Tse, Ho Man. / Thesis (M.Phil)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 166-173). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 撮要 --- p.v / Table of Content --- p.vi / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Basis of Prostate Cancer --- p.1 / Chapter 1.1.1 --- Epidemiology and Risk Factors of Prostate Cancer --- p.1 / Chapter 1.1.2 --- Pathology of Prostate Cancer --- p.2 / Chapter 1.1.3 --- Treatment Approaches for Prostate Cancer --- p.4 / Chapter 1.2 --- Sex Hormones and Prostate Cancer --- p.7 / Chapter 1.2.1 --- Prostate Development --- p.7 / Chapter 1.2.2 --- Involvement of Sex Hormones in Prostate Cancer --- p.8 / Chapter 1.2.3 --- Molecular Mechanisms of Sex Hormones --- p.13 / Chapter 1.2.4 --- Hormone Receptor Antagonists --- p.15 / Chapter 1.3 --- Involvement of microRNAs in Cancer --- p.19 / Chapter 1.3.1 --- Basis of microRNAs --- p.19 / Chapter 1.3.2 --- Aberrant microRNA Expressions in Cancers --- p.23 / Chapter 1.3.3 --- Current Understandings on Regulations of micro RN A Expressions --- p.26 / Chapter 1.3.4 --- Regulation of miRNA Expressions by Hormones --- p.29 / Chapter 1.4 --- "Effects of the Anti-estrogen ICI 182,780 on Prostate Cancer Cells" --- p.30 / Chapter 1.4.1 --- "ICI 182,780 Inhibits Cell Growth ofDU145" --- p.30 / Chapter 1.5 --- Objectives of Project --- p.32 / Chapter Chapter 2: --- Materials --- p.34 / Chapter 2.1 --- Bacteria Strain --- p.34 / Chapter 2.2 --- Tissue Culture Media --- p.34 / Chapter 2.3 --- Plasmids --- p.34 / Chapter 2.4 --- Kits and Accessories --- p.35 / Chapter 2.5 --- Reagents and Solutions --- p.36 / Chapter 2.6 --- DNA Oligos --- p.38 / Chapter 2.7 --- Equipments --- p.40 / Chapter Chapter 3: --- Methods --- p.41 / Chapter 3.1 --- Cell Culture Conditions --- p.41 / Chapter 3.2 --- miRNA Expression Profiling of DU145 --- p.41 / Chapter 3.2.1 --- RNA Isolation --- p.41 / Chapter 3.2.2 --- miRNA Microarray Profiling ofDU145 : --- p.42 / Chapter 3.2.2.1 --- Fluorescent Labeling of RNA and Microarray Hybridization --- p.42 / Chapter 3.2.2.2 --- Scanning and Analysis of Signals --- p.46 / Chapter 3.2.3 --- Confirming miR-765 Up-regulation by ICI with qRT-PCR --- p.46 / Chapter 3.2.3.1 --- Assessing ERp Dependency in miR-765 Induction --- p.48 / Chapter 3.2.4 --- Effects of ICI on ARHGEF11 Expression --- p.49 / Chapter 3.2.4.1 --- Reverse Transcription of mRNA --- p.50 / Chapter 3.2.4.2 --- Quantitative Real-Time PCR for Gene mRNA expression --- p.50 / Chapter 3.3 --- Characterizing the Promoter Region of miR-765 --- p.52 / Chapter 3.3.1 --- Cloning of miR-765 Promoter into pGL3-Basic Vector --- p.52 / Chapter 3.3.1.1 --- PCR Amplification of miR-765 Putative Promoter Region --- p.52 / Chapter 3.3.1.2 --- Ligation of the Amplified Regions in pGL3-Basic Vector --- p.55 / Chapter 3.3.1.3 --- Transformation and Screening of pGL3-765 Plasmid --- p.57 / Chapter 3.3.1.4 --- Preparation of pGL3-765 Plasmid DNA --- p.59 / Chapter 3.3.2 --- Preparation of Truncated miR- 765 Promoter Clones --- p.60 / Chapter 3.3.2.1 --- pGL3-765-Trunc#l --- p.61 / Chapter 3.3.2.2 --- pGL3-765-Trunc#2 --- p.62 / Chapter 3.3.2.3 --- pGL3-765-Trunc#3 --- p.62 / Chapter 3.3.3 --- Assessing the miR- 765 Promoter Activities --- p.63 / Chapter 3.3.3.1 --- Optimizing Transfection Conditions --- p.64 / Chapter 3.3.3.2 --- Co-transfection of pGL3-765 and pRL-CMV into DU145 Cells.. --- p.64 / Chapter 3.3.3.3 --- Measuring Luciferase Activities --- p.65 / Chapter 3.3.4 --- Computational Prediction of Transcription Factor Binding Sites on miR-765 Promoter --- p.66 / Chapter 3.4 --- Characterizing the Promoter Region of ARHGEF11.. --- p.67 / Chapter 3.4.1 --- Cloning of ARHGEF11 Promoter into pGL3-Basic Vector (pGL3-ARH) --- p.67 / Chapter 3.4.1.1 --- PCR Amplification of ARHGEF11 Putative Promoter Region --- p.67 / Chapter 3.4.1.2 --- Ligation of the Amplified Regions in pGL3-Basic Vector --- p.68 / Chapter 3.4.1.3 --- Preparation of Plasmid DNA --- p.69 / Chapter 3.4.2 --- Preparation of Truncated ARHGEF11 Promoter Clones --- p.69 / Chapter 3.4.2.1 --- pGL3-ARH-Trunc#l --- p.69 / Chapter 3.4.2.2 --- pGL3-ARH-Trunc#2 --- p.70 / Chapter 3.4.2.3 --- pGL3-ARH-Trunc#3 --- p.71 / Chapter 3.4.3 --- Assessing ARHGEF11 Promoter Activities --- p.72 / Chapter 3.5 --- Identifying Transcription Factor Binding Sites on ARHGEF11 Promoter with EMS A --- p.73 / Chapter 3.5.1 --- Computational Prediction --- p.73 / Chapter 3.5.2 --- Preparation of Biotinylated Probe for use in EMSA --- p.73 / Chapter 3.5.3 --- Preparation of Specific Competitors --- p.74 / Chapter 3.5.4 --- Preparation of DU145 Nuclear and Cytoplasmic Extracts --- p.75 / Chapter 3.5.4.1 --- Preparation of Extracts --- p.75 / Chapter 3.5.4.2 --- Measuring Protein Concentrations --- p.76 / Chapter 3.5.5 --- EMSA Detection of Interaction between Protein and Probe --- p.76 / Chapter 3.6 --- Assessing Biological Significances of miR-765 --- p.78 / Chapter 3.6.1 --- Effects of ICI on DU145 Cells Growth --- p.79 / Chapter 3.6.2 --- Effects of ICI on DU145 Migration Ability --- p.79 / Chapter 3.6.2.1 --- Monolayer Wound Healing Assay --- p.79 / Chapter 3.6.2.2 --- Transwell Migration Assay --- p.80 / Chapter 3.6.3 --- Validating Functionality of Ectopic miR- 765 --- p.81 / Chapter 3.6.3.1 --- miR-765 Recognition Sequence --- p.81 / Chapter 3.6.3.2 --- Preparation of pMIR-765 vector --- p.82 / Chapter 3.6.3.3 --- Ectopic Introduction of miR-765 into DU145 Cells --- p.84 / Chapter 3.6.3.4 --- "Verifying Functionality, of Ectopic miR-765" --- p.84 / Chapter 3.6.4 --- Effects of miR-765 on DU145 Growth --- p.86 / Chapter 3.6.5 --- Effects of miR-765 on DU145 Migration Ability --- p.86 / Chapter 3.7 --- Statistical Analysis --- p.87 / Chapter Chapter 4: --- Results --- p.88 / Chapter 4.1 --- "Identifying ICI 182,780-Regulated miRNA in DU145 Cells" --- p.88 / Chapter 4.1.1 --- miRNA Expression Profiling of DU145 with Microarray --- p.88 / Chapter 4.1.2 --- "Confirming Induction of miR-765 by ICI 182,780 with qRT-PCR" --- p.91 / Chapter 4.1.3 --- "ARHGEF11, Host Gene of miR-765" --- p.95 / Chapter 4.1.4 --- "Induction of ARHGEF 11 by ICI 182,780" --- p.96 / Chapter 4.2 --- Characterization miR-765 Promoter Region --- p.98 / Chapter 4.2.1 --- Cloning of miR- 765 Promoter Region into pGLS-Basic Vector --- p.98 / Chapter 4.2.2 --- Promoter Activity of miR-765 Promoter --- p.100 / Chapter 4.2.3 --- Deletion Mapping of miR- 765 Promoter Region --- p.102 / Chapter 4.2.4 --- Promoter Activities and Inducibitiy of Truncated miR-765 Promoters --- p.103 / Chapter 4.2.5 --- Computational Prediction of Transcription Factor Binding Sites on miR-765 Promoter --- p.105 / Chapter 4.3 --- Characterization of ARHGEF 11 Promoter Region --- p.107 / Chapter 4.3.1 --- Cloning of ARHGEF 11 Promoter --- p.107 / Chapter 4.3.2 --- Promoter Activitiy of ARHGEFll Promoter --- p.109 / Chapter 4.3.3 --- Deletion Mapping of ARHGEFll Promoter --- p.111 / Chapter 4.3.4 --- Promoter Activities and Inducibitiy of Truncated ARHGEF 11 Promoters --- p.113 / Chapter 4.4 --- Identifying Transcription Factor Binding Sites on ARHGEF 11 Promoter --- p.115 / Chapter 4.4.1 --- Computational Prediction of Transcription Factor Binding Sites onARHGEFll Promoter --- p.115 / Chapter 4.4.2 --- Preparation of Probe and Specific Competitors for EMSA --- p.117 / Chapter 4.4.3 --- Interaction between DU145 Nuclear Extract and ARHGEF 11 Promoter Region --- p.119 / Chapter 4.5 --- Biological Significances of miR-765 --- p.122 / Chapter 4.2.1 --- "Effects of ICI 182,780 on DU145 Cell growth" --- p.122 / Chapter 4.2.2 --- "Effects of ICI 182,780 on DU145 Cell Migration" --- p.124 / Chapter 4.2.3 --- Verifying Functionality of Ectopic miR-765 --- p.131 / Chapter 4.2.4 --- Effects of miR-765 on DU145 Cell Growth --- p.133 / Chapter 4.2.5 --- Effects of miR-765 on DU145 Cell Migration --- p.135 / Chapter Chapter 5: --- Discussion --- p.138 / Chapter 5.1 --- "Identifying miR-765 as an Up-regulated miRNA by ICI 182,780" --- p.139 / Chapter 5.1.1 --- "Information about ICI 182,780" --- p.139 / Chapter 5.1.2 --- miRNA Profiling of DU145 --- p.139 / Chapter 5.1.3 --- "Confirming Induction of miR-765 by ICI 182,780 and ERβ dependency with qRT-PCR" --- p.140 / Chapter 5.1.4 --- "Up-regulation of miR-765 Host Gene, ARHGEF11, by ICI" --- p.141 / Chapter 5.2 --- Regulatory Elements of miR-765 Expression --- p.143 / Chapter 5.2.1 --- Own Upstream promoter of miR- 765 --- p.144 / Chapter 5.2.2 --- Promoter of Host Gene ARHGEF11 --- p.146 / Chapter 5.2.3 --- Interaction between ARHGEF11 Promoter Critical Region and Transcription Factors --- p.147 / Chapter 5.2.4 --- Involvement of independent Promoter and Host Gene Promoter in miR-765 Regulation --- p.757 / Chapter 5.3 --- Biological Significances of miR-765 on DU145 --- p.153 / Chapter 5.4 --- Significance of Findings and Future Studies --- p.158 / Chapter 5.4.1 --- Clinical Significance --- p.158 / Chapter 5.4.2 --- Future Studies --- p.161 / Chapter Chapter 6: --- Conclusion --- p.163 / Chapter Chapter 7: --- References --- p.166

Uma nova alternativa para o tratamento do câncer foi proposta em estudos anteriores, consistindo no uso de uma nanoemulsão lipídica como transportadora de agentes quimioterápicos às células neoplásicas. A redução da toxicidade da quimioterapia promovida pelo direcionamento específico de quimioterápicos às células tumorais nos levou a testar o potencial de aplicação do sistema de nanopartículas lipídicas na terapêutica combinada do paclitaxel com a sinvastatina, um agente hipolipemiante que pode ser empregado como coadjuvante no tratamento do câncer. Nos dias 11, 14 e 19 após a inoculação de células de melanoma B16F10, camundongos C57BL/6J receberam pela via intraperitoneal soluções de oleato de paclitaxel associado à nanoemulsão lipídica 17,5µmol/kg (Nano-paclitaxel), formulação comercial do paclitaxel 17,5µmol/kg, nanoemulsão lipídica (Nanoemulsão) e solução salina (Controle). A sinvastatina 50mg/kg/dia foi administrada por gavagem do 11° ao 19° dia após a inoculação do tumor em um dos grupos de animais tratados com o Nano-paclitaxel (Nano-paclitaxel + Sinva), no grupo tratado com a formulação comercial do paclitaxel (Paclitaxel + Sinva) e como monoterapia (Sinva). Camundongos Balb-c saudáveis receberam os mesmos tratamentos para avaliação dos possíveis efeitos tóxicos dos diferentes tratamentos. A terapia combinada Nano-paclitaxel + Sinva apresentou toxicidade negligível em comparação com a terapia combinada Paclitaxel + Sinva que provocou perda de peso e mielossupressão nos animais. Nos animais portadores de tumor, o tratamento Nano-paclitaxel + Sinva inibiu 95% do crescimento tumoral, comparado à inibição de 44% promovida pelo tratamento Paclitaxel + Sinva. Além disso, apenas 37% dos animais portadores de melanoma submetidos ao tratamento com Nano-paclitaxel + Sinva apresentaram metástases, em contraste com 90% dos tratados com Paclitaxel + Sinva. A probabilidade de sobrevida também foi maior nos camundongos tratados com o Nano-paclitaxel + Sinva em comparação aos tratados com Paclitaxel + Sinva. A análise de amostras de tumores por citometria de fluxo mostrou que somente nos grupos de animais tratados com Sinva, Nano-paclitaxel ou com a combinação Nano-paclitaxel + Sinva houve aumento na expressão de p21 em comparação ao grupo Controle. Da mesma forma, apenas nos grupos Sinva e Nano-paclitaxel + Sinva houve redução na expressão de ciclina D1 em comparação ao grupo Controle. O teste de viabilidade celular com rodamina 123 mostrou despolarização da membrana mitocondrial com redução no número de células tumorais viáveis em todos os grupos de tratamentos em comparação aos grupos Nanoemulsão e Controle. A avaliação histológica dos tumores demonstrou que os grupos Nanoemulsão e Controle apresentaram alta densidade de células tumorais, diferentemente dos demais grupos de tratamento e que apenas os tumores do grupo Nano-paclitaxel + Sinva apresentaram aumento na presença de fibras de colágeno tipo I e III. Em comparação ao grupo Controle, os tumores dos grupos Sinva, Paclitaxel + Sinva, Nano-paclitaxel e Nano-paclitaxel + Sinva apresentaram redução na expressão imunohistoquímica de ICAM, MCP-1 e MMP-9 sendo que o grupo Nano-paclitaxel + Sinva apresentou a menor porcentagem de área marcada positivamente para a MMP-9. A terapia combinada com Nano-paclitaxel + Sinva é menos tóxica e mais efetiva na inibição do crescimento tumoral do que a mesma terapia com a formulação comercial do paclitaxel. / In previous studies we have proposed a novel approach for cancer treatment consisting of the use of a lipid nanoemulsion as a vehicle to direct chemotherapeutic agents to neoplastic cells. Reduction of chemotherapy toxicity promoted by specific targeting of antineoplastic agents to tumor cells led us to test the application of the lipidic nanoparticle system in combined treatment with paclitaxel and simvastatin, a cholesterol-lowering drug that can be used as coadjuvant in cancer treatment. On days 11, 14 and 19 after B16F10 melanoma cells inoculation, C57BL/6J mice were intraperitoneally injected with paclitaxel oleate associated to the lipidic nanoemulsion 17.5 µmol/kg (Nano-paclitaxel), commercial formulation of paclitaxel 17.5 µmol/kg, lipidic nanoemulsion (Nanoemulsion) or saline solution (Control). Simvastatin 50 mg/kg/day was administered by gavage from days 11 to 19 after tumor inoculation in one group of animals treated with Nano-paclitaxel (Nano-paclitaxel + Simva), in the group treated with commercial formulation of paclitaxel (Paclitaxel + Simva) and as monotherapy (Simva). Evaluation of possible toxic effects of the treatments was accessed in healthy Balb-c mice. Combined therapy with Nano-paclitaxel + Simva showed negligible toxicity as compared with the combination of Paclitaxel + Simva which resulted in animal weight loss and myelosuppression. In tumor-bearing animals, treatment with Nano-paclitaxel + Simva resulted in a remarkable tumor growth inhibition rate of 95%, compared to a 44% inhibition rate promoted by treatment with Paclitaxel + Simva. Moreover, only 37% of melanoma bearing animals treated with Nano-paclitaxel + Simva developed metastasis, in contrast to 90% of those treated with Paclitaxel + Simva. Survival rates were also higher in mice treated with Nano-paclitaxel + Simva in comparison to Paclitaxel + Simva treated animals. Analysis of tumor samples by flow cytometry showed that only animals treated with Simva, Nano-paclitaxel or Nano-paclitaxel + Simva increased the expression of p21 in comparison to Control group. Also, tumors from animals treated with Simva or Nano-paclitaxel + Simva presented a decrease in the expression of cyclin D1 in comparison to Control group. Cell viability test with rhodamine 123 showed mitochondrial membrane depolarization with reduction of tumor viable cells in all treatment groups in comparison to Nanoemulsion and Control groups. The histological study revealed that in contrast to drugs treated groups, tumors from Nanoemulsion and Control groups presented high tumor cell density and only Nano-paclitaxel + Simva treated animals presented tumors with increased presence of collagen fibers I and III. In comparison to Control group, tumors from groups Simva, Paclitaxel + Simva, Nano-paclitaxel and Nano-paclitaxel + Simva showed a reduction in immunohistochemical expression of ICAM, MCP-1 and MMP-9 and the group Nano-paclitaxel + Simva presented the lowest percentage of area positively stained for MMP-9. Combined therapy with Nano-paclitaxel + Simva was less toxic and more effective in promoting tumor growth inhibiton than the same combined therapy with the commercial formulation of paclitaxel.

Wong, Ngan Yuk. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 114-128). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.i / Declaration --- p.ii / Abstract (in English) --- p.iii / Abstract (in Chinese) --- p.v / Acknowledgments --- p.vi / Table of Contents --- p.vii / List of Abbreviations --- p.xi / List of Figures --- p.xiv / List of Tables --- p.xvi / Chapter 1. --- Literature review --- p.1 / Chapter 1.1. --- Introduction to Cordyceps --- p.1 / Chapter 1.2. --- Ingredients of Cordyceps and their related biological activities --- p.4 / Chapter 1.2.1. --- "Amino acids, peptides, proteins and polyamines" --- p.4 / Chapter 1.2.1.1. --- Proteins --- p.4 / Chapter 1.2.2. --- Saccharides and sugar derivatives --- p.7 / Chapter 1.2.2.1. --- Polysaccharides --- p.7 / Chapter 1.2.3. --- Nucleosides --- p.9 / Chapter 1.2.3.1. --- Cordycepin --- p.9 / Chapter 1.2.3.2. --- Adenosine --- p.12 / Chapter 1.2.4. --- Fatty acids and sterols --- p.14 / Chapter 1.2.5. --- Vitamins and inorganics --- p.15 / Chapter 1.3. --- Cordyceps and their related biological activities --- p.15 / Chapter 1.3.1. --- Cordyceps militaris --- p.15 / Chapter 1.3.2. --- Cordyceps sinensis --- p.17 / Chapter 1.4. --- Proteomic tools used to study the change in protein expression profiles --- p.21 / Chapter 1.4.1. --- Proteomic tools in studies of the change in protein expression --- p.21 / Chapter 1.4.2. --- Two-dimensional gel electrophoresis --- p.22 / Chapter 1.4.3. --- Mass spectrometry --- p.22 / Chapter 1.4.4. --- Current challenges --- p.23 / Chapter 2. --- Methodology --- p.25 / Chapter 2.1. --- Cultivation of Cordyceps militaris --- p.25 / Chapter 2.2. --- Preparation of Cordyceps extracts for anti-proliferation assay on cell lines --- p.26 / Chapter 2.2.1. --- Types of the extracts of Cordyceps --- p.26 / Chapter 2.2.2. --- Preparation of the Cordyceps extracts --- p.26 / Chapter 2.3. --- Anti-proliferation assay on cell lines for extract screening --- p.26 / Chapter 2.3.1. --- Cell lines and culturing condition --- p.26 / Chapter 2.3.2. --- Viable cell count using trypan blue exclusion method --- p.27 / Chapter 2.3.3. --- Anti-proliferation assay on cell lines using MTT assay --- p.28 / Chapter 2.3.4. --- Determination of the IC50 values --- p.30 / Chapter 2.3.5. --- Statistical Analysis --- p.30 / Chapter 2.4. --- Proteomic studies for HepG2 and Hs68 after the treatment of extracts --- p.30 / Chapter 2.4.1. --- Protein sample preparation of HepG2and Hs68 --- p.30 / Chapter 2.4.2. --- Protein quantitation --- p.31 / Chapter 2.4.3. --- 2D Gel electrophoresis --- p.33 / Chapter 2.4.4. --- Image analysis --- p.34 / Chapter 2.4.5. --- In gel digestion and MALDI-ToF MS --- p.35 / Chapter 2.5. --- Cell cycle analysis --- p.36 / Chapter 2.5.1. --- Cell samples preparation --- p.36 / Chapter 2.5.2. --- Popidium iodide staining --- p.36 / Chapter 2.5.3. --- Flow cytometry --- p.37 / Chapter 2.5.4. --- Statistical Analysis --- p.38 / Chapter 2.6. --- Western blotting --- p.38 / Chapter 2.6.1. --- Protein sample preparation of HepG2 and Hs68 --- p.38 / Chapter 2.6.2. --- SDS-PAGE --- p.38 / Chapter 2.6.3. --- Protein Transblotting --- p.39 / Chapter 2.6.4. --- Membrane Blocking and Antibody Incubations --- p.39 / Chapter 2.6.5. --- Detection of Proteins --- p.40 / Chapter 3. --- Results --- p.41 / Chapter 3.1. --- Investigation of anti-proliferating effect of Cordyceps extracts on HepG2 and Hs68 using MTT assays --- p.41 / Chapter 3.1.1. --- Cordyceps militaris fruiting body extract - CMFB --- p.41 / Chapter 3.1.2. --- Cordyceps militaris mycelia extract - CMM --- p.41 / Chapter 3.1.3. --- Cordyceps sinensis fruiting body extract - CSFB --- p.45 / Chapter 3.1.4. --- Cordyceps sinensis mycelia extract - CSM --- p.45 / Chapter 3.1.5. --- "Comparison of the anti-proliferation effects of the Cordyceps extracts CMFB, CMM, CSFB and CSM" --- p.49 / Chapter 3.2. --- "Investigation of anti-proliferating effect of Cordyceps militaris extracts on H292, Neuro2a and WIL2-NS using MTT assays" --- p.51 / Chapter 3.2.1. --- Cordyceps militaris fruiting body extract - CMFB --- p.51 / Chapter 3.2.2. --- Cordyceps militaris mycelia extract - CMM --- p.51 / Chapter 3.3. --- Changes in total protein expression profiles in cell lines --- p.56 / Chapter 3.3.1. --- Protein samples preparation --- p.56 / Chapter 3.3.2. --- 2D gel electrophoresis analysis of protein from cell lines --- p.56 / Chapter 3.3.2.1. --- HepG2 --- p.57 / Chapter 3.3.2.2. --- Hs68 --- p.58 / Chapter 3.3.3. --- Differentially expressed proteins identification --- p.66 / Chapter 3.3.3.1. --- HepG2 --- p.66 / Chapter 3.3.3.2. --- Hs68 --- p.67 / Chapter 3.4. --- Cell cycle analysis --- p.76 / Chapter 3.4.1. --- Cell samples preparation --- p.76 / Chapter 3.4.2. --- HepG2 --- p.76 / Chapter 3.4.3. --- Hs68 --- p.77 / Chapter 3.4.4. --- H292 --- p.77 / Chapter 3.5. --- Western blotting --- p.81 / Chapter 3.5.1. --- Protein samples preparation --- p.81 / Chapter 3.5.2. --- Detection of actin for protein loading normalization --- p.83 / Chapter 3.5.3. --- Detection of procaspase-3 and cleaved caspase-3 --- p.83 / Chapter 3.5.4. --- Detection of procaspase-7 and cleaved caspase-7 --- p.84 / Chapter 3.5.5. --- Detection of procaspase-9 and cleaved caspase-9 --- p.84 / Chapter 4. --- Discussion --- p.86 / Chapter 4.1. --- Anti-tumor proliferating effect of Cordyceps extracts --- p.86 / Chapter 4.2. --- Changes in total protein expression profiles in cell lines --- p.87 / Chapter 4.2.1. --- Differentially expressed proteins in HepG2 treated with fruiting body extract --- p.88 / Chapter 4.2.1.1. --- Heat shock 90kDa protein 1 beta (HSP90(β) --- p.89 / Chapter 4.2.1.2. --- Far upstream element-binding protein 1 (FUBP-1) --- p.90 / Chapter 4.2.1.3. --- RuvB-like 1 (RuvbLl) --- p.90 / Chapter 4.2.1.4. --- Acidic protein rich in leucine (APRIL) --- p.91 / Chapter 4.2.1.5. --- SET protein --- p.92 / Chapter 4.2.1.6. --- Enolase 1 (α-enolase) --- p.94 / Chapter 4.2.1.7. --- Aldolase A --- p.96 / Chapter 4.2.1.8. --- DNA-binding protein B --- p.96 / Chapter 4.2.1.9. --- Peroxiredoxin 1 (Prx 1) --- p.97 / Chapter 4.2.1.10. --- Proteasome activator subunit 1 iso form 1 --- p.99 / Chapter 4.2.1.11. --- Dehydrogenase/ reductase member 2 isoform 2 --- p.99 / Chapter 4.2.1.12. --- Protein disulfide isomerase- related protein 5 --- p.100 / Chapter 4.2.1.13. --- Annexin IV --- p.100 / Chapter 4.2.1.14. --- Enoyl Coenzyme A hydratase --- p.101 / Chapter 4.2.2. --- Differentially expressed proteins in HepG2 treated with mycelia extract --- p.102 / Chapter 4.2.2.1. --- Alpha actinin 4 --- p.102 / Chapter 4.2.2.2. --- SET translocation isoform 1 --- p.103 / Chapter 4.2.2.3. --- Acidic (leucine-rich) nuclear phosphoprotein 32 family member B (ANP32b) --- p.103 / Chapter 4.2.2.4. --- Endoplasmic reticulum protein 29 isoform 1 precursor (ERp29) --- p.103 / Chapter 4.2.2.5. --- Heterogeneous nuclear ribonucleoprotein H3 isoform b (hnRNP 2H9A) --- p.104 / Chapter 4.2.3. --- Differentially expressed proteins in Hs68 treated with fruiting body extract --- p.105 / Chapter 4.2.3.1. --- Lamin A/C isoform 2 --- p.105 / Chapter 4.2.3.2. --- Vimentin --- p.106 / Chapter 4.2.3.3. --- Tropomyosin 1 alpha chain isoform 4 --- p.107 / Chapter 4.2.3.4. --- Rho GDP dissociation inhibitor (GDI) alpha (RhoGDIα) --- p.109 / Chapter 4.2.3.5. --- Dihydropyrimidinase-like 2 (DRP-2) --- p.109 / Chapter 4.2.3.6. --- Keratin 7 (K7) --- p.110 / Chapter 4.2.4. --- Differentially expressed proteins in Hs68 treated with mycelia extract --- p.111 / Chapter 4.3. --- Cell cycle analysis --- p.111 / Chapter 4.4. --- Western blotting --- p.113 / Chapter 5. --- References --- p.114

This reseach firstly investigated iodine-induced apoptotic effects and the underlying mechanism in thyroid cancer cells. Results indicated that apoptosis induced by iodine, especially at high dose of iodine (100 muM), was mitochondrial-mediated, with the loss of mitochondrial membrane potential, Bak up-regulation, caspase 3 activation and cytochrome C release from mitochondria. Iodine treatment decreased the level of mutant p53 including the R273H mutant that possesses anti-apoptotic features while increased the p21 level. The block of p21 significantly prevented iodine-induced apoptosis. High doses of iodine also stimulated the transient activation of the subfamily members of MAPKs (ERK1/2, p38 and JNK1/2). The results showed the three subfamily members of MAPKs all worked as anti-apoptotic factors. Surprisingly, high doses of iodine promoted instead of suppressed the expression of anti-apoptotic protein Bcl-xL expression. The increase of Bc1-xL was likely to compensate the damage induced by iodine since the inhibition of Bc1-xL accelerated iodine-mediated apoptosis. Collectively, iodine induced mitochondrial-mediated apoptosis in thyroid cancer cells. This apoptotic pathway was involved in the activation of MAPKs pathways, which may subsequently up-regulate p21, Bc1-xL, and down-regulate anti-apoptotic mutant p53 expression. The findings provide solid molecular evidence to explain the epidemiological observation that iodine insufficiency promotes the thyroid tumor development. It may also reveal some novel molecular targets for the treatment of thyroid cancer. / Thyroid cancer is the most common endocrine malignancy and exhibits the full range of malignant behaviors from the relatively indolent occult differentiated thyroid cancer to uniformly aggressive and lethal anaplastic thyroid cancer. Iodine is a well known key element in thyroid normal function maintenance and thyroid cancer development. However, the mechanisms of iodine in thyroid cancer cells development are limited. Recent researches have indicated that iodine could induce cancer cells apoptosis, staying clear from the dysfunction of iodide-specific transportation systems in thyroid cancer cells. Thus, iodine-induced apoptosis may be an effective pathway for iodine to affect thyroid cancer development, but we know little about them. / To further explore iodine on the apoptotic effects of chemotherapeutic agents in thyroid cancer, anaplastic thyroid cancer cell line ARO was used. Anaplastic thyroid cancer is lethal because of its rapid progression and poor response to chemotherapy and radioiodine therapy. The study examined the effect of moderate dose of iodine (50 muM) on the apoptosis of ARO cells treated with doxorubicin (Dox) and histone deacetylase inhibitor sodium butyrate (NaB). The cytotoxic effect of either Dox or NaB alone was limited, but co-administration of NaB and Dox (NaB-Dox) significantly increased mitochondrial-mediated apoptosis. The effects of iodine to apoptosis-induced by the two agents were diversified. Iodine reduced the apoptosis induced by Dox or NaB-Dox but promoted apoptosis induced by NaB. To explain this diversifying finding, the experiment found that iodine exaggerated NaB-mediated Bcl-xL down-regulation. In contrast, it reduced the effect of Dox on the decrease of Bcl-xL expression. Further experiments showed that iodine regulated the level of Bcl-xL in ERK- or/and p38-related pathways. The balance between ERK and p38 may determine the iodine-modulated Bcl-xL expression. The high ERK/p38 activity ratio up-regulated Bc1-xL and enabled the tumor cells to resist chemotherapy, whereas the low ERK/p38 down-regulated Bc1-xL and sensitized the tumor cells to chemotherapy. Taken together, iodine plays a critical role in apoptosis of thyroid cancer cells induced by chemotherapeutic agents. The balance between ERK and p38 may determine cell survival and death through modulating Bcl-xL expression in thyroid cancer cells. The findings provide some new insights into the roles of iodine in chemotherapeutic agents-induced apoptosis in thyroid cancer cells. / To summarize, iodine-induced apoptotic effects on thyroid cancer cells is a key pathway for iodine to influence thyroid cancer development and chemotherapy. Meanwhile MAPKs-related mutant p53, p21 and Bcl-xL expression are critical in deciding thyroid cancer cells survival and death. Moreover, iodine can influence chemotherapeutic agents-induced apoptosis through ERK/p38-mediated Bcl-xL expression. / Liu, Xiaohong. / "December 2009." / Adviser: Charles Andrew van Hasselt. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 111-146). / 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.

Lai, Sze Tsai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 94-104). / Abstracts in English and Chinese. / Thesis Committees --- p.i / Statement --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Acknowledgments --- p.vi / List of Abbreviations --- p.vii / Table of Contents --- p.ix / List of Tables --- p.xiii / List of Figures --- p.xiv / Chapter 1 --- Literature review --- p.1 / Chapter 1.1 --- Introduction to Cordyceps --- p.1 / Chapter 1.2 --- Fungal proteomics --- p.2 / Chapter 1.2.1 --- Extraction method --- p.2 / Chapter 1.2.2 --- Proteomic study of Cordyceps --- p.3 / Chapter 1.3 --- Ingredients of Cordyceps and their related biological activities --- p.5 / Chapter 1.3.1 --- Polysaccharides --- p.5 / Chapter 1.3.2 --- Nucleosides --- p.6 / Chapter 1.3.2.1 --- Cordycepin --- p.6 / Chapter 1.3.2.2 --- Adenosine --- p.8 / Chapter 1.4 --- Cordyceps and their related biological activities --- p.9 / Chapter 1.4.1 --- Cordyceps militaris --- p.9 / Chapter 1.4.2 --- Cordyceps sinensis --- p.10 / Chapter 1.5 --- Proteomic analysis of proteome change --- p.12 / Chapter 1.5.1 --- Proteomic tools used to study the change in protein expression --- p.12 / Chapter 1.5.2 --- Two-dimensional gel electrophoresis --- p.13 / Chapter 1.5.3 --- Mass spectrometry --- p.13 / Chapter 1.6 --- Objective --- p.16 / Chapter 2 --- Methodology --- p.17 / Chapter 2.1 --- Cultivation of Cordyceps militaris --- p.17 / Chapter 2.2 --- Proteomic study on Cordyceps militaris --- p.17 / Chapter 2.2.1 --- Extraction of proteins from Cordyceps militaris --- p.17 / Chapter 2.2.2 --- Protein quantification --- p.18 / Chapter 2.2.3 --- 2D Gel electrophoresis --- p.19 / Chapter 2.2.4 --- Image analysis --- p.20 / Chapter 2.2.5 --- In gel digestion and MALDI-ToF MS --- p.20 / Chapter 2.3 --- Preparation of Cordyceps extracts for anti-proliferation assay on cell lines --- p.21 / Chapter 2.3.1 --- Types of the extracts of Cordyceps --- p.21 / Chapter 2.3.2 --- Preparation of the extracts of Cordyceps --- p.21 / Chapter 2.4 --- Anti-proliferation assay on cell lines for extract screening --- p.22 / Chapter 2.4.1 --- Cell lines and culturing condition --- p.22 / Chapter 2.4.2 --- Viable cell count using trypan blue exclusion method --- p.22 / Chapter 2.4.3 --- Anti-proliferation assay on SV7 tert using MTT assay --- p.23 / Chapter 2.4.4 --- Determination of the IC5o values --- p.24 / Chapter 2.4.5 --- Statistical Analysis --- p.24 / Chapter 2.5 --- Anti-proliferation assay on other cell lines using the two screened extracts --- p.24 / Chapter 2.5.1 --- Cell lines and culturing condition --- p.24 / Chapter 2.5.2 --- "Anti-proliferation assay on on HepG2, H292, Neuro2a，WIL2-NS cells using MTT assay" --- p.25 / Chapter 2.6 --- Proteomic studies for SV7tert and Hs68 after the treatment of extracts --- p.25 / Chapter 2.6.1 --- Protein sample preparation of SV7tert and Hs68 --- p.25 / Chapter 2.6.2 --- Protein quantification --- p.26 / Chapter 2.6.3 --- 2D Gel electrophoresis --- p.26 / Chapter 2.6.4 --- Image analysis --- p.26 / Chapter 2.7 --- Western Immunoblotting --- p.26 / Chapter 2.7.1 --- Protein sample preparation of SV7tert and Hs68 --- p.26 / Chapter 2.7.2 --- SDS-PAGE --- p.27 / Chapter 2.7.3 --- Protein Blotting --- p.27 / Chapter 2.7.4 --- Membrane Blocking and Antibody Incubations --- p.28 / Chapter 2.7.5 --- Detection of Proteins --- p.28 / Chapter 3 --- Results --- p.29 / Chapter 3.1 --- Proteins identification in Cordyceps militaris --- p.29 / Chapter 3.1.1 --- 2D gel electrophoresis analysis and resolution --- p.29 / Chapter 3.1.2 --- Identification and categorization of proteins of mycelia and fruiting body of Cordyceps militaris --- p.30 / Chapter 3.2 --- Investigation of anti-proliferating activity of extracts using MTT assays on SV7tert and Hs68 cell lines --- p.44 / Chapter 3.2.1 --- Mycelia extract from Cordyceps militaris --- p.44 / Chapter 3.2.2 --- Fruiting body extract from Cordyceps militaris --- p.44 / Chapter 3.2.3 --- Mycelia extract from Cordyceps sinensis --- p.47 / Chapter 3.2.4 --- Fruiting body extract from Cordyceps sinensis --- p.47 / Chapter 3.2.5 --- Screening of extracts --- p.50 / Chapter 3.3 --- "Investigation of anti-proliferating activity of extracts using MTT assays on HepG2，H292, Neuro2a and WIL2-NS cell lines" --- p.51 / Chapter 3.3.1 --- Mycelia extract from Cordyceps militaris --- p.51 / Chapter 3.3.2 --- Fruiting body extract from Cordyceps militaris --- p.51 / Chapter 3.4 --- Changes in total protein expression profiles in SV7tert and Hs68 cell lines --- p.56 / Chapter 3.4.1 --- Corresponding extract treatment of cell lines --- p.56 / Chapter 3.4.2 --- 2D gel electrophoresis analysis of protein from cells (SV7tert or Hs68) --- p.56 / Chapter 3.4.2.1 --- SV7tert study --- p.57 / Chapter 3.4.2.2 --- Hs68 study --- p.57 / Chapter 3.4.3 --- Protein identification --- p.65 / Chapter 3.4.3.1 --- Changes in protein expressions in SV7tert after mycelia extract treatment --- p.65 / Chapter 3.4.3.2 --- Changes in protein expressions in Hs68 after mycelia extract treatment --- p.65 / Chapter 3.4.3.3 --- Changes in protein expressions in SV7tert after fruiting body extract treatment --- p.66 / Chapter 3.4.3.4 --- Changes in protein expressions in Hs68 after fruiting body extract treatment --- p.66 / Chapter 3.5 --- Western immunoblotting --- p.71 / Chapter 3.5.1 --- Corresponding extract treatment of cell lines --- p.71 / Chapter 3.5.2 --- Normalization of protein loading using anti-actin antibody --- p.73 / Chapter 3.5.3 --- Detection of caspase 3 by use of antibody --- p.74 / Chapter 3.5.4 --- Detection of cleaved caspase 3 by use of antibody --- p.74 / Chapter 4 --- Discussion --- p.77 / Chapter 4.1 --- Identification of proteins in Cordyceps militaris --- p.77 / Chapter 4.2 --- Difficulties in identifying the proteins in Cordyceps militaris --- p.80 / Chapter 4.3 --- Investigation of anti-proliferating activity of extracts --- p.80 / Chapter 4.4 --- Changes in cell total protein expression profiles in SV7tert and Hs68 cell lines --- p.81 / Chapter 4.4.1 --- Protein alterations in SV7tert treated with mycelia extract --- p.82 / Chapter 4.4.1.1 --- Heat shock protein 27 (Hsp27) --- p.82 / Chapter 4.4.1.2 --- Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) --- p.83 / Chapter 4.4.2 --- Protein alterations in Hs68 with mycelia extract treatment --- p.84 / Chapter 4.4.2.1 --- Chain B of triosephosphate isomerase - Triosephophate isomerase 1 --- p.84 / Chapter 4.4.2.2 --- Glutathione transferase --- p.85 / Chapter 4.4.3 --- Protein alterations in SV7tert with fruiting body extract treatment --- p.86 / Chapter 4.4.3.1 --- Calreticulin precusor --- p.86 / Chapter 4.4.3.2 --- Nucleophosmin 1 isoform 2 (B23) --- p.87 / Chapter 4.4.3.3 --- Heat shock 70kDa protein 8 isoform 1 - Heat shock 70kDa protein (Hsp70) --- p.88 / Chapter 4.4.3.4 --- Voltage-dependent anion channel 2 (VDAC2) --- p.89 / Chapter 4.4.3.5 --- "Tumor protein, translationally controlled (TCTP)" --- p.90 / Chapter 4.4.3.6 --- RAN binding protein 1 (RANBP1) --- p.91 / Chapter 4.4.4 --- Protein alteration in Hs68 with mycelia extract treatment --- p.92 / Chapter 4.5 --- Conclusion --- p.93 / References --- p.94

Lin, Ka Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 108-122). / Abstracts in English and Chinese. / Abstract (In English) --- p.ii / Abstract (In Chinese) --- p.iii / Acknowledgment --- p.V / Abstracts of Publications --- p.vi / Abbreviations --- p.viii / List of Figures --- p.ix / List of Table --- p.X / Contents --- p.xi / Chapter Chapter I --- Introduction --- p.Page / Chapter 1.1 --- Pluripotent Stem Cell --- p.1 / Chapter 1.1.1 --- Characteristic of pluripotent stem cells --- p.1 / Chapter 1.1.2 --- Origin of pluripotent stem cells --- p.1 / Chapter 1.1.2.1 --- Embryonic carcinoma cells --- p.2 / Chapter 1.1.2.2 --- Embryonic stem cells --- p.2 / Chapter 1.1.2.3 --- Epiblast stem cells --- p.2 / Chapter 1.1.2.4 --- Embryonic germ cells and adult germline stem cells --- p.3 / Chapter 1.1.2.5 --- Induced pluripotent stem cells --- p.3 / Chapter 1.1.3 --- Pluripotency in Embryonic Stem Cells --- p.4 / Chapter 1.1.3.1 --- Extrinsic signal governing pluripotency --- p.5 / Chapter 1.1.3.1.1 --- LIF signaling --- p.5 / Chapter 1.1.3.1.2 --- BMP signaling --- p.6 / Chapter 1.1.3.1.3 --- Other signaling pathways --- p.6 / Chapter 1.1.3.2 --- Intrinsic sternness factors --- p.7 / Chapter 1.1.3.2.1 --- Oct4 Expression in Embryonic Stem cells --- p.7 / Chapter 1.1.3.2.2 --- Sox-2 Expression in Embryonic Stem Cells --- p.8 / Chapter 1.1.3.2.3 --- Nanog Expression in Embryonic Stem Cells --- p.9 / Chapter 1.1.3.2.4 --- "Transcriptional Regulation of Oct-4, Nanog and Sox-2 in Embryonic Stem Cells" --- p.10 / Chapter 1.1.3.2.5 --- Others pluripotent genes --- p.11 / Chapter 1.1.3.2.5.1 --- Utfl --- p.11 / Chapter 1.1.3.2.5.2 --- Rexl --- p.11 / Chapter 1.1.3.2.5.3 --- Esrrb --- p.12 / Chapter 1.1.3.2.5.4 --- Eras --- p.12 / Chapter 1.1.3.2.5.5 --- Tell --- p.12 / Chapter 1.1.3.2.5.6 --- Dnm3tl --- p.13 / Chapter 1.1.3.2.5.7 --- Dppa3 --- p.13 / Chapter 1.1.3.2.5.8 --- Dppa4 --- p.14 / Chapter 1.1.3.2.5.9 --- Dppa5 --- p.14 / Chapter 1.1.3.2.5.10 --- Klf2 --- p.15 / Chapter 1.2 --- Somatic cell reprogramming --- p.16 / Chapter 1.2.1 --- Definition of reprogramming --- p.16 / Chapter 1.2.2 --- The history of reprogramming --- p.16 / Chapter 1.2.2.1 --- Reprogramming by nuclear transfer --- p.17 / Chapter 1.2.2.2 --- Reprogramming by fusion with ES or EC cells --- p.18 / Chapter 1.2.2.3 --- Reprogramming with defined factor --- p.19 / Chapter 1.3 --- Induced pluripotent stem cells --- p.20 / Chapter 1.3.1 --- Transcription factor used for reprogramming to iPS cells --- p.20 / Chapter 1.3.1.1 --- Klf4 --- p.20 / Chapter 1.3.1.2 --- c-Myc --- p.21 / Chapter 1.3.2 --- Cornerstone of iPSC generation --- p.22 / Chapter 1.3.3 --- Major events in the reprogramming process --- p.23 / Chapter 1.3.4 --- Gene delivery systems for ips cell generation --- p.26 / Chapter 1.3.5 --- Culture system for embryonic stem cells and iPSC --- p.28 / Chapter 1.3.4.1 --- Feeder and serum used cell culture system --- p.28 / Chapter 1.3.4.2 --- Serum-free culture condition --- p.29 / Chapter 1.3.5 --- Differentiation potential of iPSC --- p.30 / Chapter 1.3.5.1 --- In vitro stringency tests --- p.30 / Chapter 1.3.5.2 --- In vivo stringency test --- p.30 / Chapter 1.3.5.3 --- In utero stringency test --- p.31 / Chapter 1.4 --- Mouse Lewis lung carcinoma-D 122 --- p.32 / Chapter 1.5 --- Dendritic cell vaccine in cancer immunotherapy --- p.33 / Chapter 1.5 --- Green Fluorescence protein Reporters --- p.35 / Chapter 1.5.1 --- GFP reporters in embryos and stem cell --- p.35 / Chapter 1.5.2 --- copGFP --- p.35 / Chapter 1.6 --- Aim of study --- p.36 / Chapter Chapter II --- Methods and Materials / Chapter 2.1 --- Materials --- p.37 / Chapter 2.1.1 --- Synthetic oligos used in polymerase chain reaction (PCR) --- p.37 / Chapter 2.1.2 --- DNA clones used in the study --- p.39 / Chapter 2.1.3 --- Materials for DNA manipulation --- p.39 / Chapter 2.1.4 --- Materials for RNA manipulation --- p.39 / Chapter 2.1.5 --- Antibodies --- p.40 / Chapter 2.1.6 --- Kits --- p.41 / Chapter 2.1.7 --- Bacteria strain and culture reagents 41 / Chapter 2.1.8 --- Culture media and reagents --- p.42 / Chapter 2.1.8.1 --- General culture media and reagents --- p.42 / Chapter 2.1.8.2 --- Traditional ES medium --- p.42 / Chapter 2.1.8.3 --- Feeder-free Serum-free ESGRO medium --- p.42 / Chapter 2.1.9 --- Cell lines used --- p.43 / Chapter 2.1.10 --- Instrumentation --- p.43 / Chapter 2.2 --- Methods --- p.44 / Chapter 2.2.1 --- Cell culture --- p.44 / Chapter 2.2.1.1 --- Routine cell culture --- p.44 / Chapter 2.2.1.2 --- Resuscitation and culture from frozen stock --- p.44 / Chapter 2.2.1.3 --- Passage of cells --- p.44 / Chapter 2.2.1.4 --- Cryopreservation of cells --- p.45 / Chapter 2.2.1.5 --- Mouse ES cells culture --- p.45 / Chapter 2.2.1.5.1 --- Passage and maintenance of SNL --- p.45 / Chapter 2.2.1.5.2 --- Inactivation and plating of SNLs (Feeder preparation) --- p.45 / Chapter 2.2.1.5.3 --- Cryopreservation (freezing) of SNLs --- p.46 / Chapter 2.2.1.6 --- Mouse ES cells culture in feeder-free culture conditions --- p.46 / Chapter 2.2.1.6.1 --- Preparation of gelatin coated plates --- p.46 / Chapter 2.2.1.6.2 --- Thawing mouse ES cells --- p.46 / Chapter 2.2.1.6.3 --- Passage of mouse ES cells --- p.47 / Chapter 2.2.1.6.4 --- Freezing mouse ES cells --- p.47 / Chapter 2.2.1.7 --- ES cells differentiation-Formation of embryoid bodies (EBs) --- p.47 / Chapter 2.2.1.8 --- Direct differentiation by retinoic acid --- p.48 / Chapter 2.2.1.9 --- Generation of iPS --- p.48 / Chapter 2.2.2 --- Cell transfections --- p.48 / Chapter 2.2.2.1 --- Lipofectamine 2000 transfection --- p.48 / Chapter 2.2.2.2 --- Nucleofection --- p.49 / Chapter 2.2.2.2.1 --- Optimization of nucleofection --- p.49 / Chapter 2.2.2.2.2 --- Nucleofection condition --- p.49 / Chapter 2.2.3 --- Nucleic acid --- p.49 / Chapter 2.2.3.1 --- Genomic DNA isolation --- p.49 / Chapter 2.2.3.2 --- Restriction Enzyme Digestion --- p.50 / Chapter 2.2.3.3 --- RNA and genomic DNA quantification --- p.50 / Chapter 2.2.3.4 --- Reversed transcription polymerase chain reaction (RT-PCR) --- p.50 / Chapter 2.2.3.4.1 --- RNA isolation and Reverse transcription (RT) --- p.50 / Chapter 2.2.3.4.2 --- Polymerase chain reaction (PCR) --- p.51 / Chapter 2.2.3.4.3 --- Real-time polymerase chain reaction (qRT- PCR) --- p.52 / Chapter 2.2.3.5 --- Agarose gel electrophoresis --- p.53 / Chapter 2.2.3.6 --- Genomic PCR for bisulfite sequencing --- p.53 / Chapter 2.2.4 --- Bacteria and Plasmid preparation --- p.54 / Chapter 2.2.4.1 --- Preparation of competent cells --- p.54 / Chapter 2.2.4.2 --- Heat-shock transformation --- p.54 / Chapter 2.2.4.3 --- Midi prep of plasmid --- p.54 / Chapter 2.2.5 --- Cell Staining --- p.55 / Chapter 2.2.5.1 --- Alkaline phosphatase staining --- p.55 / Chapter 2.2.5.2 --- Immunofluorescence --- p.55 / Chapter 2.2.6 --- Flow cytometry --- p.56 / Chapter 2.2.7 --- Animal Handling --- p.56 / Chapter Chapter III --- Results / Chapter 3.1 --- Generation of Nanog-reporter-GFP-D 122 --- p.57 / Chapter 3.2 --- Nucleofection optimization for D122 --- p.60 / Chapter 3.3 --- Generation ofD122-iPS --- p.65 / Chapter 3.3.1 --- Plasmid construct used in the study --- p.65 / Chapter 3.3.2 --- Protocol of D122-iPS generation --- p.67 / Chapter 3.3.3 --- Reprogramming Efficiency of D12´2ؤreNanog cells --- p.69 / Chapter 3.4 --- Expression of pluripotency markers upon reprogramming --- p.70 / Chapter 3.4.1 --- Alkaline Phosphatase staining --- p.70 / Chapter 3.4.2 --- Nanog-GFP expression --- p.72 / Chapter 3.4.3 --- Pluripotency gene expression upon reprogramming --- p.74 / Chapter 3.4.4 --- GFP positive D122 reNanog Colonies --- p.79 / Chapter 3.5 --- Characterization of the D122-iPS-lC --- p.80 / Chapter 3.5.1 --- Morphology of D122-iPS-lC --- p.80 / Chapter 3.5.2 --- Pluripotency gene expression --- p.82 / Chapter 3.5.3 --- Pluripotency markers SSEA-1 and Oct4 --- p.85 / Chapter 3.5.4 --- Bisulfite genomic sequencing --- p.88 / Chapter 3.5.5 --- Differentiation of the D122-iPS-lC --- p.90 / Chapter 3.5.5.1 --- Embryoid body formation by hanging drop --- p.90 / Chapter 3.5.5.2 --- Retinoic acid induced differentiation --- p.92 / Chapter Chapter IV --- Discussion / Chapter 4.1 --- General Discussion --- p.96 / Chapter 4.1.1 --- Cancer immunotherapy and dendritic cells --- p.96 / Chapter 4.1.2 --- Dendritic vaccine and tumor antigen --- p.97 / Chapter 4.1.3 --- Induced pluripotent stem cell technology and dendritic cells --- p.98 / Chapter 4.1.4 --- Tumor antigen presentation and dendritic cells --- p.98 / Chapter 4.1.5 --- D122 and cancer immunotherapy --- p.99 / Chapter 4.1.6 --- Method to introduce transcription factors for reprogramming --- p.100 / Chapter 4.1.7 --- Kinetics of reprogramming --- p.101 / Chapter 4.1.8 --- Culture condition for reprogramming D122_reNanog --- p.102 / Chapter 4.1.9 --- Reprogramming efficiency --- p.103 / Chapter 4.1.10 --- Establishment of D122-iPS-lC --- p.103 / Chapter 4.1.11 --- Differentiation of D122-iPS-1C --- p.104 / Chapter 4.2 --- Future Work --- p.106 / Chapter 4.3 --- Conclusion --- p.107 / Chapter Chapter V --- Bibliography --- p.108 / Appendix --- p.124

5-Fluorouracil (5-FU) is one of most frequently used chemotherapeutic medications for the treatment of many types of cancer in curative and palliative setting. It is important to recognize chemotherapy side effects and toxicities because some of these symptoms may indicate a clinical syndrome needing evaluation for a principal cause. We discuss a patient who developed severe mucositis requiring hospitalization after first use of Fluorouracil. Dihydropyrimidine dehydrogenase ( DPD) deficiency was suspected and was proven to be a cause of severe drug-related toxicity. Our patient is fifty six year old gentleman with stage III nasopharyngeal squamous cell carcinoma who developed two masses on right side of the neck and large posterior right nasopharyngeal mass. Patient was treated with concurrent chemotherapy with high dose of cisplatin along with radiation. Once completion of concurrent chemotherapy and radiation he was started on combination of 5-Fluorouracil and cisplatin. Three days after completion of ninety six hour continuous 5-flurouracil infusion patient developed severe mucositis. Clinical exam was consistent with swollen tongue and mouth and inability to clear oral secretions. Patient was tested for DPYD gene mutation. Testing showed heterozygous for the c.1679T>G(*13) variant in the DPYD gene consistent with predicted intermediate DPD activity (30-70% enzyme activity). About 80% of administered 5-fluorouracil is normally inactivated by DPD. A decrease in DPD enzymatic activity may lead to increased concentrations of 5-FU and elevated risk for severe toxicities. Standard dose of 5-FU was decreased by 50% with second cycle of chemotherapy. Patient tolerated the second cycle of chemotherapy well. Variants in the DPD gene can lead to reduced 5-FU catabolism resulting in severe toxicities. Some of the toxicities can cause death. It is important to screen for this deficiency and closely observe patients during chemotherapy treatment.

Partners play a critical role in making decisions about fertility preservation among young patients with cancer, yet little is known about these dyadic decisions when planning cancer treatment. Fertility preservation entails helping cancer patients preserve fertility after cancer treatment. This qualitative study investigated: 1) Heterosexual couples' responses to potential fertility loss; 2) their process in making fertility preservation decisions; 3) their ethical and legal concerns, and 4) recommendations for other couples undergoing similar treatment. Semi-structured interviews were conducted with 12 heterosexual couples whose female partners were diagnosed with cancer and had received fertility preservation consultations within the past 5 years. Interviews were recorded, transcribed, and analyzed using grounded theory methods. The results indicated that couples have varied reactions to infertility. In most, spouses let the patients take the lead in, and supported, their fertility preservation decisions. Spouses recommended support to patients. Couples face challenges in making fertility preservation decisions. Input from, and support for, both partners is essential to ensure well-informed, high-quality fertility preservation decisions.

As compared to pediatric and older adult cancer patients, young adults are the only oncology group that has not demonstrated an increase in survival rates. Low treatment adherence rates have been one explanation for this discrepancy, although this hypothesis has not been explored specifically. Guided by the biopsychosocial model of health and wellness, this study compared the treatment adherence rates of 46 young adult cancer patients (ages 18-39 years) to 46 older adult cancer patients (ages 40 years and older). Adherence was measured by a dichotomized variable, as yes/no, adhering to radiation treatment and follow-up appointments recommended by the physician, 95% of the time. Additionally, gender and race were explored in relationship to adherence to radiation treatment and follow-up appointments. Demographic data were first extracted from the Cancer Registry of a Midwestern Hospital. Then radiation appointments and follow-up appointments were examined for each patient, in paper and/or computerized charts, to determine adherence rates. McNemar's test was used to compare young adults and older adult oncology patients' adherence rates, and Chi-square analysis was used to explore gender and race in relationship to adherence. Results indicated a lower adherence rate to follow-up appointments for younger adults as compared to older adults, with older adults adhering 3 -½ times more than younger adults. Gender was also related to follow-up appointment adherence, with males adhering better than females. This study contributes to positive social change by increasing the knowledge base of healthcare providers on adherence rates of young adult patients and reducing the dollars spent on treatment for re occurrences.

The concept of patient empowerment, although acknowledged by the medical community as important, is rarely understood and seldom given priority in the illness trajectory of the cancer patient. A pilot study of a Shared Care Model amongst haematological cancer patients highlighted the fact that some patients spoke of a sense of empowerment and an overall sense of greater control when more fully included in the treatment and management of their condition. The research which forms the basis of this thesis focused on the role of empowerment in the wellbeing of cancer patients. There were three objectives to be met by completing this research. Firstly, to demonstrate that empowerment is a uniquely identifiable concept and can be measured separately from other quality of life indicators. Secondly, the study sought to explore that concept that empowerment takes into account the way in which patients act upon their prognosis and optimise the outcomes of treatment. Thus it is believed that accessing tailored resources and support structures benefit cancer patients and those who are caring for them such as close family members and friends by helping the patient achieve an individual level of empowerment. Finally, the research sought to explore the concept that empowerment improves psychological outcome in patients. The benefits are increased empowerment and an active use of coping strategies amongst patients in order to regain a measure of control over their illness. The Patient Empowerment Scale was developed to measure empowerment as an individual construct. '...' The Patient Empowerment Scale (15 items) was shown to be a reliable measure of empowerment and fitted the model well. A qualitative methodological approach sought to address and explore the second and third concepts. In addition, the concept of empowerment as it relates to motivation and self-efficacy was investigated qualitatively using in-depth interviewing technique. A phenomenological methodology was used to explore the 'lived experience of cancer patients' in regard to regaining control of their illness and the management thereof. Participants were interviewed using concepts identified for the Patient Empowerment Scale such as support strategies and use of resources. A series of interviews with breast cancer patients were conducted whereby patients responded to a number of questions. The questions explored areas such as support mechanisms in relation to cancer, their relationships with health professionals and significant others and their attitude toward and use of other resources and support systems such as support groups, spirituality, complementary therapies. In addition their views on acceptance and adaptation to their altered health status were explored. Results The research confirmed that it is feasible to measure empowerment as a separate quality of life indicator. Furthermore, that empowerment is linked to motivation and self-efficacy beliefs. The research also demonstrated that there are a number of core areas which are fundamental to regaining control and increasing empowerment for patients. These core areas are linked to support mechanisms, willingness to adapt and to access resources tailored to meet their needs. Patient empowerment emerged as a key aspect of enhanced quality of life regardless of prognosis and improved psychological outlook.