by Chung Lung Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 149-157). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abbreviations --- p.ii / Abstracts --- p.v / List of figures --- p.ix / List of tables --- p.xii / Contents --- p.xiii / Contents / General Introduction --- p.1 / Chapter CHAPTER ONE --- CISPLATIN RESISTANCE MECHANISMS / Chapter 1.1 --- INTRODUCTION --- p.3 / Chapter 1.1.1 --- History of Cisplatin as An Anticancer Drug --- p.3 / Chapter 1.1.2 --- Active Mechanisms of Cisplatin --- p.8 / Chapter 1.1.3 --- Formation of DNA Adducts --- p.8 / Chapter 1.1.4 --- Cisplatin Resistance Mechanisms --- p.9 / Chapter 1.1.4.1 --- Intracellular Accumulation of Cisplatin --- p.11 / Chapter 1.1.4.2 --- Glutathione-S-transferase and Glutathion --- p.12 / Chapter 1.1.4.3 --- Metallothionein --- p.16 / Chapter 1.1.4.4 --- Cell Cycle Perturbation --- p.16 / Chapter 1.1.4.5 --- P-glycoprotein --- p.17 / Chapter 1.1.4.6 --- Multidrug Resistant Protein --- p.19 / Chapter 1.1.4.7 --- Topoisomerase II --- p.20 / Chapter 1.1.4.8 --- DNA Repair --- p.22 / Chapter 1.1.4.9 --- Induction of Programme Cell Death --- p.23 / Chapter 1.2 --- OBJECTIVES --- p.27 / Chapter 1.3 --- MATERIALS AND METHODS / Chapter 1.3.1 --- Materials --- p.28 / Chapter 1.3.2 --- Methods --- p.31 / Chapter 1.3.2.1 --- Cell Lines --- p.31 / Chapter 1.3.2.2 --- Drug Sensitivity Assay --- p.31 / Chapter 1.3.2.3 --- Platinum Uptake --- p.32 / Chapter 1.3.2.4 --- Cell Cycle Analysis --- p.32 / Chapter 1.3.2.5 --- Western Blot Analysis --- p.33 / Chapter 1.3.2.6 --- Glutathione Content Determination --- p.36 / Chapter 1.3.2.7 --- DNA Fragmentation --- p.36 / Chapter 1.3.2.8 --- JC-1 Staining --- p.37 / Chapter 1.3.2.9 --- HE and DCF Staining --- p.38 / Chapter 1.3.2.10 --- Quantitative RT-PCR --- p.38 / Chapter 1.4 --- RESULTS / Chapter 1.4.1 --- Cisplatin Sensitivity of A431 Cells by MTT Assay --- p.40 / Chapter 1.4.2 --- Cross-resistance to Anti-cancer Drugs --- p.40 / Chapter 1.4.3 --- Quantitation of Cisplatin Accumulation in A431 Cells by AAS --- p.44 / Chapter 1.4.4 --- Drug Detoxification Agent --- p.45 / Chapter 1.4.5 --- Detection of Cell Cycle Arrest by Flow Cytometer --- p.47 / Chapter 1.4.6 --- Expression of Drug Resistance Related Genes --- p.48 / Chapter 1.4.7 --- Detection of Apoptosis by DNA Fragmentation --- p.50 / Chapter 1.4.8 --- Role of Mitochondria and Reactive Oxygen Species by Flow Cytometer --- p.52 / Chapter 1.4.9 --- Detection of Apoptotic mRNA Level by Quantitative RT-PCR --- p.57 / Chapter 1.4.10 --- Detection of Apoptotic Protein Level by Western Blot Analysis --- p.57 / Chapter 1.5 --- DISCUSSIONS --- p.59 / Chapter CHAPTER TWO: --- THE INTERACTION BETWEEN DRUG RESISTANCE MECHANISMS AND GROWTH FACTOR SIGNALLING PATHWAY / Chapter 2.1 --- INTRODUCTION --- p.63 / Chapter 2.1.1 --- Structure of EGF and EGFR --- p.64 / Chapter 2.1.2 --- Growth Factor Signal Transduction Pathway --- p.69 / Chapter 2.1.3 --- Biological Effect of EGF --- p.69 / Chapter 2.1.3.1 --- Modification of Drug Sensitivity by EGF --- p.71 / Chapter 2.2 --- OBJECTIVES --- p.74 / Chapter 2.3 --- MATERIALS AND METHODS / Chapter 2.3.1 --- Materials --- p.75 / Chapter 2.3.2 --- Methods / Chapter 2.3.2.1 --- Cell Lines --- p.76 / Chapter 2.3.2.2 --- Drug Sensitivity Assay --- p.77 / Chapter 2.3.2.3 --- Northern Blot Analysis --- p.77 / Chapter 2.3.2.4 --- Southern Blot Analysis --- p.78 / Chapter 2.3.2.5 --- Others --- p.78 / Chapter 2.4 --- RESULTS / Chapter 2.4.1 --- Sensitivity to EGF --- p.79 / Chapter 2.4.2 --- EGFR Expression Levels --- p.80 / Chapter 2.4.3 --- EGF Induced Protein Phosphorylation Pattern --- p.84 / Chapter 2.4.4 --- Effect of EGF on A431 Cells --- p.86 / Chapter 2.4.5 --- Response of Cells to Agents Targeting on EGF Signalling Pathway --- p.91 / Chapter 2.4.6 --- Response of Cells to Other Growth Factors --- p.97 / Chapter 2.4.7 --- Sensitivity of Cells to Different Anti-cancer Drugs --- p.99 / Chapter 2.4.8 --- Drug Resistance Mechanisms --- p.103 / Chapter 2.4.9 --- 5-Fluorouracil Sensitivity in A431 Cells --- p.108 / Chapter 2.4.10 --- Cisplatin Sensitivity in A431 Cells --- p.113 / Chapter 2.5 --- DISCUSSIONS --- p.117 / Chapter CHAPTER THREE: --- IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENE IN A431 CELLS BY DIFFERENTIAL DISPLAY / Chapter 3.1 --- INTRODUCTION --- p.122 / Chapter 3.2 --- MATERIALS AND METHODS / Chapter 3.2.1 --- Materials --- p.128 / Chapter 3.2.2 --- Methods / Chapter 3.2.2.1 --- Identification of Differentially Expressed Genes by RT-PCR / Chapter 3.2.2.2 --- Cloning of a Differentially Expressed cDNAs --- p.129 / Chapter 3.2.2.3 --- Screening and Sequencing of cDNA Inserts --- p.130 / Chapter 3.2.2.4 --- Rapid Amplification of cDNA Ends (RACE) --- p.131 / Chapter 3.2.2.5 --- Amplifcation Reaction --- p.131 / Chapter 3.2.2.6 --- Cloning and Sequencing of the RACE Fragment --- p.132 / Chapter 3.3 --- RESULTS / Chapter 3.3.1 --- Identification of novel cDNA by mRNA differential display --- p.133 / Chapter 3.4 --- DISCUSSIONS --- p.145 / General Conclusion --- p.147 / References --- p.149

Made available in DSpace on 2014-06-11T19:33:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2004-08-31Bitstream added on 2014-06-13T19:44:16Z : No. of bitstreams: 1 marcaccini_am_dr_arafo.pdf: 3610599 bytes, checksum: 65fbbd0cb1b0657f1b361bf4e461a54e (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os objetivos deste estudo foram avaliar o efeito de biomateriais associados ao PRP na formação óssea em defeitos padronizados de rádio e avaliar o efeito de PRP+RTG+enxerto ósseo autógeno para tratamento de lesões de furca grau II em cães. Para tal, em um primeiro estudo, foram confeccionados 5 defeitos de 5mm em cada rádio (direito e esquerdo) em 05 cães, constituindo assim espaços para preenchimento com os enxertos ou substitutos ósseos avaliados, totalizando 50 cavidades. Os materiais testados foram DFDBA, vidro bioativo, osso autógeno e osso mineral bovino, associados ou não ao PRP. Estes grupos foram avaliados e comparados com os grupos representados por coágulo sangüíneo e PRP. Ao final de 60 dias, foi realizada biópsia e preparo laboratorial para avaliação histológica e histomorfométrica. Os grupos que apresentaram melhores resultados foram coágulo, PRP e osso autógeno associado ou não a PRP; e a utilização do PRP não promoveu maior formação óssea em relação aos demais grupos, com exceção do vidro bioativo+PRP que apresentou os melhores resultados. No segundo estudo, foram criados cirurgicamente defeitos periodontais de furca nos quartos pré-molares mandibulares, bilateralmente em 5 cães, cronificados por um período de três meses e tratados por PRP/RTG/enxerto ósseo autógeno (grupo experimental) ou RTG/enxerto ósseo autógeno (grupo controle). Quatro meses após o tratamento, os cães foram sacrificados. Na área de furca, houve maior preenchimento ósseo e extensão linear de novo cemento, nova adaptação conjuntiva e regeneração periodontal no grupo experimental (p<0.005) e maior extensão linear de epitélio no grupo controle (p<0.005). A associação do PRP a RTG e enxerto ósseo autógeno proporcionou maior regeneração dos tecidos periodontais. Portanto, o PRP promoveu melhores resultados na formação óssea somente... . / The aims of this study were to evaluate the effect of biomaterials in association to PRP on bone formation, in padronized defects of radius, and evaluate the effect of PRP/GTR/autogenous bone graft in the treatment of Class II furcation lesions in dogs. For that, in a first study, five defects of 5mm each were produced in each radius (left and right) in 5 dogs, so creating spaces to be filled with grafts or the bone substitutes under study, totalling 50 cavities. The biomaterials tested were DFDBA, bioglass, autogenous bone and bovine mineral bone grafts, associated, or not, with PRP. These biomaterials were compared with coagulum and PRP. At the end of 60 days biopsy was done and histological laminas were prepared. Under the experimental condictions, coagulum, PRP and autogenous bone associated or not to PRP presented the best results, with more new bone formation; and no difference was observed between the groups with biomaterials, associated or not with PRP, excepting bioactive glass/PRP which showded the best results. In the second study, periodontal furcation defects were surgically produced bilaterally in the fourth mandibular premolars in 5 dogs, cronified for 3 months and treated with PRP/GTR/autogenous bone graft (experimental group) or GTR/autogenous bone graft (control group). Four months after the treatment, the dogs were sacrified. In the furcation area, more bone filling and linear extension of new cement, new conjunctive adaptation and periodontal regeneration were observed to occur in the experimental group (p<0.005) and more linear extension of epitelium in the control group (p<0.005). The association PRP/GTR/autogenous bone graft was observed to produce more regeneration of periodontal tissues. Therefore, the PRP association showed the best results in bone formation only at bioactive glass group; and... (Complete abstract, click electronic address below).

Orientador: Elcio Marcantonio Junior / Banca: Joni Augusto Cirelli / Banca: Enilson Antonio Sallum / Banca: Luis Carlos Spolidorio / Banca: Paulo Tambasco de Oliveira / Resumo: Os objetivos deste estudo foram avaliar o efeito de biomateriais associados ao PRP na formação óssea em defeitos padronizados de rádio e avaliar o efeito de PRP+RTG+enxerto ósseo autógeno para tratamento de lesões de furca grau II em cães. Para tal, em um primeiro estudo, foram confeccionados 5 defeitos de 5mm em cada rádio (direito e esquerdo) em 05 cães, constituindo assim espaços para preenchimento com os enxertos ou substitutos ósseos avaliados, totalizando 50 cavidades. Os materiais testados foram DFDBA, vidro bioativo, osso autógeno e osso mineral bovino, associados ou não ao PRP. Estes grupos foram avaliados e comparados com os grupos representados por coágulo sangüíneo e PRP. Ao final de 60 dias, foi realizada biópsia e preparo laboratorial para avaliação histológica e histomorfométrica. Os grupos que apresentaram melhores resultados foram coágulo, PRP e osso autógeno associado ou não a PRP; e a utilização do PRP não promoveu maior formação óssea em relação aos demais grupos, com exceção do vidro bioativo+PRP que apresentou os melhores resultados. No segundo estudo, foram criados cirurgicamente defeitos periodontais de furca nos quartos pré-molares mandibulares, bilateralmente em 5 cães, cronificados por um período de três meses e tratados por PRP/RTG/enxerto ósseo autógeno (grupo experimental) ou RTG/enxerto ósseo autógeno (grupo controle). Quatro meses após o tratamento, os cães foram sacrificados. Na área de furca, houve maior preenchimento ósseo e extensão linear de novo cemento, nova adaptação conjuntiva e regeneração periodontal no grupo experimental (p<0.005) e maior extensão linear de epitélio no grupo controle (p<0.005). A associação do PRP a RTG e enxerto ósseo autógeno proporcionou maior regeneração dos tecidos periodontais. Portanto, o PRP promoveu melhores resultados na formação óssea somente... (Resumo completo, clicar acesso eletrônico abaixo). / Abstract: The aims of this study were to evaluate the effect of biomaterials in association to PRP on bone formation, in padronized defects of radius, and evaluate the effect of PRP/GTR/autogenous bone graft in the treatment of Class II furcation lesions in dogs. For that, in a first study, five defects of 5mm each were produced in each radius (left and right) in 5 dogs, so creating spaces to be filled with grafts or the bone substitutes under study, totalling 50 cavities. The biomaterials tested were DFDBA, bioglass, autogenous bone and bovine mineral bone grafts, associated, or not, with PRP. These biomaterials were compared with coagulum and PRP. At the end of 60 days biopsy was done and histological laminas were prepared. Under the experimental condictions, coagulum, PRP and autogenous bone associated or not to PRP presented the best results, with more new bone formation; and no difference was observed between the groups with biomaterials, associated or not with PRP, excepting bioactive glass/PRP which showded the best results. In the second study, periodontal furcation defects were surgically produced bilaterally in the fourth mandibular premolars in 5 dogs, cronified for 3 months and treated with PRP/GTR/autogenous bone graft (experimental group) or GTR/autogenous bone graft (control group). Four months after the treatment, the dogs were sacrified. In the furcation area, more bone filling and linear extension of new cement, new conjunctive adaptation and periodontal regeneration were observed to occur in the experimental group (p<0.005) and more linear extension of epitelium in the control group (p<0.005). The association PRP/GTR/autogenous bone graft was observed to produce more regeneration of periodontal tissues. Therefore, the PRP association showed the best results in bone formation only at bioactive glass group; and... (Complete abstract, click electronic address below). / Doutor