O objetivo deste estudo é avaliar a eficácia de duas técnicas de acupuntura no controle da náusea e vômito em pacientes portadoras de Carcinoma Invasivo de mama, submetidas à quimioterapia anti-neoplásica. Foram incluídas no estudo 64 pacientes no período de março de 2003 a fevereiro de 2007, do Hospital A.C. Camargo com diagnóstico anatomopatológico de carcinoma invasivo da mama, candidatas à quimioterapia adjuvante com esquemas que incluem doxorrubicina e ciclofosfamida. O estudo foi realizado no primeiro ciclo de quimioterapia. As pacientes foram randomizados em 3 grupos: As pacientes do grupo A, receberam tratamento antiemético convencional com medicamentos. As pacientes do grupo B, receberam o tratamento antiemético convencional e aplicação de acupuntura clássica. As pacientes do grupo C, receberam tratamento convencional e aplicação de acupuntura auricular. As pacientes foram submetidas à auto-avaliação nos primeiros 21 dias após o início da quimioterapia, e conforme os critérios da Common toxicity criteria of the National Cancer Institute (NCI CTC), preencheram a ficha de coleta de dados em relação à náusea e vômito neste período. Quanto à náusea do primeiro ao sétimo dia, encontramos pelo teste de Kruskal-Wallis diferença significativa (p=0.040), sugerindo que a acupuntura diminui a intensidade de náusea deste período, e quanto à duração de dias de pior intensidade também foi significativo (p=0,037) ou seja a acupuntura diminui os dias de pior intensidade.Quando utilizamos o teste de Mann-Whitney encontramos diferença significativa da intensidade de náusea somente entre os grupos A e B (p=0,013), significando que a acupuntura clássica diminui a intensidade de náusea em relação ao grupo controle. Em relação aos dias de pior intensidade de náusea encontramos diferença entre os grupos A e B (p=0,043) e entre grupos B e C (p=0,010) ou seja, a acupuntura clássica diminui os dias de pior intensidade de náusea em relação aos grupos controle e acupuntura auricular. Quanto à gravidade do vômito, do primeiro ao sétimo dia, encontramos pelo teste de Kruskal-Wallis diferença significativa (p=0,036) entre os grupos, sugerindo que a acupuntura diminui a gravidade de vômitos. Pelo teste de Mann-Whitney entre os grupos A e B encontrou-se diferença significativa (p=0,017), ou seja, a acupuntura clássica diminui a gravidade de vômitos comparado ao grupo controle. Quanto ao número de dias de vômito de pior grau entre o primeiro e o sétimo dia encontramos ao aplicar o teste ANOVA diferença significativa entre os grupos (p=0,029), sendo que a acupuntura diminui o número de dias com vômito. O teste t aplicado para os grupos em pares revelou entre grupos A e B (p=0,035), portanto, a acupuntura clássica diminui o número de dias de vômito comparado ao grupo controle. Ao estudarmos a intensidade de náusea entre oitavo e vigésimo primeiro dia encontramos pelo teste de Kruskal-Wallis entre os grupos diferença significativa (p=0,022), sendo que a acupuntura diminui a náusea entre oitavo e vigésimo primeiro dia. Pelo teste de Mann-Whitney entre grupos aos pares encontrou-se diferença significativa entre os grupos A e B (p=0,016), houve diferença entre grupos A e C (p=0,049). Portanto, há diferença significativa entre grupos B e C em relação ao grupo A, ou seja, a acupuntura clássica e auricular diminuem a náusea entre oitavo e vigésimo primeiro dias em relação ao grupo controle. Conclusão: A acupuntura clássica diminui a intensidade e duração de náusea e vômito do primeiro ao sétimo dia pós-quimioterapia. A acupuntura clássica e auricular diminuem a intensidade de náusea do oitavo ao vigésimo primeiro dia pós-quimioterapia. / The purpose of this study is to evaluate the efficacy of two acupuncture techniques in the control of the nausea and vomiting in patients diagnosed of Invasive Breast Carcinoma submitted to chemotherapy. Sixty four patients with pathological diagnosis of Invasive Breast Carcinoma were included in the study during the period of March 2003 to February 2007 at Hospital A.C. Camargo. The patients received adjuvant chemotherapy including doxorubicin and ciclophosphamide. The study was developed during the first chemotherapy cycle. The patients were randomized in 3 groups: Patients of the group A received conventional antiemetic treatment with drug administration. Patients of the group B received conventional antiemetic treatment with drug administration and application of classic acupuncture. Patients of the group C received conventional antiemetic treatment with drug administration and application of auricular acupuncture. During the first 21 days, the patients performed self-reports after receiving the chemotherapy shot, according to the Common toxicity criteria of the National Cancer Institute (NCI CTC). They filled out the data collection instrument related to the nausea and vomiting in this period. As for the nausea from the first to the seventh day, we found statistical significancy (p=0.040) using the Kruskal - Wallis test, suggesting that acupuncture reduces the intensity of nausea in this period. Regarding length of days having worse intensity of nausea in this period, results were statistically significant by ANOVA test (p=0.037) with acupuncture decreasing the number of days of worse intensity. Through the Mann Whitney test, we also found significant difference regarding the intensity of nausea among the groups A and B (p=0.013), showing that classic acupuncture reduces the intensity of nausea when compared to the control group. In relation to the days of worse intensity of nausea we found difference among the groups A and B (p=0.043) and among groups B and C (p=0.010) meaning that classic acupuncture decreases the days of worse intensity of nausea when compared to control group and auricular acupuncture group. As for the severity of the vomit, from the first to the seventh day, we found through Kruskal - Wallis test a significant difference (p=0.036) among the groups, suggesting that acupuncture reduces the severity of the vomits. Using the Mann - Whitney test among the groups A and B a significant difference was found (p=0.017) meaning that classic acupuncture reduces the severity of vomits compared to the control group. As for the number of days of vomit in worse degree between the first and the seventh day, we used the ANOVA test and found significant difference among the groups (p=0.029) with acupuncture reducing the number of days with vomit. The t test applied for the groups in pairs revealed statistical significance among groups A and B (p=0.035) and therefore, classic acupuncture reduces the number of days of vomit compared to the control group. We have analysed the intensity of nausea between eighth and twentieth first day. Through the Kruskal - Wallis test, we found significant difference among the groups (p=0.022) showing that acupuncture also reduces the nausea between eighth and twentieth first day. Analyzing the groups in pairs with the Mann - Whitney test, we found significant difference among the groups A and B (p=0.016) and among groups A and C (p=0.049). Therefore, there is significant difference among groups B and C in relation to the group A. Classic acupuncture and auricular acupuncture reduce the nausea between eighth and twentieth first days when compared to control group. Conclusion: Classic acupuncture reduces the intensity and duration of nausea and vomit from the first to the seventh day after chemotherapy. Classic acupuncture and auricular acupuncture reduce the intensity of nausea from the eighth to the twentieth first day after chemotherapy.

Stroke is the third leading cause of mortality in the United States, and so far, no clinical interventions have been shown completely effective in stroke treatment. Stroke may result in hypoxia, glutamate release and oxidative stress. One approach for protecting neurons from excitotoxic damage in stroke is to attenuate receptor activity with specific antagonists. Disulfiram requires bio-activation to S-methyl N, N-diethylthiolcarbamate sulfoxide (DETC-MeSO). In vivo, DETC-MeSO is further oxidized to the sulfone which is carbamoylated forming Carbamathione, a glutathione adducts. Carbamathione proved to be useful as a pharmacological agent in the treatment of cocaine dependence with the advantage that it lacks ALDH2 inhibitory activity. Carbamathione is a partial NMDA glutamate antagonist. The purpose of this dissertation study is to evaluate the neuroprotective effects of Carbamathione drug on PC-12 cell line and to understand the protective mechanisms underlying in three stroke-related models: excessive glutamate, hypoxia/reoxygenation and bilateral carotid artery occlusion (BCAO). Carbamathione was administered 14 mg/kg subcutaneously for 4 days with the first injection occurring 30 min after occlusion in the mouse BCAO stroke model. Mice were subjected to the locomotor test, and the brain was analyzed for infarct size. Heat shock proteins, key proteins involved in apoptosis and endoplasmic reticulum (ER) stress, were analyzed by immunoblotting. Carbamathione reduced both cell death following hypoxia/reoxygenation and brain infarct size. It improved performance on the locomotor test. The level of pro-apoptotic proteins declined, and anti-apoptotic, P-AKT and HSP27 protein expressions were markedly increased. We found that Carbamathione suppresses the up- regulation of Caspase-12, Caspase-3 and significantly declined ER stress protein markers GRP 78, ATF4, XBP-1, and CHOP. Carbamathione can down- regulate ATF 4 and XBP1 expression, indicating that Carbamathione inhibits the ER stress induced by hypoxia/reoxygenation through suppressing PERK and IRE1 pathways. Carbamathione elicits neuroprotection through the preservation of ER resulting in reduction of apoptosis by increase of anti-apoptotic proteins and decrease of pro-apoptotic proteins. Carbamathione can suppress the activation of both PERK and IRE1 pathways in PC-12 cell cultures and has no inhibitory effect on ATF6 pathway. These findings provide promising and rational strategies for stroke therapy. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2017. / FAU Electronic Theses and Dissertations Collection

by Tsang Wing Pui. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 195-215). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Breast cancer is the most common cancer among females and it is the leading cause of death in mid-age women. Epidemiological studies indicate that Asian women have a lower incidence of breast cancer compared with their counterparts in the West, which soy consumption has been suggested as a contributory factor. Soy and soy-based food contain a rich amount of phytoestrogens, which are suggested to be protective against cancer. Cyclooxygenase (COX) is the rate-limiting enzyme for the conversion of arachidonic acid to prostaglandins. Recent studies have revealed that up-regulation of cyclooxygenase-2 (COX-2), an isoform of COX, plays an important role in tumorigenesis and metastasis. COX-2 may facilitate carcinogenesis in a number of means, may include altering cell proliferation and apoptosis, enhancing angiogenesis and suppressing immune surveillance. Clinical examinations of breast cancer specimens indicated that COX-2 is overexpressed. In the present study, we investigated the effect of flavonoids on COX-2 expression in human breast cells. / Our results showed that daidzein and its metabolite eqoul, genistein, butein, isoliquiritigenin (ILN) and apigenin could inhibit phorbol 12-myristate 13-acetate (PMA)-induced COX-2 expression in breast cells MCF-7 and MCF-10A. The inhibitory effects were in concentration-dependent manners. Real-time PCR and western blot analysis showed that the flavonoids suppressed the induced mRNA and protein expression. Suppression could be observed in concentration as low as 0.1 muM. Luciferase reporter assay indicated that the inhibition was at the gene transactivation level. Further investigation using truncated hCOX-2 promoter plasmids revealed that the AP-1 site (-67/-61) and cyclic AMP response element (CRE) site (-59/-53) on hCOX-2 promoter were responsible for the suppression. Electrophoretic mobility shift assay results further confirmed that the flavonoids acted through inhibiting AP-1/CREB DNA binding to suppress the expression. / To examine the possible upstream signal transduction pathways involved, inhibitors of protein kinase A (PKA), protein kinase (PKC) and mitogen activated protein kinase (MAPK) were employed. Reporter gene assay revealed a possible involvement of ERK1/2 MAP kinase in AP-1 and/or CRE activation of hCOX-2 promoter. Taken together, these results suggested that the phytochemicals down-regulated PMA-induced COX-2 expression by counteracting AP-1 and CRE sites via the modulation of MAPK pathway. The findings might have significant implications in the chemopreventive and chemotherapeutic applications of flavonoids in breast cancer. / Lau, Tak Yi. / "December 2007." / Adviser: Lai Kwok Leung. / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4734. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 142-164). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

by Po Chu, Leung. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 141-169). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abbreviation --- p.iii / Abstract --- p.vii / Chinese Abstract --- p.x / Table of Contents --- p.xii / Chapter Chapter 1: --- General Introduction / Chapter 1.1 --- Hematopoiesis --- p.1 / Chapter 1.1.1 --- The Development of Hematopoietic Progenitor Cells --- p.1 / Chapter 1.1.2 --- Hematopoietic Growth Factors --- p.3 / Chapter 1.1.3 --- Transcriptionl Factors Involved in Lineage Commitment of Hematopoietic Progenitor Cells --- p.5 / Chapter 1.2 --- Leukemia --- p.7 / Chapter 1.2.1 --- Occurrence and Classification of Leukemia --- p.7 / Chapter 1.2.2 --- The Pathological Features and Etiology of Leukemia --- p.10 / Chapter 1.2.3 --- The Molecular Basis of Leukemia --- p.13 / Chapter 1.2.4 --- Current Therapeutic Strategies --- p.14 / Chapter 1.2.4.1 --- Conventional Therapies for Leukemia --- p.14 / Chapter 1.2.4.2 --- Induction of Cell Differentiation and Apoptosis for Treatment of Leukemia --- p.16 / Chapter 1.2.5 --- The Use of Murine Myelomonocytic Leukemia WEHI-3B JCS Cells As a Model for the Study of Leukemia Cell Proliferation， Differentiation and Survival --- p.22 / Chapter 1.3 --- Taxol: A Novel Anti-cancer Agent --- p.23 / Chapter 1.3.1 --- Discovery and Action Mechanism --- p.23 / Chapter 1.3.2 --- Metabolism and Toxicity of Taxol --- p.27 / Chapter 1.3.3 --- The Biological Activities of Taxol --- p.28 / Chapter 1.3.4 --- The Anti-tumor Effects of Taxol --- p.30 / Chapter 1.3.5 --- The Effects of Taxol on Leukemia --- p.31 / Chapter 1.4 --- Aims and Scopes of This Investigation --- p.32 / Chapter Chapter 2: --- Materials and Methods / Chapter 2.1 --- Materials --- p.35 / Chapter 2.1.1 --- Mice --- p.35 / Chapter 2.1.3 --- "Culture Media,Buffer and Other Solutions" --- p.37 / Chapter 2.1.4 --- Radioisotope and Scintillation Fluid --- p.39 / Chapter 2.1.5 --- Taxol --- p.40 / Chapter 2.1.6 --- Recombinant Cytokines --- p.40 / Chapter 2.1.7 --- Vitamin Analogs --- p.42 / Chapter 2.1.8 --- Various Signal Transduction Pathway Activators and Inhibitors --- p.42 / Chapter 2.1.9 --- Monoclonal Antibodies and Buffers for Flow Cytometry --- p.43 / Chapter 2.1.10 --- Reagents and Chemicals for Gene Expression Study --- p.45 / Chapter 2.1.11 --- Chemical Solutions and Buffers for Western Blot --- p.50 / Chapter 2.1.12 --- Reagents for Colony Assay --- p.54 / Chapter 2.2 --- Methods --- p.55 / Chapter 2.2.1 --- Culture of Leukemia Cell Lines --- p.55 / Chapter 2.2.2 --- Treatment of Leukemia Cells with Various Drugs and Cytokines --- p.55 / Chapter 2.2.3 --- Cell Morphological Study --- p.55 / Chapter 2.2.4 --- Determination of Leukemia Cell Survival and Proliferation --- p.56 / Chapter 2.2.5 --- Colony Assay --- p.56 / Chapter 2.2.6 --- Flow Cytometry Analysis --- p.57 / Chapter 2.2.6.1 --- Surface Antigen Immunophenotyping --- p.57 / Chapter 2.2.6.2 --- Assay of Endocytic Activity --- p.58 / Chapter 2.2.6.3 --- Cell Cycle /DNA Content Evaluation --- p.58 / Chapter 2.2.7 --- Gene Expression Study --- p.59 / Chapter 2.2.7.1 --- Preparation of Total Cellular RNA --- p.59 / Chapter 2.2.7.2 --- Reverse Transcription --- p.60 / Chapter 2.2.7.3 --- Polymerase Chain Reaction (PCR) --- p.60 / Chapter 2.2.7.4 --- Agarose Gel Electrophoresis --- p.61 / Chapter 2.2.8 --- DNA Fragmentation Analysis --- p.61 / Chapter 2.2.9 --- Protein Expression Study --- p.62 / Chapter 2.2.9.1 --- Protein Extraction --- p.62 / Chapter 2.2.9.2 --- Quantification of the Protein --- p.62 / Chapter 2.2.9.3 --- Western Blot Analysis --- p.63 / Chapter 2.2.10 --- Statistical Analysis --- p.64 / Chapter Chapter 3: --- Results / Chapter 3.1 --- Effects of Taxol on the Proliferation and Apoptosis of the Murine Myeloid Leukemia Cells --- p.65 / Chapter 3.1.1 --- Growth-Inhibitory Effects of Taxol on Murine Myeloid Leukemia WEHI-3B JCS cells --- p.65 / Chapter 3.1.2 --- Cytotoxic Effects of Taxol on Murine Bone Marrow Cells and Myeloid Leukemia WEHI-3B JCS Cells --- p.69 / Chapter 3.1.3 --- Anti-proliferative Effect of Taxol on Different Leukemia Cell Lines --- p.72 / Chapter 3.1.4 --- Effects of Taxol on the Cell Cycle Kinetics of WEHI-3B JCS Cells --- p.81 / Chapter 3.1.5 --- Induction of DNA Fragmentation of WEHI-3B JCS cells by Taxol --- p.83 / Chapter 3.1.6 --- Effect of Taxol on the Clonogenicity of WEHI-3B JCS Cells In Vitro and Tumorigenicity In Vivo --- p.86 / Chapter 3.2 --- Effects of Taxol on the Induction of Monocytic Cell Differentiation in Murine Myeloid Leukemia Cells --- p.88 / Chapter 3.2.1 --- Morphological Changes in Taxol-Treated Murine Myelomonocytic Leukemia WEHI-3B JCS Cells --- p.88 / Chapter 3.2.2 --- Surface Antigen Immunophenotyping of Taxol-treated WE HI-3B cells --- p.91 / Chapter 3.2.3 --- Endocytic Activity of Taxol-treated WEHI-3B JCS cells --- p.95 / Chapter 3.3 --- Modulatory Effect of Taxol and Cytokines on the Proliferation of WEHI- 3B JCS Cells --- p.96 / Chapter 3.4 --- Modulatory Effect of Taxol and Physiological Differentiation Inducers on the Proliferation of WEHI-3B JCS cells --- p.103 / Chapter 3.5 --- The Possible Involvement of Protein Kinase C in the Anti-proliferative Activity of Taxol on WEHI-3B JCS Cells --- p.106 / Chapter 3.6 --- Modulation of Apoptotic Gene Expression in Taxol-treated WEHI-3B JCS cells --- p.113 / Chapter 3.7 --- Modulatory Effects of Taxol on the Protein Expression of WEHI-3B JCS Cells --- p.119 / Chapter Chapter 4: --- Discussion and Conclusions / Chapter 4.1 --- "Effects of Taxol on the Proliferation,Differentiation and Apoptosis of the Murine Myeloid Leukemia Cells" --- p.126 / Chapter 4.2 --- "Modulatory Effects of Taxol, Cytokines and Physiological Differentiation Inducers on the Proliferation of the Myelomonocytic Leukemia WEHI-3B JCS Cells" --- p.132 / Chapter 4.3 --- The Possible Involvement of Protein Kinase C in Anti-proliferative Activity of Taxol on WEHI-3B JCS Cells --- p.136 / Chapter 4.4 --- The Modulation of Apoptosis Gene Expression in Taxol-treated WEHI-3B JCS Cells --- p.137 / Chapter 4.5 --- The Modulation of Protein Expression in Taxol-treated WEHI-3B JCS Cells --- p.138 / Chapter 4.6 --- Conclusions and Future Perspectives --- p.139 / References --- p.141

Siu Pak-yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 158-174). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Contents --- p.vi / List of Figures and Tables --- p.xiii / List of Abbreviations --- p.xviii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Characteristics of Arsenic Compound --- p.1 / Chapter 1.1.1 --- Arsenic Compounds are Used as Poison --- p.1 / Chapter 1.1.2 --- Arsenic Compounds are Used as Medicine --- p.2 / Chapter 1.2 --- Arsenic Trioxide is a Traditional Chinese Medicine --- p.3 / Chapter 1.3 --- Properties of Arsenic Trioxide --- p.5 / Chapter 1.4 --- Use of Arsenic Trioxide in Cancer Treatment --- p.7 / Chapter 1.4.1 --- Arsenic Trioxide as a Therapeutic Agent in the Treatment of Acute Promyelocytic Leukemia --- p.7 / Chapter 1.4.1.1 --- Characteristics of Acute Promyelocytic Leukemia --- p.7 / Chapter 1.4.1.2 --- Treatment of Acute Promyelocytic Leukemia with All-Trans Retinoic Acid --- p.10 / Chapter 1.4.1.3 --- Treatment of Acute Promyelocytic Leukemia with Arsenic Trioxide --- p.11 / Chapter 1.4.1.4 --- Action Mechanism of Arsenic Trioxide --- p.13 / Chapter 1.4.2 --- Arsenic Trioxide as a Therapeutic Agent in the Treatment of Non-APL Leukemia --- p.15 / Chapter 1.4.3 --- Arsenic Trioxide as a Therapeutic Agent in the Treatment of Solid Tumors --- p.16 / Chapter 1.5 --- Human Hepatocellular Carcinoma --- p.16 / Chapter 1.5.1 --- The Incidence of Liver Cancer --- p.16 / Chapter 1.5.2 --- Classification of Liver Cancer --- p.17 / Chapter 1.6 --- Aim of the Project --- p.17 / Chapter 1.6.1 --- In Vitro Study of the Effect of Arsenic Trioxide on HepG2 Cells --- p.19 / Chapter 1.6.2 --- In Vivo Study of the Effect of Arsenic Trioxide by Tumor-Bearing Nude Mice Model --- p.20 / Chapter 1.6.3 --- "In Vitro Study of the Effect of Arsenic Trioxide on Multidrug-Resistant Human Hepatocellular Carcinoma Cell Line, R-HepG2" --- p.22 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.24 / Chapter 2.1.1 --- Cell Lines and Culture Medium --- p.24 / Chapter 2.1.1.1 --- Cell Lines --- p.24 / Chapter 2.1.1.2 --- Culture Medium --- p.25 / Chapter 2.1.2 --- Chemicals --- p.26 / Chapter 2.1.3 --- Reagents and Buffers --- p.27 / Chapter 2.1.3.1 --- Phosphate Buffered Saline (PBS) --- p.27 / Chapter 2.1.3.2 --- "3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) Solution" --- p.27 / Chapter 2.1.3.3 --- Reagents for DNA Fragmentation Assay --- p.21 / Chapter 2.1.3.3.1 --- DNA Lysis Buffer --- p.27 / Chapter 2.1.3.3.2 --- Tris-EDTA (TE) Buffer --- p.27 / Chapter 2.1.3.3.3 --- Tris-Acetate (TAE) Buffer --- p.28 / Chapter 2.1.3.3.4 --- Proteinase K and Ribonuclease A (RNase A) --- p.28 / Chapter 2.1.3.3.5 --- 6X DNA Loading Dye --- p.28 / Chapter 2.1.3.3.6 --- One Hundred Base-Pair DNA Ladder --- p.28 / Chapter 2.1.3.4 --- Reagents for Western Blot Analysis --- p.29 / Chapter 2.1.3.4.1 --- SDS Lysis Buffer --- p.29 / Chapter 2.1.3.4.2 --- 4X Lower Gel Buffer --- p.29 / Chapter 2.1.3.4.3 --- 4X Upper Gel Buffer --- p.29 / Chapter 2.1.3.4.4 --- 10X SDS Running Buffer --- p.29 / Chapter 2.1.3.4.5 --- 2X SDS Sample Loading Dye --- p.30 / Chapter 2.1.3.4.6 --- Electroblotting Buffer --- p.30 / Chapter 2.1.3.4.7 --- Tris-Buffered Saline with 01% Tween-20 (TBS-T) --- p.30 / Chapter 2.1.3.4.8 --- Lysis Buffer for Detection of the Release of Cytochrome C --- p.31 / Chapter 2.1.3.5 --- Propidium Iodide (PI) --- p.31 / Chapter 2.1.3.6 --- "5，5 ´ة,6,6´ة-tetrachloro-1,1',3,3 '-tetraethylbenzimidazolyl carbocyanine Iodide (JC-1)" --- p.31 / Chapter 2.1.3.7 --- Reagents for In Vivo Study --- p.32 / Chapter 2.1.3.7.1 --- Saline --- p.32 / Chapter 2.1.3.7.2 --- Homogenizing Buffer --- p.32 / Chapter 2.1.3.7.3 --- 10% Buffered Formalin --- p.32 / Chapter 2.1.3.7.4 --- Acid Alcohol --- p.32 / Chapter 2.1.3.7.5 --- Scott's Tap Water --- p.32 / Chapter 2.1.3.7.6 --- 0.5% Aqueous Eosin --- p.33 / Chapter 2.2 --- Methods --- p.33 / Chapter 2.2.1 --- MTT Assay --- p.33 / Chapter 2.2.2 --- Trypan Blue Exclusion Assay --- p.34 / Chapter 2.2.3 --- Analysis of Cell-Cycle Phase Distribution by Flow Cytometry with PI Staining --- p.34 / Chapter 2.2.4 --- DNA Fragmentation Assay --- p.35 / Chapter 2.2.5 --- Quantification of Apoptosis by Flow Cytometry with Annexin V-PI Staining --- p.36 / Chapter 2.2.6 --- Assessment of the Change in Mitochondrial Membrane Potential (ΔΦm) --- p.37 / Chapter 2.2.7 --- Western Analysis --- p.38 / Chapter 2.2.8 --- Glucose Uptake Assay --- p.40 / Chapter 2.2.9 --- ATP Production Assay --- p.41 / Chapter 2.2.10 --- In Vivo Study --- p.44 / Chapter 2.2.10.1 --- Animal Model --- p.44 / Chapter 2.2.10.2 --- Cell Line --- p.44 / Chapter 2.2.10.3 --- Treatment with Arsenic Trioxide --- p.44 / Chapter 2.2.10.4 --- Assessment of the Anti-Cancer Activity of Arsenic Trioxide --- p.45 / Chapter 2.2.10.5 --- Tissue Sample Preparation --- p.45 / Chapter 2.2.10.5.1 --- Preparation of Plasma --- p.45 / Chapter 2.2.10.5.2 --- Preparation of Liver Tissue Homogenate --- p.46 / Chapter 2.2.10.5.3 --- Preparation of Cytosolic Fraction --- p.46 / Chapter 2.2.10.6 --- Measurement of the Plasma Enzyme Activity --- p.46 / Chapter 2.2.10.6.1 --- "Plasma Creatine Kinase (CK) Activity, Plasma Lactate Dehydrogenase (LDH) Activity, Plasma Alanine Transaminase (ALT) Activity and Plasma Asparate Transaminase (AST) Activity" --- p.46 / Chapter 2.2.10.7 --- Preparation of Tissue for Light Microscopic Study --- p.48 / Chapter 2.2.10.8 --- Measurement of the Basal Reduced Glutathione (GSH) Level of Liver Tissue --- p.51 / Chapter 2.2.10.9 --- "Measurement of the Activity of Antioxidant Enzyme, Glutathione S-Transferase (GST) of Liver Tissue" --- p.53 / Chapter 2.3 --- Statistical Analysis --- p.54 / Chapter Chapter 3 --- "In Vitro Study of Arsenic Trioxide on Acute Promyelocytic Leukemia Cell Line, NB-4" / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Principle of Flow Cytometry with Annexin V-PI Staining --- p.56 / Chapter 3.3 --- The Effect of Arsenic Trioxide on Cell Proliferation of NB-4 Cells --- p.59 / Chapter 3.4 --- Study of the Action Mechanism of Arsenic Trioxide upon Treatment of NB-4 Cells --- p.61 / Chapter 3.5 --- Summary --- p.63 / Chapter Chapter 4 --- "In Vitro Study of Arsenic Trioxide on Human Hepatocellular Carcinoma Cell Line, HepG2" / Chapter 4.1 --- Introduction --- p.64 / Chapter 4.2 --- The Effect of Arsenic Trioxide on Cell Proliferation of HepG2 Cells by MTT Assay --- p.66 / Chapter 4.3 --- The Effect of Arsenic Trioxide on HepG2 Cells at Clinically Achievable Concentration --- p.68 / Chapter 4.3.1 --- The Cytotoxicity of Arsenic Trioxide on HepG2 Cells by Trypan Blue Exclusion Assay --- p.68 / Chapter 4.3.2 --- The Effect of Arsenic Trioxide on Cell-Cycle Phase Distribution --- p.71 / Chapter 4.3.3 --- The Underlying Mechanism of the Cytotoxic Effect of Arsenic Trioxide 一 Necrosis or Apoptosis? --- p.74 / Chapter 4.3.3.1 --- DNA Fragmentation Assay --- p.74 / Chapter 4.3.3.2 --- Flow Cytometry with Annexin V-PI Staining --- p.76 / Chapter 4.3.3.3 --- Brief Conclusion --- p.78 / Chapter 4.3.4 --- The Study of the Mechanism of Apoptotic Pathway --- p.78 / Chapter 4.3.4.1 --- Activation of Caspase-3 upon Arsenic Trioxide Treatment --- p.79 / Chapter 4.3.4.2 --- The Participation of Mitochondria in Arsenic Trioxide-Induced Apoptosis --- p.81 / Chapter 4.3.4.2.1 --- The Change in Mitochondrial Membrane Potential upon Arsenic Trioxide Treatment --- p.81 / Chapter 4.3.4.2.2 --- The Study of the Release of Cytochrome C from the Mitochondria to Cytosol upon Treatment with Arsenic Trioxide --- p.85 / Chapter 4.3.4.2.3 --- Brief Conclusion --- p.87 / Chapter 4.4 --- Arsenic Trioxide Mediated Its Effect via Other Action Mechanisms --- p.87 / Chapter 4.4.1 --- The Effect of Arsenic Trioxide on the Expression of Glucose Transporters 1 and2 --- p.88 / Chapter 4.4.2 --- The Effect of Arsenic Trioxide on Glucose Uptake --- p.91 / Chapter 4.4.3 --- The Effect of Arsenic Trioxide on ATP Production --- p.93 / Chapter 4.4.4 --- Brief Conclusion --- p.93 / Chapter 4.5 --- Summary --- p.95 / Chapter Chapter 5 --- In Vivo Study of Arsenic Trioxide on HepG2-Bearing Nude Mice / Chapter 5.1 --- Introduction --- p.96 / Chapter 5.2 --- Treatment with Arsenic Trioxide --- p.97 / Chapter 5.3 --- Assessment of the Anti-Tumor Effect of Arsenic Trioxide --- p.99 / Chapter 5.4 --- The Effect of Arsenic Trioxide toward Normal Tissues --- p.103 / Chapter 5.4.1 --- The Effect of Arsenic Trioxide on Liver --- p.104 / Chapter 5.4.1.1 --- Morphological Study --- p.104 / Chapter 5.4.1.2 --- Enzymatic Study --- p.107 / Chapter 5.4.1.3 --- Brief Conclusion --- p.107 / Chapter 5.4.2 --- The Effect of Arsenic Trioxide on Heart --- p.110 / Chapter 5.4.2.1 --- Morphological Study --- p.110 / Chapter 5.4.2.2 --- Enzymatic Study --- p.112 / Chapter 5.4.2.3 --- Brief Conclusion --- p.112 / Chapter 5.5 --- Involvement of the Glutathione Redox System --- p.115 / Chapter 5.5.1 --- Basal GSH Level --- p.115 / Chapter 5.5.2 --- The Activity of Glutathion S-Transferase --- p.117 / Chapter 5.5.3 --- Brief Conclusion --- p.117 / Chapter 5.6 --- Summary --- p.120 / Chapter Chapter 6 --- "In Vitro Study of Arsenic Trioxide on Multidrug-Resistant Human Hepatocellular Carcinoma Cell Line, R-HepG2" / Chapter 6.1 --- Introduction --- p.121 / Chapter 6.2 --- The Effect of Doxorubicin on the Parental HepG2 Cells and R-HepG2 Cells by MTT Assay --- p.123 / Chapter 6.3 --- The Effect of Arsenic Trioxide on Cell Proliferation of R-HepG2 Cells by MTT Assay --- p.126 / Chapter 6.4 --- The Effect of Arsenic Trioxide on Cell-Cycle Phase Distribution of R-HepG2 Cells --- p.129 / Chapter 6.5 --- Trioxide on R-HepG2 Cells ´ؤ Necrosis or Apoptosis? --- p.131 / Chapter 6.5.1 --- DNA Fragmentation Assay --- p.131 / Chapter 6.5.2 --- Flow Cytometry with Annexin V-PI Staining --- p.133 / Chapter 6.5.3 --- Brief Conclusion --- p.133 / Chapter 6.6 --- Examination of the Probable Involvement of Arsenic Trioxide as a Substrate of P-Glycoprotein --- p.135 / Chapter 6.7 --- Summary --- p.137 / Chapter Chapter 7 --- Discussion / Chapter 7.1 --- The Significance of the Study of Arsenic Trioxide in the Treatment of Arsenic Trioxide --- p.138 / Chapter 7.2 --- Comparison of Preparation of Drug in Present Study with Others --- p.140 / Chapter 7.3 --- Effect of Arsenic Trioxide on Human Hepatocellular Carcinoma --- p.142 / Chapter 7.4 --- Mechanism Study of Arsenic Trioxide --- p.142 / Chapter 7.5 --- Dosage of Arsenic Trioxide Used in In Vivo Study --- p.152 / Chapter 7.6 --- Cytotoxicity of Arsenic Trioxide toward Normal Tissues --- p.153 / Chapter 7.7 --- "Effect of Arsenic Trioxide on Multidrug-Resistant Human Hepatocellular Carcinoma Cell Line, R-HepG2" --- p.154 / Chapter 7.8 --- Conclusions and Future Prospect --- p.156 / Chapter Chapter 8 --- References / Chapter 8.1 --- English References --- p.158 / Chapter 8.2 --- Chinese References --- p.174 / Chapter 8.3 --- Online References --- p.174

Chan Ping Kei. / Thesis submitted in: December 2001. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 135-143). / Abstracts in English and Chinese. / ACKNOWLEGEMENTS --- p.i / ABSTRACT --- p.ii / TABLE OF CONTECTS --- p.v / LIST OF FIGURE --- p.viii / LIST OF ABBREVIATIONS --- p.xii / CHAPTER / Chapter 1 --- INTRODUCTION / Chapter 1.1 --- Epidemilogy --- p.1 / Chapter 1.2 --- Malaria disease --- p.1 / Chapter 1.3 --- Life cycle of Malaria --- p.1 / Chapter 1.4 --- Current measure to control Malaria --- p.6 / Chapter 1.5 --- Anti-malaria vaccine candidate --- p.7 / Chapter 1.6 --- Anti-erythrocytic malaria vaccine MSP-1 --- p.10 / Chapter 1.7 --- Baculovirus Expression System --- p.20 / Chapter 1.8 --- hexa-histidine tagged fusion protein --- p.25 / Chapter 1.9 --- IMAC --- p.26 / Chapter 1.10 --- Aim of study --- p.26 / Chapter 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.29 / Chapter 2.2 --- Methods --- p.40 / Chapter 3 --- CONSTRUCTION AND CHARACTERIZATION OF RECOMBINANT BmNPV CARRYING PfMSP-l42 / Chapter 3.1 --- Cloning of C-HisPfMSP-l42 into pBM030 --- p.71 / Chapter 3.2 --- Construction of Recombinant BmNPV Carrying PfMSP-l42 --- p.72 / Chapter 3.3 --- Purification of Recombinant BmNPVs --- p.78 / Chapter 3.4 --- In vitro expression of Recombinant --- p.80 / Chapter 3.5 --- In Vivo Expression of Recombinant PfMSP-l42 Protein --- p.80 / Chapter 4 --- PURIFICATION OF BmNPV-EXPRESSED RECOMBINANT C- TERMIAL HEXA-HIS-TAGGED PfMSP-l42 PROTEIN / Chapter 4.1 --- Nickel ion charged Chelating Sepharose Fast Flow (immobilized metal affinity chromatography) --- p.88 / Chapter 4.2 --- POROS HS/M (Strong Cation Exchanger) --- p.105 / Chapter 4.3 --- Combination of chromatographic separations --- p.107 / Chapter 5 --- CHARACTERIZATION OF RECOMBINANT C-HISPfMSP-l42 PROTEIN / Chapter 5.1 --- Proper formation of disulphide bridges in epidermal growth factor (EGF) like domains --- p.115 / Chapter 5.2 --- Characterization of the integrity of hexa-histidines residue on recombinant PfMSP-142 protein --- p.117 / Chapter 5.3 --- Immunogenicity of Recombinant C-HisPfMSP-l42 Protein --- p.117 / Chapter 6 --- DISCUSSION / Chapter 6.1 --- Construction of recombinant BmNPV carrying HisPfMSP-l42 --- p.122 / Chapter 6.2 --- Expression of recombinant HisPfMSP-l42 proteins --- p.123 / Chapter 6.3 --- Purification of recombinant C-HisPfMSP-l42 protein --- p.125 / Chapter 6.4 --- Characterization of recombinant C-HisPfMSP-l42 protein --- p.128 / Chapter 6.5 --- Future prospects --- p.130 / REFERENCE --- p.135 / APPENDICES / Chapter 1. --- Appearance of Mulberry leaves / Chapter 2. --- Biomark 2000 (Beckman) program for sandwich ELISA protocol / Chapter 3. --- Nucleotide Sequence of PfMSP-l42 3D7 Isolate / Chapter 4. --- Nucleotide sequence of PfMSP-l42 FVO isolate / Chapter 5. --- Efficiency of the mAb5.2 immunoaffinity column in purifying the recombinant PfMSP-l42 protein

Yip Nga-lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 151-164). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese Version) --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Tables --- p.xi / List of Figures --- p.xii / List of Abbreviations --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.6 / Chapter 2.1 --- Medicinal Plants and Herbs --- p.6 / Chapter 2.1.1 --- Antitumor activities --- p.8 / Chapter 2.1.1.1 --- Successful examples - Camptothecin --- p.8 / Chapter 2.1.1.2 --- Successful examples - Taxol --- p.13 / Chapter 2.2 --- Compositae and its Traditional Functions --- p.16 / Chapter 2.3 --- Antitumor Activity of Compositae --- p.19 / Chapter 2.3.1 --- Emilia sonchifolia --- p.19 / Chapter 2.3.2 --- Silymarin --- p.20 / Chapter 2.4 --- Wedelia Species --- p.22 / Chapter 2.4.1 --- Hepatoprotective effect --- p.22 / Chapter 2.4.2 --- Hypoglycemic effect --- p.23 / Chapter 2.4.3 --- Antimicrobial activity --- p.23 / Chapter 2.4.4 --- Antinociceptive activity --- p.24 / Chapter 2.4.5 --- Antifeedant and antifungal activities --- p.24 / Chapter 2.4.6 --- Trypanosomicidal effect --- p.25 / Chapter 2.4.7 --- Chemical constituents --- p.25 / Chapter 2.5 --- Sesquiterpene lactone --- p.27 / Chapter 2.6 --- Cell Cycle Control and Apoptosis --- p.30 / Chapter 2.6.1 --- Cell cycle and its control in cancer therapy --- p.30 / Chapter 2.6.2 --- Apoptosis --- p.36 / Chapter 2.7 --- Four Selected Compositae Species Used in the Study --- p.42 / Chapter 2.7.1 --- Dendranthema indicum (野菊花) --- p.42 / Chapter 2.7.2 --- Dendranthema morifolium (菊花) --- p.43 / Chapter 2.7.3 --- Taraxacum mongolicum --- p.44 / Chapter 2.7.4 --- Wedelia trilobata (三裂葉蟛蜞菊） --- p.45 / Chapter Chapter 3 --- Materials and Methods / Chapter 3.1 --- Extraction --- p.47 / Chapter 3.1.1 --- Water extraction --- p.47 / Chapter 3.1.2 --- NaOH extraction --- p.48 / Chapter 3.1.3 --- Ethanol precipitation --- p.50 / Chapter 3.1.4 --- Bioactivity guided fractionation --- p.50 / Chapter 3.1.4.1 --- Macroporous resin column (D101) --- p.50 / Chapter 3.1.4.2 --- Silica gel 60 column --- p.51 / Chapter 3.1.5 --- High Performance Liquid Chromatography (HPLC) analysis --- p.53 / Chapter 3.2 --- Characterization --- p.54 / Chapter 3.2.1 --- Chemical tests --- p.54 / Chapter 3.2.1.1 --- Alkaloids --- p.54 / Chapter 3.2.1.1.1 --- Mayer reagent --- p.54 / Chapter 3.2.1.1.2 --- Dragendorff reagent --- p.54 / Chapter 3.2.1.1.3 --- Wagner reagent --- p.55 / Chapter 3.2.1.2 --- Lactones and coumarins --- p.55 / Chapter 3.2.1.2.1 --- Ferric hydroxamine acid test --- p.55 / Chapter 3.2.1.2.2 --- Emersen reagent --- p.56 / Chapter 3.2.1.3 --- Flavonoids --- p.56 / Chapter 3.2.1.3.1 --- Shinoda test --- p.56 / Chapter 3.2.1.3.2 --- Aluminum chloride reagent --- p.56 / Chapter 3.2.1.4 --- Sterols --- p.57 / Chapter 2.2.1.4.1 --- Liebermann-Buchard test --- p.57 / Chapter 3.2.1.4.2 --- Salkowski reaction --- p.57 / Chapter 3.2.1.5 --- Saponins --- p.57 / Chapter 3.2.1.6 --- Carbohydrates --- p.58 / Chapter 3.2.1.6.1 --- Molisch reagent --- p.58 / Chapter 3.2.1.6.2 --- Aniline acetate reaction --- p.58 / Chapter 3.2.1.7 --- Terpenoids --- p.58 / Chapter 3.2.1.7.1 --- Vanillin reagent --- p.58 / Chapter 3.2.1.7.2 --- Carr-price reagent --- p.59 / Chapter 3.2.1.8 --- Anthraquinone --- p.59 / Chapter 3.2.1.8.1 --- Borntrager reaction --- p.59 / Chapter 3.2.1.8.2 --- Magnesium acetate reagent --- p.59 / Chapter 3.2.2 --- X-ray crystallography --- p.60 / Chapter 3.3 --- In vitro Assay --- p.61 / Chapter 3.3.1 --- Cell lines --- p.61 / Chapter 3.3.2 --- Maintenance of cell lines --- p.61 / Chapter 3.3.3 --- In vitro antitumor assay --- p.62 / Chapter 3.3.3.1 --- Trypan blue exclusion method --- p.63 / Chapter 3.3.3.2 --- MTT assay method --- p.65 / Chapter 3.3.3.3 --- Determination of IC50 --- p.66 / Chapter 3.3.4 --- Cytotoxicity assay on normal cell lines --- p.67 / Chapter 3.4 --- In vivo Assay --- p.68 / Chapter 3.4.1 --- Animals --- p.68 / Chapter 3.4.2 --- Maintenance of Sarcoma 180 cell line --- p.68 / Chapter 3.4.3 --- Tumor inoculation --- p.69 / Chapter 3.4.4 --- Preparation of samples --- p.69 / Chapter 3.4.5 --- In vivo antitumor assay --- p.70 / Chapter 3.4.5.1 --- Antitumor effect of WT4-4 on Sarcoma 180 solid tumor --- p.70 / Chapter 3.4.5.2 --- Effect of 5% DMSO in sterile PBS --- p.70 / Chapter 3.4.6 --- Body weight change --- p.71 / Chapter 3.5 --- DNA Agarose Gel Electrophoresis --- p.73 / Chapter 3.6 --- Statistical Analysis --- p.74 / Chapter Chapter 4 --- Results / Chapter 4.1 --- Extraction and characterization of Wedelia trilobata (WT) --- p.76 / Chapter 4.1.1 --- Percentage of yield in extraction and fractionation of WT --- p.76 / Chapter 4.1.2 --- HPLC chromatograms of WT fractions and the purified component (crystal) --- p.77 / Chapter 4.1.3 --- Phytochemical groups of WT4-4 --- p.78 / Chapter 4.1.4 --- Results on X-ray crystallography of the isolated crystal from WT4-4 A --- p.78 / Chapter 4.2 --- In vitro Antitumor Assay --- p.95 / Chapter 4.2.1 --- "Effects ofDIl, DM1, TM1 and WT1 on suspension cancer cell line" --- p.95 / Chapter 4.2.2 --- Effects of WT fractions and the purified component (crystal) on suspension cell lines --- p.95 / Chapter 4.2.3 --- Effects of WT fractions on adhesive cancer cell lines --- p.98 / Chapter 4.2.4 --- Effects of WT4-4 on normal cell lines --- p.98 / Chapter 4.3 --- In vivo Antitumor Effect of WT4-4 --- p.126 / Chapter 4.4 --- Results of DNA Agarose Gel Electrophoresis --- p.130 / Chapter Chapter 5 --- Discussion / Chapter 5.1 --- Characterization of Antitumor Fraction and Compound of Wedelia trilobata (WT) --- p.132 / Chapter 5.2 --- Antitumor Effects of WT Fractions and Purified Component (Crystal) --- p.137 / Chapter 5.2.1 --- In vitro assay --- p.137 / Chapter 5.2.2 --- DNA agarose gel electrophoresis --- p.143 / Chapter 5.2.3 --- In vivo assay --- p.144 / Chapter 5.3 --- Further Study --- p.146 / Chapter Chapter 6 --- Conclusion --- p.148 / References --- p.151

by Chung Wilson. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 126-149). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract (English) --- p.ii / Abstract (Chinese) --- p.v / Content --- p.vii / Abbreviations --- p.xii / List of Figures --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Alzheimer's disease --- p.1 / Chapter 1.1.1 --- History of Alzheimer's disease --- p.1 / Chapter 1.1.2 --- Definition of Alzheimer's disease --- p.2 / Chapter 1.1.3 --- Symptoms of Alzheimer's disease --- p.6 / Chapter 1.1.3.1 --- Memory deficit --- p.6 / Chapter 1.1.3.2 --- Difficulty in learning --- p.6 / Chapter 1.1.3.3 --- Language difficulties --- p.7 / Chapter 1.1.3.4 --- Decline in ability to perform routine tasks --- p.7 / Chapter 1.1.4 --- Prevalence of Alzheimer's disease --- p.8 / Chapter 1.2 --- Present treatment of Alzheimer's disease --- p.9 / Chapter 1.2.1 --- Acetylcholine and dementia --- p.9 / Chapter 1.2.2 --- Tacrine as first drug approved by US Food and Drug Administration --- p.9 / Chapter 1.3 --- Proposed theory of Alzheimer's disease formation --- p.10 / Chapter 1.3.1 --- The amyloid cascade hypothesis --- p.10 / Chapter 1.3.1.1 --- The amyloid precursor protein --- p.10 / Chapter 1.3.1.2 --- The processing of amyloid precursor protein --- p.12 / Chapter 1.3.1.3 --- Neurotoxic effect of amyloid plaque --- p.15 / Chapter 1.3.1.4 --- Genetic factors --- p.15 / Chapter 1.3.1.4.1 --- The amyloid precursor protein --- p.15 / Chapter 1.3.1.4.2 --- Apolipoprotein E (ApoE) --- p.16 / Chapter 1.3.1.4.3 --- Presenilin genes --- p.17 / Chapter 1.3.2 --- Tau and tangle hypothesis --- p.19 / Chapter 1.3.2.1 --- Tau protein --- p.19 / Chapter 1.3.2.2 --- Paired helical filaments (PHF) --- p.20 / Chapter 1.3.2.3 --- Tau protein kinase --- p.20 / Chapter 1.3.2.3.1 --- Glycogen synthase kinase-3 (GSK-3) --- p.21 / Chapter 1.3.2.3.2 --- Cyclin-dependent kinase 5 (CDK5) --- p.21 / Chapter 1.3.2.4 --- Tangle leads to dementia --- p.22 / Chapter 1.4 --- Cross-talk between the two hypotheses --- p.24 / Chapter 1.5 --- β -secretase (BACE) --- p.24 / Chapter 1.5.1 --- Discovery of β-secretase (BACE) --- p.24 / Chapter 1.5.2 --- Detailed structure of BACE --- p.25 / Chapter 1.5.3 --- Comparsion of human and mouse BACE --- p.27 / Chapter 1.5.4 --- Comparsion of BACE-1 with BACE-2 --- p.27 / Chapter 1.5.5 --- Properties of BACE-1 --- p.28 / Chapter 1.5.6 --- Expression of BACE in E.coli --- p.29 / Chapter 1.5.7 --- Expression of BACE in mammalian cells --- p.30 / Chapter 1.6 --- Objectives of the present study --- p.32 / Chapter Chapter 2 --- Materials and Methods --- p.34 / Chapter 2.1 --- Recombinant DNA techniques --- p.34 / Chapter 2.1.1 --- Amplification of genes by PCR techniques --- p.34 / Chapter 2.1.2 --- Agarose gel electrophoresis --- p.34 / Chapter 2.1.3 --- Extraction of DNA from agarose gel --- p.35 / Chapter 2.1.4 --- Digestion of various vectors and inserts --- p.36 / Chapter 2.1.5 --- Ligation of DNA fragments --- p.36 / Chapter 2.1.6 --- Preparation of Escherichia coli competent cells --- p.37 / Chapter 2.1.7 --- Bacterial transformation --- p.38 / Chapter 2.1.8 --- Minipreparation of plasmid DNA --- p.38 / Chapter 2.1.9 --- Large scale preparation of plasmid DNA --- p.39 / Chapter 2.1.10 --- Strain storage and revival --- p.40 / Chapter 2.1.11 --- Plasma DNA purification by High Pure plasmid isolation kit --- p.41 / Chapter 2.1.12 --- DNA sequencing --- p.42 / Chapter 2.1.13 --- Quantitation of DNA by spectrophotometric method --- p.43 / Chapter 2.2 --- Prokaryotic protein expression --- p.43 / Chapter 2.2.1 --- Selection of appropriate clones for recombinant protein expression using conventional method --- p.43 / Chapter 2.2.2 --- Selection of appropriate clones for recombinant protein expression using modified method --- p.44 / Chapter 2.2.3 --- Large -scale expression of recombinant human BACE protein using modified method --- p.45 / Chapter 2.2.4 --- Preparation of inclusion body from the bacterial expression culture --- p.46 / Chapter 2.2.5 --- Refolding of human BACE --- p.47 / Chapter 2.2.6 --- Purification of recombinant human BACE by immobilized metal ion affinity chromatography (IMAC) --- p.47 / Chapter 2.2.7 --- Protein concentration determination --- p.48 / Chapter 2.2.8 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.48 / Chapter 2.2.9 --- Western blotting --- p.50 / Chapter 2.2.10 --- Plasmid stability test --- p.50 / Chapter 2.3 --- Mammalian cell expression --- p.51 / Chapter 2.3.1 --- Transient transfection --- p.51 / Chapter 2.3.2 --- Measuring transfection efficiency --- p.52 / Chapter 2.3.3 --- Stable transfection --- p.52 / Chapter 2.3.4 --- Preparation of membrane extracts from CHO cells --- p.53 / Chapter 2.4 --- HPLC analysis --- p.53 / Chapter 2.4.1 --- Preparation of samples --- p.53 / Chapter 2.4.2 --- Reverse phase HPLC --- p.54 / Chapter 2.5 --- Fluorometric assay --- p.54 / Chapter 2.6 --- Immunohistochemistry --- p.55 / Chapter 2.7 --- Reagents and buffers --- p.55 / Chapter 2.7.1 --- Medium for bacterial culture --- p.56 / Chapter 2.7.2 --- Reagents for preparation of plasmid DNA --- p.56 / Chapter 2.7.3 --- Buffers for agarose gel electrophoresis --- p.57 / Chapter 2.7.4 --- Buffers for SDS-PAGE --- p.58 / Chapter 2.7.5 --- Buffer for purification of protein --- p.60 / Chapter 2.7.6 --- Buffer for Western Blotting --- p.61 / Chapter 2.7.7 --- Culturing medium of CHO cells --- p.62 / Chapter 2.7.8 --- Solutions for estimating transfection efficiency --- p.63 / Chapter 2.7.9 --- Reagents for HPLC --- p.64 / Chapter 2.7.10 --- Reagents for fluorometric assays --- p.65 / Chapter 2.7.11 --- Reagents for Immunohistochemistry --- p.66 / Chapter Chapter 3 --- Results --- p.67 / Chapter 3.1 --- Expression of BACE in E. coli --- p.67 / Chapter 3.1.1 --- Cloning of truncated human and mouse BACE into pRSET --- p.67 / Chapter 3.1.2 --- Expression of BACE in BL21(DE3)LysS cells --- p.70 / Chapter 3.1.2.1 --- Expression of truncated mouse and human BACEin BL21(DE3)LysS cells using conventional method --- p.70 / Chapter 3.1.2.2 --- Expression of truncated mouse and human BACEin BL21(DE3)LysS cells using modified method --- p.72 / Chapter 3.1.3 --- Analysis of BACE activity of purified recombinant proteins --- p.76 / Chapter 3.1.3.1 --- Fluorometric analysis --- p.76 / Chapter 3.2 --- Expression of BACE in mammalian cells --- p.81 / Chapter 3.2.1 --- "Cloning of full length mouse and human BACE into pCDNA3, pCDNA4HisMax" --- p.81 / Chapter 3.2.2 --- Transient transfection --- p.84 / Chapter 3.2.2.1 --- Western blot analysis --- p.86 / Chapter 3.2.2.2 --- Fluorometric analysis --- p.88 / Chapter 3.2.2.3 --- HPLC --- p.91 / Chapter 3.2.3 --- Stable transfection --- p.100 / Chapter 3.2.3.1 --- Western blot analysis --- p.101 / Chapter 3.2.3.2 --- Fluorometric analysis --- p.103 / Chapter 3.2.3.3 --- HPLC --- p.105 / Chapter 3.2.3.4 --- Immunohistochemistry --- p.112 / Chapter Chapter 4 --- Discussion --- p.115 / References --- p.126 / Appendix --- p.i / Chapter A1 --- Vector circle map --- p.i / Chapter A1-1 --- Vector circle map of pBluescript II- --- p.i / Chapter A1-2 --- Vector circle map of pCDNA3 --- p.ii / Chapter A1-3 --- Vector circle map of pCDNA4HisMax --- p.iii / Chapter A1-4 --- Vector circle map of pRSET --- p.iv / Chapter A2 --- Primer lists --- p.v / Chapter A3 --- Chemical structure of fluorophore and quench used in fluorometric assay --- p.vi

by Wong Che-cheuk, Dobe. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 98-112). / Abstract also in Chinese. / Acknowledgements --- p.i / Abstracts --- p.ii / List of Abbreviation --- p.vi / Table of Contents --- p.vii / Chapter 1. --- Introduction / Chapter 1.1 --- Stages of Cancer Development --- p.1 / Chapter 1.2 --- Comparison of Breast Cancer in Hong Kong & the United States --- p.2 / Chapter 1.2.1 --- Statistics of Breast Cancer in the United States --- p.2 / Chapter 1.2.2 --- Statistics of Breast Cancer in Hong Kong --- p.2 / Chapter 1.3 --- Factors for Breast Cancer --- p.6 / Chapter 1.3.1 --- Genetic Factor --- p.6 / Chapter 1.3.2 --- Hormonal Factor --- p.7 / Chapter 1.3.3 --- Genetic Bias --- p.9 / Chapter 1.3.4 --- Influence of Diet --- p.10 / Chapter 1.3.5 --- Obesity --- p.14 / Chapter 1.3.6 --- Xenoestrogen --- p.14 / Chapter 1.4 --- Hormonal Therapy in Breast Cancer --- p.15 / Chapter 1.4.1 --- Antiestrogen --- p.15 / Chapter 1.4.2 --- Progestin Antagonist --- p.19 / Chapter 1.4.3 --- Aromatase Inhibitor --- p.20 / Chapter 1.4.4 --- Gonadotropin Releasing Hormone (GnRH) Analogue --- p.23 / Chapter 1.5 --- Metabolism of Estrogen --- p.25 / Chapter 1.6 --- Substance with Chemopreventive Properties towards Breast Cancer --- p.29 / Chapter 1.7 --- Aryl Hydrocarbon Receptor --- p.33 / Chapter 1.8 --- Cytochrome P450s --- p.34 / Chapter 1.9 --- Yuehchukene --- p.36 / Chapter 1.10 --- Objectives of the Present Study --- p.38 / Chapter 2. --- Materials and Methods / Chapter 2.1 --- Animals --- p.40 / Chapter 2.2 --- Animal Treatment --- p.40 / Chapter 2.3 --- Preparation of Crude Microsomal Fraction --- p.41 / Chapter 2.4 --- Protein Assay --- p.41 / Chapter 2.5 --- Ethoxyresorufm-O-deethylase Assay --- p.41 / Chapter 2.6 --- Methoxyresorufin-O-deethylase Assay --- p.42 / Chapter 2.7 --- Estradiol-2-hydroxylase Assay --- p.42 / Chapter 2.8 --- Progesterone Hydroxylase Assay --- p.43 / Chapter 2.9 --- Hepatic Aromatase Activity Assay --- p.43 / Chapter 2.10 --- Inhibition of Ethoxyresorufm-O-deethylase and Estradiol-2-hydroxylase --- p.44 / Chapter 2.11 --- Free Radicals Scavenging Assay --- p.44 / Chapter 2.12 --- Chemicals --- p.45 / Chapter 3. --- Result / Chapter 3.1 --- Optimization of Condition --- p.47 / Chapter 3.1.1 --- Dosage --- p.47 / Chapter 3.1.2 --- Time for Sacrifice --- p.47 / Chapter 3.2 --- "Effect of Isoprenyl Compounds on the Body Weight, Liver Weight and Hepatic Microsomal Protein Content" --- p.50 / Chapter 3.3 --- Hepatic Enzyme Activities --- p.54 / Chapter 3.3.1 --- Ethoxyresorufm-O-deethylase --- p.54 / Chapter 3.3.2 --- Methoxyresorufm-O-deethylase --- p.57 / Chapter 3.3.3 --- Estradiol-2-hydroxylase --- p.60 / Chapter 3.3.4 --- Progesterone Hydroxylase --- p.62 / Chapter 3.3.5 --- Aromatase --- p.65 / Chapter 3.4 --- Effect of Inhibitors in Ethoxyresorufin-O-deethylase and Estradiol-2-hydroxylase Activity --- p.65 / Chapter 3.5 --- Free Radical Scavenging Activity --- p.72 / Chapter 4. --- Discussion --- p.77 / Chapter 5. --- Conclusion --- p.95 / Chapter 6. --- References --- p.98 / Chapter 7. --- Appendix --- p.113