Introdução: Comportamentos alimentares inadequados, inatividade física e maior tempo despendido na frente da televisão e/ou computadores são frequentes entre adolescentes e aumentam o risco para o ganho de peso. Destaca-se a importância em abordar adolescentes com estratégias de intervenção para a promoção da saúde. Objetivo: avaliar o efeito da intervenção sobre o estado nutricional, ingestão dietética, atividade física e comportamentos sedentários de lazer. Métodos: É um ensaio comunitário randomizado controlado com adolescentes do sexo feminino, matriculadas em 10 escolas técnicas públicas da cidade de São Paulo. O estudo está registrado na plataforma ClinicalTrials.gov e segue o checklist do CONSORT. A intervenção foi adaptada do estudo NEAT Girls em referência a Teoria Social Cognitiva e teve duração de seis meses. Foram avaliados o peso, a estatura e a circunferência da cintura, índice de massa corporal (IMC) e IMC z-score. O consumo alimentar foi avaliado com o questionário de frequência alimentar validado. A atividade física pelo questionário que avalia os três níveis de atividade física de lazer e os comportamentos sedentários de lazer por um questionário auto-relatado. Foi realizada estatística descritiva dos dados, os testes do qui-quadrado, t-student independe e o modelo linear misto para as análises dos dados com nível alpha de significância de 5 por cento . Todas as analises seguiram o princípio de intenção-tratamento. Resultados: Participaram 253 adolescentes (16.05 [EP 0.05] anos), 142 no grupo intervenção e 111 no grupo controle. Após os seis meses da intervenção, houve diferenças estatisticamente significantes a favor do grupo intervenção para o indicador da circunferência da cintura (-1.09cm; [0.51], p=0.027); consumo do grupo das frutas (0.23porção [0.11], p=0.006), das verduras e legumes (1.15porção [0.55], p=0.005) e dos açúcares e doces (-0.67porção [0.35], p=0.0.036); tempo despendido na frente dos computadores durante a semana (-0.39horas [0.21], p=0.039) e aos finais de semana (-0.54horas [0.22], p=0.030) e a soma de todas as atividades de tela durante o final de semana (-0.79horas [0.32], p=0.007). As diferenças proporcionais entre os níveis de atividade física leve (x2=20.95 p=0.007) e vigorosa (x2=8.09, p=0.017) favoreceram as meninas da intervenção. No geral, as meninas ficaram satisfeitas com o programa e o workshop de nutrição foi o componente que elas mais gostaram. Conclusão: O Hábitos Saudáveis, Meninas Saudáveis Brasil apresentou melhoria para o indicador circunferência da cintura, no consumo das frutas, verduras e legumes, dos doces, tempo despendido na frente dos computadores e na atividade física. / Introduction: Inadequate eating behaviors, physical inactivity and increase time spent on TV and computers are often among adolescents and increase the risk for weight gain. It highlights the importance to target adolescent in intervention strategies to promote health. Objective: evaluate the intervention effect on the nutritional status, dietary intake, physical activity and leisure sedentary behaviors. Methods: It is a group randomized controlled trial with female adolescents attending 10 public schools from São Paulo. The study is registered in ClinicalTrials.gov and follows the CONSORT checklist. The intervention was adapted from the NEAT Girls study in reference of the Social Cognitive Theory with 6 months duration. It was accessed weight, height, waist circumference, body mass index and body mass index Z-score. The food consumption was evaluated using a food frequency validated questionnaire. Physical activity was evaluated by a questionnaire that assesses the three levels of leisure-time physical activity and leisure sedentary behaviors using a self-reported questionnaire. Descriptive statistics, chi-squared, student-t and linear mixed models were used to analyze the data with a level of significance of 5 per cent . All analyses followed intention-to-treat principles. Results: Participated 254 adolescents (16.05 [EP 0.05] years), 142 and 111 from intervention and control groups, respectively. After six-month intervention, significant findings favoring intervention group were found for the waist circumference indicator (- 1.09cm; [0.51], p=0.027), fruits (0.23serving [0.11], p=0.006), vegetables (1.15serving [0.55], p=0.005), sweets (-0.67serving [0.35], p=0.0.036), time spent on computers during the weekdays (-0.39hours [0.21], p=0.039) and weekends (-0.54hours [0.22], p=0.030) and sum of the screen activities on the weekends (-0.79hours [0.32], p=0.007). Proportional differences in light (x2=20.95 p=0.007) and vigorous (x2=8.09, p=0.017) physical activity significantly favored the interventions girls. In general, girls were satisfied with the program and the nutrition workshop was the component most enjoyable. Conclusion: The Healthy Habits, Healthy Girls Brazil demonstrated an improvement on waist circumference indicator, fruit and vegetables, sweets, time spent on computers and physical activity levels.

Chu Wai Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 87-96). / Abstracts in English and Chinese. / Acknowledgments --- p.ii / Publication --- p.iii / List of Abbreviations --- p.iv / List of Tables --- p.v / List of Figures --- p.vi / Abstract --- p.vii / 摘要 --- p.x / Table of Contents --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Epidemiology of gastric cancer --- p.1 / Chapter 1.2 --- Risk factors associated with gastric cancer --- p.7 / Chapter 1.3 --- Prevention of Gastric Cancer --- p.9 / Chapter 1.4 --- H. pylori eradication and gastric cancer development --- p.11 / Chapter 1.5 --- Non-steroidal anti-inflammatory drugs and gastric cancer prevention --- p.13 / Chapter 1.6 --- COX-2 independent pathway --- p.14 / Chapter 1.7 --- Animal model of gastric cancer --- p.15 / Chapter 1.8 --- Microarray system --- p.16 / Chapter 1.9 --- Hypothesis --- p.18 / Chapter 1.10 --- Aim of study --- p.19 / Chapter Chapter 2 --- Chemoprevention of gastric cancer by celecoxib --- p.20 / Chapter 2.1 --- Introduction --- p.20 / Chapter 2.2 --- Material and Methods --- p.22 / Chapter 2.2.1 --- Animals --- p.22 / Chapter 2.2.2 --- Chemicals --- p.22 / Chapter 2.2.3 --- Study design --- p.23 / Chapter 2.2.4 --- Cell Culture --- p.24 / Chapter 2.2.5 --- Celecoxib treatment --- p.24 / Chapter 2.2.6 --- Cell proliferation assay --- p.25 / Chapter 2.3 --- Results --- p.26 / Chapter 2.3.1 --- Chemoprevention of gastric cancer by celecoxib in rats --- p.26 / Chapter 2.3.2 --- Effects of celecoxib on growth of human gastric cancer cells --- p.29 / Chapter 2.4 --- Discussion --- p.30 / Chapter 2.4.1 --- MNNG induced gastric cancer effectively --- p.30 / Chapter 2.4.2 --- Celecoxib significantly suppressed gastric carcinogenesis in rats --- p.31 / Chapter 2.4.3 --- Celecoxib inhibited the growth of MKN 45 in a concentration-dependent manner --- p.31 / Chapter 2.4.4 --- Celecoxib may exert its anti-tumor property through COX independent pathway --- p.32 / Chapter Chapter 3 --- Gene expression profiles of celecoxib treated rat gastric tumor and human gastric cells --- p.34 / Chapter 3.1 --- Introduction --- p.34 / Chapter 3.2 --- Material and Methods --- p.34 / Chapter 3.2.1 --- RNA extraction --- p.34 / Chapter 3.2.2 --- Target preparation and Array hybridization --- p.35 / Chapter 3.2.3 --- Post-hybridization processing and Scanning --- p.36 / Chapter 3.2.4 --- Microarray data analysis --- p.36 / Chapter 3.2.5 --- Quantitative RT-PCR --- p.37 / Chapter 3.3 --- Results --- p.39 / Chapter 3.3.1 --- Gene expression profiles of rat gastric tumors --- p.39 / Chapter 3.3.1.1 --- Genes differentially expressed in MNNG induced gastric tumors --- p.39 / Chapter 3.3.1.2 --- Genes differentially expressed in celecoxib treated group --- p.42 / Chapter 3.3.1.3 --- Mechanisms underlying chemoprevention of celecoxib --- p.43 / Chapter 3.3.2 --- Verification of gene expression by quantitative RT-PCR --- p.55 / Chapter 3.3.3 --- Confirmation of the gene expression profiles in human by quantitative RT-PCR --- p.59 / Chapter 3.4 --- Discussions --- p.63 / Chapter Chapter 4 --- Effects of celecoxib on Akt pathway in gastric cancer cells --- p.68 / Chapter 4.1 --- Introduction --- p.68 / Chapter 4.2 --- Material and methods --- p.72 / Chapter 4.2.1 --- Protein extraction --- p.72 / Chapter 4.2.2 --- Western blotting --- p.72 / Chapter 4.3 --- Results --- p.74 / Chapter 4.3.1 --- Expression of the Akt pathway after treatment with celecoxib --- p.74 / Chapter 4.4 --- Discussions --- p.78 / Chapter Chapter 5 --- Conclusion --- p.82 / References --- p.87

To Man Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 85-105). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / (in Chinese) --- p.iv / Acknowledgements --- p.vi / Publications --- p.viii / Table of Contents --- p.ix / List of Tables --- p.xii / List of Figures --- p.xiii / List of Abbreviations --- p.xiv / Chapter CHAPTER 1: --- General Introduction --- p.1 / Chapter Section 1.1 --- Background and Clinical Application of Anthracylines --- p.1 / Chapter Section 1.2 --- DOX-induced Cardiotoxicity --- p.3 / Chapter 1.2.1 --- Types of Cardiotoxicity --- p.4 / Chapter 1.2.1.1 --- Acute Cardiotoxicity --- p.4 / Chapter 1.2.1.2 --- Chronic Cardiotoxicity --- p.5 / Chapter 1.2.2 --- Subcellular Effects of DOX --- p.6 / Chapter 1.2.2.1 --- Ultrastructural Lesions --- p.6 / Chapter 1.2.2.2 --- Effects on Mitochondrial Functions --- p.7 / Chapter 1.2.2.3 --- Effects on Sarcoplasmic reticulum (SR) Functions --- p.8 / Chapter Section 1.3 --- Mechanisms of DOX-induced Cardiotoxicity --- p.8 / Chapter 1.3.1 --- Formation of Free Radicals --- p.9 / Chapter 1.3.1.1 --- Generation of Free Radicals by DOX --- p.10 / Chapter 1.3.1.2 --- Cardiac damage by Free radicals --- p.12 / Chapter 1.3.2 --- Induction of Apoptosis --- p.14 / Chapter 1.3.2.1 --- Characteristics and Pathway of Apoptosis --- p.14 / Chapter 1.3.2.2 --- Mitochondria and Apoptosis --- p.15 / Chapter 1.3.2.3 --- Caspases and Apoptosis --- p.17 / Chapter 1.3.2.4 --- Apoptosis and DOX-induced Cardiotoxicity --- p.18 / Chapter Section 1.4 --- Strategies to Reduce DOX-induced Cardiotoxicity --- p.19 / Chapter 1.4.1 --- Dosage optimization and Schedule modification --- p.19 / Chapter 1.4.2 --- Anthracycline Analogues --- p.21 / Chapter 1.4.3 --- Cardioprotective Agents --- p.21 / Chapter Section 1.5 --- Thrombopoietin --- p.23 / Chapter CHAPTER 2: --- Hypotheses and Objectives --- p.30 / Chapter CHAPTER 3: --- Methodology --- p.33 / Chapter Section 3.1 --- Methods --- p.33 / Chapter 3.1.1 --- Culture of Rat H9C2 Myoblast Cell Line and Primary Neonatal Rat Cardiomyocytes --- p.33 / Chapter 3.1.1.1 --- Maintenance of Cell Line --- p.33 / Chapter 3.1.1.2 --- Culture of Primary Neonatal Rat Cardiomyocytes --- p.34 / Chapter 3.1.2 --- Effects of Thrombopoietin on Cell Viability of Rat H9C2 Myoblast Cell Line and Beating Rates of Primary Rat Cardiomyocytes --- p.35 / Chapter 3.1.2.1 --- Cell Viability assay --- p.35 / Chapter 3.1.2.2 --- Beating Rate of Primary Beating Cardiomyocytes --- p.36 / Chapter 3.1.3 --- Effects of Thrombopoietin on the Protection against DOX-induced Heart Injury In Vivo --- p.36 / Chapter 3.1.3.1 --- Animals --- p.36 / Chapter 3.1.3.2 --- Experimental Protocol --- p.37 / Chapter 3.1.3.3 --- Echocardiography --- p.38 / Chapter 3.1.3.4 --- Blood Cell Counts --- p.39 / Chapter 3.1.3.5 --- Histopathology --- p.39 / Chapter 3.1.4 --- Effects of Thrombopoietin on Apoptosis and Mitochondrial Integrity of Rat H9C2 Myoblast Cell Line and Apoptosis In Vivo --- p.40 / Chapter 3.1.4.1 --- Determination of Externalized Phosphatidylserine --- p.40 / Chapter 3.1.4.2 --- Determination of Active Caspase-3 Expression --- p.41 / Chapter 3.1.4.3 --- Assessment of Mitochondrial Integrity --- p.42 / Chapter 3.1.4.4 --- TUNEL assay --- p.43 / Chapter 3.1.5 --- Statistical Analysis --- p.44 / Chapter CHAPTER 4: --- Effects of Thrombopoietin on Cell Viability of Rat H9C2 Myoblast Cell Line and Beating Rates of Primary Neonatal Rat Cardiomyocytes --- p.46 / Chapter Section 4.1 --- Results --- p.46 / Chapter 4.1.1 --- Effects of TPO on DOX-induced Cell Death --- p.46 / Chapter 4.1.2 --- Effects of TPO on the Beating Rates of Primary Cardiomyocytes --- p.47 / Chapter Section 4.2 --- Discussion --- p.47 / Chapter CHAPTER 5： --- Effects of Thrombopoietin on the Protection Against DOX-induced Heart Injury In Vivo --- p.54 / Chapter Section 5.1 --- Results --- p.54 / Chapter 5.1.1 --- General Observations and Survival --- p.54 / Chapter 5.1.2 --- Blood Cell Counts --- p.55 / Chapter 5.1.3 --- Cardiac Functions by Echocardiography --- p.56 / Chapter 5.1.4 --- Gross Anatomic Changes and Pathology of the Myocardium --- p.57 / Chapter Section 5.2 --- Discussion --- p.58 / Chapter CHAPTER 6: --- Effects of Thrombopoietin on Apoptosis and Mitochondrial Integrity of H9C2 Cell Line and Apoptosis In Vico --- p.69 / Chapter Section 6.1 --- Results --- p.69 / Chapter 6.1.1 --- Determination of Externalized Phosphatidylserine --- p.69 / Chapter 6.1.2 --- Determination of Active Caspase-3 Activity --- p.70 / Chapter 6.1.3 --- Assessment of Mitochondrial Membrane Potential --- p.70 / Chapter 6.1.4 --- Determination of Apoptosis by TUNEL assay --- p.72 / Chapter Section 6.2 --- Discussion --- p.72 / Chapter CHAPTER 7： --- General Discussion and Conclusion --- p.83 / References --- p.85

Submitted by Filipe dos Santos (fsantos@pucsp.br) on 2016-11-29T11:27:18Z No. of bitstreams: 1 Juliana Vaz.pdf: 3364230 bytes, checksum: 3d8d38bd51667b7aa95a158dc47949cc (MD5) / Made available in DSpace on 2016-11-29T11:27:18Z (GMT). No. of bitstreams: 1 Juliana Vaz.pdf: 3364230 bytes, checksum: 3d8d38bd51667b7aa95a158dc47949cc (MD5) Previous issue date: 2016-09-22 / Introduction: The nurse is the responsible one for acting in the prevention and control of infections in Health Services. Objectives: To assess in the undergraduate nursing course with active methodologies, the approach of the subject infections in Health Services; identify through the perspective of professors and students the approach of infections in Health Services and verify the teaching and learning methods that include this subject. Materials and Methods: A descriptive and exploratory, documentary and quanti-qualitative study held at the Undergraduate Nursing Course at the Pontifical Catholic University of São Paulo. The documentary research covered the Bank of Tutorials Problems and Material Support of the virtual environment. Participated in 12 (66.6%) teachers and 60 students (57.6%) who answered a questionnaire containing a Likert Scale and open questions. Results: The documentary study showed that the content is approached gradually, and meets the specificities of the axis of each year of graduation. The instruments are reliable, Cronbach's alpha value of 0.90 for professors and 0.91 for students. In characterizing the sample, teachers were 100% female. Students were 95% female and 5% male. 24 (40%) of the students are workers and eight (13.3%) perform paid internship. In the Structure dimension professors feel prepared to address the issue and do not provide material in the virtual environment. In Process they agreed that the practical activities allow the learning and there was no consensus for the approach of the subject as a learning objective in the study of problems. In Results they indicated that the student performs procedures applying the concepts. Students claimed that the structure of the materials used in practical activities are sufficient for learning and internship sites offer learning opportunities. In Process they agreed that the time spent on the subject is sufficient and had participated in activities addressing the subject. In the Result dimension they recognize that are applying the concepts in practice and claim that teaching activities developed in the course lead to the improvement of care quality. In comparing the three dimensions (analysis students instrument), the one that obtained the highest favorability score was Process (64%) and the worst, Structure (36%). Professors and students converged their views on the importance of the theme approach and suggest it to be further explored in the learning objectives and further deepening of the subject, respectively. Discussion: The findings allowed to verify the potential of active methods for teaching prevention of infections in Health Services and show the points that deserve more emphasis / Introdução: O Enfermeiro é o responsável em atuar na prevenção e controle de infecção em serviços de saúde. Objetivos: Avaliar no curso de graduação de enfermagem com metodologias ativas, a abordagem do tema infecções em Serviços de Saúde; identificar sob a perspectiva do docente e discente a abordagem de infecção em serviços de saúde e verificar as modalidades de ensino aprendizagem que contemplam essa temática. Material e Método: Estudo exploratório-descritivo, documental e quantiqualitativo realizado no Curso de Enfermagem da Pontifícia Universidade Católica de São Paulo. A pesquisa documental foi no Banco de Problemas Tutoriais e Material de Apoio do ambiente virtual. Participaram 12 (66,6%) docentes e 60 discentes (57,6%) que responderam um questionário, contendo uma escala de Likert e perguntas abertas. Resultados: O estudo documental mostrou que o conteúdo é abordado de forma gradativa, atende as especificidades do eixo de cada ano. Os instrumentos mostraram-se confiáveis, valor de Alfa de Cronbach de 0,90 dos professores e 0,91 dos alunos. Na caracterização, os docentes 100% eram do sexo feminino. Os discentes eram 95% do sexo feminino e 5% do sexo masculino. 24 (40%) são alunos trabalhadores e oito (13,3%) realizam estágio remunerado. Na dimensão Estrutura os docentes sentem-se preparados para abordar o tema e não disponibilizam material no ambiente virtual. No Processo concordaram que as atividades práticas permitem o aprendizado e não houve consenso quanto a abordagem do tema como objetivo de aprendizagem nos problemas. Em Resultado referiram que o aluno realiza procedimentos aplicando os conceitos. Os alunos alegaram que na Estrutura os materiais utilizados nas atividades práticas são suficientes para o aprendizado e que os locais de estágio oferecem oportunidade de aprendizado. No Processo concordaram que o tempo dispendido sobre o assunto é suficiente e que participaram de atividade sobre a temática. Na dimensão Resultado reconhecem que aplicam os conceitos na prática e que as atividades de ensino desenvolvidas no curso acarretam melhoria na qualidade da assistência. Na comparação das três dimensões (análise instrumento dos alunos), a que obteve maior escore de favorabilidade foi a de Processo (64%) e a pior o Estrutura (36%). Docentes e alunos confluíram suas opiniões quanto a importância da abordagem do tema e sugerem que seja mais explorado nos objetivos de aprendizagem e maior aprofundamento respectivamente. Discussão: Os achados permitiram verificar a potencialidade das metodologias ativas para o ensino de prevenção de infecções nos serviços de saúde e mostrar os pontos que merecem maior ênfase

Estudo transversal, descritivo e exploratório, com abordagem metodológica qualitativa, cujo objetivo foi identificar e avaliar a percepção da vulnerabilidade à doença, entre mulheres com diagnóstico avançado do câncer do colo do útero, tendo como referencial teórico, a vulnerabilidade, baseada no conceito proposto por Ayres et al. (2003). Os sujeitos do estudo foram doze mulheres que estavam em atendimento no Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo, para tratamento do câncer do colo do útero avançado, entre 08 de abril e 28 de maio de 2009. Os dados foram coletados por entrevistas. A análise dos dados se deu segundo os pressupostos da análise de conteúdo, proposta por Bardin (2006). Os resultados revelaram-se em 2 categorias centrais: Percebendo-se vulnerável e Enfrentando a doença e o tratamento. A vulnerabilidade se apresentou para as participantes deste estudo a partir do aparecimento dos primeiros sinais e sintomas da doença, crescendo à medida que o diagnóstico foi descoberto e o tratamento se fez necessário, trazendo dificuldades e sofrimento. Ao mesmo tempo, proporcionou a oportunidade de as mulheres encontrarem formas de enfrentamento e superação dos problemas, à medida do possível. Os componentes individual, social e programático da vulnerabilidade, contidos no conceito proposto por Ayres et al. (2003), mostraram-se nos fatores relacionados à cliente, aos profissionais, aos serviços, entre outros, e tornaram a mulher suscetível aos problemas e danos de sua saúde, relativos ao câncer cérvico-uterino, exacerbando sua vulnerabilidade à doença. Ficou clara a necessidade de se superar algumas deficiências no modelo de assistência e na humanização do atendimento, no grau de compromisso e na qualidade das instituições, dos recursos, do gerenciamento e do monitoramento dos programas de prevenção e detecção do câncer do colo do útero, nos diferentes níveis de atenção. / This cross-sectional, descriptive, exploratory and qualitative study aimed to identify and evaluate the perception of the vulnerability to the disease among women with delayed diagnosis of uterine cervical cancer. The theoretical framework used was the vulnerability, based in the concept proposed by Ayres et al. (2003). The subjects of the study were twelve women who were under follow-up at the Hospital das Clínicas of the University of São Paulo at Ribeirão Preto Medical School, for treatment of advanced uterine cervical cancer, between April 08th and May 28th 2009. Data were collected by interviews. The data analysis was done according to the presuppositions of content analysis, proposed by Bardin (2006). Two central categories emerged from the results: Perceiving yourself vulnerable and Facing the disease and the treatment. The vulnerability arose to the participants of this study through the emergence of the first signs and symptoms of the disease, increasing as the diagnosis was determined and treatment was needed, bringing difficulties and suffering. At the same time, women had the opportunity to find ways to face and overcome problems, to the extent possible. The individual, social and programmatic compounds of the vulnerability, included in the concept proposed by Ayres et al. (2003), were shown in the factors related to clients, professionals and services, among others, and made the women susceptible to the problems and damages of their health, related to the uterine cervical cancer, aggravating their vulnerability to the disease. The need to overcome some deficiencies in the model of care and in the humanization of care was evidenced, as well as in the level of commitment and in the quality of the institutions, resources, management, and monitoring of the programs of prevention and detection of uterine cervical cancer, in the different levels of care.

Wang Huan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 148-158). / Abstracts in English and Chinese. / TABLE OF CONTENTS --- p.I / LIST OF FIGURES AND TABLES --- p.VIII / ABSTRACT --- p.1 / 摘要 --- p.3 / Chapter CHAPTER 1 --- GENERAL INTRODUCTION / Chapter I. --- The essential factors related to cancer --- p.5 / Chapter a. --- Carcinogens --- p.5 / Chapter b. --- Carcinogenesis pathways --- p.7 / Chapter c. --- DNA adducts formation and breast cancer --- p.7 / Chapter II. --- Cytochrome P450 I enzyme family --- p.8 / Chapter a. --- CYP450 superfamily --- p.8 / Chapter b. --- CYP1A1 --- p.10 / Chapter c. --- CYP1B1 --- p.11 / Chapter III. --- Transactivation of CYP1 enzymes by aryl hydrocarbon receptor (AhR) --- p.12 / Chapter IV. --- Phase II enzyme UGT and cancer prevention --- p.13 / Chapter V. --- Estrogen metabolism and the hormone-dependent breast cancer --- p.15 / Chapter a. --- Estrogen and breast cancer initiation --- p.15 / Chapter b. --- Estrogen Receptor (ER) --- p.15 / Chapter c. --- Estradiol hydroxylation pathways --- p.15 / Chapter VI. --- Phytochemicals and cancer prevention --- p.18 / Chapter VII. --- Outline of this study --- p.20 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter I. --- Chemicals --- p.21 / Chapter II. --- Cell culture and treatments --- p.21 / Chapter 1. --- Maintenance of cells --- p.21 / Chapter 2. --- Preparation of cell stock --- p.22 / Chapter 3. --- Cell recovery from liquid nitrogen stock --- p.22 / Chapter 4. --- Measurement of cell viability --- p.22 / Chapter 5. --- Preparation of cell lysates --- p.23 / Chapter 6. --- XRE-luciferase gene reporter assay --- p.23 / Chapter a. --- Transient transfection of cell using lipofectamine PLUS reagent --- p.23 / Chapter b. --- Dual Luciferase Assay --- p.24 / Chapter III. --- Enzyme Activities --- p.24 / Chapter 1. --- Isolation of microsomes --- p.24 / Chapter 2. --- EROD activities in intact cells --- p.24 / Chapter 3. --- EROD inhibition assay --- p.25 / Chapter IV. --- Manipulation of Nuclear Acid --- p.26 / Chapter 1. --- Preparation of transfected DNA --- p.26 / Chapter a. --- Separation and purification of DNA from agarose gel --- p.26 / Chapter b. --- Restriction digestion --- p.26 / Chapter c. --- Ligation of DNA fragments --- p.27 / Chapter d. --- Transformation of DH5a --- p.27 / Chapter e. --- Small scale plasmid purification from DH5a (mini prep) --- p.28 / Chapter f. --- Large scale plasmid isolation from DH5a (maxi-prep) --- p.28 / Chapter g. --- Construction of XRE activated luciferase reporter gene --- p.29 / Chapter 2. --- Measurement of DMBA-DNA adduct formation --- p.29 / Chapter 3. --- Semi-quantitative RT-PCR Assay --- p.30 / Chapter a. --- Isolation of RNA using TRIzol® Reagent --- p.30 / Chapter b. --- RT-PCR --- p.31 / Chapter V. --- Phase II enzyme-UGT activity assay --- p.32 / Chapter VI. --- HPLC for estradiol-hydroxylation analysis --- p.33 / Chapter 1. --- HPLC condition for hydroxyestradiol separation and measurement --- p.33 / Chapter 2. --- Determination of microsomal estradiol hydroxylase activity --- p.34 / Chapter 3. --- Assay of estradiol metabolism in MCF-7 cells --- p.34 / Chapter VII. --- Statistical Analysis --- p.35 / Chapter CHAPTER 3 --- CHALCONES ANTAGONIZE DMBA-INDUCED CARCINOGENESIS BY MODULATION OF CYP1A1/1B1 AND UGT ACTIVITIES / Chapter Part One --- Introduction --- p.36 / Chapter Part Two --- Results --- p.40 / Chapter Section One --- Chalcones antagonize DMBA carcinogenesis by inhibiting CYP1A1 and CYP1B1 activities --- p.40 / Chapter I. --- Chalcones inhibited DMBA-induced EROD activities in MCF-7 cells --- p.40 / Chapter II. --- Inhibition of chalcones on microsomal CYP1A1 & 1B1 enzyme activities --- p.43 / Chapter III. --- Reduction of DMBA-induced DNA adduct by chalcones --- p.52 / Chapter IV. --- Chalcones antagonized CYP1A1 XRE transactivation --- p.54 / Chapter V. --- Chalcones suppressed DMBA-induced CYP1 gene expression --- p.56 / Chapter Section Two --- Chalcones modulate DMBA carcinogenesis by regulating UGT activities --- p.63 / Chapter I . --- Chalcones regulated UGT1A1 gene expression in MCF-7 cells --- p.63 / Chapter II. --- Chalcones affected UGT enzyme activity in HepG2 cells --- p.70 / Chapter III. --- Chalcones regulated UGT1A1 gene expression in HepG2 cells --- p.73 / Chapter Part Three --- Discussion --- p.80 / Chapter I . --- Chalcones are potential chemopreventive agents --- p.80 / Chapter II. --- Chalcones modulated Phase I enzyme activities --- p.80 / Chapter III. --- Chalcones regulated Phase II enzyme activities --- p.82 / Chapter IV. --- Chalcones suppressed DMBA-induced DNA-adduct formation in MCF-7 cells --- p.82 / Chapter V. --- The anti-carcinogenic properties of chalcones and their structures --- p.83 / Chapter CHAPTER 4 --- EFFECTS OF PERILLYL ALCOHOL AND LIMONENE ON CYP1 AND UGT ENZYMES / Chapter Part One --- Introduction --- p.85 / Chapter Part Two --- Results --- p.87 / Chapter I. --- Perillyl alcohol and limonene modulated DMBA-induced CYP1A1/1B1 activities in MCF-7 cells --- p.87 / Chapter II. --- Perillyl alcohol and limonene regulated microsomal CYP1A1/1B1 activities --- p.89 / Chapter III. --- Perillyl alcohol and limonene regulated DMBA-induced DNA adduct formation in MCF-7 cells --- p.93 / Chapter IV. --- Perillyl alcohol and limonene regulated CYP1A1 & CYP1B1 gene expressions in MCF-7 cells --- p.95 / Chapter V. --- Effect of perillyl alcohol on CYP1A1 XRE transactivation --- p.97 / Chapter VI. --- Cytotoxic effect of perillyl alcohol and limonene on MCF-7 cells --- p.98 / Chapter VII. --- Perillyl alcohol and limonene modulated UGT1A1 gene expression in MCF-7 cells --- p.99 / Chapter VIII. --- Perillyl alcohol and limonene modulated UGT enzyme in HepG2 cells --- p.101 / Chapter Part Three --- Discussion --- p.106 / Chapter CHAPTER 5 --- LYCOPENE MEDIATED DMBA-INDUCED PHASE I & PHASE II ENZYME ACTIVITIES AND GENE EXPRESSIONS / Chapter Part Three --- Introduction --- p.109 / Chapter I. --- Biochemical properties of lycopene --- p.109 / Chapter II. --- Bioavailability of lycopene --- p.110 / Chapter III. --- Lycopene and cancers in hormonal sensitive tissues --- p.110 / Chapter Part Two --- Results --- p.111 / Chapter I . --- Lycopene modulated DMBA-induced CYP1A1/1B1 activities in MCF-7 cells --- p.111 / Chapter II. --- Lycopene competitively inhibited microsomal CYP1A1 & CYP1B1 activities --- p.113 / Chapter III. --- Lycopene suppressed DMBA-induced DNA adduct formation in MCF-7 cells --- p.115 / Chapter IV. --- Lycopene regulated CYP1A1 & CYP1B1 gene expression in MCF-7 cells --- p.116 / Chapter V. --- Effect of lycopene on CYP1A1 XRE trasactivation --- p.117 / Chapter VI. --- Cytotoxic effect of lycopene on MCF-7 cells --- p.118 / Chapter VII. --- Lycopene modulated UGT enzyme in MCF-7 cells --- p.119 / Chapter VIII. --- Lycopene modulated UGT enzyme in HepG2 cells --- p.121 / Chapter Part Three --- Discussion --- p.123 / Chapter CHAPTER 6 --- CHALCONES AND PERILLYL ALCOHOL REGULATEDCYP1A1 & CYP1B1 MEDIATED ESTRADIOL METABOLIZING PATHWAYS / Chapter Part One --- Introduction --- p.125 / Chapter I . --- Estrogen hydroxylation and human breast cancer risk --- p.125 / Chapter II. --- CYP1 enzymes catalyze estradiol-hydroxylation in human breast cancer cells --- p.126 / Chapter III. --- Phytochemicals mediate estrogen-hydroxylation pathways --- p.126 / Chapter Part Two --- Estrogen metabolite detection and separation by HPLC --- p.127 / Chapter Part Three --- Results --- p.129 / Chapter I . --- Perillyl alcohol modulated CYP1A1 & CYP1B1-mediated Estradiol hydroxylation --- p.129 / Chapter II. --- Kinetics assays of chalcones on CYP1A1 & CYP1B1 microsomes induced estradiol hydroxylation --- p.131 / Chapter III. --- Chalcones suppressed Estradiol-hydroxylase activities in MCF-7 cells --- p.137 / Chapter Part Four --- Discussion --- p.140 / Chapter CHAPTER 7 --- SUMMARY / Chapter I . --- Chalcones displayed inhibitory effects on DMBA-induced carcinogenesis --- p.142 / Chapter II. --- Perillyl alcohol and limonene modulated DMBA-induced carcinogenesis --- p.143 / Chapter III. --- Lycopene also possessed chemoproventive properties --- p.143 / APPENDIX 1 ABBREVIATIONS --- p.144 / APPENDIX 2 REAGENTS --- p.145 / APPENDIX 3 PRIMER LISTS --- p.147 / REFERENCE --- p.148

Cheng Yee Wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 95-112). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Table of Contents --- p.vi / List of Figures --- p.xi / List of Plates --- p.xiv / List of Tables --- p.xvi / Abbreviations --- p.xviii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Legionella pneumophila --- p.1 / Chapter 1.1.1 --- Bacterial morphology and ultrastructure --- p.2 / Chapter 1.1.2 --- Microbial ecology and natural habitats --- p.4 / Chapter 1.1.2.1 --- Association with amoeba --- p.5 / Chapter 1.1.2.2 --- Association with biofilm --- p.5 / Chapter 1.2 --- Legionnaires' disease and clinical significance --- p.6 / Chapter 1.2.1 --- Epidemiology --- p.6 / Chapter 1.2.1.1 --- Worldwide distribution --- p.6 / Chapter 1.2.1.2 --- Local situation --- p.7 / Chapter 1.2.2 --- Clinical presentation --- p.7 / Chapter 1.2.3 --- Route of infection and pathogenesis --- p.8 / Chapter 1.2.4 --- Diagnosis --- p.10 / Chapter 1.2.4.1 --- Culture of Legionella --- p.10 / Chapter 1.2.4.2 --- Direct fluorescent antibody (DFA) staining --- p.13 / Chapter 1.2.4.3 --- Serologic tests --- p.13 / Chapter 1.2.4.4 --- Urine antigen testing --- p.14 / Chapter 1.2.4.5 --- Detection of Legionella nucleic acid --- p.15 / Chapter 1.2.5 --- Risk factors --- p.15 / Chapter 1.2.6 --- Treatment for Legionella infection --- p.16 / Chapter 1.3 --- Detection of Legionella in environment --- p.16 / Chapter 1.4 --- Disinfection methods --- p.17 / Chapter 1.4.1 --- Physical methods --- p.19 / Chapter 1.4.1.1 --- Filtration --- p.19 / Chapter 1.4.1.2 --- UV-C irradiation --- p.20 / Chapter 1.4.1.3 --- Thermal eradication (superheat-and-flush) --- p.21 / Chapter 1.4.2 --- Chemical methods --- p.21 / Chapter 1.4.2.1 --- Chlorination --- p.21 / Chapter 1.4.2.2 --- Copper-silver ionization --- p.22 / Chapter 1.4.3 --- Effect of biofilm and other factors on disinfection --- p.23 / Chapter 1.5 --- Photocatalytic oxidation (PCO) --- p.24 / Chapter 1.5.1 --- Generation of strong oxidants --- p.24 / Chapter 1.5.2 --- Disinfection mechanism(s) --- p.27 / Chapter 1.5.3 --- Major factors affecting the process --- p.28 / Chapter 2. --- Objectives --- p.30 / Chapter 3. --- Materials and Methods --- p.31 / Chapter 3.1 --- Chemicals --- p.31 / Chapter 3.2 --- Bacterial strains and culture --- p.31 / Chapter 3.3 --- Photocatalytic reactor --- p.33 / Chapter 3.4 --- PCO efficacy tests --- p.33 / Chapter 3.5 --- PCO sensitivity tests --- p.35 / Chapter 3.6 --- Optimisation of PCO conditions --- p.35 / Chapter 3.6.1 --- Optimization of TiO2 concentration --- p.36 / Chapter 3.6.2 --- Optimization of UV intensity --- p.36 / Chapter 3.6.3 --- Optimization of depth of reaction mixture --- p.36 / Chapter 3.6.4 --- Optimization of stirring rate --- p.37 / Chapter 3.6.5 --- Optimization of initial pH --- p.37 / Chapter 3.6.6 --- Optimization of treatment time and initial cell concentration --- p.37 / Chapter 3.6.7 --- Combinational optimization --- p.37 / Chapter 3.7 --- Transmission electron microscopy (TEM) --- p.38 / Chapter 3.8 --- Fatty acid profile analysis --- p.40 / Chapter 3.9 --- Total organic carbon (TOC) analysis --- p.42 / Chapter 3.10 --- UV-C irradiation --- p.44 / Chapter 3.11 --- Hyperchlorination --- p.44 / Chapter 3.12 --- Statistical analysis and replication --- p.45 / Chapter 3.13 --- Safety precautions --- p.45 / Chapter 4. --- Results --- p.46 / Chapter 4.1 --- Efficacy test --- p.46 / Chapter 4.2 --- PCO sensitivity --- p.47 / Chapter 4.3 --- Optimization of PCO conditions --- p.48 / Chapter 4.3.1 --- TiO2 concentration --- p.48 / Chapter 4.3.2 --- UV intensity --- p.48 / Chapter 4.3.3 --- Depth of reaction mixture --- p.51 / Chapter 4.3.4 --- Stirring rate --- p.56 / Chapter 4.3.5 --- Effect of initial pH --- p.56 / Chapter 4.3.6 --- Effect of treatment time and initial concentrations --- p.56 / Chapter 4.3.7 --- Combinational effects --- p.63 / Chapter 4.4 --- Transmission electron microscopy (TEM) --- p.66 / Chapter 4.4.1 --- Morphological changes induced by PCO --- p.66 / Chapter 4.4.2 --- Comparisons with changes caused by UV-C irradiation and chlorination --- p.67 / Chapter 4.5 --- Fatty acid profile analysis --- p.71 / Chapter 4.6 --- Total organic carbon (TOC) analysis --- p.73 / Chapter 4.7 --- UV-C irradiation --- p.74 / Chapter 4.8 --- Hyperchlorination --- p.74 / Chapter 5. --- Discussion --- p.76 / Chapter 5.1 --- Efficacy test --- p.76 / Chapter 5.2 --- PCO sensitivity --- p.76 / Chapter 5.3 --- Optimization of PCO conditions --- p.77 / Chapter 5.3.1 --- Effect of TiO2 concentration --- p.77 / Chapter 5.3.2 --- Effect of UV intensity --- p.78 / Chapter 5.3.3 --- Effect of depth of reaction mixture --- p.79 / Chapter 5.3.4 --- Effect of stirring rate --- p.79 / Chapter 5.3.5 --- Effect of initial pH --- p.80 / Chapter 5.3.6 --- Effect of treatment time and initial concentrations --- p.81 / Chapter 5.3.7 --- Combinational effect --- p.82 / Chapter 5.4 --- Transmission electron microscopy (TEM) --- p.83 / Chapter 5.4.1 --- Morphological changes induced by PCO --- p.83 / Chapter 5.4.2 --- Comparisons with changes caused by UV-C irradiation and chlorination --- p.85 / Chapter 5.5 --- Fatty acid profile analysis --- p.85 / Chapter 5.6 --- Total organic carbon (TOC) analysis --- p.86 / Chapter 5.7 --- Comparisons of the three disinfection methods --- p.88 / Chapter 6. --- Conclusion --- p.91 / Chapter 7. --- References --- p.95 / Chapter 8. --- Appendix --- p.113

強心苷是一類多年普遍用於心力衰竭治療的化合物,包括蟾蜍靈和地高辛。鈉泵（也可稱為鈉鉀ATP酶）是強心苷的受體。最近流行病學研究，體外實驗，動物實驗和臨床試驗表明，強心苷具有癌症治療的強大潛力。 / 大腸癌是全球第三大殺手，約有一半的大腸癌患者需要手術切除後的輔助治療。因此，通過化療殺死腫瘤細胞，是一個可行的辦法來治療大腸癌患者。在本課題的研究中，強心苷抗人結腸癌的作用在HT-29和Caco-2細胞上進行了評價與闡釋。在結腸癌細胞研究模型中，蟾蜍靈誘導caspase非依賴性的細胞死亡，伴隨沒有早期凋亡，沒有聚（ADP-核糖）聚合酶(PARP)與caspase-3裂解，這些發現與強心苷誘發其它類腫瘤細胞凋亡的機製完全不同。相反，蟾蜍靈激活自噬途徑，促進LC3-II積累和自噬流動。此外，其它強心苷如地高辛與烏本苷也促使LC3-II在HT-29細胞內聚集。沉默ATG5和Beclin-1顯著降低蟾蜍靈誘導的LC3- II積累和細胞死亡。蟾蜍靈誘導的自噬與活性氧（ROS）產生和JNK活化相關。我們的研究結果揭示了蟾蜍靈藥物對抗結腸癌細胞的一種新的機制，開闢了強心苷通過自噬途徑來治療大腸癌的可能性。 / 最近的研究表明，強心苷誘導多種癌細胞系的細胞包括促使凋亡與自噬的細胞週期阻滯在G2／M期。然而，沒有詳細的信息闡述強心苷如何阻滯細胞週期進展。在本課題研究中，我們研究了強心苷介導的細胞週期阻滯的分子機制。蟾蜍靈處理的HeLa H2B-YFP細胞被阻滯在前中期，伴隨姐妹染色單體凝聚，染色體未排列在赤道板，未退出有絲分裂期。這一結果被蟾蜍靈誘導的四倍DNA含量細胞既不在四倍體G1期也不在胞質分裂期進一步證明。此後，我們檢測了紡錘體組裝和染色體分離所需的Aurora激酶和Polo-like kinase 1 (Plk1)。結果發現，在HT-29和HeLa細胞上，蟾蜍靈和其它強心苷能顯著降低總蛋白質和磷酸化的Aurora激酶與Plk1。此外，我們還發現，蟾蜍靈通過PI3K下調有絲分裂酶的活性。這些結果已經通過沉默鈉泵α做了驗證。總之，我們的結果表明, 蟾蜍靈和其它強心苷鈉鉀泵抑製劑強有力的抑制細胞在前中期是通過PI3K/HIF-1α/NF-κB途徑下調Aurora激酶的蛋白質和磷酸化水平和Plk1的蛋白質水平。我們的研究發現在了解如何利用強心苷的潛能治療癌症以及認知鈉泵在細胞週期中的功能方面提供了有用的信息。 / The sodium pump (also known as Na+/K+-ATPase) is the receptor for cardiac glycosides, a group of compounds including bufalin and digoxin which have been commonly used for heart failure treatment for many years. Recent epidemiological studies, in vitro studies, animal studies and clinical trials have shown that cardiac glycosides have potential applications for cancer treatment. / Colorectal cancer is the third leading cause of cancer death worldwide and about half of the patients with colorectal cancer require adjuvant therapy after surgical resection. Therefore, the eradication of cancer cells via chemotherapy constitutes a viable approach to treat patients with colorectal cancer. In this study, the effects of cardiac glycosides were evaluated and characterized in HT-29 and Caco-2 human colon cancer cells. Contrary to their well documented apoptosis-promoting activity in other cancer cells, bufalin did not cause caspase-dependent cell death in colon cancer cells, as indicated by the absence of significant early apoptosis, as well as poly(ADP-ribose) polymerase (PARP) and caspase-3 cleavage. Instead, bufalin activated an autophagy pathway, as characterized by the accumulation of LC3-II and the stimulation of autophagic flux. Moreover, other cardiac glycosides digoxin and ouabain could also induce the accumulation of LC3-II in HT-29 cells. The silencing of ATG5 and Beclin-1 significantly reduced bufalin-induced LC3-II accumulation and cell death. The induction of autophagy by bufalin was linked to the generation of reactive oxygen species (ROS) and JNK activation. My findings unveil a novel mechanism of drug action by bufalin in colon cancer cells and open up the possibility of treating colorectal cancer by cardiac glycosides through an autophagy pathway. / Recent studies have revealed that cardiac glycosides induce G2/M phase arrest in many cancer cells, which include apoptosis- and autophagy-promoting cells. However, no detailed information is available on how cardiac glycosides arrest cell cycle progression. In this study, I studied the molecular mechanisms of cell cycle arrest mediated by cardiac glycosides. Bufalin-treated HeLa H2B-YFP cells were arrested at prometaphase, as characterized by the presence of sister chromatid cohesion, absence of chromosomes alignment on the metaphase plate, and failure to exit mitosis. This result was further confirmed by bufalin-induced cells with 4N DNA content in neither tetraploid G1 phase nor cytokinesis. Thereafter, I detected the Aurora kinases and Polo-like kinase 1 (Plk1), which are required for both spindle assembly and chromosome segregation. It was found that bufalin and other cardiac glycosides could significantly reduce the total protein and phosphorylation of Aurora kinases and Plk1 in HT-29 and HeLa cells. In addition, I found that PI3K was responsible for the bufalin-induced downregulation of the activities of mitotic kinases. This result was validated by silencing of sodium pump alpha. Taken together, my results demonstrate that bufalin and other cardiac glycoside inhibitors of the sodium pump potently arrest cancer cells at prometaphase by downregulating the total protein and phosphorylation of Aurora kinases and the total protein of Plk1 through the PI3K/HIF-1α/NF-κB pathway. My findings provide useful information in understanding how cardiac glycosides could be exploited for their potentials in treating cancer and in identifying the function of sodium pump in cell cycle progression. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Xie, Chuanming. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 133-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Declaration of Originality --- p.i / Acknowledgements --- p.iii / Abstract --- p.vi / Abstract (in Chinese) --- p.viii / List of Abbreviations --- p.xiv / List of Figures --- p.xvi / List of Tables --- p.xix / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Cancer --- p.1 / Chapter 1.2 --- The chemical structure of cardiac glycosides --- p.2 / Chapter 1.3 --- The traditional use of cardiac glycosides in cardiology --- p.4 / Chapter 1.4 --- The role of cardiac glycosides in cancer treatment --- p.4 / Chapter 1.5 --- The mechanisms of action by cardiac glycosides in cancer --- p.5 / Chapter 1.5.1 --- The structure and functions of cardiac glycosides receptor sodium pump --- p.5 / Chapter 1.5.2 --- Sodium pump as anticancer target --- p.6 / Chapter 1.5.3 --- The signal pathways involved in anticancer effect of cardiac glycosides --- p.7 / Chapter 1.6 --- The role of cardiac glycosides in apoptosis and autophagy --- p.8 / Chapter 1.7 --- Objectives of this project --- p.12 / Chapter Chapter 2 --- Bufalin induces autophagy but not apoptosis in human colon cancer cells --- p.17 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Materials and Methods --- p.19 / Chapter 2.2.1 --- Reagents and antibodies --- p.19 / Chapter 2.2.2 --- Cell culture --- p.19 / Chapter 2.2.3 --- Cell viability and cell death assay --- p.20 / Chapter 2.2.4 --- Annexin V and PI staining --- p.20 / Chapter 2.2.5 --- Cell cycle analysis --- p.21 / Chapter 2.2.6 --- Analysis of cleaved caspase-3-positive cells by flow cytometry --- p.21 / Chapter 2.2.7 --- Western blot analysis --- p.21 / Chapter 2.2.8 --- Immunofluorescence analysis of LC3 distribution --- p.22 / Chapter 2.2.9 --- RNA isolation and RT-PCR --- p.22 / Chapter 2.2.10 --- siRNAs transfection and treatments --- p.23 / Chapter 2.2.11 --- Transmission electron microscopy --- p.23 / Chapter 2.2.12 --- Statistical analysis --- p.24 / Chapter 2.3 --- Results --- p.24 / Chapter 2.3.1 --- Bufalin induces cell death and cell cycle arrest at G2/M phase in colon cancer cells --- p.24 / Chapter 2.3.2 --- Bufalin induces caspase-independent cell death in colon cancer cells --- p.28 / Chapter 2.3.3 --- Bufalin induces autophagy in colon cancer cells --- p.30 / Chapter 2.3.4 --- Bufalin-induced autophagy is dependent on ATG5 and Beclin-1 --- p.37 / Chapter 2.3.5 --- Increased autophagy is responsible for bufalin-induced cell death --- p.40 / Chapter 2.4 --- Discussion --- p.42 / Chapter Chapter 3 --- Bufalin mediates autophagic cell death through ROS generation and JNK activation --- p.44 / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Materials and Methods --- p.46 / Chapter 3.2.1 --- Reagents and antibodies --- p.46 / Chapter 3.2.2 --- Cell culture --- p.47 / Chapter 3.2.3 --- Cell viability and cell death assay --- p.47 / Chapter 3.2.4 --- Western blot analysis --- p.47 / Chapter 3.2.5 --- Quantification of cells with > 5 LC3 punctate staining --- p.47 / Chapter 3.2.6 --- siRNAs transfection and treatments --- p.48 / Chapter 3.2.7 --- RNA isolation and RT-PCR --- p.48 / Chapter 3.2.8 --- ROS analysis --- p.48 / Chapter 3.2.9 --- JC-1 staining --- p.49 / Chapter 3.2.10 --- Statistical analysis --- p.49 / Chapter 3.3 --- Results --- p.50 / Chapter 3.3.1 --- Bufalin induces autophagy-mediated cell death via ROS generation --- p.50 / Chapter 3.3.2 --- Activation of JNK is required for the upregulation of ATG5 and Beclin-1, and subsequent autophagy-mediated cell death in response to bufalin --- p.54 / Chapter 3.3.3 --- ROS generation is upstream of JNK activation in bufalin-induced cell death --- p.59 / Chapter 3.3.4 --- Bufalin-induced ROS generation is derived from mitochondria --- p.62 / Chapter 3.4 --- Discussion --- p.66 / Chapter Chapter 4 --- Bufalin arrests cells at prometaphase --- p.69 / Chapter 4.1 --- Introduction --- p.69 / Chapter 4.2 --- Materials and Methods --- p.70 / Chapter 4.2.1 --- Reagents and antibodies --- p.70 / Chapter 4.2.2 --- Cell synchronization --- p.70 / Chapter 4.2.3 --- Mitotic index analysis of phosphorylation of MPM2 --- p.71 / Chapter 4.2.4 --- Cell cycle analysis --- p.71 / Chapter 4.2.5 --- Time-lapse experiments --- p.71 / Chapter 4.2.6 --- Immunofluorescence analysis of phospho-histone H3 (Ser10) --- p.72 / Chapter 4.2.7 --- Western blot analysis --- p.73 / Chapter 4.3 --- Results --- p.73 / Chapter 4.3.1 --- Bufalin reduces mitotic marker phosphorylation of histone H3 and MPM2 and increases cells with 4N DNA content --- p.73 / Chapter 4.3.2 --- Increased cells with 4N DNA content after bufalin treatment are in neither a tetraploid G1 phase nor a cytokinesis arrest --- p.77 / Chapter 4.3.3 --- Bufalin-treated cells can enter prophase, but fail to pass through metaphase --- p.80 / Chapter 4.4 --- Discussion --- p.83 / Chapter Chapter 5 --- Bufalin induces prometaphase arrest through downregulating mitotic kinases --- p.87 / Chapter 5.1 --- Introduction --- p.87 / Chapter 5.2 --- Materials and Methods --- p.89 / Chapter 5.2.1 --- Reagents and antibodies --- p.89 / Chapter 5.2.2 --- Cell synchronization --- p.90 / Chapter 5.2.3 --- Immunofluorescence staining --- p.90 / Chapter 5.2.4 --- siRNAs transfection and treatments --- p.91 / Chapter 5.2.5 --- Western blot analysis --- p.91 / Chapter 5.2.6 --- Statistic analysis --- p.91 / Chapter 5.3 --- Results --- p.92 / Chapter 5.3.1 --- Bufalin downregulates Aurora A and B in protein and phosphorylation levels --- p.92 / Chapter 5.3.2 --- Bufalin prevents Aurora A recruitment to mitotic centrosomes and Aurora B recruitment to unattached kinetochores --- p.97 / Chapter 5.3.3 --- Bufalin prevents Plk1 recruitment to mitotic centrosomes and unattached kinetochores through downregulation of protein levels of Plk1 --- p.101 / Chapter 5.3.4 --- Bufalin decreases the activities of Aurora A, Aurora B and Plk1 through PI3K pathway --- p.105 / Chapter 5.3.5 --- HIF-1α and NF-κB pathways are involved in sodium pump-mediated the regulation of mitotic kinases --- p.109 / Chapter 5.4 --- Discussion --- p.112 / Chapter Chapter 6 --- General discussion --- p.115 / Chapter 6.1 --- Potential toxicity of bufalin --- p.115 / Chapter 6.2 --- Cardiac glycosides induced programmed cell death --- p.115 / Chapter 6.3 --- Signal pathways involved in cardiac glycosides-mediated autophagy --- p.117 / Chapter 6.4 --- The relationship between ROS and JNK in cardiac glycosides-induced autophagy --- p.120 / Chapter 6.5 --- The role of ROS in apoptosis and autophagy --- p.121 / Chapter 6.6 --- The role of cardiac glycosides in cell cycle arrest --- p.122 / Chapter 6.7 --- Application of cardiac glycosides in combination with chemotherapy and radiotherapy --- p.125 / Chapter Chapter 7 --- Conclusions and future perspectives --- p.127 / References --- p.133 / Appendices --- p.153 / Publication --- p.153

After investigating the injury mechanism, this dissertation aims to design an intelligent sprain-free sports shoe for the prevention of sport-related ankle sprain injury. It consists of a three-step mechanism: (1) Sensing, (2) Identification, and (3) Correction. The progress of this dissertation covers two options of sensing and identification. The first option is to monitor the ankle spraining motion with the ankle inversion angle. A method to employ two tiny inertial and magnetic sensors at the shank and foot segment is adopted as the sensing system. A laboratory study is conducted to obtain ankle inversion magnitude and velocity during various sport motions. Together with the article kinematics reported in the accidental sprain case report, a standard for identifying ankle sprain is established. The second option is to monitor the ankle spraining motion with the ankle supination torque. A feasible method to estimate the complete ground reaction forces with the information from pressure insoles is presented. Then, a tiny in-shoe three-pressure-sensor system is devised to monitor the ankle supination torque during sport motions. With the information of failure torque at the ankle joint reported by previous cadaveric study, an identification system of the ankle supination torque is devised. / Ankle sprain injury is the most common single type of sport-related musculoskeletal trauma which causes rupture and tear to the anterior talofibular ligament and ankle instability. In this dissertation, a new paradigm, "Orthopaedic Sport Biomechanics", is proposed to present the role of biomechanics in the practice of orthopaedics sports medicine, including the analysis of injury mechanism, and the design of prophylactic equipment for injury prevention. To directly investigate the injury, a case report of an accidental injury event in laboratory is presented. It was found that an internally rotated ankle orientation at foot strike may predispose the ankle joint to a supination sprain injury, by triggering lateral drift of the rearfoot and the subsequent vigorous inversion. At injury, the ankle joint reached an inversion of 48 degrees and an internal rotation of 10 degrees. / Future studies on the sensing and identification process, the correction, and the final evaluation are suggested. We hope that we could really invent the sprain-free shoe to help reducing the incidence of ankle sprain injury in sports in future. / Fong, Tik Pui Daniel. / Adviser: Kai-Ming Chan. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3635. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 102-127). / 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.

Conclusions: In the same number of people treated, the number of CVD events avoided for the overall risk approach is always larger than that of the blood pressure approach. The additional benefits of overall risk approach compared with the blood pressure approach decreases as the percentage of people from the total population is increased. If the current practice and hypertension guidelines in China are shifted to the overall risk approach, many more CVD events could be avoided with the same resources used. / Methods: The sample used in the analyses includes a subsample of 38,673 persons from the 2002 China National Nutrition and Health Survey, who were 30-74 years old, without previous CVD, and had data on all major CVD risk factors. CVD risks of the patients selected by each approach are predicted using suitable risk prediction equation. The RRR of anti-hypertensive drug treatment derived from meta-analyses of RCTs. The difference in the absolute effectiveness between the two approaches is used to quantify how many more CVD events can be prevented in 1000 people treated by the ORA as compared to the BPA. / Objective: To estimate and compare the number of major cardiovascular events that could be avoided by shifting the blood pressure approach to the overall risk approach if the same percentage of people in a large, representative Chinese population is treated with anti-hypertensive drugs. / Results: When 2.5%, 5.5%, 10.1%, 15.5%, 20.7%, 25.7% or 33.0% of the 38,673 subjects were treated by anti-hypertensive drugs by using the two approaches respectively, 22 (95%CI: 17∼28), 13 (11∼16), 9 (8∼10), 7 (6∼8), 6 (5∼7), 5 (4∼6), or 4 (3∼4) more CVD events could be avoided in every 1000 people treated if the blood pressure approach is shifted to the overall risk approach, which is in general a 15% to 25% increase in CVD events prevented. / Qin, Ying. / Adviser: Jin Ling Tang. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 116-121). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.