Les biothérapies (anticorps monoclonaux, récepteurs solubles) ciblant les cytokines IL-6 etTNF-alpha pour le traitement des maladies inflammatoires chroniques ont constitué un succèsmajeur de l’industrie pharmaceutique. Elles présentent néanmoins des inconvénientsimportants : résistances, mode d’administration contraignant, coût élevé.Notre équipe travaille sur l’identification de petites molécules inhibant directement cescytokines, afin d’élargir l’offre thérapeutique existante. Administrées par voie orale, ellesconstitueraient une alternative particluièrement favorable aux patients.Durant ma thèse, j’ai réalisé le criblage expérimental (tests cellulaires et tests biochimiquesde liaison) des meilleurs composés identifiés par criblage virtuel d’un grande chimiothèque dediversité, ainsi que de composés dérivés de pyridazine issus d’une chimiothèque médicinale. J’aiainsi pu identifier plusieurs inhibiteurs directs du TNF-alpha et de l’IL-6. De plus, mon travail apermis d’affiner les procédures de criblage du Laboratoire.Ces travaux ouvrent de nouvelles pistes pour le développement de médicaments anti-cytokines. / Anti-cytokine biologics (monoclonal antibodies, soluble receptors) targeting TNF-alpha and IL-6in chronic inflammatory diseases have been a major success for pharmaceutical industry.However, they exhibit several drawbacks : resistance, difficult administration, high costs.Our team works on the discovery of small molecule inhibitors of cytokines suck as TNF-alphaand IL-6, in order to widen the range of therapeutic drugs. Orally active drugs would represent ahighly beneficial alternative for patients.During my PhD, I have performed an experimental screening (using cellular and biochemicalbinding testings) of the best compounds identified through virtual screening of a large chemicallibrary, and on pyridazine compounds of a medicinal chemical library. I have been able toidentify several small molecules inhibiting the interaction of TNF-! and IL-6 with their receptor.Moreover, my work will have an impact on the laboratory screening strategies.Overall, this work opens new avenues for anti-cytokine drug discovery.

Thesis (M.Sc.)--Memorial University of Newfoundland, Faculty of Medicine, 1999. / Typescript. Bibliography: leaves 113-124.

Les biothérapies (anticorps monoclonaux, récepteurs solubles) ciblant les cytokines IL-6 etTNF-alpha pour le traitement des maladies inflammatoires chroniques ont constitué un succèsmajeur de l’industrie pharmaceutique. Elles présentent néanmoins des inconvénientsimportants : résistances, mode d’administration contraignant, coût élevé.Notre équipe travaille sur l’identification de petites molécules inhibant directement cescytokines, afin d’élargir l’offre thérapeutique existante. Administrées par voie orale, ellesconstitueraient une alternative particluièrement favorable aux patients.Durant ma thèse, j’ai réalisé le criblage expérimental (tests cellulaires et tests biochimiquesde liaison) des meilleurs composés identifiés par criblage virtuel d’un grande chimiothèque dediversité, ainsi que de composés dérivés de pyridazine issus d’une chimiothèque médicinale. J’aiainsi pu identifier plusieurs inhibiteurs directs du TNF-alpha et de l’IL-6. De plus, mon travail apermis d’affiner les procédures de criblage du Laboratoire.Ces travaux ouvrent de nouvelles pistes pour le développement de médicaments anti-cytokines. / Anti-cytokine biologics (monoclonal antibodies, soluble receptors) targeting TNF-alpha and IL-6in chronic inflammatory diseases have been a major success for pharmaceutical industry.However, they exhibit several drawbacks : resistance, difficult administration, high costs.Our team works on the discovery of small molecule inhibitors of cytokines suck as TNF-alphaand IL-6, in order to widen the range of therapeutic drugs. Orally active drugs would represent ahighly beneficial alternative for patients.During my PhD, I have performed an experimental screening (using cellular and biochemicalbinding testings) of the best compounds identified through virtual screening of a large chemicallibrary, and on pyridazine compounds of a medicinal chemical library. I have been able toidentify several small molecules inhibiting the interaction of TNF-! and IL-6 with their receptor.Moreover, my work will have an impact on the laboratory screening strategies.Overall, this work opens new avenues for anti-cytokine drug discovery.

The successful regeneration of muscle tissue is dependent upon the infiltration of phagocytic CD14++CD16- monocytes that support the proliferation and differentiation of myogenic precursor cells. Physiologically, the magnitude of the cellular response following resistance exercise is dictated by the level of receptor expression on the plasma membrane of the monocyte, as well as the secretion of their cognate ligands from tissue resident cells. However, it remains unclear whether the innate pro-inflammatory immune response varies with different resistance training protocols, and how it may impact recovery and the muscle remodeling process. Therefore, the purpose of this investigation was to examine temporal changes in the expression of chemotactic and adhesion receptors following an acute bout of high-volume, moderate-intensity (VOL) versus high-intensity, low-volume (HVY) lower-body resistance exercise in experienced, resistance trained men. Changes in receptor expression were assessed in conjunction with plasma concentrations of MCP-1, TNF?, and cortisol. Ten resistance-trained men (90.1 ± 11.3 kg; 176.0 ± 4.9 cm; 24.7 ± 3.4 yrs; 14.1 ± 6.1% body fat) performed each resistance exercise protocol in a random, counterbalanced order. Blood samples were obtained at baseline (BL), immediately (IP), 30 minutes (30P), 1 hour (1H), 2 hours (2H), and 5 hours (5H) post-exercise. Analysis of target receptor expression on CD14++CD16- monocytes was completed at BL, IP, 1H, 2H and 5H time points via flow cytometric analysis. Plasma concentrations of myoglobin, and LDH AUC were significantly greater following HVY compared to VOL (p = 0.003 and p = 0.010 respectively). Changes in plasma TNF?, MCP-1, and expression of CCR2, CD11b, and GCR on CD14++CD16- monocytes were similar following HVY and VOL. When collapsed across groups, TNF? was significantly increased at IP, 30P, 1H and 2H post-exercise (p = 0.001 – 0.004), while MCP-1 was significantly elevated at all post-exercise time points (p = 0.002 – 0.033). CCR2 expression was significantly lower at IP, 1H, 2H and 5H post-exercise (p = 0.020 – 0.040). In contrast, CD11b receptor expression was significantly greater at 1H relative to BL (p = 0.001), while GCR expression was not significantly different from baseline at any time point. As expected, plasma cortisol concentrations were significantly higher following VOL compared to HVY (p = 0.001), although this did not appear to be related to changes in receptor expression. Plasma testosterone concentrations and TNFr1 receptor expression did not appear to be affected by resistance exercise. Our results do not support a role for cortisol in the modulation of CCR2 receptors in vivo, while the degree of muscle damage does not appear to influence plasma concentrations of TNF?, or MCP-1. It is therefore likely that both HVY and VOL protocols constitute an exercise stimulus that is sufficient enough to promote a robust pro-inflammatory response, which is similar in timing and magnitude.

Master of Science / Department of Animal Sciences and Industry / J. Ernest Minton / It has been well documented that omega-3 PUFA (n-3 PUFA) can confer a wide variety of health benefits to humans and animals. The current study was designed to evaluate the ability of n-3 PUFA to modulate the innate immune response in two diverse breeds of horses. Ten Quarter Horse and 10 American Miniature Horse mares were assigned to either an n-3 PUFA supplemented or control diet (5 full-sized and 5 miniature mares/treatment) for 56 d. The treatment diet was designed to deliver 64.4 mg/kg BW combined eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) daily. Whole blood (20 mL) was collected via jugular veinipuncture into heparinized tubes on 0 d, 28 d, and 56 d. Serum PUFA analysis was conducted by gas chromatography. Peripheral blood mononuclear cell (PBMC) production of tumor necrosis factor-alpha (TNFalpha) in response to toll-like receptor (TLR) ligands lipopolysaccharide (LPS), flagellin, and lipoteichoic acid (LTA) was estimated using an equine-specific ELISA. Peripheral blood samples from d 56 were also analyzed for total and differential leukocyte counts and subjected to flow cytometric analysis. Body type did not affect basal or TLR stimulated TNFα production. Serum PUFA analysis revealed a decrease in linoleic acid (LA) and substantial increases in arachidonic acid (ARA), EPA, DHA, and docosapentaenoic acid (DPA) at both d 28 and 56 in horses fed n-3 PUFA (P less than 0.0001 for all). Dietary n-3 PUFA supplementation reduced (P less than 0.05) un-stimulated basal, but not TLR stimulated TNFalpha production by PBMC’s. Supplementation with n-3 PUFA did not affect total or differential leukocyte counts, nor selected cell surface markers. These results suggest that n-3 PUFA supplementation in the horse can modify circulating PUFA and alter the inflammatory response by reducing basal TNFalpha production. Furthermore, under conditions of the current study and considering the endpoints evaluated, the American Miniature Horse could potentially be used as a model for full-sized horse breeds.

In many chronic inflammatory diseases, patients suffer from skeletal muscle weakness, exacerbating their symptoms. Serum levels of tumor necrosis factor-alpha (TNF) and sphingomyelinase are increased, suggesting their possible role in the progression of this weakness. This dissertation focuses on the role that reactive oxygen species (ROS) and nitric oxide (NO) play in mediating TNF-induced skeletal muscle weakness and to what extent sphingolipid signaling mediates cellular response to TNF. The first aim of this work was to identify which endogenous oxidant species stimulated by TNF contributes to skeletal muscle weakness. In C57BL/6 mice (n=38), intraperitoneal injection of TNF elicited a 25% depression of diaphragm contractile function. In separate experiments, diaphragm fiber bundles harvested from mice (n=39) and treated with TNF ex vivo showed a 38% depression of contractile function compared to untreated controls. Using ROS and NO-sensitive fluorescence microscopy in parallel with a genetic knockout animal model, TNF-induced contractile dysfunction was found to be mediated by NO generated by a specific isoform of nitric oxide synthase (NOS), nNOS. Basal levels of ROS were necessary co-mediators, but were not sufficient to elicit TNF-induced diaphragm weakness. The second aim of this dissertation was to investigate the extent to which sphingolipids could serve as a signaling cascade post-TNF stimulus leading to the generation of NO in skeletal muscle. The effects of TNF exposure in C2C12 skeletal muscle cells were studied in vitro using mass spectroscopy to measure sphingolipid metabolism and fluorescent microscopy to quantify oxidant production. TNF exposure was associated with significant mean increases in sphingosine (+52%), general oxidant activity (+33%), and NO production (+14%). These increases were due to specific modulation of nNOS as demonstrated by siRNA knockdown of neutral ceramidase and nNOS, and confirmed by pharmacologic inhibition using N-Oleoylethanolamine and di-methylsphingosine. In summary, these findings confirm NO as a major causative oxidant contributing to TNF’s deleterious phenotype in skeletal muscle. Moreover, the work suggests a new role for sphingosine in skeletal muscle and warrants further study of the enzymatic regulation of sphingosine to advance the discovery of new therapies for patients suffering from chronic inflammation.

Il existe une variabilité interindividuelle de la relation dose - effet chez les patients atteints de maladies inflammatoires rhumatismales traités par les inhibiteurs du Tumor Necrosis Factor-alpha (TNF-a). Dans la première partie de cette thèse, la physiopathologie du TNF-a dans le processus inflammatoire est présentée. Ensuite, le travail se concentre sur la relation concentration-effet en utilisant la modélisation pharmacocinétique-pharmacodynamique (PK-PD) modèles. À la fin, après une discussion sur les biomarqueurs d'imagerie, la thèse traite de l'utilité d'une nouvelle technique permettant de détecter la réponse précoce au traitement, à savoir la tomographie par émission de positons (TEP). En résumé, ce travail décrit la relation PK-PD dans les maladies inflammatoires rhumatismales traitées par anticorps monoclonaux en utilisant les marqueurs cliniques et biologiques, et démontre également l'influence de fortes concentrations d'anticorps monoclonaux pour la maintenance au traitement. La TEP est une technique prometteuse pour identifier la réponse précoce aux antagoistes du TNF-a. / There is an interindividual variability of the dose - response relationship in patients with inflammatory rheumatic diseases treated by Tumor Necrosis Factor-alpha (TNF-a) inhibitors. In the first part of this thesis, the pathophysiology of TNF-a in inflammatory processes is presented. Then, the work focuses on the concentration-effect relationship using pharmacokinetic-pharmacodynamic (PK-PD) models. At the end, after discussion on imaging biomarkers, the thesis discusses the usefulness of a new technique to detect the early response to treatment, namely the positron emission tomography (PET). In summary, this work describes the PK-PD relationship in rheumatic inflammatory diseases treated by monoclonal antibodies using clinical and biological markers and demonstrates also the influence of high concentrations of monoclonal antibodies on maintenance to treatment. PET is a promising technique to identify early response to TNF-a antagonists.

A periodontite agressiva (PAg) compreende um grupo de doenças periodontais raras caracterizadas por rápida destruição dos tecidos periodontais, em indivíduos jovens e que geralmente não apresentam doenças sistêmicas. Estudos em populações e em famílias indicaram que fatores genéticos possuem influência na susceptibilidade a periodontite agressiva. Os polimorfismos genéticos da interleucina-1 (IL-1) e do fator de necrose tumoral-? (TNF-?) foram associados com o aumento da severidade da periodontite crônica. O objetivo deste estudo foi avaliar a associação entre o polimorfismo dos genes IL-1A (-889) e TNFA (-308) e a periodontite agressiva. Foram selecionados 60 indivíduos não fumantes, sendo 30 portadores de periodontite agressiva e os outros 30 sem doença periodontal. O polimorfismo genético foi analisado utilizando-se a técnica da reação em cadeia da polimerase e análise do polimorfismo de comprimento dos fragmentos de restrição (PCR-RFLP). Foi observado que a freqüência do genótipo 1/1 para IL-1A foi de 63,3% no grupo controle e de 56,7% no grupo teste. A avaliação do genótipo 1/2 mostrou uma freqüência de 26,7% no grupo controle e de 40% no grupo teste. O genótipo 2/2 ocorreu com uma freqüência de 10% no grupo controle e de 3,3% no grupo teste. O genótipo 1/1 para TNFA estava presente em 73,3% do grupo controle e em 80% do grupo teste. O genótipo 1/2 ocorreu com freqüência de 20% em ambos os grupos. O genótipo 2/2 foi encontrado em 6,7% dos controles e não foi detectado no grupo teste. Em relação aos alelos, o alelo 1 apresentou freqüência de 76,7% e o alelo 2 de 23,3% para ambos os grupos, para o gene IL-1A (-889). E para o gene TNFA (- 308) o alelo 1 ocorreu com freqüência de 83,3% no grupo controle e de 90% no grupo teste e o alelo 2 de 16,7% no grupo controle e 10% no grupo teste. A análise estatística revelou que não houve diferença significativa na distribuição dos genótipos, para ambos os genes, entre os dois grupos estudados. Não foi encontrada associação entre a periodontite agressiva e o polimorfismo dos genes IL- 1A (-889) e TNFA (-308) na população estudada. / Agressive periodontitis (AgP) is a relatively uncommon form of periodontal disease characterized by a rapid destruction of the periodontal supporting tissues in young adults who are usually systemically well. The results of population and family studies indicate that genetic factors seem to have a strong influence on susceptibility to AP. Genetic polymorphism at the interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFA) were associated with the increase on the severity of chronic periodontitis. The aim of this study was to explore a possible association between IL-1A and TNFA genotypes in Brazilian white Caucasian patients with aggressive periodontitis. Sixty nonsmoking subjects, 30 patients and 30 periodontal healthy controls were included in the study. All subjects were systemically healthy. Two polymorphisms, IL-1A (-889) and TNFA (-308), were analyzed by means of polymerase chain reaction-restriction fragment length polymorphism. The 1/1 genotype for IL-1A was present in 63.3% of the controls and in 56.7% of the aggressive periodontitis patients. The genotype 1/2 was present in 26.7% of the controls and in 40% of the patients. The 2/2 genotype was present in 10% of the controls and in 3.3% of the diseased subjects. The 1/1 genotype for the TNFA was present in 73.3% of the controls and in 80% of the patients. The genotype 1/2 was present in the 20% of both groups. The genotype 2/2 was present in 6.7% of the controls and was not detected in the patients group. With regard to the IL-1A (-889) genotype, 76.7% of controls and patients were positive for allele 1. Allele 1 of the TNFA (-308) polymorphism was carried by 83.3% of the controls and 90% of the patients and allele 2 was carried by 16.7% of the controls and 10% of the patients. Statistical analysis revealed no significant difference in the distribution of genotypes for both genes between the two groups. No association was found between AgP and the IL-1A (-889) and TNFA (-308) polymorphisms investigated in the population presented here.

Tsoi Lo Ming. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 99-109). / Abstracts in English and Chinese. / Acknowledgements --- p.I / Abstract --- p.II / Abstract in Chinese --- p.III / List of Abbreviations --- p.IV / List of Figures --- p.V / List of Tables --- p.VI / Table of Contents --- p.VII / Chapter Chapter 1 : --- Introduction / Chapter 1.1. --- Apolipoprotein and Lipoprotein Metabolism --- p.1 / Chapter 1.2. --- Molecular Information of ApoE --- p.2 / Chapter 1.3. --- Tissue Distribution of ApoE --- p.2 / Chapter 1.4. --- Functions of ApoE --- p.4 / Chapter 1.5. --- Genetic Polymorphism of ApoE --- p.7 / Chapter 1.6. --- Protein Structure and Characteristics of ApoE Isoforms --- p.9 / Chapter 1.7. --- Plasma and Cellular Expression Level of ApoE Isoforms --- p.12 / Chapter 1.8. --- Association between ApoE Isoforms and Plasma Lipid Profiles --- p.13 / Chapter 1.9. --- ApoE Polymorphisms and Pathophysiological Conditions / Chapter 1.9.1. --- Type III Hyperlipoproteinemia (Type III HLP) --- p.14 / Chapter 1.9.2. --- Alzheimer's Disease --- p.15 / Chapter 1.9.3. --- Atherosclerosis / Chapter 1.9.3.1. --- Atherosclerosis - An Inflammatory Process --- p.15 / Chapter 1.9.3.2. --- Role of ApoE in Atherosclerosis --- p.18 / Chapter (a) --- Functions Associated to Lipid Metabolism --- p.19 / Chapter (b) --- Functions Independent to Lipid Metabolism --- p.20 / Chapter 1.9.3.3. --- TNF-α and IL-6 in Atherosclerosis --- p.25 / Chapter 1.10. --- Macrophage Cytokine Expression and MAPKs / Chapter 1.10.1. --- Organization of MAPKs Signaling Pathway --- p.26 / Chapter 1.10.2. --- Lipopolysaccharide and MAPKs in Macrophage Cytokine Expression --- p.28 / Chapter 1.10.3. --- Regulation of Macrophage Cytokine Expression / Chapter 1.10.3.1. --- ERK1/2 and p38 MAPK Pathway --- p.30 / Chapter 1.10.3.2. --- Arachidonic Acid Metabolism --- p.30 / Chapter 1.11. --- Aim and Hypothesis --- p.31 / Chapter Chapter 2 : --- Materials and Methods / Materials / Chapter 2.1 --- Culture of ApoE-isoform-expressing J774A.1 Macrophage Cell Line --- p.32 / Chapter 2.2 --- RNA Extraction and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.33 / Chapter 2.3 --- Protein Extraction and Quantification --- p.37 / Chapter 2.4 --- Enzyme-linked Immunosorbent Assay (ELISA) --- p.38 / Chapter 2.5 --- Western Blotting --- p.39 / Chapter 2.6 --- LPS Treatment --- p.42 / Chapter 2.7 --- MAPK Inhibitor Experiment --- p.43 / Methods / Chapter 2.8 --- Study on the Effect of Endogenously Expressed ApoE Isoforms on Macrophage Cytokine Secretion / Chapter 2.8.1. --- Establishment of ApoE-isoform-expressing Macrophages --- p.44 / Chapter 2.8.2. --- Semi-quantification of ApoE mRNA Level by RT-PCR / Chapter 1) --- Isolation of Total RNA --- p.45 / Chapter 2) --- RT-PCR --- p.46 / Chapter 2.8.3. --- Determination of ApoE Protein Expression Level by ELISA and Western Blot --- p.47 / Chapter 1) --- Quantification of Total Proteins --- p.48 / Chapter 2) --- ELISA --- p.48 / Chapter 3) --- Western Blot --- p.49 / Chapter 2.8.4. --- LPS Treatment --- p.51 / Chapter 2.8.5. --- MEK1/2 Inhibitor Experiment --- p.53 / Chapter 2.8.6. --- p38 Inhibitor Experiment --- p.54 / Chapter 2.9 --- Study on the Effect of Exogenous ApoE Isoform on Macrophage Cytokine Secretion --- p.55 / Chapter 2.10 --- Statistical Analysis --- p.55 / Chapter Chapter 3: --- Results / Changes of Inflammatory Properties Associated with Endogenous ApoE Isoform Expression in Macrophages / Chapter 3.1 --- Characterization of ApoE-isoform-expressing Macrophages --- p.56 / Chapter 3.1.1. --- Cell Lines with Stable Expression of ApoE Isoforms --- p.56 / Chapter 3.2 --- Cell Morphology Study --- p.58 / Chapter 3.3 --- Changes of IL-6 and TNF-α Secretion Associated with Endogenous ApoE Isoforms Expression / Chapter 3.3.1. --- In the Presence of Lipoproteins --- p.60 / Chapter 3.3.2. --- Serum/Lipoprotein-independent Effects of ApoE Isoforms --- p.63 / Chapter 3.4 --- The Effects of Endogenous ApoE Isoform Expression on the Activities of MAPK Signaling Pathways / Chapter 3.4.1. --- Study on the Activation Status and Expression of MAPKs --- p.66 / Chapter 1) --- ERK1/2 MAPK Pathway --- p.66 / Chapter 2) --- p38 MAPK Pathway --- p.69 / Chapter 3.4.2. --- IL-6 and TNF-a Secretion Among ApoE Isoforms in the Presence of MEK1/2 mhibitor --- p.72 / Chapter 3.4.3. --- IL-6 and TNF-α Secretion Among ApoE Isoforms in the Presence of p38 Inhibitor --- p.75 / Chapter Chapter 4 : --- Discussions / Chapter 4.1. --- Mouse Peritoneal Macrophage Cell Line J774A.1 as Cell Model --- p.79 / Chapter 4.2. --- Inflammatory Properties Associated with Endogenous ApoE Isoform Expression in Macrophages / Chapter 4.2.1. --- Expression Level of ApoE Isoform Transgenes in Mouse Peritoneal Macrophages --- p.80 / Chapter 4.2.2. --- Macrophage Activation by LPS --- p.81 / Chapter 4.2.3. --- Effect of Endogenous ApoE Isoform Expression on Cytokine Secretion and Signal Transduction in Macrophages --- p.82 / Chapter 4.3. --- Conclusions and Future Prospects / Chapter 4.3.1. --- Conclusions --- p.90 / Chapter 4.3.2. --- Future Prospects --- p.91 / Chapter Chapter 5 : --- Appendices / Chapter 5.1 --- Changes of Inflammatory Properties of Macrophages Supplemented with Exogenous ApoE Isoforms / Chapter 5.1.1. --- Changes of IL-6 and TNF-a Secretion in Macrophages Supplemented with Exogenous ApoE Isoforms --- p.92 / Chapter 5.1.2. --- Changes of Signal Transduction in Macrophages Supplemented with Exogenous ApoE Isoforms / Chapter 5.1.2.1. --- Study on the Activation Status and Expression of MAPKs / Chapter 1) --- ERK1/2 MAPK Pathway --- p.95 / Chapter 2) --- p38 MAPK Pathway --- p.97 / Chapter Chapter 6: --- Bibliography --- p.99

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