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Cancer/testis (CT) antigens are encoded by genes that are normally expressed only in the human germ-line, but are also expressed in various tumor types. CT antigens are also being studied for their roles in carcinogenesis as well as for their potentials as targets for anti-cancer therapy. A novel CT gene, NYD-SP8, (Accession No. AY014285.1) has recently been identified. It is located to human chromosome 19q13.31 and encodes a 27 kDa glucosylphosphatidylinositol (GPI) anchored cell surface protein, which shows structural homology to urokinase plasminogen activator receptor (uPAR). This thesis describes the characterization and functional roles of NYD-SP8 involved in cancer development. / In summary, the present findings have demonstrated the roles of NYD-SP8 in multi-step cancer development. Further investigations of NYD-SP8 in cancer development may provide new insights and ground for potential use of CT antigens in anti-cancer therapy. (Abstract: 428 words) / In the first set of experiments, the possible role(s) and underlying mechanism(s) of NYD-SP8 in regulating cell proliferation and apoptosis were investigated. Flow cytometric analysis, cell proliferation assay and Western blot analysis showed that NYD-SP8 promoted cell proliferation and protected cells against TNFalpha-induced apoptosis in Human embryonic kidney cells (HEK293) and human hepatocellular carcinoma cells (hHCC). In vitro studies showed that NYD-SP8 enhanced anchorage-independent growth of hHCC, further suggesting the pro-survival effect of NYD-SP8. These data demonstrated important functions of NYD-SP8 in promoting cell growth and preventing apoptosis during cancer development. / In the last part of thesis, the involvement of NYD-SP8 in epithelial-mesenchymal transitions (EMTs) was demonstrated. Upon TGFbeta stimulation or TGFbeta/TNFalpha co-stimulation, the mRNA and protein expression of NYD-SP8 was decreased in LIM1863 cells. Cell adhesion assay showed that the attachment ability of hHCC-SP8 was lowered in laminin and fibronectin coated plate, suggesting the possible role of NYD-SP8 in affecting cell-matrix interaction. These data indicate that NYD-SP8 is involved in the EMTs process and may serve as potential EMTs markers during cancer development. / In the second sets of experiments, the possible role(s) and underlying mechanism(s) of NYD-SP8 in regulating cancer invasion and metastasis were investigated. The results showed that NYD-SP8 could suppress multiple "tumor associated" proteases. Overexpression of NYD-SP8 resulted in reducing activities of the three major classes of proteases known to be involved in ECM degradation, including uPA, matrix metalloproteinases (MMPs) and cathepsin B, leading to suppression of both in vitro and in vivo cancer cell invasion and metastasis. Co-immunoprecipitation experiments showed binding of NYD-SP8 to uPA/uPAR complexes and interfering with active uPA production. These data demonstrated an important function of NYD-5P8 in regulating ECM degradation, providing a novel mechanism that modulates uPA/uPAR signaling in the suppression of cancer progression. / Chung, Chin Man. / "December 2009." / Adviser: H.C. Chan. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 124-150). / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

隨著社會發展, 各種病毒、環境致癌物, 以及不健康食等多種因素導致諸種頑症高發, 並以人類獲得牲免疫缺陷綜合症(艾滋病)和各種腫瘤為代表。從天然產物, 特別是傳統中藥中, 篩選藥用有效成分是治療這類疾病的有效途徑之一。研究發現, 核糖體失活蛋白, 核糖核酸酶, 凝集素, 蛋白酪抑制劑等具有良好的藥用開發前景。本論文著重於從不同植物種于中篩選藥用蛋白, 並對其藥用機制進行研究。 / 是吹研究共純化出六種藥用蛋白。第一, 從苦瓜種子中純化出一種二型核糖體失活蛋白MCL。體外細胞試驗和體內裸鼠試驗顯示MCL 能夠有效抑制鼻咽癌細胞CNE-l 和CNE-2 生長。第二, 苦瓜種于中一個新的核糖核酸酪RNase MC2被分離出來。RNase MC2 通過調控半胱氨酸依賴性細胞死亡蛋白晦(Caspase)信號途徑和絲裂原活化蛋白激酶(MAPKs) 信號途徑誘導乳腺癌MCF-7 細胞凋亡。第三, 從宮粉羊蹄甲種子中提取一種具有抑制腫瘤細胞生長的蛋白酪抑制劑BvvTI 。第四, 從紅花羊蹄甲種子中提取出一與BvvTI 類似的具有抗腫瘤生長的蛋白酶抑制劑BPLTI。 第五，特長秋紫莢豆中存在一個血液凝集素EAPl。EAPL具有制止HIV-l 逆轉錄酶活性, 抗腫瘤, 誘導一氧化氮生成功效。第六, 從另外一種四季豆, 藍虎王, 中提取出一個血液凝集素BTKL。BTKL 通過誘導肝癌HepG2細胞出現DNA 斷裂, 細胞核破壞, 升高線粒體膜通透性, 誘導一氧化氮和細胞因予的表達, 從而導致其凋亡。 / 總之, 上述實驗結果表明, 這六種蛋白具有一定的藥用前景, 可以作為治療艾滋病和不同腫瘤的候造藥物或者候選輔助藥物。進一步體內實驗和臨床實驗評價其療效值得開展。 / Viral pathogens, environmental carcinogens, and unhealthy diets cause severe damage to humans, leading to the acquirement of different stubborn diseases exemplified by AIDS/HIV and neoplasms. Screening of new drugs from natural products, especially from traditional Chinese medicine, provides a promising strategy for these patients. Proteins with potential medicinal applications include ribosome-inactivating proteins (RIPs), ribonucleases, lectins, protease inhibitors and others. The intent of this research proposal is to isolate and characterize proteins with therapeutic potential from plant seeds. / In this project, six medicinal proteins of different origins have been purified by liquid chromatography. One of them is Momordica charantia lectin (MCL), which is a type 11 RIP from the seeds of bitter gourd (M. charantia, BG), with antitumor activity toward human nasopharyngeal carcinoma cells in vitro and in vivo. We have purified a new ribonuclease, named RNase MC2, from BG seeds. It selectively induces apoptosis in breast cancer cells associated with caspase pathways induction and MAPKs activation. Two Kunitz-type trypsin inhibitors, termed BvvTI and BPLTI, have been purified and characterized from the seeds of Bauhinia variegata var. variegata, and B. purpurea L., respectively. EAPL, a lectin with anti-HIV-l reverse transcriptase, antitumor, and nitric oxide (NO) inducing activities was purified from seeds of Phaseolus vulgaris cv. Extralong autumn purple bean. Finally, BTKL is a new P. vulgaris lectin that induced selective toxicity on human liver carcinoma Hep G2 cells. / In conclusion, the above results evince that these proteins are good candidates for the exploration of anti-HIV and/or antitumor drugs or adjuvants. Further research on their efficacies in in vivo as well as clinical trials is warranted. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Fang, Fei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 162-187). / 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.ii / 中文摘要 (CHINESE ABSTRACT) --- p.iii / ACKNOWLEDGEMENTS --- p.iv / PUBLICATIONS --- p.v / LIST OF ABBREViATIONS --- p.ix / LIST OF FIGURES --- p.x / LIST OF TABLES --- p.xii / Chapter CHAPTER1 --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Prelude --- p.2 / Chapter 1.2 --- Literature review of bitter gourd --- p.4 / Chapter 1.2.1 --- Anti-diabetic property of BG --- p.5 / Chapter 1.2.2 --- Anti-HIV activity of BG --- p.15 / Chapter 1.2.3 --- Antitumor activity of BG --- p.18 / Chapter 1.2.4 --- Looking forward --- p.25 / Chapter 1.2.5 --- Conclusions --- p.29 / Chapter 1.3 --- Research rationale, design, and brief results --- p.35 / Chapter CHAPTER2 --- PURIFICATION AND CHARACTERIZATION OF RIBOSOME INACTIVATING PROTEIN --- p.39 / Chapter 2.1 --- Momordica charantia lectin, a type 11 ribosome inactivating protein, exhibits antitumor activity toward human nasopharyngeal carcinoma cells in vitro and in vivo --- p.40 / Chapter 2.1.1 --- Introduction --- p.40 / Chapter 2.1.2 --- Materials and methods --- p.42 / Chapter 2.1.3 --- Results --- p.46 / Chapter 2.1.4 --- Discussion --- p.59 / Chapter CHAPTER 3 --- PURIFICATION AND CHARACTERIZATION OF RIBONUCLEASE --- p.64 / Chapter 3.1 --- RNase MC2: A new Momordica charantia ribonuclease that selectively induces apoptosis in breast cancer cells associated with MAPKs activation and caspase pathways induction --- p.66 / Chapter 3.1.1 --- Introduction --- p.66 / Chapter 3.1.2 --- Materials and methods --- p.67 / Chapter 3.1.3 --- Results --- p.69 / Chapter 3.1.4 --- Discussion --- p.79 / Chapter CHAPTER 4 --- PURIFICATION AND CHARACTERIZATION OF PROTEASE INHIBITORS --- p.84 / Chapter 4.1 --- Bauhinia variegata var variegata trypsin inhibitor: from isolation to potential medicinal applications --- p.86 / Chapter 4.1.1 --- Introduction --- p.86 / Chapter 4.1.2 --- Materials and methods --- p.86 / Chapter 4.1.3 --- Results --- p.89 / Chapter 4.1.4 --- Discussion --- p.97 / Chapter 4.2 --- A potential human hepatocellular carcinoma inhibitor from Bauhinia purpurea L.seeds: From purification to mechanism exploration --- p.99 / Chapter 4.2.1 --- Introduction --- p.99 / Chapter 4.2.2 --- Materials and methods --- p.100 / Chapter 4.2.3 --- Results --- p.101 / Chapter 4.2.4 --- Discussion --- p.112 / Chapter CHAPTER 5 --- PURIFICATION AND CHARACTERIZATION OF MEDICINAL LECTINS --- p.114 / Chapter 5.1 --- A Lectin with Anti-HIV-l Reverse Transcriptase, Antitumor and Nitric Oxide Inducing Activities from Seeds of Phaseolus vulgaris cv Extra-long Autumn Purple Bean --- p.116 / Chapter 5.1.1 --- Introduction --- p.118 / Chapter 5.1.2 --- Materials and methods --- p.119 / Chapter 5.1.3 --- Results --- p.123 / Chapter 5.1.4 --- Discussion --- p.132 / Chapter 5.2 --- A new Phaseolus vulgaris lectin induces selective toxicity on human liver carcinoma Hep G2 cells --- p.136 / Chapter 5.2.1 --- Introduction --- p.136 / Chapter 5.2.2 --- Materials and methods --- p.137 / Chapter 5.2.3 --- Results --- p.138 / Chapter 5.2.4 --- Discussion --- p.150 / Chapter CHAPTER6 --- CONCLUSION AND FUTURE PERSPECTiVES --- p.154 / Chapter 6.1 --- Conclusions --- p.156 / Chapter 6.2 --- Future perspectives --- p.159 / References --- p.162

Zhang, Xinran. / Thesis submitted in: December 2007. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 80-87). / Abstracts in English and Chinese. / TITLE PAGE --- p.i / ABSTRACT --- p.ii / 中文摘要 --- p.v / ACKNOWLEDGEMENTS --- p.vii / TABLE OF CONTENTS --- p.viii / LIST OF FIGURES --- p.xi / LIST OF TABLES --- p.xiv / ABBREVIATIONS --- p.xv / Chapter CHAPTER 1. --- Introduction / Chapter 1.1. --- Rationale of the Study --- p.1 / Chapter 1.2. --- Current technologies for formulating long-acting parenteral protein deliver system --- p.3 / Chapter 1.2.1. --- Chemical Modification --- p.3 / Chapter 1.2.2. --- Sustained-release formulation --- p.4 / Chapter 1.2.2.1. --- Phase separation method --- p.4 / Chapter 1.2.2.2. --- Solvent evaporation/extraction method --- p.5 / Chapter 1.2.2.3. --- Spray drying method --- p.6 / Chapter 1.2.2.4. --- Causes for protein instability --- p.6 / Chapter 1.2.2.4.1. --- Water/organic solvent interface --- p.6 / Chapter 1.2.2.4.2. --- Lyophilization --- p.8 / Chapter 1.2.2.4.3. --- Polymer --- p.11 / Chapter 1.2.2.4.4. --- Stabilizing additive --- p.13 / Chapter 1.3. --- Aqueous-aqueous emulsion technology --- p.17 / Chapter 1.3.1. --- Background --- p.17 / Chapter 1.3.2. --- Basic Principle --- p.17 / Chapter 1.3.3. --- Phase diagram --- p.18 / Chapter 1.3.4. --- Formation of aqueous-aqueous emulsion --- p.19 / Chapter 1.3.4.1. --- Introduction of a water-soluble charged polymer as stabilizer --- p.19 / Chapter 1.3.4.2. --- Freezing-induced phase separation --- p.20 / Chapter 1.3.5. --- General Protocol --- p.21 / Chapter 1.3.5.1. --- Introduction of a water-soluble charged polymeric stabilizer --- p.22 / Chapter 1.3.5.2. --- Freezing-induced phase separation --- p.22 / Chapter 1.3.6. --- Merits and limitations of the aqueous-aqueous emulsion technology --- p.23 / Chapter 1.3.7. --- Protein selection for the sustained release formulation --- p.25 / Chapter 1.4. --- Aims and scope of study --- p.26 / Chapter "CHAPTER 2," --- Materials and Methods / Chapter 2.1. --- Materials --- p.28 / Chapter 2.1.1. --- Proteins --- p.28 / Chapter 2.1.2. --- Polymers --- p.28 / Chapter 2.1.3. --- Media for TF-1 Cell Culture --- p.28 / Chapter 2.1.4. --- Chemicals and Solvents for Cell Proliferation Assay --- p.29 / Chapter 2.1.5. --- Other Chemicals and Solvents --- p.29 / Chapter 2.1.6. --- Materials for Cell Culture --- p.29 / Chapter 2.1.7. --- Materials for Reagent Kits --- p.30 / Chapter 2.2. --- Methods --- p.30 / Chapter 2.2.1. --- Determination of the Partition Coefficients of Proteins Between PEG and Dextran --- p.30 / Chapter 2.2.2. --- Preparation of Glassy Particles --- p.31 / Chapter 2.2.2.1. --- Standard Stable Aqueous-aqueous Emulsion Method --- p.31 / Chapter 2.2.2.2. --- Freezing-induced Phase Separation --- p.32 / Chapter 2.2.3. --- Preparation of Protein-loaded and Blank Microspheres Using S-o-w Solvent Extraction Technique --- p.32 / Chapter 2.2.4. --- Optical Microscopy and Scanning Electron Microscopy --- p.33 / Chapter 2.2.5. --- Determination of Protein Loading --- p.34 / Chapter 2.2.5.1. --- Within Dextran Particles --- p.34 / Chapter 2.2.5.2. --- Within PLGA microspheres --- p.34 / Chapter 2.2.6. --- Evaluation of Protein Structural Integrity and Bioactivity in Dextran Particles and PGLA Microspheres --- p.35 / Chapter 2.2.7. --- In vitro Release Study --- p.36 / Chapter 2.2.8. --- RhIFN Stability Determination under Simulated In Vitro Release Conditions --- p.37 / Chapter 2.2.8.1. --- In the Absence of PLGA --- p.37 / Chapter 2.2.8.2. --- In the Presence of PLGA --- p.37 / Chapter 2.2.9. --- MicroBCÁёØ Protein Assay --- p.38 / Chapter 2.2.10. --- Size Exclusion Chromatography (SEC) - High Performance Liquid Chromatography (HPLC) --- p.38 / Chapter 2.2.11. --- ELISA --- p.39 / Chapter 2.2.12. --- Bioactivity Assay --- p.40 / Chapter 2.2.12.1. --- RhIFN --- p.40 / Chapter 2.2.12.2. --- RhGM-CSF --- p.41 / Chapter CHAPTER 3. --- Results and Discussions / Chapter 3.1. --- Sustained-release RhIFN Formulation --- p.45 / Chapter 3.1.1. --- Partition Coefficient of RhIFN --- p.45 / Chapter 3.1.2. --- Formulation Based on the Standard Aqueous-aqueous Emulsion (SA-AE) Method With Sodium Alginate as Stabilizer --- p.45 / Chapter 3.1.2.1. --- Surface Morphology --- p.45 / Chapter 3.1.2.2. --- Formulation Characterization --- p.46 / Chapter 3.1.2.3. --- In Vitro Release of RhIFN from PLGA Microsheres --- p.54 / Chapter 3.1.3. --- Formulation Based on the Freezing-induced Phase Separation (FIPS) Technique without Sodium Alginate --- p.56 / Chapter 3.1.3.1. --- Formulation Characterization --- p.56 / Chapter 3.1.3.2. --- In Vitro Release of RhIFN from PGLA Microsphees --- p.59 / Chapter 3.2. --- RhIFN Stability Assessment under Simulated In Vitro Release Conditions --- p.63 / Chapter 3.2.1. --- In the Absence of PLGA --- p.63 / Chapter 3.2.2. --- In the Presence of PLGA --- p.65 / Chapter 3.3. --- Sustained-release RhGM-CSF Formulation --- p.68 / Chapter 3.3.1. --- Partition Coefficient Determination of RhGM-CSF Between PEG and Dextran --- p.68 / Chapter 3.3.2. --- Formulation Based on Freezing-induced Phase Separation --- p.68 / Chapter 3.3.2.1. --- Validation of MTT Assay Conditions --- p.69 / Chapter 3.3.2.2. --- Formulation Characterization --- p.71 / Chapter 3.3.2.3. --- In Vitro Release of RhGM-CSF from PLGA Microspheres --- p.75 / Chapter CHAPTER 4. --- Conclusion and Future Studies / Chapter 4.1. --- Conclusion --- p.78 / Chapter 4.2. --- Future Studies --- p.79 / References --- p.80

3C-like protease (3CLpro) of severe acute respiratory syndrome-coronavirus (SARS-CoV) is required for autoprocessing of the polyproteins 1a and 1ab, and is a potential target for treating coronaviral infection. To obtain a thorough understanding of its substrate preference, we created a substrate library of 19 x 8 variants by performing saturation mutagenesis on the autocleavage sequence at P5 to P3' positions. The substrate sequences were inserted between cyan and yellow fluorescent proteins so that the cleavage rates were monitored by in vitro fluorescence resonance energy transfer (FRET). The relative cleavage rate for different substrate sequences was correlated with various structural properties. P5 and P3 positions prefer residues with high beta-sheet propensity P4 prefers small hydrophobic residues: P2 prefers hydrophobic residues without beta-branch. Gln is the best residue at P1 position, but observable cleavage can be detected with His and Met substitutions. P1' position prefers small residues, while P2' and P3' positions have no strong preference on residue substitutions. Noteworthy, solvent exposed sites such as P5, P3 and P3' positions favour positively charged residues over negatively charged one, suggesting that electrostatic interactions may play a role in catalysis. A super-active substrate, which combined the preferred residues at P5 to P1 positions, was found to have 2.8 fold higher activity than the wild-type sequence. / Inhibition of SARS-CoV 3CLpro proteolytic activity suppresses virion replication and virus-induced cytopathic effects. Peptidomimetic inhibitors with nitrile warheads, which inhibit Cys protease activity, have been applied for clinical therapy. To investigate whether the nitrile group can target 3CLpro, a series of nitrile-based peptidomimetic inhibitors with various protective groups, peptide length and peptide sequences were synthesized. Inhibitor potency in terms of IC50 and Ki values was determined by FRET assay. Most of these nitrile-based inhibitors in micromolar range can significantly reduce 3CLpro activity. The most potent inhibitor is the tetrapeptidomimetie inhibitor linked with carbobenzyloxy (cbz) group 'cbz-AVLQ-CN' with IC50 and Ki values of 5.9 +/- 0.6 muM and 0.62 +/- 0.11 muM respectively. Crystal structures of 3CLpro-inhibitor complexes demonstrated that nitrite warhead covalently bonded to Cys145, while P1 -- P4 residues interacted with 3CLpro as substrate bound. The cbz group in 'cbz-AVLQ-CN' flipped into a cavity of Gu166 -- Pro168, providing an extra binding force to enhance inhibitor potency. In conclusion, the nitrile-based peptidomimetic inhibitor with cbz group is a convincing model for drug development. / Substrate specificities of various 3CLpro were further investigated by using the substrate library of SARS-CoV 3CLpro. Among various viral strains, the proteases of HCoV-NL63, HCoV-OC43 and infectious bronchitis virus (IBV) were selected from group I, IIa and III respectively for specificity profiling. Their proteolytic rates against 19 x 8 variants were obtained by FRET assay, and correlated with structural properties of substituting residues. Like SARS-CoV 3CLpro in group IIb, these 3CLpro consistently prefer small hydrophobic P4 residues, positively charged P3 residues, hydrophobic P2 residues without beta-branch, P1-Gln and small P1' residues. These proteases also tend to accommodate P5 and P3' residues with positive charge, and P2' residues with small size. In contrast, their preferences on secondary structure are diverse. Correlation was found between IBV 3Clpro activity and beta-sheet propensity at P5 position, while no strong correlation with secondary structure propensities was observed in HCoV-NL63 and HCoV-0C43. Collectively, all 3CLpro share universal preferences on charge, side chain volume and hydrophobicity, but not secondary structure. Their relative activities against universal and specific super-active substrates were elevated to 1.4 -- 4.3, showing synergetic effects by combining preferred residues. These substrates were examined by group I HCoV-229E and group IIa HCoV-HKU1 in parallel. Their activities were highly comparable to those of other group members. / Chuck, Chi Pang. / Adviser: Chi-Cheong Wan. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves [179]-187). / 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.

by Liu Fang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 159-178). / ACKNOWLEGEMENTS --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.v / LIST OF TABLES --- p.ix / LIST OF FIGURES --- p.xi / ABBREVIATIONS --- p.xiv / Chapter Chapter 1. --- General Introduction --- p.1 / Chapter Chapter 2. --- Literature Review --- p.7 / Chapter 2.1. --- Biologically active polysaccharides --- p.7 / Chapter 2.2. --- Antitumor activities of polysaccharides --- p.11 / Chapter 2.2.1. --- In vivo studies --- p.11 / Chapter 2.2.2. --- In vitro studies --- p.15 / Chapter 2.3. --- Antitumor mechanisms of polysaccharides --- p.17 / Chapter 2.4. --- Structure and antitumor activities of polysaccharides --- p.25 / Chapter 2.4.1. --- The effect of molecular mass --- p.26 / Chapter 2.4.2. --- The impact of branching configuration --- p.21 / Chapter 2.4.3. --- The relationship between antitumor activity and conformation --- p.28 / Chapter 2.4.4. --- Improvement of antitumor activity by chemical modification --- p.29 / Chapter 2.5. --- Other biological activities --- p.30 / Chapter 2.5.1. --- Antiviral activity --- p.30 / Chapter 2.5.2. --- Antimicrobial activity --- p.31 / Chapter 2.5.3. --- Free radical scavenging activity --- p.32 / Chapter 2.5.4. --- Hepatic protective effect --- p.32 / Chapter Chapter 3. --- Isolation and Characterization of a Polysaccharide-Protein Complex (PSPC) from Tricholoma lobayense --- p.34 / Chapter 3.1. --- Introduction --- p.34 / Chapter 3.2. --- Materials and methods --- p.36 / Chapter 3.2.1. --- Strain --- p.36 / Chapter 3.2.2. --- Culture conditions --- p.36 / Chapter 3.2.3. --- Extraction of T. lobayense --- p.39 / Chapter 3.2.4. --- Purification of polysaccharide-protein complex --- p.40 / Chapter 3.2.5. --- Molecular mass determination --- p.43 / Chapter 3.2.6. --- High performance liquid chromatography --- p.43 / Chapter 3.2.7. --- SDS-polyacrylamide gel electrophoresis --- p.44 / Chapter 3.2.8. --- Ultraviolet scanning --- p.44 / Chapter 3.2.9. --- Chemical analysis --- p.45 / Chapter 3.2.10. --- Experimental animals --- p.47 / Chapter 3.2.11. --- In vivo antitumor assay --- p.48 / Chapter 3.2.12. --- Safety tests --- p.49 / Chapter 3.2.13. --- Statistical analysis --- p.51 / Chapter 3.3. --- Results --- p.51 / Chapter 3.3.1. --- Extraction and purification --- p.51 / Chapter 3.3.2. --- Biochemical analysis --- p.52 / Chapter 3.3.3. --- Chemical analysis --- p.60 / Chapter 3.3.4. --- In vivo antitumor activity --- p.68 / Chapter 3.3.5. --- Safety evaluation --- p.68 / Chapter 3.4. --- Discussion --- p.75 / Chapter 3.5. --- Summary --- p.84 / Chapter Chapter 4. --- "Immunomodulating, Antitumor and other Biological Activities of Polysaccharide-Protein Complex (PSPC) from Tricholoma lobayense" --- p.85 / Chapter 4.1. --- Introduction --- p.85 / Chapter 4.2. --- Materials and methods --- p.87 / Chapter 4.2.1. --- Experimental animals --- p.87 / Chapter 4.2.2. --- Cultivation of tumor cells --- p.87 / Chapter 4.2.3. --- Preparation of peritoneal exudate cells and splenocytes --- p.87 / Chapter 4.2.4. --- Mitogenic response of T cells --- p.89 / Chapter 4.2.5. --- Responses of peritoneal exudate cells --- p.89 / Chapter 4.2.6. --- In vitro antitumor assay --- p.92 / Chapter 4.2.7. --- Transmission electron microscope --- p.93 / Chapter 4.2.8. --- Evaluation of other biological activities --- p.94 / Chapter 4.2.9. --- Statistical analysis --- p.99 / Chapter 4.3. --- Results --- p.99 / Chapter 4.3.1. --- Immunomodulating activity --- p.99 / Chapter 4.3.2. --- In vitro antitumor action --- p.107 / Chapter 4.3.3. --- Observation on tumor regression induced by PSPC --- p.107 / Chapter 4.3.4. --- Other biological actions --- p.112 / Chapter 4.4. --- Discussion --- p.121 / Chapter 4.4.1. --- Immunomodulating activity --- p.121 / Chapter 4.4.2. --- Antitumor activity --- p.125 / Chapter 4.4.3. --- Other biological activities --- p.127 / Chapter 4.5. --- Summary --- p.130 / Chapter Chapter 5. --- Induction of Gene Expression of Immunomodulating Cytokines by Polysaccharide-Protein Complex (PSPC) from Tricholoma lobayense --- p.132 / Chapter 5.1. --- Introduction --- p.132 / Chapter 5.2. --- Materials and methods --- p.135 / Chapter 5.2.1. --- Experimental animals --- p.135 / Chapter 5.2.2. --- Preparation of peritoneal exudate cells and splenocytes --- p.136 / Chapter 5.2.3. --- RNA extraction --- p.137 / Chapter 5.2.4. --- Reverse transcription- polymerase chain reaction --- p.137 / Chapter 5.2.5. --- Dot blot --- p.138 / Chapter 5.2.6. --- Hybridization --- p.141 / Chapter 5.3. --- Results --- p.142 / Chapter 5.3.1. --- mRNA phenotyping of cytokines and cytokine receptors in normal mice --- p.142 / Chapter 5.3.2. --- mRNA phenotyping of cytokines and cytokine receptors in tumor-bearing mice --- p.142 / Chapter 5.4. --- Discussion --- p.150 / Chapter 5.5. --- Summary --- p.153 / Chapter Chapter 6. --- General Discussion and Conclusion --- p.155 / REFERENCES --- p.159

Polyelectrolytes of opposite charge in aqueous solution can undergo a liquid-liquid phase separation known as complex coacervation. Complex coacervation of ampholytic proteins with oppositely charged polyelectrolytes is of increasing interest as it results in a protein rich phase that has potential applications in food science, protein therapeutics, protein purification, and biocatalysis. However, many globular proteins do not phase separate when mixed with an oppositely charged polyelectrolyte, and those that do phase separate do so over narrow concentration, pH, and ionic strength ranges. Much of the work that has been done on complex coacervates looks at polymer-polymer systems. While there have been some initial studies showing that proteins can undergo complex coacervation, the major design factor studied to date has been overall protein charge. The tools of genetic engineering, which allow the precise tuning and placement of charge have not been used to more fully understand the design criteria for protein complex coacervation. In this dissertation, we developed a model protein library based on green fluorescent protein (GFP) to study the impact of protein net charge and charge distribution on protein phase separation with polyelectrolytes. We developed a short, ionic polypeptide sequence (6-18 amino acids) that can drive the liquid-liquid phase separation of globular proteins. We characterize the phase behavior of the protein library with a homopolymer and diblock copolymer of similar chemistry to elucidate how protein design impacts macro- and microphase separation. In these phase characterization studies, differences in the nature of phase separation as well as the salt stability of the protein coacervates with the different polymer species are identified. We finally used this model protein library to study the effects of the protein design and phase separation behavior for coacervate-based applications including intracellular protein delivery, purification, and protein stabilization.

Microbiome research in the past decade has revealed an astounding prevalence of bacteria in various tissues in the human body. Concurrent progress in synthetic biology has generated a converging interest in the genetic programming of bacteria to locally produce therapeutic payloads and supplant physiological niches. This dissertation presents the development of bioengineering tools that address several key challenges for the clinical translation of therapeutic bacteria. In particular, we focus on the engineering of bacteria for tumor and granuloma applications. Bacteria have been demonstrated to selectively grow within solid tumors, primarily due to the reduced immune surveillance in the necrotic and hypoxic cores. This natural tropism to tumors presents a unique opportunity to engineer bacteria as drug delivery vehicles for cancer therapy. While the recent advancement in microbial engineering has constructed ranges of therapeutic bacteria, a universal bottleneck for clinical development is the lack of tools to rapidly characterize therapeutic candidates in a complex physiological environment. To recapitulate bacterial tumor colonization in vitro, we developed a method that selectively grows bacteria within the necrotic core of tumor spheroids. This platform enabled high-throughput cocultures and predicted in vivo therapeutic outcomes, identifying potent anticancer proteins deliverable by tumor-homing Salmonella typhimurium. To ensure safety when using bacteria that produce cytotoxic payloads, we prevented bacterial spread to unintended locations by confining bacterial growth in a tumor-specific environment. We constructed hypoxia, pH, and lactate sensors and regulated bacterial growth based on sensor activation. To improve tumor specificity, we engineered gene circuits to sense hypoxia and lactate in an AND-logic gate manner. Leveraging the coculture platform, we characterized sensor activities and circuit functionalities in tumor spheroids. This engineered strain showed improved tumor specificity in an animal tumor model. Moving towards clinical applications, a key challenge is to ensure bacterial delivery to tumors without activating adverse immune responses. Approaches such as surface decoration can evade immune systems, but static modification may result in bacterial overgrowth. We developed a genetically-encoded microbial encapsulation system with a tunable, dynamic expression of capsular polysaccharides. We constructed an inducible gene circuit to regulate encapsulation, which exhibited tunable protection of the probiotic Escherichia coli Nissle 1917 (EcN) from host immune factors. By dynamically balancing low immunogenicity and protection, transient encapsulation increased the maximum tolerated dose of bacteria by approximately 10-fold when systemically injected in vivo. This strategy enhanced antitumor efficacy in multiple tumor models. Building on our work of therapeutic bacteria for cancer, we explored the use of engineered bacteria to infiltrate other pathogenic regions in the body. Specifically, we discovered that probiotic EcN colonizes granulomas, pathological features that develop at infection sites including tuberculosis. Granulomas share key similarities with solid tumors, including hypoxia and necrosis, and pose significant challenges for delivering therapeutic agents to eradicate the pathogen Mycobacterium tuberculosis within. We engineered the probiotics to locally produce antimicrobial proteins against Mycobacterium within granulomas. We developed a novel dual lysis mechanism to simultaneously enhance therapeutic protein release and limit bacterial overgrowth. To improve specificity, we constructed hypoxia-dependent bacterial growth coupled with quorum-mediated gene activation. Finally, we showed that our engineered probiotics reduced levels of Mycobacterium strains. Altogether, the presented technologies utilize a multiscale framework from circuit design to in vitro and in vivo models and advance bacteria as next-generation drug delivery vehicles capable of sensing and responding to diseases in the body.

Background: The impairment of articular cartilage caused by trauma or degenerative pathology is one of the most challenging issues in clinical Orthopedics because of the limited intrinsic regenerative capability of this tissue. Hypoxia is a major stimulus to initiate gene programs in regulating chondrogenic lineage cell functions during cartilage development and regeneration. Hypoxia-inducible factor-1α (HIF-1α), the key transcription factor to sense oxygen fluctuations of cells, is abundantly expressed in the cartilage and considered as a potential therapeutic target for cartilage tissue homeostasis or repair. However, the molecular mechanisms and therapeutic efficacy of targeting the HIF-1α pathway remain to be well defined. / Methods: Osteochondral defect mouse model was generated to examine the hypoxia states during articular cartilage repair with the Hypoxyprobe. Specific HIF-1α deletion in the repairing tissue was established to determine its regulatory role during cartilage restoration. Deferoxamine (DFO), stabilizing HIF-1α from proteolysis by inhibiting the prolyl hydroxylases (PHDs), was investigated systemically on the function of chondroprogenitors and mesenchymal stem cells (MSCs) in vitro. Alcian blue staining determined the proteoglycan synthesis. HIF components, chondrogenic related genes and proteins were examined by quantitative PCR, western blotting and immunohistochemistry, respectively. The proliferation, differentiation and migration assays were performed to determine the influence of DFO onchondroprogenitors and MSCs. The recruitment or engraftment of MSCs in the injured site was traced by transplantation of GFP-labeled MSCs adjacent to the defect region, and examined by immunofluorescence staining. DFO incorporated in a 3D alginate-gelfoam scaffold was analyzed for its therapeutic effects on the articular cartilage regeneration. At 6 and 12 weeks following surgery, the cartilage tissue repair was scored and the expression of proliferating cell nuclear antigen (PCNA), Sox9 and collagen typeⅡ(Col2) was examined by immunohistochemistry. / Results: Hypoxia states and the expression of HIF-1α in the repair tissue were ubiquitously existed in the osteochondral defect model. DFO significantly upregulated HIF-1α expression and nuclear localization, and increased the levels of PHDs. DFO increased chondroprogenitor cell proliferation as visualized by colony forming unit assay, which was in accord with the upregulation of cyclin D1. DFO significantly induced chondrogenic differentiation indexed by increased Col2 and Sox9 protein expression and elevated proteoglycan synthesis. With sustained upregulation of HIF-1α DFO was supposed to effectively promote chondrogenesis in mimic of hypoxic microenvironment. DFO also increased the migration of MSCs, and elevated the expression of tissue inhibitor metalloproteinase-3 (TIMP3) through transcriptional control by HIF-1α. Furthermore, DFO initiated MSCs membrane protrusion through regulating the expression and interaction of the key focal adhesion proteins vinculin and paxillin. In vivo study showed that DFO dramatically facilitated the recruitment and functional engraftment of MSCs to the lesion site compared with the controls. Alginate-gelfoam scaffold incorporated with DFO enhanced articular cartilage repair through increasing chondrogenic cell proliferation, differentiation and proteoglycan synthesis. The enhanced therapeutic effect of DFO on articular cartilage repair was eliminated following HIF-1α deletion in the repairing cells of the cartilage lesion. The results indicate that the positive effect of DFO on articular cartilage repair is at least partially mediated by HIF-1α. / Conclusion: HIF-1α is an essential mediator during articular cartilage repair. Activation of HIF-1α by PHD inhibitor DFO increases chondroprogenitor cell proliferation, differentiation and migration in vitro. DFO enhances articular cartilage repair through coordinating MSCs migration, chondrogenic differentiation and functional engraftment. The results provide proof of principle that targeting the HIF-1α pathway may serve as a novel approach for promoting articular cartilage regeneration. / 背景：关节软骨自愈能力非常有限，由创伤或退行性病变引起关节软骨损伤的治疗是骨科领域的一大难题。在软骨发育和再生过程中，低氧条件对启动基因表达及调控软骨系细胞功能至关重要。低氧诱导因子-1α（HIF-1α）作为关键的转录因子可感应细胞外氧含量变化，广泛存在于软骨组织中，并被认为对维持软骨组织内稳态及促进软骨修复有重要作用。然而，以HIF-1α 通路为靶点的小分子靶向药物的分子机制与治疗效果尚不明确。 / 方法：本课题系统性地研究了HIF 信号通路激活剂去铁胺（DFO）对软骨损伤的作用。我们构建了骨软骨缺损模型，应用缺氧探针检测了软骨缺损过程中修复组织的低氧状态，并特异性敲除软骨修复组织中HIF-1α 表达，研究其在软骨再生过程中的调节作用。我们用阿利新蓝染色检测软骨细胞蛋白多糖的合成及分泌。通过实时荧光定量聚合酶链式反应，免疫印迹以及免疫组化等方法检测了HIF 家族成员和软骨分化标志物的基因和蛋白含量变化。通过增殖及迁移实验检测了DFO 对软骨细胞或者骨髓间充质干细胞（MSC）功能的影响。另外，我们还将GFP 标记的MSC 注射到与小鼠软骨缺损区域相邻的软骨下骨中，观察其在软骨缺损模型中的募集及功能性植入。我们以藻酸盐和明胶海绵复合物为给药系统，包载DFO 并作用于关节软骨缺损部位。术后6 周及12 周取材，以番红O 染色检测DFO 对小鼠关节软骨缺损的修复效果，并通过免疫组化检测增殖细胞核抗原（PCNA），Sox9 以及Col2 等蛋白的表达。 / 结果：低氧状态和HIF-1α 在骨软骨缺损模型中的软骨缺损区域广泛存在和表达。DFO 显著提高了HIF-1α 蛋白表达及转运入核，增加了脯氨酸羟化酶（PHD）表达。在软骨祖细胞中，DFO 可提高其增殖、克隆能力，并增加周期蛋白D1的表达。同时，DFO 能明显促进软骨祖细胞分化，增加软骨分化标志物基因以及Sox9 和Col2 蛋白表达，提高蛋白多糖分泌。通过持续性激活HIF-1α，DFO可模仿低氧微环境来提高软骨细胞增殖、分化能力。分子机制研究发现，DFO激活HIF-1α 后，HIF-1α 作用在靶基因金属蛋白酶组织抑制剂-3 启动子上，增加其转录和蛋白表达，进而提高MSC 的迁移能力。另外，激活HIF-1α 蛋白可增加黏着斑蛋白，桩蛋白表达以及它们的相互作用，促进MSC 伪足延伸。体内实验中，通过追踪小鼠体内GFP 标记的MSC 发现， DFO 可在软骨损伤早期（7 天及14 天）提高受损部位MSC 募集数量，并促进其向软骨细胞谱系分化。通过增加软骨系细胞增殖、分化、蛋白多糖合成，包载DFO 的藻酸盐明胶海绵给药系统显著提高了软骨缺损组织的修复效果。而在软骨修复组织中特异性敲除HIF-1α 蛋白后，明显降低了DFO 对软骨缺损的治疗效果，提示DFO对软骨修复的作用至少部分由HIF-1α 介导。 / 结论：HIF-1α 是关节软骨修复过程中的重要调控因子。PHD 抑制剂DFO 可以激活HIF-1α 表达，增加软骨祖细胞增殖、分化和迁移。DFO 通过调控MSC 募集、软骨细胞谱系分化以及功能性植入，明显改善关节软骨再生修复的效果。本研究为HIF-1α 信号通路作为一种新的治疗靶点促进关节软骨再生提供了重要证据。 / Shu, Yinglan. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 155-181). / Abstracts also in Chinese. / Title from PDF title page (viewed on 09, September, 2016). / Shu, Yinglan. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Prostate cancer remains one of the most commonly diagnosed cancers in men and is a leading cause of cancer death. While great success has been achieved at curing early stage prostate cancer, limited success has been obtained when treating late-stage hormone independent prostate cancer. This is due to the increased propensity for skeletal and non-skeletal metastases. Thus development of novel effective therapeutic modalities against late stage prostate cancer is of critical importance. / Towards these objectives, I have focused my attention on the role of prostate secretory protein (PSP-94) which is expressed in normal individuals and in patients with early stage prostate cancer. Using our well established in vivo models of prostate cancer, I have evaluated the ability of PSP-94 and its amino acids 31-45 required (PCK3145) to decrease tumor growth and skeletal metastases in vivo and evaluated the potential mechanism(s) associated with PCK3145 anti-cancer actions. / Prostatic cancer can also develop as a result of epigenetic activation of tumor promoting genes. To evaluate the role of methylation in prostate cancer, late stage prostate cancer cells were treated with the universal methylating agent S-adenosylmethionine (SAM) and an anti-sense oligonucleotide directed against MBD2 (AS). Scrambled oligonucleotide was included as a control (S). Both SAM and MBD2-AS resulted in inhibition in uPA, MMP-2 and VEGF production leading to decreased tumor cell invasive capacity. However, SAM and MBD2-AS were not able to either further repress partially methylated genes (GSTP1) or reactivate already methylated genes (AR). Furthermore, SAM and MBD2-AS treatment resulted in significant reduction in tumor growth in vivo . Immunohistochemical and RT-PCR analyses carried out on SAM and MBD2-AS tumors revealed decreased protein and mRNA expression of uPA and MMP-2 which was partially due to increased methylation of the respective promoters even after 10 weeks post in vitro treatment as analyzed by bisulfate sequencing. In addition decreased levels of angiogenesis and tumor survival markers were observed. / Collectively, these studies are aimed at the development of novel reliable approached to diagnose and treat advanced, hormone refractory prostate cancer to reduce tumor associated morbidity and mortality.