molecular mechanism of the anti-cancer activity of a hot water extract containing a polysaccharide-protein complex isolated from Pleurotus pulmonarius (PP) in liver cancer cells. / Our results indicated that exposure of liver cancer cells to PP not only significantly reduced the in vitro cancer cell proliferation and invasion but also enhanced the drug-sensitivity to the chemotherapeutic drug Cisplatin. Both oral administration and intraperitoneal injection of PP significantly inhibited the tumor growth in xenograft BALB/c nude mice. PP triggered a marked suppression of the PI3K/AKT signaling pathway in liver cancer cells in vitro and in vivo, and overexpression of the constitutively active form of AKT, Myr-AKT, abrogated this effect and the inhibited proliferation and invasion by PP. Both western blot and ELISA results showed that PP-treated liver cancer cells had reduced expression and secretion of vascular endothelial growth factor (VEGF). Addition of recombinant human VEGF attenuated the inhibitory effects of PP on PI3K/AKT pathway and the cancer phenotypes. / Our results demonstrated that PP suppressed the proliferation, invasion, and drug-resistance of liver cancer cells in vitro and in vivo, mediated by the inhibition of autocrine VEGF-induced PI3K/AKT signaling pathway. All these results suggest the potential therapeutic implication of PP in the treatment of human liver cancer. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Xu, Wenwen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 83-99). / Abstracts also in Chinese. / Thesis Committee --- p.i / English Abstract --- p.ii / Chinese Abstract --- p.iv / Acknowledgements --- p.v / List of Tables --- p.vi / List of Figures --- p.vii / Abbreviations --- p.x / Content page --- p.xiv / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Mushroom as functional foods --- p.1 / Chapter 1.1.1 --- Introduction of functional food --- p.1 / Chapter 1.1.2 --- Functional food and cancer --- p.1 / Chapter 1.1.3 --- Edible Mushroom as functional food --- p.4 / Chapter 1.1.4 --- Pleurotus pulmonarius and its function --- p.7 / Chapter 1.2 --- Hepatocellular carcinoma --- p.9 / Chapter 1.2.1 --- Liver and hepatocellular carcinoma --- p.9 / Chapter 1.2.2 --- Carcinogenesis of liver cancer --- p.12 / Chapter 1.2.2.1 --- Hallmarks of cancer --- p.12 / Chapter 1.2.2.2 --- Cell cycle --- p.13 / Chapter 1.2.2.3 --- Apoptosis --- p.15 / Chapter 1.2.2.4 --- Angiogenesis --- p.17 / Chapter 1.2.2.5 --- Invasion and metastasis --- p.19 / Chapter 1.2.2.6 --- Drug resistance --- p.21 / Chapter 1.2.3 --- The role of PI3K/AKT pathway --- p.23 / Chapter 1.2.4 --- The role of growth factor Vascular endothelial growth factor (VEGF) in HCC --- p.25 / Chapter 1.3 --- Research objectives --- p.27 / Chapter 1.3.1 --- Hypothesis and objectives --- p.27 / Chapter 1.3.2 --- Experimental design --- p.28 / Chapter Chaper 2 --- Materials and Methods --- p.29 / Chapter 2.1 --- Materials --- p.29 / Chapter 2.1.1 --- Mushroom Pleurotus pulmonarius --- p.29 / Chapter 2.1.2 --- Drugs and cell lines --- p.29 / Chapter 2.1.3 --- Antibodies list --- p.30 / Chapter 2.1.4 --- Animal models --- p.32 / Chapter 2.2 --- Sample preparation and structure investigation --- p.32 / Chapter 2.2.1 --- Polysaccharide extraction from mushroom --- p.32 / Chapter 2.2.2 --- Endotoxin test --- p.32 / Chapter 2.2.3 --- Determination of monosaccharide profile by gas chromatography and mass spectrometry (GC/MS) --- p.33 / Chapter 2.2.3.1 --- Sample preparation for gas chromatography analysis --- p.33 / Chapter 2.2.3.1.1 --- Acid depolymerisation --- p.33 / Chapter 2.2.3.1.2 --- Neutral sugar derivatization --- p.33 / Chapter 2.2.3.1.3 --- External monosaccharide standard preparation --- p.34 / Chapter 2.2.3.2 --- Gas chromatography-mass spectrometry (GC/MS) --- p.34 / Chapter 2.2.4 --- Determination of total sugar by phenol-sulfuric acid method (Dubois, 1956) --- p.36 / Chapter 2.2.5 --- Determination of protein content by Lowry-Folin method (Lowry et al.,1951) --- p.37 / Chapter 2.3 --- Biological assays --- p.38 / Chapter 2.3.1 --- In vitro assays --- p.38 / Chapter 2.3.1.1 --- MTT assay --- p.38 / Chapter 2.3.1.2 --- Colony formation assay --- p.38 / Chapter 2.3.1.3 --- Plasmid transfection --- p.39 / Chapter 2.3.1.4 --- In vitro cell invasion assay --- p.39 / Chapter 2.3.1.5 --- Cell cycle analysis --- p.39 / Chapter 2.3.1.6 --- Western blot analysis --- p.40 / Chapter 2.3.1.7 --- VEGF ELISA Kit --- p.42 / Chapter 2.3.2 --- In vivo assays --- p.43 / Chapter 2.3.2.1 --- Tumor xenograft nude mouse model --- p.43 / Chapter 2.3.2.2 --- Immunohistochemistry --- p.45 / Chapter 2.3.2.3 --- H&Estaining --- p.45 / Chapter 2.3.3 --- Statistical analysis --- p.45 / Chapter Chaper 3 --- Results and discussion --- p.46 / Chapter 3.1 --- The yield and chemical characteristic of PP --- p.46 / Chapter 3.1.1 --- The yield of PP from mushroom Pleurotus pulmonarius --- p.46 / Chapter 3.1.2 --- Total carbohydrate and protein content --- p.47 / Chapter 3.1.3 --- Monosaccharide composition by GC-MS --- p.48 / Chapter 3.2 --- Toxicity of the PP water by Limulus amebocyte lysate (LAL) test --- p.48 / Chapter 3.2.1 --- Limulus amebocyte lysate (LAL) test --- p.48 / Chapter 3.3 --- Effects of PP on the proliferation of liver cancer cell lines --- p.50 / Chapter 3.3.1 --- MTT assay --- p.50 / Chapter 3.3.2 --- Colony-formation assay --- p.51 / Chapter 3.3.3 --- Cytotoxic effects of PP against normal liver cell --- p.52 / Chapter 3.3.4 --- The anti-proliferative effect of PP on other cancer types --- p.53 / Chapter 3.3.5 --- Cell cycle analysis by flow cytometry of PP treated liver cancer cells --- p.54 / Chapter 3.3.6 --- Protein expression by western blot analysis of P treated liver cancer cells --- p.56 / Chapter 3.4 --- Anti-cancer effect of PP on liver cancer cells through inactivation of PI3K/AKT signaling pathway --- p.57 / Chapter 3.4.1 --- Effect of PP on inactivation of PI3K/AKT pathway --- p.57 / Chapter 3.4.2 --- The abrogated inhibitory effect of PP on Huh7 with overexpression of AKT. --- p.59 / Chapter 3.4.3 --- The abrogated inhibitory effect of PP on PI3K/AKT signal pathway with overexpression of the constitutively active form of AKT, Myr-AKT --- p.60 / Chapter 3.5 --- Inhibition of VEGF expression and secretion by PP --- p.62 / Chapter 3.5.1 --- ELISA result of PP on VEGF secretion --- p.62 / Chapter 3.5.2 --- The attenuated inhibitory effect of PP on cell proliferation with addition of rhVEGF --- p.63 / Chapter 3.5.3 --- The attenuated inhibitory effect of PP on PI3K/AKT signal pathway with addition of rhVEGF --- p.64 / Chapter 3.6 --- Effect of PP on enhancing the chemosensitivity of liver cancer cells to Cisplatin --- p.66 / Chapter 3.6.1 --- Synergistic effect of PP with cisplatin (DDP) in liver cancer cells --- p.66 / Chapter 3.6.2 --- The abrogated drug-resistant effect by PP by overexpression of the constitutively active form of AKT, Myr-AKT --- p.67 / Chapter 3.6.3 --- The abrogated drug-resistant effect of PP with addition of rhVEGF --- p.68 / Chapter 3.7 --- The anti-invasive potential of PP on liver cancer cells. --- p.69 / Chapter 3.7.1 --- Boyden chamber assay --- p.69 / Chapter 3.7.2 --- The attenuated anti-invasive effect of PP on liver cancer cells with overexpression of constitutively activated AKT --- p.71 / Chapter 3.7.3 --- The attenuated anti-invasive effect of PP on liver cancer cells with addition of rhVEGF --- p.72 / Chapter 3.8 --- The anti-tumor effect of PP in vivo --- p.73 / Chapter 3.8.1 --- The anti-tumor effect of PP by using tumor xenograft model --- p.73 / Chapter 3.8.2 --- Body weight of nude mice treated with PP --- p.75 / Chapter 3.8.3 --- Harmful effect of PP on nude mice --- p.76 / Chapter 3.8.4 --- Immunohistochemist analysis of mice tumor xenograft treated with PP --- p.77 / Chapter 3.8.5 --- Western blot anaylysis using the tumor tissues harvested from mice xenograftes treated with PP --- p.78 / Chapter Chapter 4 --- Conclusion and future Plan --- p.81 / Reference --- p.83 / Related Publication List --- p.100

Chau, Hiu Yan Anita. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 109-122). / Abstracts in English and Chinese. / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Introduction on mushroom life cycle --- p.1 / Chapter 1.2 --- Introduction of mushroom sclerotium --- p.2 / Chapter 1.3 --- Different extraction methods of mushroom polysaccharides --- p.3 / Chapter 1.4 --- Bioactivities of mushroom polysaccharides and factors affecting their biological activities --- p.4 / Chapter 1.4.1 --- Molecular weight --- p.4 / Chapter 1.4.2 --- Linkages --- p.5 / Chapter 1.4.3 --- Branching rate --- p.5 / Chapter 1.4.4 --- Conformation --- p.6 / Chapter 1.5 --- Mechanisms for antitumor activites of mushrooms polysaccharides.… --- p.7 / Chapter 1.5.1 --- Cancer-preventing activity --- p.7 / Chapter 1.5.2 --- Immuno-enhancing activity (BRM) --- p.8 / Chapter 1.5.3 --- Direct tumor inhibition activity --- p.8 / Chapter 1.6 --- Cell cycle regulation and induction of apoptosis --- p.9 / Chapter 1.6.1 --- The cell cycle machinery --- p.9 / Chapter 1.6.2 --- Cell cycle arrest and regulation --- p.11 / Chapter 1.6.3 --- Apoptosis and regulation --- p.13 / Chapter 1.7 --- Literature review on Pleurotus tuber-regium --- p.16 / Chapter 1.7.1 --- Introduction of Pleurotus tuber-regium --- p.16 / Chapter 1.7.2 --- Antitumor effect of mushroom polysaccharides isolated from different developmental stages of Pleurotus tuber-regium --- p.17 / Chapter 1.7.2.1 --- Sclerotium --- p.17 / Chapter 1.7.2.2 --- Mycelium --- p.19 / Chapter 1.7.2.3 --- Culture medium --- p.19 / Chapter 1.7.2.4 --- Fruiting body --- p.20 / Chapter 1.8 --- Literature review on Size reduction process --- p.21 / Chapter 1.8.1 --- Introduction of micron technology --- p.21 / Chapter 1.8.1.1 --- Ball milling --- p.21 / Chapter 1.8.1.2 --- Jet milling --- p.22 / Chapter 1.8.1.3 --- High-pressure micronizing --- p.22 / Chapter 1.8.1.4 --- Oscillatory milling --- p.23 / Chapter 1.8.2 --- Effect of particle sizes on physicochemical properties and biological activities of plant materials --- p.23 / Chapter 1.8.2.1 --- Physicochemical properties --- p.24 / Chapter 1.8.2.2 --- Biochemical activities --- p.24 / Chapter 1.9 --- Objectives --- p.26 / Chapter Chapter 2. --- Materials and methods --- p.28 / Chapter 2.1 --- Materials --- p.28 / Chapter 2.1.1 --- Mushroom sclerotia --- p.28 / Chapter 2.1.2 --- Micronisation --- p.29 / Chapter 2.1.3 --- Cell lines --- p.31 / Chapter 2.1.4 --- Antibodies --- p.33 / Chapter 2.1.5 --- Animal model --- p.33 / Chapter 2.2 --- Methods --- p.34 / Chapter 2.2.1 --- Micronisation --- p.34 / Chapter 2.2.2 --- Hot water extraction for mushroom sclerotia --- p.35 / Chapter 2.2.3 --- Measurement of monosaccharide profile --- p.36 / Chapter 2.2.3.1 --- Acid deploymerisation --- p.36 / Chapter 2.2.3.2 --- Neutral sugar derivatization --- p.36 / Chapter 2.2.3.3 --- Gas chromatography (GC) --- p.37 / Chapter 2.2.4 --- Total sugar content by Phenol-sulphuric acid Method --- p.38 / Chapter 2.2.5 --- Acidic sugar content by measuring uronic acid content --- p.39 / Chapter 2.2.6 --- Protein content by Lowry-Folin Method --- p.40 / Chapter 2.2.7 --- Size exclusion chromatography by high pressure liquid chromatograhy (HPLC) --- p.41 / Chapter 2.2.8 --- In vitro antitumor assay --- p.41 / Chapter 2.2.8.1 --- Trypan blue exclusion assay --- p.42 / Chapter 2.2.8.2 --- MTT Assay --- p.42 / Chapter 2.2.9 --- Cell cycle analysis by Flow Cytometry --- p.43 / Chapter 2.2.10 --- Protein expression involved in apoptosis --- p.45 / Chapter 2.2.10.1 --- Cell lysates preparation --- p.45 / Chapter 2.2.10.2 --- Determination of protein concentrations --- p.46 / Chapter 2.2.10.3 --- Western blot --- p.46 / Chapter 2.2.11 --- In vivo antitumor assay --- p.50 / Chapter 2.2.11.1 --- BALB/c mice --- p.50 / Chapter 2.2.11.2 --- Athymic nude mice --- p.50 / Chapter 2.2.12 --- Statistical methods --- p.51 / Chapter Chapter 3 --- Results and Discussion --- p.52 / Chapter 3.1 --- Yield of hot water-soluble extracts from Pleurotus tuber-regium --- p.52 / Chapter 3.2 --- Chemical composition of hot water-soluble extracts from PTR --- p.56 / Chapter 3.2.1 --- Total carbohydrate content --- p.56 / Chapter 3.2.2 --- Uronic acid content --- p.57 / Chapter 3.2.3 --- Protein content --- p.58 / Chapter 3.3 --- Monosaccharide profiles of hot water-soluble extracts from PTR by gas chromatography (GC) --- p.61 / Chapter 3.4 --- Molecular weight profile of hot water-soluble extracts from PTR by size exclusion chromatography (SEC) --- p.64 / Chapter 3.5 --- Antitumor effects of mushroom sclerotial polysaccharides --- p.72 / Chapter 3.5.1 --- In vitro antiproliferation study --- p.72 / Chapter 3.5.1.1 --- In vitro antiproliferation study by HL-60 --- p.72 / Chapter 3.5.1.2 --- In vitro antiproliferation study by THP-1 --- p.75 / Chapter 3.5.1.3 --- In vitro antiproliferation study by MCF-7 --- p.77 / Chapter 3.5.1.4 --- In vitro antiproliferation study by K562 --- p.77 / Chapter 3.5.1.5 --- In vitro antiproliferation study by SI80 --- p.79 / Chapter 3.5.1.6 --- In vitro antiproliferation study by normal cells --- p.79 / Chapter 3.5.1.7 --- Dose-response relationship between hot water-soluble extract from PTR and tumor cell inhibition --- p.80 / Chapter 3.5.2 --- In vivo antitumor study --- p.83 / Chapter 3.5.2.1 --- BALB/c mice --- p.83 / Chapter 3.5.2.2 --- Athymic nude mice --- p.84 / Chapter 3.6 --- Flow cytometric analysis of tumor cells treated by various hot wter-soluble extracts from PTR --- p.88 / Chapter 3.6.1 --- Antiproliferative effect of various hot water-soluble extracts from 10PTR on HL-60 --- p.88 / Chapter 3.6.2 --- Antiproliferative effect of various hot water-soluble extracts from 10PTR on THP-1 --- p.93 / Chapter 3.7 --- Effects of various hot water-soluble extracts from 10PTR on expression of Bcl-2 and Bax proteins in HL-60 cells --- p.99 / Chapter 3.8 --- "Correlation between particle size, structure and antitumor activity of mushroom sclerotial extracts" --- p.101 / Chapter Chapter 4. --- Conclusions and Future Works --- p.105 / List of References --- p.109 / Related Publications --- p.123

published_or_final_version / Anatomy / Master / Master of Philosophy

Ph.D. / Ligands bis(pyrazolyl)acetic acid (L1) and bis(3,5-dimethylpyrazolyl)acetic acid (L2) were synthesised by reacting pyrazoles and dibromoacetic acid under phase transfer conditions, by using benzyltriethylammonium chloride as the catalyst. Ligands L1 and L2 were characterised by a combination of 1H, 13C{1H} NMR, IR spectroscopy and microanalysis. Esterification of L1 and L2 led to formation of bis(pyrazolyl)ethyl acetate (L3) and bis(3,5-dimethylpyrazolyl)ethyl acetate (L4). Ligands L3 and L4 were also characterised by a combination of 1H, 13C{1H} NMR, IR spectroscopy and microanalysis. Subsequently, new pyrazolyl palladium(II) and platinum(II) compounds, [PdCl2(L1)] (1), [PdCl2(L2)] (2), [PtCl2(L1)] (3a) and [PtCl2(L2)] (4) were prepared by reacting bis(pyrazolyl)acetic acid ligands (L1-L2) with K2[PdCl4] or K2[PtCl4] respectively. The structures of complex 1 and 2 reveal distorted square planar geometries. The bond angles of N-Pd-N, N-Pd-Cl, N-Pd-Cl, for 1 and 2 are between 85.8(3)o and 90.81(4)o). The platinum compound, K2[Pt4Cl8(L1)2(deprotonated-L1)2].2H2O (3b), crystallised from aqueous solutions containing 3a when such solutions were left to stand overnight. Each platinum coordination environment consists of two cis-Cl ligands and one K2-N^N(L1) unit (L1 = bis(pyrazolyl)acetic acid), with two ligand moieties in 3b that are deprotonated with two K+ counter ions. Reaction of bis(pyrazolyl)acetic acid ligands (L1-L2) with [HAuCl4].4H2O gave gold(III) complexes [AuCl2(L1)]Cl (5a) and [AuCl2(L2)]Cl (6a). The spectroscopic, mass spectroscopy and microanalysis data were used to confirm the formation of the desired complexes. However, attempts to crystallise 5a and 6a led to formation of [AuCl2(pz)(pzH)] (5b) and [AuCl2(3,5-Me2pz)(3,5-Me2pzH)] (6b). This was confirmed by the structural characterisation of 5b, which has a distorted square-planar geometry. When complexes 1-6a were screened for their anti-tumour activity against CHO-22 cells, they showed no appreciable biological activities against CHO-22 cells. Substitution reactions of complexes 1-6a with L-cysteine performed to probe any relationship between the observed antitumour activities and the rates of ligand substitution of these complexes were inconclusive. Dithiocarbamate ligands L5-L8 were synthesised as potassium salts by introducing a CS2 group in positions 1 of pyrazole, 3,5-dimethylpyrazole, indazole and imidazole. The reaction of L5-L8 with [AuCl(PPh3)], [Au2Cl2(dppe)], [Au2Cl2(dppp)] and [Au2Cl2(dpph)], led to isolation of complexes [Au(L)(PPh3)] (13-16), [Au2(L)2(dppe)] (17a-19), [Au2(L)2(dppp)] (20-22) and [Au2(L)2(dpph)] (23-25) (dppe = bis(diphenylphosphino)ethane, dppp = bis(diphenylphosphino)propane, dpph = bis(diphenylphosphino)hexane; L = anions of L5-L8). The mononuclear molecular structure of 15 features a near linear geometry with a P(1)-Au(1)-S(1) angle of 175.36(2) o. The binuclear gold(I) complexes 20-22 and 23-25 have two P-Au-S moieties as evident in the solid state structure of 25. Attempts to crystallise complex 17a led to the formation of a gold(I) cluster complex [Au18S8(dppe)6]2+ (17b) as confirmed by X-ray crystallography. Cluster 17b features weak Au···Au interactions (2.9263(7)-3.1395(7) Å). Complexes 13-16 and 20-25 were tested in vitro for anticancer activity on HeLa cells. The activities of gold(I) complexes 13-16 were comparable to that of cisplatin. Dinuclear gold(I) complexes 20-25 also showed appreciable antitumour activity against HeLa cells. However, the dpph gold(I) compounds (23-25) were highly active, with 24 showing the highest activity against HeLa cells (IC50 = 0.1 μM). The tumour specificity (TS) factors for 23 and 24 were 31.0 and 70.5, respectively.

Malaria is one of the three most deadly diseases in Africa. Although there are available treatments, their efficacy has been greatly reduced over the past two decades due to the development of resistance to currently available drugs. This has necessitated the search for new and effective antimalarial agents. This project approached the search for new antimalarial compounds in two ways: (i) by screening natural products isolated from marine algae against the Plasmodium parasite and (ii) by modification of selected isolated active compounds to target 1-deoxY-đ-xylulose 5-phosphate reductoisomerase (DXR), an enzyme found in the nonmevalonate isoprenoid biosynthetic pathway of Plasmodium Jalciparum. It was envisaged that such a compound would exhibit dual action on the Plasmodium parasite. Extracts obtained from 22 marine algae were prefractionated by solvent partitioning and were screened for anti plasmodial activity against the chloroquine sensitive (CQS) P. Jalciparum D 10 strain. Overall, 50% of the algae screened produced at least one crude fraction with activity against P. Jalciparum. Extracts of the algae Sargassum heterophyllum, Plocamium cornutum, Amphiroa ephedrea and Pterosiphonia cloiophylla gave the most promising results. Fractionation of S. heterophyllum afforded three tetraprenyltoluquinols (3.1, 3.2 and 3.5) and an all-trans-fucoxanthin (3.6). Three new compounds (4.5, 4.6 and 4.7) and two known halogenated monoterpenes (4.1 and 4.4) were isolated from P. cornutum. Each of the isolated compounds from both S. heterophyllum and P. cornutum showed antiplasmodial activity with IC₅₀ values ranging from 2.0 - 15.3 μM for S. heterophyllum and 13 - 230 μM for P. cornutum. Attempts to synthetically modify halogenated monoterpene 4.4 by dihydroxylation and phosphorylation in order to inhibit the DXR enzyme was unsuccessful. However, the hemiterpene analogue (5.42) of the halogenated monoterpenes was successfully phosphorylated and dihydroxylated to give compound 5.45 which showed promising activity against DXR. The result obtained indicated that the proposed phosphorylation and dihydroxylation of the halogenated monoterpene 4.4 would result in the synthesis of a potent DXR inhibitor and therefore a potential antimalarial agent with dual mode of action on the Plasmodium parasite.