Being the most abundant carbon-containing terrestrial biopolymer, lignocellulose serves as one of the best candidate feedstocks for biofuel production. The current cost-ineffective method for lignocellulose pretreatment is one of the major barriers that hinder the development of biofuel production. This leads to an exploration in the potential application of lignocellulolytic enzymes in this biorefinery process. Taking advantage of the strong activity of ligninolytic enzymes in L. edodes, we aimed at cloning and heterologously expressing these enzymes. The present project applied a yeast expression system, Pichia pastoris, as a laboratory-scale platform for heterologous expression of one of our target ligninolytic enzymes, manganese peroxidase (MnP). We successfully cloned and expressed recombinant MnP. Its enzymatic activity was the highest when grown in the presence of hemoglobin. Our long-term goal is to establish a platform for the large-scale production of recombinant lignocellulotyic enzymes at low-cost, which would strengthen their application in biofuel production. / The shitake mushroom, Lentinula edodes, is one of the most commonly consumed edible mushrooms in Asian countries. It is a saprophyte that naturally colonizes dead wood. As a member of wood-decaying white rot basidiomycete, L. edodes is able to depolymerize lignin and hydrolyze wood polysaccharides. However, the enzymatic mechanism for its lignocellulolytic system is poorly understood. Examination on the L. edodes genome and transcriptome revealed a unique lignocellulolytic system. L. edodes has a diverse enzymatic arsenal for lignin degradation. The enzymes include laccase, manganese peroxidase, cellobiose dehydrogenase and various lignin degrading auxiliary enzymes. When compared to another white rot fungus Phanerochaete chrysosporium, L. edodes possesses more hemicellulase- and pectinase-coding genes, and fewer genes encoding cellulases, suggesting that it preferentially attacks non-cellulosic polysaccharides. The transcription analysis on genes related to antioxidative mechanisms also offers insights to the oxidative stress encountered by mycelium during the free radical-mediated lignin degradation. / Kwok, Sze Wai. / Adviser: Hoi-Shan Kwan. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 141-160). / 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.

Tjia Wai Mui. / Thesis submitted in: July 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 123-140). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknowledgement --- p.iv / Abbreviations --- p.v / Table of Contents --- p.vi / List of Figures --- p.x / List of Tables --- p.xii / Chapter Chapter One --- Literature Review / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Growth of L. edodes --- p.3 / Chapter 1.2.1 --- Life cycle of L. edodes --- p.3 / Chapter 1.2.2 --- Growth parameters of L. edodes --- p.6 / Chapter 1.2.2.1 --- Temperature --- p.6 / Chapter 1.2.2.2 --- Relative humidity --- p.7 / Chapter 1.2.2.3 --- Moisture content in substrate --- p.7 / Chapter 1.2.2.4 --- Light --- p.8 / Chapter 1.2.2.5 --- pH --- p.8 / Chapter 1.3 --- Cultivation of L. edodes --- p.9 / Chapter 1.3.1 --- History and development of artificial cultivation --- p.9 / Chapter 1.3.2 --- Use of forced fruiting --- p.11 / Chapter 1.4 --- Molecular studies of stress on fungi --- p.12 / Chapter 1.4.1 --- Studies of temperature stress in mushroom --- p.12 / Chapter 1.4.2 --- Studies of molecular chaperones in fungi --- p.13 / Chapter 1.4.2.1 --- Role of molecular chaperones --- p.13 / Chapter 1.4.2.2 --- Heat shock protein 70 (Hsp70) and their cochaperones --- p.13 / Chapter 1.4.2.3 --- Other chaperones --- p.15 / Chapter 1.4.2.4 --- Molecular chaperones and development --- p.16 / Chapter 1.5 --- Prospectus --- p.19 / Chapter Chapter Two --- The Effect of Stress on the Growth of L. edodes / Chapter 2.1 --- Introduction --- p.23 / Chapter 2.2 --- Materials and Methods --- p.24 / Chapter 2.2.1 --- Strain and culture conditions --- p.24 / Chapter 2.2.2 --- Stress treatments --- p.24 / Chapter 2.2.3 --- Data collection --- p.25 / Chapter 2.2.4 --- Data analysis --- p.25 / Chapter 2.3 --- Results --- p.27 / Chapter 2.3.1 --- Reliability analysis --- p.27 / Chapter 2.3.2 --- Descriptive statistics --- p.28 / Chapter 2.3.3 --- Independent t-test (ANOVA) --- p.33 / Chapter 2.4 --- Discussion --- p.37 / Chapter Chapter Three --- Sequence Analysis of selected Stress Genes / Chapter 3.1 --- Introduction --- p.39 / Chapter 3.2 --- Materials and Methods --- p.40 / Chapter 3.2.1 --- Isolation of stress genes --- p.40 / Chapter 3.2.1.1 --- Construction of primordial cDNA library --- p.40 / Chapter 3.2.1.2 --- Screening of cDNA clones --- p.40 / Chapter 3.2.2 --- Sequence analyses of stress genes --- p.41 / Chapter 3.2.2.1 --- Amplification and purification of cDNA insert --- p.41 / Chapter 3.2.2.2 --- Full length DNA cycle sequencing --- p.42 / Chapter 3.2.2.3 --- Sequence analyses --- p.43 / Chapter 3.2.3 --- Screening of LeSSA (Inducible HSP70) --- p.45 / Chapter 3.2.3.1 --- PCR screening of LeSSA by degenerate primers and LeSSB specific primers --- p.45 / Chapter 3.2.3.2 --- Screening of LeSSA from cDNA library by hybridization --- p.49 / Chapter 3.3 --- Results --- p.51 / Chapter 3.3.1 --- Sequence analyses --- p.51 / Chapter 3.3.1.1 --- LeSSB --- p.51 / Chapter 3.3.1.2 --- LeMge1 --- p.57 / Chapter 3.3.1.3 --- LeSTI1 --- p.62 / Chapter 3.3.1.4 --- LeTCP1β --- p.69 / Chapter 3.3.1.5 --- LeTCP1γ --- p.74 / Chapter 3.3.2 --- Failure of isolating LeSSA (Inducible HSP70) --- p.80 / Chapter 3.4 --- Discussion --- p.82 / Chapter 3.4.1 --- Sequence analyses --- p.82 / Chapter 3.4.2 --- Screening of LeSSA --- p.84 / Chapter Chapter Four --- Characterization of stress genes upon different stresses / Chapter 4.1 --- Introduction --- p.86 / Chapter 4.2 --- Materials and Methods --- p.87 / Chapter 4.2.1 --- Strain and culture conditions --- p.87 / Chapter 4.2.2 --- Stress treatments --- p.87 / Chapter 4.2.3 --- Isolation of total RNAs --- p.87 / Chapter 4.2.4 --- Reverse transcriptase-polymerase chain reaction (RT-PCR) --- p.88 / Chapter 4.2.4.1 --- Reverse transcription --- p.88 / Chapter 4.2.4.2 --- PCR amplification by specific primers of stress genes --- p.89 / Chapter 4.2.5 --- Northern blot analyses --- p.91 / Chapter 4.2.5.1 --- RNA fractionation by formaldehyde gel electrophoresis --- p.91 / Chapter 4.2.5.2 --- Northern blotting --- p.91 / Chapter 4.2.5.3 --- Preparation of probes --- p.92 / Chapter 4.2.5.4 --- Hybridization and stringency washes --- p.93 / Chapter 4.2.6 --- Isolation of total protein --- p.94 / Chapter 4.2.7 --- Quantification of protein by Bradford method --- p.95 / Chapter 4.2.8 --- Western blot analyses --- p.95 / Chapter 4.2.8.1 --- Sodium dodecyl sulfate ´ؤ polyacrylamide gel electrophoresis (SDS-PAGE) --- p.95 / Chapter 4.2.8.2 --- Western blotting --- p.96 / Chapter 4.2.8.3 --- Immunodetection --- p.98 / Chapter 4.2.8.4 --- ECL detection --- p.98 / Chapter 4.3 --- Results --- p.99 / Chapter 4.3.1 --- Reverse transcriptase-polymerase chain reaction (RT-PCR) --- p.99 / Chapter 4.3.2 --- Northern blot hybridization --- p.106 / Chapter 4.3.2.1 --- Establishing an internal control --- p.106 / Chapter 4.3.2.2 --- Dig-labelling of stress genes --- p.106 / Chapter 4.3.2.3 --- Northern blot hybridizaton of stress genes --- p.106 / Chapter 4.3.3 --- Western blot hybridization --- p.111 / Chapter 4.4 --- Discussions --- p.113 / Chapter Chapter Five --- General Discussions --- p.118 / References --- p.123

by Ng Tak Pan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 151-168). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Abbreviations --- p.v / Table of Contents --- p.vi / List of Figures --- p.x / List of Tables --- p.xiii / Chapter Chapter One --- Literature Review / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Nutraceutical and Medicinal Properties of L. edodes --- p.4 / Chapter 1.2.1 --- Nutritional value --- p.4 / Chapter 1.2.2 --- Hypocholesterolaemic Effect --- p.5 / Chapter 1.2.3 --- Anti-tumor Effect --- p.5 / Chapter 1.2.4 --- Anti-viral Effect --- p.6 / Chapter 1.2.5 --- Immunopotentiating Effect --- p.6 / Chapter 1.3 --- Life cycle of L. edodes --- p.7 / Chapter 1.4 --- Environmental factors affecting mycelial growth and fruit body --- p.11 / Chapter 1.4.1 --- Nutrient requirement --- p.11 / Chapter 1.4.2 --- Physical and chemical factors --- p.12 / Chapter 1.5 --- Molecular studies on mushroom development --- p.15 / Chapter 1.5.1 --- Mating-type genes --- p.15 / Chapter 1.5.2 --- Hydrophobins --- p.19 / Chapter 1.5.3 --- Fruiting regulatory genes --- p.23 / Chapter 1.5.4 --- Molecular studies on fruit body development of I. edodes --- p.24 / Chapter 1.5.4.1 --- Identification of L. edodes genes --- p.24 / Chapter 1.5.4.2 --- Functional characterization of L. edodes genes --- p.27 / Chapter 1.5.4.3 --- Transformation in L. edodes --- p.28 / Chapter Chapter Two --- Expressed Sequence Tags (ESTs) of L. edodes / Chapter 2.1 --- Introduction --- p.30 / Chapter 2.2 --- Materials and Methods --- p.33 / Chapter 2.2.1 --- Generation of expressed sequence tag --- p.33 / Chapter 2.2.1.1 --- Mushroom cultivation and RNA extraction --- p.33 / Chapter 2.2.1.2 --- Construction of primordium cDNA library --- p.34 / Chapter 2.2.1.3 --- Mass excision of pBK-CMV plasmid --- p.34 / Chapter 2.2.1.4 --- Random screening of mass excised cDNA clone --- p.38 / Chapter 2.2.1.5 --- Isolation of recombinant plasmid --- p.38 / Chapter 2.2.1.6 --- Generation of 3´ة end partially sequence --- p.39 / Chapter 2.2.1.7 --- Sequence analysis --- p.40 / Chapter 2.2.2 --- Reverse dot-blot Hybridization --- p.40 / Chapter 2.2.2.1 --- PCR amplification of cDNA clone --- p.40 / Chapter 2.2.2.2 --- Membrane preparation --- p.40 / Chapter 2.2.2.3 --- cDNA probe preparation --- p.41 / Chapter 2.2.2.4 --- Hybridization --- p.42 / Chapter 2.2.2.5 --- Stringent washing and autoradiography --- p.43 / Chapter 2.3 --- Results --- p.44 / Chapter 2.3.1 --- Construction of primordium cDNA library --- p.44 / Chapter 2.3.2 --- Screening of recombinant clone --- p.44 / Chapter 2.3.3 --- Isolation and reconfirmation of recombinant plasmid --- p.46 / Chapter 2.3.4 --- Generation of EST --- p.47 / Chapter 2.3.5 --- EST identity --- p.47 / Chapter 2.3.6 --- Reverse dot-blot hybridization --- p.56 / Chapter 2.3.7 --- Analysis of hybridization signal --- p.60 / Chapter 2.4 --- Discussion --- p.71 / Chapter Chapter Three --- Sequence Analysis and Transcriptional Profiling of Genes Encoding GTP-binding Proteins / Chapter 3.1 --- Introduction --- p.78 / Chapter 3.2 --- Materials and Methods --- p.82 / Chapter 3.2.1 --- Sequence manipulation --- p.82 / Chapter 3.2.2 --- Northern blot hybridization --- p.82 / Chapter 3.2.2.1 --- RNA fragmentation by formaldehyde gel electrophoresis --- p.82 / Chapter 3.2.2.2 --- RNA fixation by capillary method --- p.83 / Chapter 3.2.2.3 --- Probe preparation --- p.84 / Chapter 3.2.2.4 --- Hybridization --- p.85 / Chapter 3.2.2.5 --- Stringent washing and autoradiography --- p.85 / Chapter 3.2.3 --- Real-Time SYBR Green RT-PCR --- p.85 / Chapter 3.2.3.1 --- Primer design --- p.85 / Chapter 3.2.3.2 --- RT-PCR reaction --- p.86 / Chapter 3.3 --- Results --- p.88 / Chapter 3.3.1 --- Sequence manipulation --- p.88 / Chapter 3.3.2 --- Transcriptional analysis --- p.103 / Chapter 3.4 --- Discussion --- p.108 / Chapter 3.4.1 --- Heterotrimeric G proteins --- p.108 / Chapter 3.4.2 --- Ras-related protein Rab7 --- p.112 / Chapter 3.4.3 --- Developmentally regulated GTP-binding protein --- p.113 / Chapter Chapter Four --- Yeast Complementation and Over-expression tests of Le.Gβ1 and Le.Gγ1 / Chapter 4.1 --- Introduction --- p.115 / Chapter 4.2 --- Materials and Methods --- p.120 / Chapter 4.2.1 --- "Yeast strains, media and yeast vectors" --- p.120 / Chapter 4.2.2 --- Primer design --- p.121 / Chapter 4.2.3 --- RT-PCR Amplification of Le.Gβ1 and Le.Gγ1 --- p.121 / Chapter 4.2.4 --- Purification of PCR products --- p.122 / Chapter 4.2.5 --- Enzymatic digestion and purification --- p.122 / Chapter 4.2.6 --- Ligation and E. coli transformation --- p.122 / Chapter 4.2.7 --- PCR screening of E. coli transformants --- p.124 / Chapter 4.2.8 --- Plasmids extraction --- p.124 / Chapter 4.2.9 --- Yeast transformation --- p.124 / Chapter 4.2.10 --- Mating test --- p.125 / Chapter 4.3 --- Results --- p.129 / Chapter 4.3.1 --- Cloning of Le.Gβ1 and Le.Gγ1 --- p.129 / Chapter 4.3.2 --- Yeast transformation --- p.129 / Chapter 4.3.3 --- Mating test --- p.130 / Chapter 4.4 --- Discussion --- p.141 / Chapter Chapter Five --- General Discussion --- p.144 / References --- p.151

In this study, a novel compound was isolated and purified from the solid culture medium (potato dextrose agar) of shiitake 1358 strain through series of methods, such as ethanol precipitation, macroporous resin column separation, semi-preparative high performance liquid chromatography separation and preparative thin-layer chromatography separation. Analyzing spectra from fourier transform infra-red spectroscopy, gas chromatography-mass spectrometry, 1-dimension and 2-dimension nuclear magnetic resonance, the chemical structure of the novel compound was determined and named as 4-amino-5,6-dihydrobenzo[d]oxonine-2,7(1H,4H)-dione. It could inhibit the proliferation of HL-60 leukemia cells significantly and with an IC50 of 1.56 mug/ml (7.123 mumol/L) in the 72-hour treatment. From the results, it is suggested that this compound could activate the G2 phase checkpoint control of the cell cycle to arrest the cell cycle in G2 phase. In addition, it could suppress the replicative DNA synthesis to inhibit the proliferation of HL-60 leukemia cells. The more important is that this compound can induce the apoptosis of HL-60 leukemia cells significantly through intrinsic and extrinsic apoptotic pathways. The compound could induce intrinsic and extrinsic apoptosis through the regulation of the apoptosis-related proteins, such as Fas ligand, Bax, Bcl-2, Caspase 8, Caspase 9, and Caspase 3. For intrinsic pathway, the compound might upregulate Bax, downregulated Bcl-2, activated the Caspase 9, subsequently activated Capase 3, and ultimately led to cell death. For extrinsic pathway, the compound upregulated the Fas ligand, cleaved and activated Procaspase 8 to active Caspase 8, further cleaved and activated Procaspase 3 to active Caspase 3 to commit the cells to apoptosis. / Leukemia is a malignant cancer that involves the bone marrow and blood circulation systems. Leukemia results in the uncontrolled growth of abnormal (leukemic) white blood cells and may also invade other organs, including the liver, spleen, lymph nodes, testes, and brain. In 2007, about 44,240 new cases of leukemia were diagnosed and 21,790 patients died from all types of leukemias in USA. / Shiitake was first cultivated in China more than 800 years ago. It is the second most commonly cultivated edible mushrooms in the world nowadays. For a long time, shiitake has been valued for its unique taste and flavor and as a medicinal invigorant. According to ancient Chinese medicinal theory, consumption of shiitake was in favor of long life and good health. In China and Japan, shiitake has been used as both a food and a medicinal herb for thousands of years. It is the source of several well-studied preparations with proven pharmacological properties, especially the polysaccharide lentinan. Currently, most researches concentrate on the anticancer activities of the extracts from the fruiting body of shiitake, especially polysaccharides. Report about the anti-cancer effects of other components from the shiitake mushroom is scarce. The objectives of this investigations were: (1) to study the anticancer activities of brownish substances obtained during the solid medium culture of shiitake on specific cancer cell unes, especially HL60 cancer cell line; (2) to isolate and characterize the active compound(s) in the brown mushroom exudates; and (3) to propose the possible mechanism of actions, especially the function of the bcl-2 family genes and proteins. / by Guo, Yuming. / Adviser: Chung Hale Yin. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3314. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 188-199). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.