by Yuen Wai Fan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 211-232). / Abstracts in English and Chinese. / Acknowledgements --- p.i / List of Publications and Abstracts --- p.ii / Abbreviations --- p.iv / Abstract --- p.xi / Abstract in Chinese --- p.xiv / List of Figures --- p.xvii / List of Tables --- p.xxiii / Contents --- p.xxiv / Chapter Chapter 1. --- General Introduction --- p.1 / Chapter 1.1 --- Hyperthermia --- p.2 / Chapter 1.1.1 --- History of Hyperthermia --- p.2 / Chapter 1.1.2 --- Biological Functions of Hyperthermia --- p.3 / Chapter 1.1.3 --- Clinical Application of Hyperthermia --- p.4 / Chapter 1.1.3.1 --- Whole-body Hyperthermia --- p.4 / Chapter 1.1.3.2 --- Regional Hyperthermia --- p.4 / Chapter 1.1.3.3 --- Local Hyperthermia --- p.5 / Chapter 1.1.4 --- Combination Therapy --- p.5 / Chapter 1.1.4.1 --- Combined treatment with Hyperthermia and Radiotherapy --- p.6 / Chapter 1.1.4.2 --- Combined treatment with Hyperthermia and Chemotherapy --- p.6 / Chapter 1.2 --- Tumour Necrosis Factor --- p.9 / Chapter 1.2.1 --- History of Tumour Necrosis Factor --- p.9 / Chapter 1.2.2 --- Sources of TNF-α and TNF-β --- p.9 / Chapter 1.2.3 --- Biological Roles of TNF --- p.10 / Chapter 1.2.3.1 --- Receptors of TNF-α --- p.11 / Chapter 1.2.4 --- Signaling Pathway of TNF --- p.12 / Chapter 1.2.4.1 --- Activation of Death Domain --- p.12 / Chapter 1.2.4.2 --- Activation of Sphingomyelin Pathway --- p.13 / Chapter 1.2.4.3 --- Activation of NF-kB pathway --- p.13 / Chapter 1.3 --- Types of Cell Death: Necrosis and Apoptosis --- p.16 / Chapter 1.3.1 --- Necrosis --- p.16 / Chapter 1.3.2 --- Apoptosis --- p.16 / Chapter 1.4 --- Signaling Pathway in Apoptosis --- p.19 / Chapter 1.4.1 --- Factors Involved in Apoptotic Pathway --- p.19 / Chapter 1.4.1.1 --- Caspases --- p.19 / Chapter 1.4.1.2 --- Death Substrates --- p.20 / Chapter 1.4.1.3 --- Bcl-2 Protein Family --- p.21 / Chapter 1.4.1.4 --- Role of Mitochondria --- p.23 / Chapter 1.5 --- Objectives of the Project --- p.26 / Chapter Chapter 2. --- Materials and Methods --- p.28 / Chapter 2.1 --- Materials --- p.29 / Chapter 2.1.1 --- Culture of Cells --- p.34 / Chapter 2.1.1.1 --- "TNF-α Sensitive Cell Line, L929" --- p.34 / Chapter 2.1.1.2 --- "TNF-α Resistance Cell Line, L929-11E" --- p.34 / Chapter 2.1.1.3 --- Preservation of Cells --- p.35 / Chapter 2.1.2 --- Culture Media --- p.36 / Chapter 2.1.2.1 --- RPMI 1640 (Phenol Red Medium) --- p.36 / Chapter 2.1.2.2 --- RPMI 1640 (Phenol Red-Free Medium) --- p.36 / Chapter 2.1.3 --- Buffers and Reagents --- p.37 / Chapter 2.1.3.1 --- Preparation of Buffers --- p.37 / Chapter 2.1.3.2 --- Buffer for Common Use --- p.37 / Chapter 2.1.3.3 --- Reagents for Annexin-V-FITC/PI assay --- p.37 / Chapter 2.1.3.4 --- Reagents for Cytotoxicity Assay --- p.37 / Chapter 2.1.3.5 --- Reagents for Molecular Biology Work --- p.38 / Chapter 2.1.3.6 --- Reagents for Western Blotting Analysis --- p.38 / Chapter 2.1.4 --- Chemicals --- p.40 / Chapter 2.1.4.1 --- Recombinant Murine TNF-α --- p.40 / Chapter 2.1.4.2 --- Dye for Cytotoxicity Assay --- p.41 / Chapter 2.1.4.3 --- Fluorescence Dyes --- p.41 / Chapter 2.1.4.4 --- Chemicals Related to Mitochondrial Studies --- p.41 / Chapter 2.1.4.5 --- Inhibitors of Caspases --- p.42 / Chapter 2.1.4.6 --- Antibodies for Western Blotting --- p.42 / Chapter 2.1.4.7 --- Other Chemicals --- p.43 / Chapter 2.2 --- Methods --- p.44 / Chapter 2.2.1 --- Treatment with TNF-α --- p.44 / Chapter 2.2.2 --- Treatment with Hyperthermia --- p.44 / Chapter 2.2.3 --- In vitro Cell Cytotoxicity Assay --- p.45 / Chapter 2.2.4 --- Flow Cytometry --- p.46 / Chapter 2.2.4.1 --- Introduction --- p.46 / Chapter 2.2.4.2 --- Analysis by FCM --- p.48 / Chapter 2.2.4.3 --- Determination of Apoptotic and Late Apoptotic/Necrotic Cells with Annexin-V-FITC/PI Cytometric Analysis --- p.50 / Chapter 2.2.4.4 --- Determination of Mitochondrial Membrane Potential (ΔΨm) --- p.51 / Chapter 2.2.4.5 --- Determination of Hydrogen Peroxide (H202) Release --- p.52 / Chapter 2.2.4.6 --- Determination of Intracellular Free Calcium ([Ca2+]i) Level --- p.52 / Chapter 2.2.4.7 --- Determination of the Relationship of ΔΨm and [Ca2+]i Level --- p.53 / Chapter 2.2.5 --- Western Blotting Analysis --- p.53 / Chapter 2.2.5.1 --- Preparation of Proteins from Cells --- p.53 / Chapter 2.2.5.2 --- SDS Polyacrylamide Gel Electophoresis (SDS- PAGE) --- p.56 / Chapter 2.2.5.3 --- Electroblotting of Proteins --- p.57 / Chapter 2.2.5.4 --- Probing Antibodies for Proteins --- p.57 / Chapter 2.2.5.5 --- Enhanced Chemiluminescence (ECL) assay --- p.58 / Chapter 2.2.6 --- Reverse Transcriptase Polymerase Chain Reaction --- p.58 / Chapter 2.2.6.1 --- Extraction of RNA by Trizol Reagent --- p.59 / Chapter 2.2.6.2 --- Determination of the Amount of RNA --- p.60 / Chapter 2.2.6.3 --- Agarose Gel Electrophoresis --- p.60 / Chapter 2.2.6.4 --- Reverse Transcription --- p.63 / Chapter 2.2.6.5 --- Polymerase Chain Reaction (PCR) --- p.63 / Chapter 2.2.6.6 --- Design of Primers for Different Genes --- p.64 / Chapter 2.2.6.7 --- Determination of the Number of Cycles in PCR for Different Genes --- p.67 / Chapter 2.2.7 --- Caspase Fluorescent Assay --- p.67 / Chapter 2.2.7.1 --- Caspase-3 or ´ؤ8 Assay --- p.67 / Chapter Chapter 3. --- Results --- p.59 / Chapter 3.1 --- Studies of the Characteristics of L929 and L929-11E cells --- p.70 / Chapter 3.1.1 --- Determination of the Growth Curve of L929 and L929-11E Cells --- p.70 / Chapter 3.2 --- Studies on the Effect of TNF-α on L929 and L929-11E Cells --- p.73 / Chapter 3.2.1 --- TNF-α Induced Cell Death in L929 Cells but not in L929- 11E Cells --- p.73 / Chapter 3.2.2 --- TNF-α Induced Apoptosis in a Time-dependent Manner in L929Cells but not in L929-11E Cells --- p.80 / Chapter 3.2.3 --- TNF-α Induced Mitochondrial Membrane Depolarization in a Time-dependent Manner in L929 Cells but notin L929-11E Cells --- p.87 / Chapter 3.2.4 --- TNF-α Induced Cytochrome c Release in a Time- dependent Manner in L929 Cells but not in L929-11E Cells --- p.92 / Chapter 3.3 --- Effect of Hyperthermia on L929 and L929-11E Cells --- p.96 / Chapter 3.3.1 --- Introduction --- p.95 / Chapter 3.3.2 --- Hyperthermia Induced Apoptosis in L929 and L929-11E Cells --- p.96 / Chapter 3.3.3 --- Effect of Hyperthermia on Mitochondrial Membrane Depolarization --- p.100 / Chapter 3.3.4 --- Hyperthermia Induced Cyto c Release in a Time-dependent Manner in L929 and L929-11E Cells --- p.105 / Chapter 3.4 --- Relationship of Hyperthermia and TNF-α with PTP in L929 Cells --- p.107 / Chapter 3.5 --- Effect of TNF-α and Hyperthermia on the Level of Hydrogen Peroxide (H202) in L929 and L929-11E Cells --- p.114 / Chapter 3.5.1 --- Introduction --- p.114 / Chapter 3.5.2 --- TNF-α Enhanced the Level of H202 in L929 cells but not in L929-11E Cells --- p.115 / Chapter 3.5.3 --- Hyperthermia Enhanced the Level of H202 in L929 and L929-11E cells --- p.117 / Chapter 3.6 --- Effect of TNF-α and Hyperthermia on the Level of Intracellular Calcium in L929 and L929-11E Cells --- p.122 / Chapter 3.6.1 --- Increase in the Intracellular Calcium Level Induced by TNF-α Was Related to the Mitochondrial Membrane Depolarization in L929 Cells but not in L929-11E Cells --- p.122 / Chapter 3.6.2 --- Hyperthermia Increased the Level of [Ca2+]i in L929 and L929-11E Cells in a Time-dependent Manner --- p.124 / Chapter 3.7 --- Effect of Combined Hyperthermia and TNF-α Treatment on the Induction of Apoptosis in L929 and L929-1 1E Cells --- p.129 / Chapter 3.7.1 --- Combined Treatment with Hyperthermia and TNF- α Induced Apoptosis in Both L929 and L929-11E cells --- p.129 / Chapter 3.7.2 --- Hyperthermia and Its Combined Treatment with TNF-α Induced Mitochondrial Membrane Depolarization in L929 and L929-11E Cells --- p.135 / Chapter 3.8 --- Investigation of the Downstream Apoptotic Pathway in L929 and L929-11E Cells Upon Hyperthermia and TNF-a treatment --- p.142 / Chapter 3.8.1 --- Introduction --- p.142 / Chapter 3.8.2 --- Effect ofTNF-α and Hyperthermia on p53 Expression --- p.142 / Chapter 3.8.3 --- Effect of Hyperthermia and TNF-α on PARP --- p.146 / Chapter 3.8.4 --- Effect of Hyperthermia and TNF-α on Caspase-3 Activity --- p.149 / Chapter 3.8.5 --- Effect of Hyperthermia and TNF-α on Bid protein --- p.158 / Chapter 3.8.6 --- Effect of Hyperthermia and TNF-α on Caspase-8 Activity --- p.165 / Chapter 3.8.7 --- Effect ofTNF-α on TNFR1 Expression --- p.169 / Chapter Chapter 4. --- Discussion / Chapter 4.1 --- TNF-α Induced Apoptosis and Changed the Mitochondrial Activities in L929 Cells --- p.176 / Chapter 4.2 --- L929-11E cells Possessed Resistance Towards TNF-α --- p.187 / Chapter 4.3 --- Hyperthermia Triggered Apoptosis and Changed Mitochondrial Activities in L929 and L929-11E cells --- p.190 / Chapter 4.4 --- Combined hyperthermia and TNF-α treatment induced cell death and changed mitochondria activities in L929 and L929-11E cells --- p.195 / Chapter 4.5 --- Reversal of the TNF-α resistance and Enhancement of Sensitivity Towards Hyperthermia in L929-11E cells --- p.197 / Chapter 4.6 --- Proposed Pathway in the TNF-α- and Hyperthermia-mediated Apoptosis --- p.200 / Chapter 4.7 --- Application of TNF-α and Hyperthermia on Clinical Cancer Treatment --- p.203 / Chapter Chapter 5. --- Future Perspective of the Project --- p.206 / References --- p.210

Orientador: Célio Percinoto / Banca: Elisa Maria Aparecida Giro / Banca: Marcelo José Strazzeri Bönecker / Banca: Robson Frederico Cunha / Banca: Alberto Carlos Botazzo Delbem / Resumo: A necessidade de melhorar a qualidade dos materiais utilizados no tratamento endodôntico tem estimulado o estudo de vários materiais buscando melhores condições de trabalho, biocompatibilidade e capacidade de estimular a mineralização dos tecidos. Este trabalho teve a finalidade de avaliar a capacidade de mineralização do MTA, Sealapex e Sealapex plus (combinação entre MTA e Sealapex) usando o implante de tubos de dentina preenchidos com os materiais em tecido subcutâneo de ratos e a citotoxicidade e a produção de citocinas (IL-1β e IL-6) por fibroblastos de camundongos estimulados por estes cimentos. Para avaliação da capacidade de mineralização foram utilizados 32 ratos, os quais receberam individualmente dois implantes de tubos de dentina contendo os cimentos ou tubos vazios que serviram de controle. Os períodos de avaliação foram 7 e 30 dias. Após cada período experimental, quatro animais foram sacrificados e os tubos de dentina juntamente com o tecido que o circunda foram removidos, fixados e processados para análise em microscopia óptica. As peças foram incluídas em uma mistura de parafina e cera de carnaúba (5%). As secções foram feitas seriadamente com 10mm de espessura e foram coradas de acordo com a técnica Von Kossa ou permaneceram sem coloração para serem visualizadas sob luz polarizada em microscópio óptico. Os critérios de avaliação foram: cápsula fibrosa fina quando < 150μm e espessa quando > 150μm. Necrose e calcificação foram registradas como presente ou ausente. Os resultados foram analisados estatisticamente pelo teste Qui-quadrado. A formação de tecido mineralizado e granulações birrefringentes à luz polarizada foram observadas em todos os materiais nos tempos de 7 e 30 dias. Para o teste de citotoxicidade foram utilizados fibroblastos de camundongos (L929) que foram incubados em placas... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The quality improvement of the endodontic materials is a need that has been stimulating the study of several materials aiming better work conditions, biocompatibility and mineralized tissue stimulation ability. The present work aimed to evaluate the mineralization ability of MTA, Sealapex and Sealapex plus (combination between MTA and Sealapex) using the implant of dentin tubes filled with the materials into the subcutaneous tissue of rats, the cytotoxicity, and cytokines production (IL-1β and IL-6) by fibroblasts stimulated by the materials. Thirty two rats were used for the tissue reaction test which received two implants of dentine tubes filled with the materials or empty tubes were used as control. The periods of evaluation were 7 and 30 days. After each period of evaluation, four animals were killed and the tubes and the surrounding tissue were removed, fixed and processed to be evaluated in optic microscope. The specimens were embedded in a mixture of paraffin and carnauba wax (5%). The sections were serially cut with 10mm and stained with Von Kossa or remained without staining to be evaluated under polarized light. The evaluation criteria were: fibrous capsule thin < 150 μm and thick > 150 μm. Necrosis and calcification were recorded as present or absent. The results were statistically analyzed using Qui-square test. Mineralization and birrefringent granulations to polarized light were observed with all materials and in both periods of observation. For the cytotoxicity test, fibroblasts from mice (L929) were incubated in 24 well plates and stimulated with polyethylene tubes filled with the materials. Cells cultured without the materials stimulation were used as control. After 24 hours of stimulation, the cytotoxicity was evaluated using the reduction colorimetric test (MTT). The results were analyzed using ANOVA and Bonferroni's correction... (Complete abstract click electronic access below) / Doutor

Made available in DSpace on 2014-06-11T19:33:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-03-10Bitstream added on 2014-06-13T19:22:58Z : No. of bitstreams: 1 gomes_ac_dr_araca.pdf: 755020 bytes, checksum: f24a6520c92284993a7187c85d96cf70 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A necessidade de melhorar a qualidade dos materiais utilizados no tratamento endodôntico tem estimulado o estudo de vários materiais buscando melhores condições de trabalho, biocompatibilidade e capacidade de estimular a mineralização dos tecidos. Este trabalho teve a finalidade de avaliar a capacidade de mineralização do MTA, Sealapex e Sealapex plus (combinação entre MTA e Sealapex) usando o implante de tubos de dentina preenchidos com os materiais em tecido subcutâneo de ratos e a citotoxicidade e a produção de citocinas (IL-1β e IL-6) por fibroblastos de camundongos estimulados por estes cimentos. Para avaliação da capacidade de mineralização foram utilizados 32 ratos, os quais receberam individualmente dois implantes de tubos de dentina contendo os cimentos ou tubos vazios que serviram de controle. Os períodos de avaliação foram 7 e 30 dias. Após cada período experimental, quatro animais foram sacrificados e os tubos de dentina juntamente com o tecido que o circunda foram removidos, fixados e processados para análise em microscopia óptica. As peças foram incluídas em uma mistura de parafina e cera de carnaúba (5%). As secções foram feitas seriadamente com 10mm de espessura e foram coradas de acordo com a técnica Von Kossa ou permaneceram sem coloração para serem visualizadas sob luz polarizada em microscópio óptico. Os critérios de avaliação foram: cápsula fibrosa fina quando < 150μm e espessa quando > 150μm. Necrose e calcificação foram registradas como presente ou ausente. Os resultados foram analisados estatisticamente pelo teste Qui-quadrado. A formação de tecido mineralizado e granulações birrefringentes à luz polarizada foram observadas em todos os materiais nos tempos de 7 e 30 dias. Para o teste de citotoxicidade foram utilizados fibroblastos de camundongos (L929) que foram incubados em placas... / The quality improvement of the endodontic materials is a need that has been stimulating the study of several materials aiming better work conditions, biocompatibility and mineralized tissue stimulation ability. The present work aimed to evaluate the mineralization ability of MTA, Sealapex and Sealapex plus (combination between MTA and Sealapex) using the implant of dentin tubes filled with the materials into the subcutaneous tissue of rats, the cytotoxicity, and cytokines production (IL-1β and IL-6) by fibroblasts stimulated by the materials. Thirty two rats were used for the tissue reaction test which received two implants of dentine tubes filled with the materials or empty tubes were used as control. The periods of evaluation were 7 and 30 days. After each period of evaluation, four animals were killed and the tubes and the surrounding tissue were removed, fixed and processed to be evaluated in optic microscope. The specimens were embedded in a mixture of paraffin and carnauba wax (5%). The sections were serially cut with 10mm and stained with Von Kossa or remained without staining to be evaluated under polarized light. The evaluation criteria were: fibrous capsule thin < 150 μm and thick > 150 μm. Necrosis and calcification were recorded as present or absent. The results were statistically analyzed using Qui-square test. Mineralization and birrefringent granulations to polarized light were observed with all materials and in both periods of observation. For the cytotoxicity test, fibroblasts from mice (L929) were incubated in 24 well plates and stimulated with polyethylene tubes filled with the materials. Cells cultured without the materials stimulation were used as control. After 24 hours of stimulation, the cytotoxicity was evaluated using the reduction colorimetric test (MTT). The results were analyzed using ANOVA and Bonferroni’s correction... (Complete abstract click electronic access below)

The immunological mechanisms involved in virus-induced hepatitis were examined by measuring the cytotoxic capabilities and the morphological and antigenic phenotypes of leukocytes isolated from the livers of virus-infected mice. Large granular lymphocytes (LGL) of both natural killer (NK) cell and cytotoxic T lymphocyte (CTL) phenoytpes [phenotypes] accumulated in livers of mice infected with lymphocytic choriomeningitis virus (LCMV) of either the nonhepatotropic Armstrong strain (LCMV-ARM) or the hepatotropic WE strain (LCMV-WE). NK cell activity and LGL number increased 3- to 4-fold between days 1 and 5 postinfection (p.i.). These LGL were characterized as NK cells on the basis of cell surface antigens, kinetics of appearance, target cell range, and morphology. By day 7 p.i., virus-specific, H-2-restricted, Thy-1+, Lyt-2+CTL activity was present in the liver, and its appearance correlated with a second wave of LGL accumulation. Total CTL activity, leukocyte numbers, and CTL/LGL numbers were at least 5-fold higher in the livers of LCMV-WE-infected mice than in the livers of LCMV-ARM-infected mice. Mice infected with the cytopathic viruses, mouse hepatitis virus and murine cytomegalovirus, experienced greater increases in NK/LGL by day 3 p.i. than did mice either infected with LCMV or injected with poly I:C. The early and late accumulations of LGL in the virus-infected liver were associated with the appearance of two waves of LGL with blast cell morphology expressing the phenotypes of NK cells and CTL, respectively. Thus, the organ-associated accumulation, blastogenesis, and in situ proliferation of cytotoxic LGL provide a means for the localization and site-specific augmentation of a host's cell-mediated antiviral defenses. The mechanism of inhibition of virus synthesis in vivo by immune splenocytes containing virus-specific CTL was examined in mice dually infected with two different viruses and then adoptively immunized with spleen cells immune to one of the two viruses. Only the titer of the virus to which the splenocytes were immune was reduced in titer, and no nonspecific antiviral effect was seen on the titer of the 'bystander' heterologous virus. These data are consistent with an in vivo mechanism of CTL-mediated antiviral resistance involving direct cytotoxicity rather than release and dissemination of antigen-nonspecific antiviral factors, such as interferon, following recognition of appropriate viral antigen.

Lau Wing-Ngar Vivian. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 135-144). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abbreviations --- p.vi / List of figures --- p.viii / List of tables --- p.xi / Contents --- p.xiii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- Roadside air pollution worldwide and in Hong Kong --- p.2 / Chapter 1.2.1 --- Air quality in Hong Kong --- p.3 / Chapter 1.3 --- Characteristics of particulate matter --- p.9 / Chapter 1.4 --- Composition and sources of particulate matter --- p.11 / Chapter 1.5 --- Toxic effects of particulate matter --- p.12 / Chapter 1.5.1 --- Lung injury --- p.12 / Chapter 1.5.2 --- Cardiovascular injury --- p.15 / Chapter 1.5.3 --- Mutagenesis and carcinogenesis --- p.16 / Chapter 1.6 --- Aims of my study --- p.16 / Chapter 2 --- Toxic Effects of Heavy Metals Ions on Selected Cultured Cell-lines --- p.18 / Chapter 2.1 --- Introduction --- p.18 / Chapter 2.1.1 --- Metals --- p.18 / Chapter 2.1.1.1 --- Cadmium --- p.22 / Chapter 2.1.1.2 --- Chromium --- p.23 / Chapter 2.1.1.3 --- Lead --- p.25 / Chapter 2.1.1.4 --- Zinc --- p.26 / Chapter 2.1.2 --- Metallothioneins --- p.28 / Chapter 2.1.3 --- p53 --- p.31 / Chapter 2.1.4 --- Tumor Necrosis Factor-alpha (TNF-α) --- p.32 / Chapter 2.1.5 --- Aims of this chapter --- p.32 / Chapter 2.2 --- Materials and methods --- p.35 / Chapter 2.2.1 --- Reagents --- p.35 / Chapter 2.2.2 --- Cultured Cell lines --- p.35 / Chapter 2.2.2.1 --- PU5-18 --- p.36 / Chapter 2.2.2.2 --- LL24 --- p.36 / Chapter 2.2.2.3 --- HBE4-E6/E7 --- p.37 / Chapter 2.2.3 --- Cytotoxicity assays --- p.37 / Chapter 2.2.4 --- ELISA assays --- p.40 / Chapter 2.2.4.1 --- ELISA assay ofp53 levels --- p.41 / Chapter 2.2.4.2 --- ELISA assay of TNF-α levels --- p.43 / Chapter 2.2.5 --- MT gene expression studies by Luciferase assay --- p.44 / Chapter 2.2.5.1 --- PCR amplification --- p.44 / Chapter 2.2.5.2 --- 5´ة End modification of PCR amplified DNA --- p.44 / Chapter 2.2.5.3 --- Ligation of DNA fragment to linearized vector --- p.46 / Chapter 2.2.5.4 --- E. coli. transformation by heat shock --- p.46 / Chapter 2.2.5.5 --- PCR sequencing --- p.47 / Chapter 2.2.5.6 --- Transfection of plasmid into HBE4-E6/E7 cells --- p.49 / Chapter 2.2.5.7 --- Data analysis --- p.50 / Chapter 2.3 --- Results and discussion --- p.51 / Chapter 2.3.1 --- Cytotoxicity assays --- p.51 / Chapter 2.3.2 --- Combination effects of metals on cytotoxicity --- p.61 / Chapter 2.3.3 --- p53 --- p.65 / Chapter 2.3.4 --- TNF-α --- p.68 / Chapter 2.3.5 --- MT gene expression studies by Luciferase assay --- p.69 / Chapter 2.4 --- Conclusion --- p.74 / Chapter 3 --- Effects of Polycyclic Aromatic Hydrocarbons (PAHs) on Cultured Cell-lines --- p.75 / Chapter 3.1 --- Introduction --- p.75 / Chapter 3.2 --- Materials and methods --- p.79 / Chapter 3.2.1 --- Reagents --- p.79 / Chapter 3.2.2 --- Cell culture --- p.79 / Chapter 3.2.3 --- AlamarBlue assay --- p.80 / Chapter 3.2.4 --- EROD assay --- p.80 / Chapter 3.3 --- Results and discussion --- p.84 / Chapter 3.4 --- Conclusion --- p.88 / Chapter 4 --- Chemical and Biological Assays on Roadside Dust --- p.89 / Chapter 4.1 --- Introduction --- p.89 / Chapter 4.1.1 --- Composition of particulate matter in Hong Kong --- p.89 / Chapter 4.1.2 --- Metal contents of particulate matter in Hong Kong --- p.91 / Chapter 4.1.3 --- Possible adverse health impacts of particulate matter --- p.94 / Chapter 4.1.3.1 --- In vitro studies using different cell models --- p.94 / Chapter 4.1.3.2 --- In vivo studies using rodents --- p.97 / Chapter 4.1.3.3 --- Epidemiological studies --- p.98 / Chapter 4.1.4 --- Aims of this chapter --- p.100 / Chapter 4.2 --- Materials and methods --- p.101 / Chapter 4.2.1 --- Sampling of roadside dust --- p.101 / Chapter 4.2.2 --- Chemical analysis of roadside dust --- p.104 / Chapter 4.2.2.1 --- Reagents --- p.104 / Chapter 4.2.2.2 --- Total metal contents --- p.105 / Chapter 4.2.2.3 --- Extractable metal contents --- p.105 / Chapter 4.2.3 --- Biological assays --- p.105 / Chapter 4.2.3.1 --- Cell models --- p.106 / Chapter 4.2.3.2 --- Pretreatment of roadside dust --- p.106 / Chapter 4.2.3.3 --- AlamarBlue assay --- p.106 / Chapter 4.2.3.4 --- ELISA assays --- p.108 / Chapter 4.2.3.5 --- Luciferase assay --- p.108 / Chapter 4.3 --- Results and discussion --- p.110 / Chapter 4.3.1 --- Total metal contents --- p.110 / Chapter 4.3.2 --- Extractable metal contents --- p.113 / Chapter 4.3.3 --- AlamarBlue assay --- p.116 / Chapter 4.3.4 --- p53 --- p.122 / Chapter 4.3.5 --- TNF-α --- p.122 / Chapter 4.3.6 --- Luciferase assay --- p.126 / Chapter 4.4 --- Conclusion --- p.129 / Chapter 5 --- General discussion and conclusion --- p.130 / Chapter 6 --- References --- p.135

No description available.

<p>Uobičajeni pristup i testiranju biolo&scaron;kog pona&scaron;anja materijala je da se počne sa jednostavnim in vitro testovima baziranim na ćelijskim kulturama. In vitro testovi citotoksičnosti su danas jedan od osnovnih načina za procenu biolo&scaron;kog odgovora na materijal jer su brži, lak&scaron;i za ponavljanje, ocenjivanje i jeftiniji u odnosu na eksperimente koji se izvode na životinjama i ljudima. Koriste se kao neka vrsta skrining testova za procenu biolo&scaron;ke sigurnosti materijala. Za razliku od ćelijskih kultura, istraživanja koja uključuju eksperimentalne životinje pružaju bolji uvid u biokompatibilnost materijala, zbog mogućnosti praćenja kompleksnog imunolo&scaron;kog odgovora živog organizma. Smatraju se neophodnim za ocenu bilo&scaron;kih odgovora na novi materijal, pre nego &scaron;to se on upotrebi na ljudima. Mnogi aspekti biolo&scaron;kog odgovora ne mogu biti reprodukovani in vitro testovima uključujući krvne interakcije, zarastanje rana, reakcije preosetljivosti, karcinogenezu, hroničnu inflamaciju. Eksperimenti na životinjama pružaju informacije o ovim tipovima efekata bez izlaganja ljudi riziku. Cilj ovog istraživanja bio je da se oceni biokompatibilnost objekata od polimera na bazi epoksi smole Accura&reg; ClearVue&trade; (hemijski sastav: 4,4&rsquo;- izopropilidendicikloheksanol, produkti oligomerne reakcije sa 1-hlor-2,3- epoksipropanom(40-65%), sme&scaron;а triaril-sulfonijum soli (50% propilen-karbonata i 50% triaril-sulfonijum heksafluoroantimonatnih soli) (1-10%) i 3-etil-3hidroksimetil-oksetan(10-20%). U oceni citotoksičnosti materijala Accura&reg; ClearVue&trade; kori&scaron;ćeni su agar diguzioni i MTT test. Oba testa rađena sun a ćelijskim kulturama L929 (mi&scaron;iji fibroblasti) i MRC-5 (humani fibroblasti). Ocena biokompatibilnosti testiranog materijala vr&scaron;ena je na osnovu urađenog testa iritacije oralne mukoze na modelu bukalne kesice hrčka, &scaron;to je definisano standardom ISO 10993-10:2010. Biokompatibilnost materijala ispitana je i implantacijom uzoraka u potkožno tkivo dorzuma pacova soja Wistar.</p> / <p>Uobičajeni pristup i testiranju biolo&scaron;kog pona&scaron;anja materijala je da se počne sa jednostavnim in vitro testovima baziranim na ćelijskim kulturama. In vitro testovi citotoksičnosti su danas jedan od osnovnih načina za procenu biolo&scaron;kog odgovora na materijal jer su brži, lak&scaron;i za ponavljanje, ocenjivanje i jeftiniji u odnosu na eksperimente koji se izvode na životinjama i ljudima. Koriste se kao neka vrsta skrining testova za procenu biolo&scaron;ke sigurnosti materijala. Za razliku od ćelijskih kultura, istraživanja koja uključuju eksperimentalne životinje pružaju bolji uvid u biokompatibilnost materijala, zbog mogućnosti praćenja kompleksnog imunolo&scaron;kog odgovora živog organizma. Smatraju se neophodnim za ocenu bilo&scaron;kih odgovora na novi materijal, pre nego &scaron;to se on upotrebi na ljudima. Mnogi aspekti biolo&scaron;kog odgovora ne mogu biti reprodukovani in vitro testovima uključujući krvne interakcije, zarastanje rana, reakcije preosetljivosti, karcinogenezu, hroničnu inflamaciju. Eksperimenti na životinjama pružaju informacije o ovim tipovima efekata bez izlaganja ljudi riziku. Cilj ovog istraživanja bio je da se oceni biokompatibilnost objekata od polimera na bazi epoksi smole Accura&reg; ClearVue&trade; (hemijski sastav: 4,4&rsquo;- izopropilidendicikloheksanol, produkti oligomerne reakcije sa 1-hlor-2,3- epoksipropanom(40-65%), sme&scaron;a triaril-sulfonijum soli (50% propilen-karbonata i 50% triaril-sulfonijum heksafluoroantimonatnih soli) (1-10%) i 3-etil-3hidroksimetil-oksetan(10-20%). U oceni citotoksičnosti materijala Accura&reg; ClearVue&trade; kori&scaron;ćeni su agar diguzioni i MTT test. Oba testa rađena sun a ćelijskim kulturama L929 (mi&scaron;iji fibroblasti) i MRC-5 (humani fibroblasti). Ocena biokompatibilnosti testiranog materijala vr&scaron;ena je na osnovu urađenog testa iritacije oralne mukoze na modelu bukalne kesice hrčka, &scaron;to je definisano standardom ISO 10993-10:2010. Biokompatibilnost materijala ispitana je i implantacijom uzoraka u potkožno tkivo dorzuma pacova soja Wistar.</p> / <p>The usual approach in testing biological behavior of materials is to start with simple in vitro tests based on cell cultures. In vitro cytotoxicity tests are one of the basic methods of assessing the biological response to material because they are faster, cheaper, easier for repeating and evaluating compared to experiments carried out on animals and humans. They are used as a kind of screening test for evaluating the biosafety of materials. Unlike cell culture, studies involving experimental animals provide better insight into the biocompatibility of materials due to the possibility of monitoring the complex immune response of a living organism. They are considered necessary for assessing the biological response to new material before it is used on humans. Many aspects of a biological response cannot be reproduced with in vitro tests, including blood interaction, wound healing, hypersensitivity reactions, carcinogenesis, chronic inflammation. Animal experiments provide information about these types of effects without exposing humans to risk.&nbsp; The aim of this study was to evaluate the biocompatibility of polymer objects on the basis of epoxy resins Accura&reg; ClearVue &trade; (chemical composition: 4.4&#39; Isopropylidenedicyclohexanol, oligomeric reaction products with 1-chloro-2.3-epoxypropane (40-65%), a mixture of triaryl sulfonium salt (50% propylene carbonate and 50% of a triarylsulfonium hexafluoroantimonate salt) (1- 10%) and 3-ethyl-3-hydroxymethyl-oxetane (10-20%). In the assessment of the cytotoxicity of materials Accura&reg; ClearVue &trade; agar diffusion and MTT tests were used. Both tests were conducted on cell cultures L929 (mouse fibroblasts) and MRC-5 (human fibroblasts). An assessment of the biocompatibility of the tested material was done on the basis of an oral mucosa irritation test on a hamster cheek pouch as defined by ISO 10993-10: 2010. The biocompatibility of the material was also tested with the implantation of a samples into the dorsal subcutaneous tissue of a Wistar rats. The subcutaneous implantation test, as one of the most reliable methods for assessing the biocompatibility of dental materials, is defined by ISO 10993-6: 2010. The study was conducted on 30 rats which were sacrificed in groups</p>