Tang Yin Nei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves xv-xxv). / Abstracts in English and Chinese. / ACKNOWLEDGEMENT --- p.i / ABBREVIATIONS --- p.ii-iii / INDEX FOR FIGURES --- p.iv-v / INDEX FOR TABLES --- p.vi / ABSTRACT (Chinese and English) --- p.vii-xi / TABLE OF CONTENTS --- p.xii-xiv / Chapter CHAPTER I 226}0ؤ --- INTRODUCTION --- p.1 / Chapter 1.1 --- Background --- p.2 / Chapter 1.2 --- Tendon / Chapter 1.2.1 --- Structure and function --- p.3 / Chapter 1.2.2 --- Tendon fibroblast --- p.6 / Chapter 1.2.3 --- Components of the extracellular matrix --- p.7 / Chapter 1.2.3.1 --- Collagen --- p.8 / Chapter 1.2.3.2 --- Proteoglycan --- p.9 / Chapter 1.2.3.3 --- Non-collagenous structural glycoprotein --- p.10 / Chapter 1.3 --- Inflammation disorders of tendon / Chapter 1.3.1 --- Inflammation --- p.11 / Chapter 1.3.2 --- Treatment --- p.12 / Chapter 1.3.2.1 --- Glucocorticoid as an anti-inflammatory agent --- p.12 / Chapter 1.3.2.2 --- Dexamethasone --- p.14 / Chapter 1.3.3 --- Clinical occurrence of tendon rupture --- p.15 / Chapter 1.3.4 --- Animal research related to glucocorticoids and tendon rupture --- p.18 / Chapter 1.4 --- Platelet-derived growth factor isoform B (PDGFBB) / Chapter 1.4.1 --- Structure and function --- p.21 / Chapter 1.4.2 --- PDGFbb effects on connective tissue --- p.22 / Chapter CHAPTER II 226}0ؤ --- AIM OF THE STUDY --- p.23 / Chapter 2.1 --- Limitations of the past researches --- p.24 / Chapter 2.2 --- Hypothesis of this study --- p.25 / Chapter 2.3 --- Objectives --- p.26 / Chapter 2.4 --- Long term significance --- p.26 / Chapter CHAPTER III 226}0ؤ --- METHODOLOGY --- p.27 / Chapter 3.1 --- Chemicals and materials used / Chapter 3.1.1 --- Chemicals --- p.28 / Chapter 3.1.2 --- Materials --- p.28 / Chapter 3.2 --- Specimen collection and preparation / Chapter 3.2.1 --- Collection --- p.29 / Chapter 3.2.2 --- Preparation and isolation --- p.30 / Chapter 3.2.3 --- Cell culture --- p.31 / Chapter 3.3 --- Reagent preparation / Chapter 3.3.1 --- Charcoal-stripped serum --- p.32 / Chapter 3.3.2 --- Phenol-red free DMEM --- p.33 / Chapter 3.3.3 --- MTT --- p.33 / Chapter 3.3.4 --- Dexamethasone --- p.34 / Chapter 3.3.5 --- PDGFbb --- p.34 / Chapter 3.3.6 --- Trypan blue --- p.35 / Chapter 3.3.7 --- TCA/Tannic acid --- p.35 / Chapter 3.3.8 --- Collagenase buffer --- p.35 / Chapter 3.4 --- Morphology / Chapter 3.4.1 --- Inverted phase contrast light microscopy --- p.36 / Chapter 3.4.2 --- Scanning electron microscopy --- p.36 / Chapter 3.5 --- Biological assays / Chapter 3.5.1 --- "MTT (3-[4,5-Dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide) assay" --- p.38 / Chapter 3.5.1.1 --- Correlation between MTT assay and trypan blue dye method --- p.38 / Chapter 3.5.1.2 --- Growth kinetics for tendon fibroblasts --- p.41 / Chapter 3.5.1.3 --- Cell viability --- p.43 / Chapter 3.5.2 --- Brdu (5-bromo-2'-deoxyuridine) assay --- p.44 / Chapter 3.5.3 --- Flow cytometry --- p.45 / Chapter 3.5.4 --- Apoptosis --- p.47 / Chapter 3.5.5 --- 3H-Proline incorporation assay --- p.48 / Chapter 3.5.6 --- 35Sulfate incorporation assay --- p.51 / Chapter 3.5.7 --- Immunocytochemistry (PDGF-β receptor) --- p.54 / Chapter 3.6 --- Statistical analysis / Chapter 3.6.1 --- Dose-response curve of dexamethasone on cell viability and proliferation --- p.55 / Chapter 3.6.2 --- Comparison among various treatments of fibroblasts --- p.55 / Chapter CHAPTER I´Vؤ --- RESULTS --- p.56 / Chapter 4.1 --- In vitro effect of dexamethasone on rat tendon fibroblasts / Chapter 4.1.1 --- Viable cell number between two sexes --- p.57 / Chapter 4.2 --- In vitro effect of dexamethasone and PDGFBB on human tendon fibroblasts / Chapter 4.2.1 --- Gross morphology --- p.58 / Chapter 4.2.2 --- Cell cycle --- p.60 / Chapter 4.2.3 --- Apoptosis --- p.61 / Chapter 4.2.4 --- Viable cell number / Chapter 4.2.4.1 --- Effect of dexamethasone --- p.62 / Chapter 4.2.4.2 --- Effect of PDGFBB --- p.63 / Chapter 4.2.5 --- Cell proliferation / Chapter 4.2.5.1 --- Effect of dexamethasone --- p.65 / Chapter 4.2.5.2 --- Effect of PDGFbb --- p.67 / Chapter 4.2.6 --- Collagen synthesis --- p.68 / Chapter 4.2.7 --- Proteoglycan synthesis --- p.72 / Chapter 4.2.8 --- PDGF-rβ expression --- p.74 / Chapter CHAPTER V 226}0ؤ --- DISCUSSION --- p.75 / Chapter 5.1 --- Dexamethasone and PDGFBB induced change of cell morphology --- p.77 / Chapter 5.2 --- Dexamethasone retarded cell growth of human tendon fibroblast --- p.80 / Chapter 5.3 --- Dexamethasone inhibited collagen synthesis --- p.82 / Chapter 5.4 --- Dexamethasone inhibited proteoglycan synthesis --- p.86 / Chapter 5.5 --- PDGFbb could counteract the inhibitory effects of dexamethasone --- p.88 / Chapter 5.6 --- Expression of PDGF-(3 receptor is regulated by dexamethasone and PDGFBB --- p.90 / Chapter 5.7 --- Limitations of this study / Chapter 5.7.1 --- Not enough sample to differentiate different between two sexes --- p.92 / Chapter 5.7.2 --- Small sample size and few assays --- p.92 / Chapter 5.7.3 --- Limitations of the cell culture model --- p.93 / Chapter 5.7.4 --- Difficult to further in vivo study on human --- p.93 / Chapter 5.8 --- Contributions of this study / Chapter 5.8.1 --- Improve the limitation of the past research --- p.94 / Chapter 5.8.1.1 --- Human tendon specimen --- p.94 / Chapter 5.8.1.2 --- In vitro system --- p.94 / Chapter 5.8.2 --- Understand the effect of dexmaethasone on human tendon fibroblasts --- p.95 / Chapter 5.8.3 --- Counteract the deleterious effects of dexamethasone by PDGFBB --- p.95 / Chapter CHAPTER VÍؤ --- CONCLUSION & FUTURE STUDY --- p.96 / Chapter 6.1 --- Conclusion --- p.97 / Chapter 6.2 --- Future study --- p.98 / Chapter 6.2.1 --- Study the balance between matrix synthesis and degradation --- p.98 / Chapter 6.2.2 --- Determine collagen typing --- p.99 / Chapter 6.2.3 --- Further explore the effect of glucocorticoid in organ culture model --- p.100 / Chapter 6.2.4 --- Investigate molecular mechanism of dexamethasone and PDGFBB --- p.100 / REFERENCES --- p.xv-xxv / APPENDIX --- p.xxvi

Chiu Lai-Ching. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 137-152). / Abstracts in English and Chinese. / Acknowledgement --- p.II / Abstract --- p.IV / Contents --- p.VIII / Abbreviations --- p.XIV / Chapter Chapter One --- General introduction / Chapter 1.1 --- Haemopoiesis --- p.1 / Chapter 1.1.1 --- Background --- p.1 / Chapter 1.1.2 --- Regulation --- p.2 / Chapter 1.1.2.1 --- Stromal cells --- p.2 / Chapter 1.1.2.2 --- Haemopoietic regulator --- p.3 / Chapter 1.1.2.3 --- Haemopoietic regulator receptors and signal transduction --- p.5 / Chapter 1.2 --- Disorder of haemopoiesis --- p.9 / Chapter 1.2.1 --- Causes --- p.9 / Chapter 1.2.2 --- Types of leukemia --- p.9 / Chapter 1.2.3 --- Treatment of leukemia --- p.10 / Chapter 1.3 --- Prostaglandins --- p.13 / Chapter 1.3.1 --- Introduction --- p.13 / Chapter 1.3.2 --- Types and biosynthesis --- p.14 / Chapter 1.3.3 --- Prostaglandin receptors --- p.15 / Chapter 1.3.4 --- Prostaglandins and cell differentiation --- p.17 / Chapter 1.3.4.1 --- PGD2 and cell differentiation --- p.19 / Chapter 1.3.4.2 --- PGE2 and cell differentiation --- p.20 / Chapter 1.3.4.3 --- PGJ2 and cell differentiation --- p.22 / Chapter 1.4 --- WEHI-3B JCS cells --- p.25 / Chapter 1.5 --- Aims of study --- p.27 / Chapter Chapter Two --- Roles of Prostaglandin D2，E2 and J2 in WEHI-3B JCS myeloid leukemia cell differentiation / Chapter 2.1 --- Introduction --- p.28 / Chapter 2.1.1 --- Morphological studies of JCS cells --- p.28 / Chapter 2.1.2 --- Methods in determining cell proliferation --- p.29 / Chapter 2.1.3 --- Methods in determining differentiated cells --- p.31 / Chapter 2.2 --- Materials --- p.33 / Chapter 2.2.1 --- Cell line --- p.33 / Chapter 2.2.2 --- Chemicals --- p.33 / Chapter 2.2.3 --- Solutions and buffers --- p.34 / Chapter 2.3 --- Methods --- p.36 / Chapter 2.3.1 --- Microscopic studies of the JCS cells --- p.36 / Chapter 2.3.1.1 --- Histochemical staining of JCS --- p.36 / Chapter 2.3.1.2 --- Transmission electronic microscopic --- p.36 / Chapter 2.3.2 --- [3H]-thymidine incorporation assay --- p.37 / Chapter 2.3.3 --- MTT assay --- p.37 / Chapter 2.4 --- Results --- p.38 / Chapter 2.4.1 --- Histochemical staining of JCS cells --- p.38 / Chapter 2.4.2 --- Electron microscopy --- p.40 / Chapter 2.4.3 --- "Effect of PGD2, E2 and J2 on JCS cells proliferation" --- p.44 / Chapter 2.4.4 --- "Effect of PGD2, E2 and J2 on JCS cells differentiation" --- p.48 / Chapter 2.5 --- Discussion --- p.53 / Chapter 2.5.1 --- Morphological differentiation of JCS cells --- p.53 / Chapter 2.5.2 --- The ultra-structures of JCS cells --- p.53 / Chapter 2.5.3 --- "Effect of PGD2, E2 and J2 on JCS cells proliferation" --- p.54 / Chapter 2.5.4 --- "Effect of PGD2, E2 and J2 on JCS cells differentiation" --- p.55 / Chapter Chapter Three --- Roles of Prostaglandin E2 in normal haemopoiesis and the detection of PGE2 receptors expression in JCS and bone marrow cells / Chapter 3.1 --- Introduction --- p.57 / Chapter 3.1.1 --- Colony assay --- p.57 / Chapter 3.1.2 --- The use of RT-PCR --- p.58 / Chapter 3.1.3 --- Prostaglandin E receptors --- p.59 / Chapter 3.2 --- Materials --- p.62 / Chapter 3.2.1 --- Bone marrow cells --- p.62 / Chapter 3.2.2 --- Cell line --- p.62 / Chapter 3.2.3 --- Chemicals --- p.62 / Chapter 3.2.4 --- Primers --- p.63 / Chapter 3.2.5 --- Solutions and buffers --- p.64 / Chapter 3.2.6 --- Enzymes and reagents --- p.65 / Chapter 3.3 --- Methods --- p.66 / Chapter 3.3.1 --- Titration of mouse IL-3 --- p.66 / Chapter 3.3.2 --- Determination of suitable IL-3 concentration for growth of bone marrow cells in colony assay --- p.66 / Chapter 3.3.2.1 --- Preparation of bone marrow cells --- p.66 / Chapter 3.3.2.2 --- Preparation of culture medium for colony assay --- p.67 / Chapter 3.3.3 --- Investigation of the effect of PGE2 on normal haemopoiesis by colony assay --- p.68 / Chapter 3.3.4 --- Detection of PGE2 receptors expression on JCS cells and bone marrow cells --- p.68 / Chapter 3.3.4.1 --- Preparation of cell lysates --- p.68 / Chapter 3.3.4.2 --- Preparation of total RNA of JCS cells and bone marrow cells --- p.68 / Chapter 3.3.4.3 --- RT-PCR --- p.69 / Chapter 3.4 --- Results --- p.71 / Chapter 3.4.1 --- Titration of mouse IL-3 --- p.71 / Chapter 3.4.2 --- Effect of mouse IL-3 on normal haemopoiesis --- p.73 / Chapter 3.4.3 --- Effect of PGE2 on mouse IL-3 driven normal bone marrow cell differentiation --- p.76 / Chapter 3.4.4 --- Analysis of total RNA prepared from uninduced JCS cells and bone marrow cells --- p.79 / Chapter 3.4.5 --- "Expression of gapdh in heart, liver, spleen, JCS and bone marrow cells" --- p.81 / Chapter 3.4.6 --- "Expression of PGE2 receptors in heart, liver, spleen, JCS and bone marrow cells" --- p.82 / Chapter 3.5 --- Discussion --- p.84 / Chapter 3.5.1 --- Effect of PGE2 on IL-3 driven normal bone marrow cells differentiation --- p.84 / Chapter 3.5.2 --- "Expression of PGE2 receptors in heart, liver, spleen, JCS and bone marrow cells" --- p.85 / Chapter Chapter Four --- Gene expression profile of JCS cells under 5 hours of PGE2 induction / Chapter 4.1 --- Introduction --- p.88 / Chapter 4.1.1 --- Review of methods studying differential gene expression --- p.88 / Chapter 4.1.2 --- The choice of method studying differential gene expression --- p.92 / Chapter 4.1.3 --- The microarray --- p.93 / Chapter 4.2 --- Materials --- p.95 / Chapter 4.2.1 --- Cell line --- p.95 / Chapter 4.2.2 --- Kits --- p.95 / Chapter 4.2.3 --- Chemicals --- p.95 / Chapter 4.2.4 --- Solutions and buffers --- p.96 / Chapter 4.2.5 --- Reagents --- p.97 / Chapter 4.3 --- Methods --- p.98 / Chapter 4.3.1 --- Preparation of total RNA from PGE2 induced JCS cells --- p.98 / Chapter 4.3.2 --- Preparation of cDNA probes --- p.98 / Chapter 4.3.2.1 --- Probe synthesis from total RNA --- p.98 / Chapter 4.3.2.2 --- Column chromatography --- p.99 / Chapter 4.3.3 --- Hybridizing cDNA probes to the Atlas Array --- p.99 / Chapter 4.4 --- Results --- p.101 / Chapter 4.4.1 --- Spectrophotometric analysis of total RNA after ethanol precipitation --- p.101 / Chapter 4.4.2 --- Hybridization of cDNA probes to Atlas Array --- p.102 / Chapter 4.5 --- Discussion --- p.121 / Chapter 4.5.1 --- Genes with increased expression --- p.121 / Chapter 4.5.2 --- Genes with decrease expression --- p.127 / Chapter 4.5.3 --- Study of gene expression profile by microarray --- p.128 / Chapter Chapter Five --- General discussion / Chapter 5.1 --- Introduction --- p.131 / Chapter 5.2 --- Roles of PGE2 in JCS cells differentiation --- p.131 / Chapter 5.3 --- Roles of PGE2 in normal haemopoiesis --- p.134 / Chapter 5.4 --- Further studies --- p.135 / References --- p.137

Chong Lung-wun. / Thesis (M.Sc.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 49-58). / Abstracts in English and Chinese. / Acknowledgement --- p.1 / Contents --- p.2 / Abstract --- p.4 / Chapter I. --- Introduction --- p.6 / Chapter 1. --- Nitrate therapy for angina patients --- p.7 / Chapter 1.1 --- The mode of action of nitrates --- p.8 / Chapter 1.2 --- Hypotheses for the nitrate tolerance --- p.9 / Chapter 1.2.1 --- The Sulfhydryl depletion hypothesis --- p.9 / Chapter 1.2.2 --- Neurohormonal hypothesis --- p.10 / Chapter 1.2.3 --- Plasma volumc expansion hypothesis --- p.10 / Chapter 1.2.4 --- Free-radical hypothesis --- p.11 / Chapter 2. --- Testosterone therapy for angina patients --- p.12 / Chapter 3. --- Nitric Oxide and Steroidogenesis --- p.13 / Chapter II. --- Materials and Methods --- p.14 / Chapter 1. --- Subjects --- p.14 / Chapter 2. --- Method for measuring nitrite and nitrate in plasma --- p.16 / Chapter 3. --- Method for the determination of total and free testosterone in blood --- p.24 / Chapter 3.1 --- Reagent preparation --- p.24 / Chapter 3.2 --- Assay procedure for total testosterone --- p.28 / Chapter 3.3 --- Assay procedure for free testosterone --- p.29 / Chapter 3.4 --- Determination of working antiserum for free testosterone assay --- p.30 / Chapter 4. --- Method for the determination of serum Cortisol --- p.31 / Chapter III. --- Result --- p.33 / Chapter 1. --- Summary of nitric oxide assay performance --- p.33 / Chapter 2. --- Summary of total testosterone assay performance --- p.34 / Chapter 3. --- Summary of free testosterone assay performance --- p.34 / Chapter 4. --- Data analysis --- p.35 / Chapter IV. --- Discussion --- p.43 / Chapter V. --- Conclusion --- p.47 / Chapter VI. --- References --- p.49

Submitted by Fabíola Silva (fabiola.silva@famerp.br) on 2017-03-16T11:48:07Z No. of bitstreams: 1 anamarciasdealmeidavianna_dissert.pdf: 923997 bytes, checksum: 91cfee711fbecdf2ade7a86724cc75a5 (MD5) / Made available in DSpace on 2017-03-16T11:48:07Z (GMT). No. of bitstreams: 1 anamarciasdealmeidavianna_dissert.pdf: 923997 bytes, checksum: 91cfee711fbecdf2ade7a86724cc75a5 (MD5) Previous issue date: 2015-06-29 / Breast cancer is the second most frequent type of cancer, only preceded by skin cancer. Although considered of good prognosis, it is still a stigmatizing condition. Objective: To characterize demographic aspects and quality of life (QoL) of women with breast cancer before and after treatment with neo-adjuvant chemotherapy at a teaching hospital in the state of São Paulo. Methodology: descriptive, cross-sectional study. Patients: participants were 60 adult women starting neo-adjuvant chemotherapy AC (Adriamycin + Cyclophosphamide). For data collection the following instruments were used: Identification Card, Interview Guide and Functional Assessment of Cancer Therapy (FACT-B). Data were qualitative and quantitatively analyzed with descriptive statistics. For comparative analysis between variables the Wilcoxon test was used with significance level of p<0.05. Results: Participants ages ranged from 27 to 83 years old (M = 52.42 years, SD = 12.66 years), married (61.7%), with children and attending primary school (75%). For the physical, and functional domain, TOI (Trial Outcome Index), FACT-G (Functional Assessment of Cancer Therapy – General) and FACT-B there was a significant difference in the assessment of QoL pre and post-QT. Feeling unattractive (58.3%), sexual dissatisfaction (56.6%), insomnia (53.3%), alopecia (52.3%), fatigue (43.3%), inability to experience pleasure in life (40%) and feelings of sadness (41.6%) and inability to work (35%) were the most frequent symptoms post-QT. Conclusion: QT has an important impact on physical and functional QoL, impairing several roles played by the patient and affecting sexual and affective relationships. / Câncer de mama é o segundo tipo de neoplasia mais frequente, apenas precedido do câncer de pele. Apesar de ser considerado com bom prognóstico, ainda assim é uma condição estigmatizante. Objetivo: caracterizar aspectos sócios demográficos e avaliar a qualidade de vida (QV) de mulheres com câncer de mama pré e pós-quimioterapia neoadjuvante (QT), atendidas no Hospital de Base de São José do Rio Preto-SP. Metodologia: estudo descritivo, transversal. Casuística: participaram 60 mulheres adultas que iniciaram a QT neoadjuvante AC (Adriamicina + Ciclofosfamida). Para a coleta dos dados foram utilizados: Ficha de Identificação, Roteiro de Entrevista e Functional Assessment of Cancer Therapy - Breast (FACT-B). Os questionários foram aplicados em dois momentos: antes do início e após o término da QT. Os dados foram analisados de forma quali e quantitativa, por meio de estatística descritiva e para análise de comparação entre as variáveis o Teste de Wilcoxon com nível de significância p<0,05. Resultados: A idade das participantes variou entre 27 e 83 anos (M=52,42 anos; DP = 12,66 anos), casadas (61,7%), com filhos e cursando ensino fundamental (75%). Nos domínio físico, funcional, TOI (Trial Outcome Index), FACT-G (Functional Assessment of Cancer Therapy – General) e FACT-B houve diferença significante na avaliação da QV pré e pós-QT. Não sentir-se atraente (58,3%), insatisfação sexual (56,6%), insônia (53,3%), alopécia (52,3%), fadiga (43,3%), incapacidade de sentir prazer na vida (40%) e sentimentos de tristeza (41,6%) e incapacidade para trabalhar (35%), foram sintomas mais freqüentes pós-QT. Conclusão: A QT é um tratamento de grande impacto e sofrimento físico-funcional na QV, com prejuízo no desempenho de papéis e relações afetivo-sexuais.

A quimioterapia é uma das abordagens terapêuticas para o tratamento do câncer e, no caso específico das crianças e adolescentes, a mais frequente, de forma individual ou associada a outras modalidades. Tem sido fonte de preocupações, questionamentos e dúvidas, tanto por parte das crianças e adolescentes quanto de seus familiares, por seus efeitos colaterais físicos e psicológicos. O presente estudo tem por objetivo compreender como as crianças e os adolescentes com câncer vivenciam a experiência da quimioterapia, a partir de seus próprios relatos, visto que, ao conhecer suas demandas e sentimentos, podemos incorporá-los ao plano de cuidados de enfermagem e contribuir para uma assistência qualificada e dirigida à qualidade de vida da criança, do adolescente e de sua família. Exploratório e com análise qualitativa dos dados este estudo contou com a participação de 10 crianças e adolescentes entre oito e 18 anos, em diferentes momentos da terapêutica quimioterápica. Para a coleta dos dados, utilizamos como instrumentos a entrevista semiestruturada e a observação livre, complementadas com dados do prontuário dos sujeitos em questão. A análise do material empírico seguiu a técnica de análise de conteúdo. Após a caracterização dos sujeitos da pesquisa, os resultados foram agrupados ao redor de três grandes temas, seguidos dos respectivos subtemas: a doença, compreendendo a trajetória até o diagnóstico e o conhecimento sobre a doença; a quimioterapia, a partir do impacto do tratamento, as características dos quimioterápicos e a visão do processo e, por fim, a rede de apoio que incluiu a família, os amigos e a religião. O estudo nos permitiu compreender que, para as crianças e adolescentes, a experiência da quimioterapia está intimamente ligada à experiência do câncer, sendo impossível entender a dimensão da terapêutica sem antes compreender a base desta questão, o câncer infanto-juvenil. Lembrada, principalmente por seus efeitos colaterais e o sofrimento, a quimioterapia causa, de imediato, um forte impacto, deixando sobressair o medo. Passado este primeiro impacto, principalmente das alterações físicas, as preocupações dirigem-se à recuperação da doença, ou seja, à cura. Mencionam que o câncer interrompe uma vida até então normal e provoca dúvidas quanto ao futuro e mesmo o presente. Em contrapartida, com o tempo, relatam que o processo terapêutico, incluindo a quimioterapia, levou-os a valorizar mais a vida. Brincar, ter a família e os amigos por perto, buscar Deus, conhecer a doença e enfrentá-la, se envolver em todo o processo e, acima de tudo, acreditar no sucesso foram estratégias utilizadas durante a quimioterapia, terapêutica sofrida, restritiva, mas acima de tudo, responsável pela cura da criança e do adolescente. / Chemotherapy is one of the therapies used in cancer treatment. Separately or associated to other therapies, it is the most frequently used to treat children and adolescents. Due to its physical and psychological collateral effects, it has been source of preoccupations, questions and doubts, both for children and adolescents as well as their relatives. This exploratory and qualitative study aimed to understand how children and adolescents with cancer experience chemotherapy, from their own reports. Knowing their demands and feelings permits to incorporate them to the nursing care plan and contributes to a quality care, targeting the quality of life of children, adolescents and their families. Participants were 10 children and adolescents between 8 and 18 years of age, on different moments of chemotherapy. Data collection was done through semi-structured interview and free observation, added by data from subjects patient files. Analysis of the empirical material was done through content analysis. After characterization of the research subjects, results were grouped into three themes and their respective subthemes: disease, understanding the trajectory to diagnosis and knowledge about the disease; chemotherapy, the treatment impact, characteristics of chemotherapeutic drugs and the view of the process and, lastly, the support network, which includes family, friends and religion. The study permitted to understand that, for children and adolescents, the chemotherapy experience is closely related to the cancer experience, and it is impossible to understand the dimension of the therapy without previously understanding the basis of the issue: child and juvenile cancer. Chemotherapy is mainly reminded by its collateral effects and suffering. It has a strong impact on participants, showing the fear caused by it. After the initial impact, mainly due to physical changes, preoccupations are related to disease recovery, that is, cure. They mention that cancer interrupts what was so far a normal life and causes doubts regarding the future and even present life. On the other hand, according to their reports, treatment, including chemotherapy, made them value life more. Playing, being close to family and friends, searching for Good, knowing and facing the disease, getting involved in the process and, above all, believing in success, were the strategies used during chemotherapy, which is a restrictive therapy, that causes suffering, but which is primarily responsible for children and adolescent cure.

Gao, Si. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 77-85). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / Abstract (in Chinese) --- p.iv / List of Abbreviations --- p.v / List of Figures --- p.vii / Table of Contents --- p.ix / Chapter Chapter One: --- Introduction --- p.1 / Chapter 1.1. --- Cancer --- p.1 / Chapter 1.2. --- Chemotherapy --- p.2 / Chapter 1.3. --- Cisplatin --- p.3 / Chapter 1.4. --- Cisplatin-induced nephrotoxicity --- p.4 / Chapter 1.5. --- Mechanisms of cisplatin-induced nephrotoxicity --- p.5 / Chapter 1.5.1. --- Apoptosis in cisplatin-induced nephrotoxicity --- p.5 / Chapter 1.5.2. --- MAPK activation in cisplatin-induced nephrotoxicity --- p.7 / Chapter 1.5.3. --- Oxidative stress in cisplatin-induced nephrotoxicity --- p.8 / Chapter 1.6. --- Polyphenols --- p.10 / Chapter 1.7. --- Anthocyanins --- p.10 / Chapter 1.8. --- Rose --- p.11 / Chapter 1.9. --- Cyanidin --- p.12 / Chapter 1.10. --- Objectives of this project --- p.13 / Chapter Chapter Two: --- Materials and Methods --- p.15 / Chapter 2.1. --- Materials --- p.15 / Chapter 2.2. --- Cell culture --- p.15 / Chapter 2.3. --- Drug treatment --- p.16 / Chapter 2.4. --- MTT assay --- p.16 / Chapter 2.5. --- Lactate dehydrogenase (LDH) assay --- p.16 / Chapter 2.6. --- TUNEL assay and DAPI staining --- p.17 / Chapter 2.7. --- Flow cytometric analysis --- p.17 / Chapter 2.8. --- Determination of caspase-3 activity --- p.18 / Chapter 2.9. --- Measurement of ROS generation --- p.18 / Chapter 2.10. --- Evaluation of mitochondrial membrane potential --- p.19 / Chapter 2.11. --- Single Cell Gel Electrophoresis (Comet Assay) --- p.19 / Chapter 2.12. --- Western blot analysis --- p.20 / Chapter 2.13. --- Statistical analysis --- p.21 / Chapter Chapter Three: --- Results --- p.22 / Chapter 3.1. --- Cyanidin attenuates cisplatin-induced cytotoxicity in HK-2 cells --- p.22 / Chapter 3.1.1. --- Cytotoxicity induces by cisplatin in HK-2 cells --- p.22 / Chapter 3.1.2. --- Rose extract attenuates cisplatin-induced cytotoxicity and LDH leakage --- p.26 / Chapter 3.1.3. --- Cyanidin attenuates cisplatin-induced cytotoxicity and LDH leakage --- p.26 / Chapter 3.1.4. --- Cyanidin did not affect cisplatin-induced cytotoxicity in Hela cell --- p.30 / Chapter 3.2. --- Cyanidin rescues HK-2 cells from cisplatin-induced apoptosis --- p.31 / Chapter 3.2.1. --- Cisplatin induces cell apoptosis in HK-2 cells --- p.31 / Chapter 3.2.2. --- Rose extract rescues HK-2 cells from cisplatin-induced apoptosis --- p.31 / Chapter 3.2.3. --- Cyanidin rescues HK-2 cells from cisplatin-induced apoptosis --- p.32 / Chapter 3.3. --- Cyanidin suppresses cisplatin-induced activation of caspase and cleavage of PARP --- p.38 / Chapter 3.3.1. --- Cisplatin induces activation of caspase-3 --- p.38 / Chapter 3.3.2. --- Rose extract suppresses cisplatin-induced activation of caspase-3 --- p.38 / Chapter 3.3.3. --- Cyanidin suppresses cisplatin-induced activation of caspase-3 --- p.38 / Chapter 3.3.4. --- Rose extract suppresses cisplatin-induced caspase activation and PARP cleavage --- p.41 / Chapter 3.3.5. --- Cyanidin suppresses cisplatin-induced caspase activation and PARP cleavage --- p.43 / Chapter 3.4. --- Cyanidin rescues HK-2 cells from cisplatin-induced mitochondrial dysfuntion by regulating the expression of Bcl-2 family proteins --- p.43 / Chapter 3.4.1. --- Cyanidin prevents cisplatin-induced loss of mitochondrial membrane potential (A^m) --- p.43 / Chapter 3.4.2. --- Cyanidin regulates the expression of Bcl-2 family proteins to prevent cisplatin-induced mitochondrial dysfunction --- p.44 / Chapter 3.5. --- Cyanidin reduces cisplatin-induced apoptosis by suppressing the activation of p53 --- p.46 / Chapter 3.6. --- Cyanidin inhibits ROS-mediated DNA damage in HK-2 cells --- p.48 / Chapter 3.6.1. --- Cyanidin prevents cisplatin-induced DNA damage --- p.48 / Chapter 3.6.2. --- Cyanidin inhibits cisplatin-induced accumulation of ROS --- p.48 / Chapter 3.7. --- "Cyanidin suppresses cisplatin-induced apoptosis by activation of AKT, JNK and ERK" --- p.52 / Chapter 3.7.1. --- Cisplatin activates ERK and AKT pathways --- p.52 / Chapter 3.7.2. --- Cyanidin suppresses cisplatin-induced activation of MAPKs and AKT pathways --- p.52 / Chapter 3.7.3. --- AKT and ERK Inhibitors attenuates cisplatin-induced apoptosis in HK-2 cells --- p.53 / Chapter Chapter Four: --- Discussion --- p.60 / Chapter 4.1. --- Cell model and cisplatin treatment --- p.60 / Chapter 4.2. --- Cisplatin nephrotoxicity and its renoprevention --- p.61 / Chapter 4.3. --- Rose extract prevents cisplatin-induced apoptosis in HK-2 cells --- p.62 / Chapter 4.3.1. --- Rose extract prevents cisplatin-induced apoptosis in HK-2 cells --- p.63 / Chapter 4.3.2. --- Rose extract inhibits cisplatin-induced caspase activation and PARP cleavage --- p.64 / Chapter 4.4. --- Cyanidin prevents cisplatin-induced apoptosis in HK-2 cells --- p.66 / Chapter 4.4.1. --- Cyanidin will not affect cisplatin-induced cell death in HeLa cells --- p.66 / Chapter 4.4.2. --- Cyanidin prevents cisplatin-induced apoptosis by inhibiting caspase activation and PARP cleavage in HK-2 cells --- p.66 / Chapter 4.4.3. --- Cyanidin prevents the cisplatin-induced loss of mitochondrial membrane potential by regulating Bcl-2 proteins in HK-2 cells --- p.67 / Chapter 4.4.4. --- Cyanidin suppresses cisplatin-induced total and phosphorylated p53 activation --- p.68 / Chapter 4.4.5. --- Cyanidin prevents the cisplatin-induced overproduction of intracellular ROS and subsequent DNA damage in HK-2 cells --- p.69 / Chapter 4.4.6. --- Cyanidin suppresses the cisplatin-induced activation of MAPKs and AKT pathways in HK-2 cells --- p.71 / Chapter Chapter Five: --- Conclusion --- p.74 / References --- p.77

Yip, Hon Yan Kelvin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 218-242). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.vii / CHINESE ABSTRACT (摘要） --- p.xi / PUBLICATIONS --- p.xiv / TABLE OF CONTENTS --- p.xv / Chapter CHAPTER 1: --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Neuroblastoma --- p.2 / Chapter 1.1.1 --- An overview of neuroblastoma --- p.2 / Chapter 1.1.2 --- Epidemiology of neuroblastoma --- p.3 / Chapter 1.1.3 --- Clinical presentations of neuroblastoma --- p.5 / Chapter 1.1.4 --- Diagnosis and clinical assessment of neuroblastoma --- p.8 / Chapter 1.1.5 --- Staging of neuroblastoma --- p.10 / Chapter 1.1.6 --- Genetic aberrations of neuroblastoma --- p.12 / Chapter 1.1.7 --- Therapies of neuroblastoma --- p.15 / Chapter 1.2 --- Banlangen alkaloids --- p.20 / Chapter 1.2.1 --- An overview of Banlangen alkaloids --- p.20 / Chapter 1.2.2 --- "Pharmacokinetics of indirubin, tryptanthrin and their derivatives" --- p.24 / Chapter 1.2.2.1 --- Bioavailability of indirubin and its derivatives --- p.24 / Chapter 1.2.2.2 --- Toxicity of indirubin and its derivatives --- p.25 / Chapter 1.2.2.3 --- Bioavailability of tryptanthrin --- p.26 / Chapter 1.2.2.4 --- Toxicity of tryptanthrin --- p.27 / Chapter 1.2.3 --- "Pharmacological effects of indirubin, tryptanthrin and their derivatives" --- p.28 / Chapter 1.2.3.1 --- Selective inhibitor on kinases --- p.29 / Chapter 1.2.3.2 --- Anti-inflammatory activities --- p.31 / Chapter 1.2.3.3 --- Anti-tumour activities --- p.32 / Chapter 1.2.3.3.1 --- Anti-leukemic activity --- p.32 / Chapter 1.2.3.3.2 --- Apoptosis-inducing activity --- p.34 / Chapter 1.2.3.4 --- Anti-viral properties --- p.37 / Chapter 1.2.3.5 --- Anti-microbial properties --- p.37 / Chapter 1.3 --- Aims and Scope of This Study --- p.39 / Chapter CHAPTER 2: --- MATERIALS AND METHODS --- p.41 / Chapter 2.1 --- Materials --- p.42 / Chapter 2.1.1 --- Cell lines --- p.42 / Chapter 2.1.2 --- "Cell culture media, reagents and buffers" --- p.43 / Chapter 2.1.3 --- General staining solutions --- p.46 / Chapter 2.1.4 --- Drugs and chemicals --- p.47 / Chapter 2.1.5 --- Reagent for primary cultures preparation --- p.48 / Chapter 2.1.6 --- Reagents for cell proliferation assay --- p.48 / Chapter 2.1.7 --- Reagents for DNA extraction --- p.50 / Chapter 2.1.8 --- Reagents for gel electrophoresis of nucleic acids --- p.51 / Chapter 2.1.9 --- Reagents and buffers for flow cytometry --- p.53 / Chapter 2.1.10 --- Reagents and buffers for measuring caspase activity --- p.54 / Chapter 2.1.11 --- "Reagents, buffers and materials for Western blot analysis" --- p.58 / Chapter 2.1.12 --- Reagent for Hoechst staining --- p.68 / Chapter 2.2 --- Methods --- p.69 / Chapter 2.2.1 --- Culture of cell lines --- p.69 / Chapter 2.2.2 --- Determination of cell viability --- p.70 / Chapter 2.2.3 --- Determination of cell proliferation by tritiated thymidine ([ 3H]-TdR) incorporation assay --- p.72 / Chapter 2.2.4 --- "Isolation, culture and cytotoxicity test of murine peritoneal macrophages" --- p.73 / Chapter 2.2.5 --- "Isolation, culture and cytotoxicity test of murine bone marrow cells" --- p.74 / Chapter 2.2.6 --- Cytotoxicity test of primary cortical neurons from SD rats --- p.75 / Chapter 2.2.7 --- Determination of colony forming ability --- p.75 / Chapter 2.2.8 --- Analysis of cell cycle profile /DNA content by flow cytometry --- p.76 / Chapter 2.2.9 --- Detection of DNA fragmentation by agarose gel electrophoresis --- p.77 / Chapter 2.2.10 --- Quantitative detection of DNA fragmentation by Cell Death ELISAplus kit --- p.78 / Chapter 2.2.11 --- Detection of intracellular reactive oxygen species (ROS) generation --- p.80 / Chapter 2.2.12 --- Measurement of caspase activity --- p.81 / Chapter 2.2.13 --- Hoechst 33342 staining --- p.83 / Chapter 2.2.14 --- Cell morphological study --- p.83 / Chapter 2.2.15 --- Analysis of morphological changes by flow cytometry --- p.84 / Chapter 2.2.16 --- Assay for acetylcholine esterase (AChE) activity --- p.85 / Chapter 2.2.17 --- Protein expression study --- p.86 / Chapter 2.2.18 --- Statistical analysis --- p.89 / Chapter CHAPTER 3: --- IN VITRO STUDIES ON THE ANTI PROLIFERATIVE EFFECT OF INDIRUBIN-3'-OXIME AND TRYPTANTHRIN ON NEUROBLASTOMA CELLS --- p.90 / Chapter 3.1 --- Introduction --- p.91 / Chapter 3.2 --- Results --- p.95 / Chapter 3.2.1 --- Effects of indirubin-3'-oxime and tryptanthrin on the proliferation of human and the murine neuroblastoma cells --- p.95 / Chapter 3.2.2 --- Kinetic and reversibility studies of the anti-proliferative effect of indirubin-3'-oxime and tryptanthrin on the murine neuroblastoma Neuro-2a BU-1 cells --- p.107 / Chapter 3.2.3 --- Cytotoxic effect of indirubin-3'-oxime and tryptanthrin on the murine neuroblastoma Neuro-2a BU-1 cells --- p.115 / Chapter 3.2.4 --- Effects of indirubin-3'-oxime and tryptanthrin on the clonogenicity of the murine neuroblastoma Neuro-2a BU-1 cells --- p.120 / Chapter 3.2.5 --- Cytotoxicity of indirubin-3'-oxime and tryptanthrin on primary cells --- p.123 / Chapter 3.2.6 --- Effects of tryptanthrin on the cell cycle profile and expression of cyclins and cyclin-dependent kinases (CDKs) in the murine neuroblastoma Neuro-2a BU-1 cells --- p.132 / Chapter 3.2.7 --- Effect of indirubin-3'-oxime on the cell cycle profile in the murine neuroblastoma Neuro-2a BU-1 cells --- p.133 / Chapter 3.3 --- Discussion --- p.142 / Chapter CHAPTER 4: --- IN VITRO STUDIES ON THE APOPTOSIS-INDUCING EFFECT OF INDIRUBIN-3'-OXIME ON NEUROBLASTOMA CELLS --- p.150 / Chapter 4.1 --- Introduction --- p.151 / Chapter 4.2 --- Results --- p.156 / Chapter 4.2.1 --- Induction of DNA fragmentation in the indirubin-3'-oxime-treated murine neuroblastoma Neuro-2a BU-1 cells --- p.156 / Chapter 4.2.2 --- Induction of chromatin condensation in the indirubin-3 '-oxime-treated murine neuroblastoma Neuro-2a BU-1 cells --- p.160 / Chapter 4.2.3 --- Induction of caspase activities in the indirubin-3 '-oxime-treated murine neuroblastoma Neuro-2a BU-1 cells --- p.162 / Chapter 4.2.4 --- Induction of Reactive Oxygen Species (ROS) in the indirubin-3' -oxime-treated murine neuroblastoma Neuro-2a BU-1 cells --- p.169 / Chapter 4.2.5 --- Expression of pro-apoptotic and anti-apoptotic proteins in the indirubin-3 '-oxime-treated murine neuroblastoma Neuro-2a BU-1 cells --- p.173 / Chapter 4.3 --- Discussion --- p.177 / Chapter CHAPTER 5: --- STUDIES ON THE DIFFERENTIATION-INDUCING ACTIVITY OF TRYPTANTHRIN ON NEUROBLASTOMA CELLS --- p.188 / Chapter 5.1 --- Introduction --- p.189 / Chapter 5.2 --- Results --- p.193 / Chapter 5.2.1 --- Effects of tryptanthrin on the cell size and cellular complexity of the murine neuroblastoma Neuro-2a BU-1 cells --- p.193 / Chapter 5.2.2 --- Morphological studies on tryptanthrin-treated murine neuroblastoma Neuro-2a BU-1 cells --- p.196 / Chapter 5.2.3 --- Effect of tryptanthrin on the acetylcholine esterase (AChE) activity in the murine neuroblastoma Neuro-2a BU-1 cells --- p.198 / Chapter 5.2.4 --- Effects of tryptanthrin on the expression of tau protein and other mediators involved in the differentiation pathway --- p.200 / Chapter 5.3 --- Discussion --- p.204 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.209 / REFERENCES --- p.218

Tse, Yuk Tin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 141-149). / Abstracts in English and Chinese. / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- The influenza virus --- p.1 / Chapter 1.1.1 --- Influenza NA and its inhibitors --- p.3 / Chapter 1.1.2 --- Follow-up on the use of NAIs --- p.9 / Chapter 1.2 --- Sources of NA for experimental studies --- p.11 / Chapter 1.2.1 --- Viral sources --- p.11 / Chapter 1.2.2 --- NA isolation --- p.12 / Chapter 1.2.3 --- Recombinant NA expressed in cell lines --- p.12 / Chapter 1.2.4 --- Glycosylation on NA functionality --- p.13 / Chapter 1.2.5 --- Recombinant NA expressed in yeast --- p.15 / Chapter 1.3 --- Research objectives --- p.16 / Chapter 2 --- Cloning of Influenza Neuraminidase and Expression in P. pastoris --- p.17 / Chapter 2.1 --- Background --- p.17 / Chapter 2.1.1 --- Full-length cloning of the A/HongKong/483/97(H5N 1) NA --- p.17 / Chapter 2.1.2 --- Identification of the H274 equivalent --- p.19 / Chapter 2.1.3 --- The experiment --- p.22 / Chapter 2.2 --- Materials and methods --- p.23 / Chapter 2.2.1 --- Preparation of chemically competent Escherichia coli --- p.23 / Chapter 2.2.1.1 --- Reagents --- p.23 / Chapter 2.2.1.2 --- Reagent setup --- p.23 / Chapter 2.2.1.3 --- Equipment --- p.23 / Chapter 2.2.1.4 --- Procedure --- p.23 / Chapter 2.2.2 --- Amplification of N1 NA and EGFP genes --- p.24 / Chapter 2.2.2.1 --- Reagents --- p.24 / Chapter 2.2.2.2 --- Reagent setup --- p.25 / Chapter 2.2.2.3 --- Equipment --- p.25 / Chapter 2.2.2.4 --- Procedure --- p.26 / Chapter 2.2.2.4.1 --- Amplification of the full-length N1 NA gene from cDNA --- p.26 / Chapter 2.2.2.4.2 --- Amplification of the EGFP gene --- p.26 / Chapter 2.2.3 --- TA cloning of PCR products --- p.27 / Chapter 2.2.3.1 --- Reagents --- p.27 / Chapter 2.2.3.2 --- Reagent setup --- p.27 / Chapter 2.2.3.3 --- Equipment --- p.28 / Chapter 2.2.3.4 --- Procedure --- p.28 / Chapter 2.2.3.4.1 --- TA cloning of PCR products --- p.28 / Chapter 2.2.3.4.2 --- Site-directed mutagenesis by overlapping PCR --- p.30 / Chapter 2.2.4 --- Construction of P. pastoris expression vectors --- p.31 / Chapter 2.2.4.1 --- Reagents --- p.31 / Chapter 2.2.4.2 --- Reagent setup --- p.31 / Chapter 2.2.4.3 --- Procedure --- p.32 / Chapter 2.2.4.3.1 --- Generation of N1 NA expression vectors --- p.32 / Chapter 2.2.4.3.2 --- Generation of EGFP expression vectors --- p.34 / Chapter 2.2.5 --- Transformation of P. pastoris --- p.37 / Chapter 2.2.5.1 --- Reagents --- p.37 / Chapter 2.2.5.2 --- Reagent setup --- p.37 / Chapter 2.2.5.3 --- Equipment --- p.38 / Chapter 2.2.5.4 --- Procedure --- p.38 / Chapter 2.2.5.4.1 --- Preparation and transformation of electrocompetent P. pastoris --- p.38 / Chapter 2.2.5.4.2 --- PCR analysis of P. pastoris transformants (colony PCR) --- p.39 / Chapter 2.2.6 --- Expression of N1 NA and EGFP in P. pastoris --- p.40 / Chapter 2.2.6.1 --- Reagents --- p.40 / Chapter 2.2.6.2 --- Reagent setup --- p.40 / Chapter 2.2.6.3 --- Procedure --- p.41 / Chapter 2.2.6.3.1 --- Small-scale protein expression in P. pastoris --- p.41 / Chapter 2.2.6.3.2 --- Sequence alignment --- p.42 / Chapter 2.2.6.3.3 --- Data processing --- p.42 / Chapter 2.3 --- Results and Discussion --- p.43 / Chapter 2.3.1 --- Cloning of NA and EGFP into the pPICZB expression vector --- p.43 / Chapter 2.3.2 --- Growth of P. pastoris transformants --- p.51 / Chapter 3 --- Physical Characterization of Influenza Neuraminidase Expressed in P. pastoris --- p.53 / Chapter 3.1 --- Background --- p.53 / Chapter 3.1.1 --- Structural significance of disulphide bonds in NA --- p.53 / Chapter 3.1.2 --- Localization of recombinant N1 NA in P. pastoris --- p.55 / Chapter 3.1.3 --- The experiment --- p.56 / Chapter 3.2 --- Materials and methods --- p.57 / Chapter 3.2.1 --- Differential centrifugation --- p.57 / Chapter 3.2.1.1 --- Reagents --- p.57 / Chapter 3.2.1.2 --- Reagent setup --- p.57 / Chapter 3.2.1.3 --- Equipment --- p.57 / Chapter 3.2.1.4 --- Procedures --- p.58 / Chapter 3.2.1.4.1 --- Cell harvesting and lysis --- p.58 / Chapter 3.2.1.4.2 --- Preparation of crude membrane --- p.58 / Chapter 3.2.1.4.3 --- Preparation of plasma membrane --- p.58 / Chapter 3.2.2 --- Sodium dodecyl sulphate polyaciylamide gel electrophoresis (SDS-PAGE)… --- p.59 / Chapter 3.2.2.1 --- Reagents --- p.59 / Chapter 3.2.2.2 --- Reagent setup --- p.60 / Chapter 3.2.2.3 --- Equipment --- p.61 / Chapter 3.2.2.4 --- Procedure --- p.61 / Chapter 3.2.3 --- Immunoblotting --- p.61 / Chapter 3.2.3.1 --- Reagents --- p.61 / Chapter 3.2.3.2 --- Reagent setup --- p.62 / Chapter 3.2.3.3 --- Equipment --- p.62 / Chapter 3.2.3.4 --- Procedure --- p.62 / Chapter 3.2.3.4.1 --- Electroblotting --- p.62 / Chapter 3.2.3.4.2 --- Blocking and probing --- p.63 / Chapter 3.2.3.4.3 --- Immunodetection --- p.63 / Chapter 3.2.3.4.4 --- Molecular weight determination --- p.63 / Chapter 3.2.4 --- Confocal microscopy --- p.64 / Chapter 3.2.4.1 --- Equipment --- p.64 / Chapter 3.2.4.2 --- Procedure --- p.64 / Chapter 3.2.4.2.1 --- Image acquisition --- p.64 / Chapter 3.2.4.2.2 --- Image processing --- p.65 / Chapter 3.3 --- Results --- p.66 / Chapter 3.3.1 --- Localization of recombinant N1 NA in P. pastoris sub-cellular fractions --- p.66 / Chapter 3.3.2 --- Molecular weight determination for the N1 NA expressed in P. pastoris --- p.69 / Chapter 3.3.3 --- Cellular localization of recombinant N1 NA in P. pastoris --- p.71 / Chapter 3.4 --- Discussion --- p.77 / Chapter 3.4.1 --- Molecular weight determination for N1 NA expressed in P. pastoris --- p.77 / Chapter 3.4.2 --- Disulphide bond formation in N1 NA expressed in P. pastoris --- p.78 / Chapter 3.4.3 --- Cell-surface association of recombinant N1 NA in P. pastoris --- p.79 / Chapter 3.5 --- Conclusion --- p.81 / Chapter 4 --- Functional Characterization of Influenza Neuraminidase Expressed in P. pastor --- p.is / Chapter 4.1 --- Background --- p.82 / Chapter 4.1.1 --- Fluorometric NA activity assay --- p.82 / Chapter 4.1.2 --- Colorimetric assay of NA activity --- p.84 / Chapter 4.1.3 --- The experiment --- p.85 / Chapter 4.2 --- Materials and methods --- p.86 / Chapter 4.2.1 --- Fluorometric assay of N1 NA expressed in P. pastoris --- p.86 / Chapter 4.2.1.1 --- Reagents --- p.86 / Chapter 4.2.1.2 --- Reagent setup --- p.86 / Chapter 4.2.1.3 --- Equipment --- p.86 / Chapter 4.2.1.4 --- Procedure --- p.87 / Chapter 4.2.1.4.1 --- Calibrating cell density with viable cell counts --- p.87 / Chapter 4.2.1.4.2 --- End-point measurement of NA activity --- p.87 / Chapter 4.2.1.4.2.1 --- Determination of expression yield --- p.89 / Chapter 4.2.1.4.2.2 --- End-point assay of NAI sensitivity --- p.89 / Chapter 4.2.1.4.3 --- Kinetic measurement of NA activity --- p.90 / Chapter 4.2.1.4.3.1 --- Derivation ofV0 --- p.92 / Chapter 4.2.1.4.3.2 --- Graphical determination of KM --- p.93 / Chapter 4.2.1.4.3.3 --- Graphical determination of KI --- p.94 / Chapter 4.2.2 --- Colorimetric assay of N1 NA expressed in P. pastoris --- p.96 / Chapter 4.2.2.1 --- Reagents --- p.96 / Chapter 4.2.2.2 --- Reagent setup --- p.96 / Chapter 4.2.2.3 --- Equipment --- p.96 / Chapter 4.2.2.4 --- Procedure --- p.96 / Chapter 4.3 --- Results --- p.98 / Chapter 4.3.1 --- CFU determination --- p.98 / Chapter 4.3.2 --- Fluorescent NA activity assay for N1 NA expressed in P. pastoris --- p.98 / Chapter 4.3.2.1 --- End-point measurement of NA activity --- p.98 / Chapter 4.3.2.1.1 --- Course of N1 NA expression in P. pastoris --- p.102 / Chapter 4.3.2.1.1.1 --- NA activity per unit cell mass --- p.102 / Chapter 4.3.2.1.1.2 --- Yield of NA --- p.102 / Chapter 4.3.2.1.2 --- End-point assay for NAI sensitivity --- p.105 / Chapter 4.3.2.2 --- Kinetic measurement of NA activity and NAI sensitivity --- p.107 / Chapter 4.3.2.2.1 --- Graphical determination of KM --- p.107 / Chapter 4.3.2.2.2 --- Graphical determination of KI --- p.107 / Chapter 4.3.2.3 --- Colorimetric NA activity assay --- p.111 / Chapter 4.4 --- Discussion --- p.114 / Chapter 4.4.1 --- Fluorescent NA activity assay of N1 NA expressed in P. pastoris --- p.115 / Chapter 4.4.1.1 --- End-point measurement of NA activity --- p.115 / Chapter 4.4.1.1.1 --- Time course of expression --- p.115 / Chapter 4.4.1.1.2 --- Effect of H275Y mutation on NA activity and NAI sensitivity --- p.117 / Chapter 4.4.1.1.3 --- Effect of C-terminal tags on NA activity and NAI sensitivity --- p.117 / Chapter 4.4.1.2 --- Kinetic measurement of NA activity --- p.118 / Chapter 4.4.1.2.1 --- Graphical determination of KM --- p.119 / Chapter 4.4.1.2.2 --- Graphical determination of KI --- p.120 / Chapter 4.4.1.3 --- Comparison of fluorometric NA activity assays for use with whole P pastoris cells --- p.122 / Chapter 4.4.2 --- Colorimetric NA activity assay --- p.124 / Chapter 4.5 --- Conclusion --- p.126 / Chapter 5 --- Conclusions and Discussions --- p.127 / Chapter 5.1 --- General conclusions --- p.127 / Chapter 5.2 --- Follow-up --- p.127 / Chapter 5.2.1 --- Studies of influenza NA with enhanced activity --- p.128 / Chapter 5.2.2 --- NAI screening using yeast-expressed NA --- p.132 / Appendix --- p.134 / References --- p.141

Wong, Wing Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 90-105). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / Abstract (Chinese Version) --- p.v / Table of Contents --- p.vi / List of Figures --- p.ix / List of Abbreviations --- p.xi / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Overview of cancer --- p.1 / Chapter 1.2 --- Acute lymphoblastic leukemia --- p.3 / Chapter 1.2.1 --- T-cell acute lymphoblastic leukemia --- p.5 / Chapter 1.2.1.1 --- Chemotherapy / Chapter 1.2.1.2 --- Induction therapy / Chapter 1.2.1.3 --- Intensification therapy / Chapter 1.2.1.4 --- Maintenance therapy --- p.6 / Chapter 1.2.2 --- Chemoresistance in T-ALL / Chapter 1.3 --- Apoptosis and cancer --- p.7 / Chapter 1.3.1 --- Chemoresistance --- p.9 / Chapter 1.4 --- Caspase-dependent apoptosis --- p.10 / Chapter 1.4.1 --- Regulation of caspase-dependent apoptosis / Chapter 1.4.2 --- Initiation of apoptosis --- p.11 / Chapter 1.4.3 --- Exrtinsic pathway / Chapter 1.4.4 --- Intrinsic mitochondrial pathway --- p.15 / Chapter 1.4.4.1 --- Regulation of apoptosis by Bcl-2 family proteins --- p.16 / Chapter 1.4.4.2 --- Reactive Oxygen Species --- p.19 / Chapter 1.5 --- Selenium --- p.23 / Chapter 1.5.1 --- Importance of Se to human health --- p.25 / Chapter 1.5.2 --- Cancer chemoprevention by Se --- p.27 / Chapter 1.5.3 --- Preclinical studies --- p.28 / Chapter 1.5.4 --- Clinical investigations / Chapter 1.5.5 --- Mechanisms of action by selenocompounds --- p.29 / Chapter 1.6 --- Aims of current study --- p.31 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Cell culture --- p.32 / Chapter 2.2 --- Measurement of growth and survival of T-ALL cell lines / Chapter 2.3 --- Induction and quantification of apoptosis --- p.34 / Chapter 2.4 --- Western blotting / Chapter 2.4.1 --- Protein extraction and determination of protein concentration / Chapter 2.4.2 --- SDS-PAGE and immunodetection --- p.35 / Chapter 2.5 --- Analysis of mitochondrial membrane potential --- p.36 / Chapter 2.6 --- Measurement of ROS generation --- p.37 / Chapter 2.7 --- Verification of ROS generation via the addition of N-Acetyl-L-cysteine and glutathione / Chapter 2.8 --- Statistical analysis --- p.38 / Chapter Chapter 3 --- Results / Chapter 3.1 --- SeC induces prominent growth inhibition on Sup-T1 --- p.39 / Chapter 3.2 --- SeC induces S-phase arrest in cell cycle and triggers apoptosis in Sup-T1 --- p.44 / Chapter 3.3 --- SeC triggers DNA fragmentation in Sup-T1 --- p.48 / Chapter 3.4 --- SeC induces PARP cleavage in Sup-T1 --- p.52 / Chapter 3.5 --- SeC activates caspases in Sup-T1 --- p.53 / Chapter 3.6 --- SeC abrogates mitochondrial membrane potential in Sup-T1 cells --- p.56 / Chapter 3.7 --- SeC modulates expressions of Bcl-2 members and activates Bim and Bid in Sup-T1 --- p.61 / Chapter 3.8 --- SeC induces ROS production in Sup-T1 --- p.64 / Chapter 3.9 --- Antioxidants protect Sup-T1 cells from SeC-induced growth inhibition --- p.66 / Chapter 3.10 --- Antioxidants protect Sup-T1 cells from SeC-induced apoptosis --- p.69 / Chapter 3.11 --- Antioxidants effectively block SeC-induced ROS generation in Sup-T1 cells --- p.72 / Chapter 3.12 --- SeC induces mitochondrial membrane permeabilization via ROS-mediated mechanisms Sup-T1 cells --- p.75 / Chapter Chapter 4 --- Discussion --- p.79 / Conclusion --- p.87 / References --- p.90

Ng, Chor Fung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 130-144). / Abstracts in English and Chinese. / ABSTRACT (ENGLISH VERSION) --- p.i / ABSTRACT (CHINESE VERSION) --- p.iv / ACKNOWLEDGEMENTS --- p.vi / TABLE OF CONTENTS --- p.viii / LIST OF TABLES AND FIGURES --- p.xii / ABBREVIATIONS --- p.xiv / Chapter CHAPTER 1. --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Definition of asthma --- p.1 / Chapter 1.2 --- Asthma epidemiology --- p.2 / Chapter 1.3 --- Pathogenesis of Asthma --- p.3 / Chapter 1.3.1 --- Gene-environment interaction --- p.3 / Chapter 1.3.2 --- Allergens and atopic sensitization --- p.4 / Chapter 1.3.3 --- Other environmental factors --- p.5 / Chapter 1.4 --- House dust mite (HDM) --- p.5 / Chapter 1.4.1 --- Characteristics of HDM allergens --- p.5 / Chapter 1.4.2 --- HDM and asthma --- p.6 / Chapter 1.5 --- Pathophysiology of asthma --- p.8 / Chapter 1.5.1 --- Airway inflammation --- p.8 / Chapter 1.5.1.1 --- Cellular mechanism --- p.8 / Chapter 1.5.1.2 --- Characteristics of chronic inflammation --- p.9 / Chapter 1.5.1.3 --- Inflammatory cells in airway inflammation --- p.10 / Chapter 1.5.1.3.1 --- Mast cell --- p.10 / Chapter 1.5.1.3.2 --- Macrophages --- p.11 / Chapter 1.5.1.3.3 --- T lymphocytes --- p.12 / Chapter 1.5.1.3.4 --- Eosinophils --- p.12 / Chapter 1.5.1.3.5 --- Epithelial cells --- p.13 / Chapter 1.5.1.4 --- Cytokines in asthma --- p.14 / Chapter 1.5.1.4.1 --- Inflammatory cytokines --- p.14 / Chapter 1.5.1.4.1.1 --- Interleukin-4 --- p.14 / Chapter 1.5.1.4.1.2 --- Interleukin-5 --- p.14 / Chapter 1 5.1.4.1.3 --- Interleukin-6 --- p.15 / Chapter 1.5.1.4.1.4 --- Granulocyte Monocyte Colony Stimulating Factor (GM-CSF) --- p.15 / Chapter 1.5.1.4.1.5 --- Tumor Necrosis Factor-α (TNF-α) --- p.16 / Chapter 1.5.1.4.2 --- Anti-inflammatory cytokines --- p.17 / Chapter 1.5.1.4.2.1 --- Interleukin-10 --- p.17 / Chapter 1.5.1.4.2.2 --- Interferon-γ(IFN-γ) --- p.17 / Chapter 1.5.2 --- Airway hyperresponsiveness (AHR) --- p.18 / Chapter 1.5.3 --- A irway remodeling --- p.19 / Chapter 1.6 --- Asthma therapy --- p.21 / Chapter 1.6.1 --- β2-agonists --- p.21 / Chapter 1.6.2 --- Cromolyn and nedocromil --- p.21 / Chapter 1.6.3 --- Theophylline --- p.22 / Chapter 1.6.4 --- Leukotriene modifiers --- p.22 / Chapter 1.6.5 --- Corticosteroids --- p.23 / Chapter 1.7 --- Traditional Chinese Medicine --- p.24 / Chapter 1.7.1 --- Introduction --- p.24 / Chapter 1.7.2 --- Traditional Chinese Medicine (TCM) --- p.24 / Chapter 1.7.3 --- "Chinese herbal formula, CU Formula 2 (CUF2) and Kuan Dong Hua" --- p.26 / Chapter 1.8 --- Objectives of our studies --- p.28 / Chapter CHAPTER 2. --- ESTABLISHMENT OF A HDM-INDUCED ASTHMATIC ANIMAL MODEL IN SD RATS --- p.32 / Chapter 2.1 --- Introduction --- p.32 / Chapter 2.2 --- Materials and methods --- p.33 / Chapter 2.2.1 --- Buffers and solutions --- p.33 / Chapter 2.2.2 --- Animals --- p.33 / Chapter 2.2.3 --- Preparation of aluminum hydroxide gel --- p.34 / Chapter 2.2.4 --- HDMAllergen --- p.34 / Chapter 2.2.5 --- Sensitization Procedure --- p.35 / Chapter 2.2.6 --- Intratracheal instillation challenge --- p.35 / Chapter 2.2.7 --- Bronchoalveolar lavage (BAL) and BAL Cell counting --- p.36 / Chapter 2.2.8 --- Lung Histopathological Analysis --- p.37 / Chapter 2.2.9 --- Measurement of cytokine and chemokine by Enzyme-Linked Immunosorbent Assay (ELISA) --- p.39 / Chapter 2.2.10 --- Statistical Analysis --- p.40 / Chapter 2.3 --- Results --- p.41 / Chapter 2.3.1 --- Cellular Analysis of BALF --- p.41 / Chapter 2.3.2 --- Histopathology --- p.42 / Chapter 2.3.3 --- Cytokine and chemokine --- p.43 / Chapter 2.4 --- Discussion --- p.44 / Chapter CHAPTER 3. --- IMMUNOMODULATORY EFFECT OF CUF2 AND KUAN DONG HUA IN A RAT MODEL OF HDM-INDUCED ASTHMA --- p.65 / Chapter 3.1 --- Introduction --- p.65 / Chapter 3.2 --- Materials and methods --- p.67 / Chapter 3.2.1 --- Herbal materials and extraction method --- p.67 / Chapter 3.2.2 --- "Antigen sensitization, challenge, and treatment" --- p.68 / Chapter 3.2.3 --- Bronchoalveolar lavage and cell differential counts --- p.69 / Chapter 3.2.4 --- Histological Studies --- p.69 / Chapter 3.2.5 --- Measurement of BALF cytokines and chemokines --- p.70 / Chapter 3.2.6 --- "Body weight, thymus index and spleen index" --- p.70 / Chapter 3.2.7 --- Statistical analysis --- p.70 / Chapter 3.3 --- Results --- p.71 / Chapter 3.3.1 --- Effect of herbs and DXA on total cells and eosinophils in BALF --- p.71 / Chapter 3.3.2 --- Effect of herb and DXA on lung histology --- p.72 / Chapter 3.3.3 --- Effect of herbs and DXA on cytokine and chemokine level in BALF --- p.73 / Chapter 3.3.4 --- "Effect of herb and DXA on body weight, thymus index and spleen index" --- p.75 / Chapter 3.4 --- Discussion --- p.77 / Chapter CHAPTER 4. --- IMMUNOMODULATORY EFFECT OF KUAN DONG HUA ON HUMAN MAST CELLS (HMC-1) --- p.109 / Chapter 4.1 --- Introduction --- p.109 / Chapter 4.2 --- Materials and methods --- p.110 / Chapter 4.2.1 --- Reagents --- p.110 / Chapter 4.2.2 --- Cell line and Cell Culture --- p.111 / Chapter 4.2.3 --- Herb and extraction procedure --- p.111 / Chapter 4.2.4 --- Cell Viability Assay --- p.112 / Chapter 4.2.5 --- Assay of cytokine secretion --- p.113 / Chapter 4.2.6 --- Quantitative Analysis of cytokines --- p.113 / Chapter 4.2.7 --- Bacterial endotoxin contamination --- p.114 / Chapter 4.2.8 --- Statistical analysis --- p.115 / Chapter 4.3 --- Results --- p.116 / Chapter 4.3.1 --- Effect of Kuan Dong Hua on cell viability of HMC-I --- p.116 / Chapter 4.3.2 --- Effect of Kuan Dong Hua on cytokine release from HMC-I --- p.116 / Chapter 4.3.3 --- Effect of endotoxin contamination in the extract --- p.117 / Chapter 4.4 --- Discussion --- p.118 / Chapter CHAPTER 5. --- GENERAL CONCLUSION --- p.125 / Chapter 5.1 --- Conclusion --- p.125 / Chapter 5.2 --- Limitations of this study and Future work --- p.128 / REFERENCES --- p.130 / APPENDICES --- p.145 / Appendix A. Wright-Giemsa Stain for cytospin preparations --- p.145 / Appendix B. Hematoxylin & eosin (H&E) staining --- p.145 / Appendix C. Congo Red staining --- p.146 / Appendix D. Periodic acid-Schiff (PAS) staining --- p.146