Kar Wing To. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 200-223). / Abstracts in English and Chinese. / Acknowledgements --- p.i / List of Abbreviations --- p.ii / Abstract --- p.v / 撮要 --- p.viii / List of Tables --- p.x / List of Figures --- p.xi / Contents --- p.xv / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- The General Characteristics of Glial Cells --- p.1 / Chapter 1.1.1 --- Astrocytes --- p.1 / Chapter 1.1.2 --- Oligodendrocytes --- p.5 / Chapter 1.1.3 --- Microglial --- p.6 / Chapter 1.2 --- Brain Injury and Astrocyte Proliferation --- p.6 / Chapter 1.3 --- Reactive Astrogliosis and Glial Scar Formation --- p.9 / Chapter 1.4 --- Astrocytes and Immune Response --- p.10 / Chapter 1.5 --- Cytokines --- p.10 / Chapter 1.5.1 --- Cytokines and the Central Nervous System (CNS) --- p.12 / Chapter 1.5.2 --- Cytokines and brain injury --- p.13 / Chapter 1.5.3 --- Cytokines-activated astrocytes in brain injury --- p.13 / Chapter 1.5.4 --- Tumour Necrosis Factor-a --- p.14 / Chapter 1.5.4.1 --- Types of TNF-α receptor and their sturctures --- p.16 / Chapter 1.5.4.2 --- Binding to TNF-α --- p.17 / Chapter 1.5.4.3 --- Different Roles of the TNF-a Receptor Subtypes --- p.17 / Chapter 1.5.4.4 --- Role of TNF-α and Brain Injury --- p.19 / Chapter 1.5.4.5 --- TNF-α Stimulates Proliferation of Astrocytes and C6 Glioma Cells --- p.23 / Chapter 1.5.5 --- Interleukin-1 (IL-1) --- p.26 / Chapter 1.5.5.1 --- Interleukin-1 and Brain Injury --- p.27 / Chapter 1.5.6 --- Interleukin-6 (IL-6) --- p.28 / Chapter 1.5.6.1 --- Interleukin-6 and brain injury --- p.29 / Chapter 1.5.7 --- γ-Interferon (γ-IFN) --- p.30 / Chapter 1.5.7.1 --- γ-Interferon and Brain Injury --- p.30 / Chapter 1.6 --- Ion Channels and Astrocytes --- p.31 / Chapter 1.6.1 --- Roles of Sodium Channels in Astrocytes --- p.33 / Chapter 1.6.2 --- Role of Potassium Channels in Astrocytes --- p.33 / Chapter 1.6.3 --- Importance of Calcium Ion Channels in Astrocytes --- p.34 / Chapter 1.6.3.1 --- Function of Cellular and Nuclear Calcium --- p.34 / Chapter 1.6.3.2 --- Nuclear Calcium in Cell Proliferation --- p.36 / Chapter 1.6.3.3 --- Nuclear Calcium in Gene Transcription --- p.36 / Chapter 1.6.3.4 --- Nuclear Calcium in Apoptosis --- p.38 / Chapter 1.6.3.5 --- Spatial and Temporal Changes of Calcium-Calcium Oscillation --- p.39 / Chapter 1.6.3.6 --- Calcium Signalling in Glial Cells --- p.39 / Chapter 1.6.3.7 --- Calcium Channels in Astrocytes --- p.41 / Chapter 1.6.3.8 --- Relationship Between [Ca2+]i and Brain Injury --- p.43 / Chapter 1.6.3.9 --- TNF-α and Astrocyte [Ca2+]i --- p.45 / Chapter 1.6.3.10 --- Calcium-Sensing Receptor (CaSR) --- p.46 / Chapter 1.7 --- Protein Kinase C (PKC) Pathways --- p.49 / Chapter 1.7.1 --- PKC and Brain Injury --- p.50 / Chapter 1.7.2 --- Role of Protein Kinase C Activity in TNF-α Gene Expression in Astrocytes --- p.51 / Chapter 1.7.3 --- PKC and Calcium in Astrocytes --- p.52 / Chapter 1.8 --- Intermediate Early Gene (IEGs) --- p.54 / Chapter 1.8.1 --- IEGs Expression and Brain Injury --- p.54 / Chapter 1.8.2 --- IEGs Expression and Calcium --- p.55 / Chapter 1.9 --- The Rat C6 Clioma Cells --- p.56 / Chapter 1.10 --- The Aim of This Project --- p.58 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.61 / Chapter 2.1.1 --- Sources of the Chemicals --- p.61 / Chapter 2.1.2 --- Materials Preparation --- p.65 / Chapter 2.1.2.1 --- Rat C6 Glioma Cell Line --- p.65 / Chapter 2.1.2.2 --- C6 Glioma Cell Culture --- p.65 / Chapter 2.1.2.2.1 --- Complete Dulbecco's Modified Eagle Medium (CDMEM) --- p.65 / Chapter 2.1.2.2.2 --- Serum-free Dulbecco's Modified Eagle Medium --- p.66 / Chapter 2.1.2.3 --- Phosphate Buffered Saline (PBS) --- p.66 / Chapter 2.1.2.4 --- Recombinant Cytokines --- p.67 / Chapter 2.1.2.5 --- Antibodies --- p.67 / Chapter 2.1.2.5.1 --- Anti-TNF-Receptor 1 (TNF-R1) Antibody --- p.67 / Chapter 2.1.2.5.2 --- Anti-TNF-Receptor 2 (TNF-R2) Antibody --- p.67 / Chapter 2.1.2.6 --- Chemicals for Signal Transduction Study --- p.68 / Chapter 2.1.2.6.1 --- Calcium Ionophore and Calcium Channel Blocker --- p.68 / Chapter 2.1.2.6.2 --- Calcium-Inducing Agents --- p.68 / Chapter 2.1.2.6.3 --- Modulators of Protein Kinase C (PKC) --- p.69 / Chapter 2.1.2.7 --- Reagents for Cell Proliferation --- p.69 / Chapter 2.1.2.8 --- Reagents for Calcium Level Measurement --- p.70 / Chapter 2.1.2.9 --- Reagents for RNA Extraction and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.71 / Chapter 2.1.2.10 --- Sense and Antisense Used --- p.72 / Chapter 2.1.2.11 --- Reagents for Electrophoresis --- p.74 / Chapter 2.2 --- Methods --- p.74 / Chapter 2.2.1 --- Maintenance of the C6 Cell Line --- p.74 / Chapter 2.2.2 --- Cell Preparation for Assays --- p.75 / Chapter 2.2.3 --- Determination of Cell Proliferation --- p.76 / Chapter 2.2.3.1 --- Determination of Cell Proliferation by [3H]- Thymidine Incorporation --- p.76 / Chapter 2.2.3.2 --- Measurement of Cell Viability Using Neutral Red Assay --- p.77 / Chapter 2.2.3.3 --- Measurement of Cell Proliferation by MTT Assay --- p.77 / Chapter 2.2.3.4 --- Protein Assay --- p.78 / Chapter 2.2.3.5 --- Data Analysis --- p.79 / Chapter 2.2.3.5.1 --- The Measurement of Cell Proliferation by [3H]- Thymidine Incorporation --- p.79 / Chapter 2.2.3.5.2 --- The Measurement of Cell growth in Neutral Red and MTT Assays --- p.79 / Chapter 2.2.3.5.3 --- The Measurement of Cell Proliferationin Protein Assay --- p.79 / Chapter 2.2.4 --- Determination of Intracellular Calcium Changes --- p.80 / Chapter 2.2.4.1 --- Confocal Microscopy --- p.80 / Chapter 2.2.4.1.1 --- Procedures for Detecting Cell Activity by CLSM --- p.81 / Chapter 2.2.4.1.2 --- Precautions of CLSM --- p.82 / Chapter 2.2.5 --- Determination of Gene Expression by Reverse- Transcription Polymerase Chain Reaction (RT-PCR) --- p.83 / Chapter 2.2.5.1 --- RNA Preparation --- p.83 / Chapter 2.2.5.1.1 --- RNA Extraction --- p.83 / Chapter 2.2.5.1.2 --- Measurement of RNA Yield --- p.84 / Chapter 2.2.5.2 --- Reverse Transcription (RT) --- p.84 / Chapter 2.2.5.3 --- Polymerase Chain Reaction (PCR) --- p.85 / Chapter 2.2.5.4 --- Separation of PCR Products by Agarose Gel Electrophoresis --- p.85 / Chapter 2.2.5.5 --- Quantification of Band Density --- p.86 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- Effects of Different Drugs on C6 Cell Proliferation --- p.87 / Chapter 3.1.1 --- Effects of Cytokines on C6 Cell Proliferation --- p.87 / Chapter 3.1.1.1 --- Effect of TNF-α on C6 Proliferation --- p.88 / Chapter 3.1.1.2 --- Effects of Other Cytokines on C6 Cell Proliferation --- p.92 / Chapter 3.1.2 --- The Signalling Pathway of TNF-α induced C6 Cell Proliferation --- p.92 / Chapter 3.1.2.1 --- The Involvement of Calcium Ions in TNF-α-induced C6Cell Proliferation --- p.95 / Chapter 3.1.2.2 --- The Involvement of Protein Kinase C in TNF-α- induced C6 Cell Proliferation --- p.96 / Chapter 3.1.3 --- Effects of Anti-TNF Receptor Subtype Antibodies on C6 Cell Proliferation --- p.102 / Chapter 3.2 --- The Effect of in Tumour Necrosis Factor-α on Changesin Intracellular Calcium Concentration --- p.102 / Chapter 3.2.1 --- Release of Intracellular Calcium in TNF-α-Treated C6 Cells --- p.104 / Chapter 3.2.2 --- Effects of Calcium Ionophore and Calcium Channel Blocker on TNF-α-induced [Ca2+]i Release --- p.107 / Chapter 3.2.3 --- Effects of Other Cytokines on the Change in [Ca2+]i --- p.109 / Chapter 3.2.4 --- The Role of PKC in [Ca2+]i release in C6 Glioma Cells --- p.109 / Chapter 3.2.4.1 --- Effects of PKC Activators and Inhibitors on the Changes in [Ca2+]i --- p.114 / Chapter 3.3 --- Determination of Gene Expression by RT-PCR --- p.114 / Chapter 3.3.1 --- Studies on TNF Receptors Gene Expression --- p.117 / Chapter 3.3.1.1 --- Effect of TNF-α on TNF Receptors Expression --- p.117 / Chapter 3.3.1.2 --- Effects of Other Cytokines on the TNF Receptors Expression --- p.119 / Chapter 3.3.1.3 --- Effects of Anti-TNF Receptor Subtype Antibodies on the TNF-a-induced Receptors Expression --- p.121 / Chapter 3.3.1.4 --- Effect of Calcium Ions on TNF Receptors Expression --- p.121 / Chapter 3.3.1.4.1 --- Effect of Calcium Ionophore on TNF Receptors Expression --- p.126 / Chapter 3.3.1.4.2 --- Effect of TNF-α Combination with A23187 on the Expression of TNF Receptors --- p.128 / Chapter 3.3.1.4.3 --- Effects of Calcium Ionophore and Channel Blocker on TNF Receptors Expression --- p.130 / Chapter 3.3.1.4.4 --- Effects of Calcium-Inducing Agents on TNF Receptors Gene Expressions --- p.130 / Chapter 3.3.1.5 --- Effects of PKC Activator and Inhibitor on TNF-α- induced TNF Receptors Expressions --- p.135 / Chapter 3.3.1.6 --- Effect of PKC and Ro31-8220 on IL-l-induced TNF Receptors Expressions --- p.138 / Chapter 3.3.2 --- Expression of Calcium-sensing Receptor upon Different Drug Treatments --- p.138 / Chapter 3.3.2.1 --- Effect of TNF-α on the Calcium-sensing Receptor Expression --- p.141 / Chapter 3.3.2.2 --- Effects of Other Cytokines on CaSR Expression --- p.143 / Chapter 3.3.2.3 --- Effect of A23187 on CaSR Expression --- p.143 / Chapter 3.3.2.4 --- Effect of TNF-α and A23187 Combined Treatment on CaSR Expression --- p.146 / Chapter 3.3.2.5 --- Effects of Calcium-inducing Agents on CaSR Expression --- p.149 / Chapter 3.3.2.6 --- Effects of PKC Activator and PKC Inhibitor on CaSR Expression --- p.149 / Chapter 3.3.3 --- Expression of PKC Isoforms upon Treatment with Different Drugs --- p.153 / Chapter 3.3.3.1 --- Effect of TNF-α on the PKC Isoforms Expression --- p.155 / Chapter 3.3.3.2 --- Effect of A23187 on the PKC Isoforms Expression --- p.155 / Chapter 3.3.3.3 --- Effect of TNF-α and Calcium Ionophore Combined Treatment on PKC Isoforms Expression --- p.157 / Chapter 3.3.3.4 --- Effects of Calcium-inducing Agents on PKC Isoforms Expression --- p.159 / Chapter 3.3.4 --- Expression of the Transcription Factors c-fos and c-junin Drug Treatments --- p.161 / Chapter 3.3.4.1 --- Effect of TNF-a on c-fos and c-jun Expression --- p.163 / Chapter 3.3.4.2 --- Effect of A23187 on c-fos and c-jun Expression --- p.163 / Chapter 3.3.4.3 --- Effect of TNF-a in Combination with A23187 on c- fos and c-jun Expression --- p.165 / Chapter 3.3.4.4 --- Effects of Calcium-inducing Agents on c-fos and c- jun Expression --- p.167 / Chapter 3.3.5 --- Effects of Different Drugs on Endogenous TNF-α Expression --- p.167 / Chapter 3.3.5.1 --- Effect of TNF-α on Endogenous TNF-α Expression --- p.169 / Chapter 3.3.5.2 --- Effect of A23187 on Endogenous TNF-α Expression --- p.169 / Chapter 3.3.5.3 --- Effect of TNF-α in Combination with A23187 on the Expression of Endogenous TNF-α --- p.172 / Chapter 3.3.5.4 --- Effects of Calcium-inducing Agents on Endogenous TNF-α Expression --- p.172 / Chapter 3.3.6 --- Effect of Different Drugs on LL-1 Expression --- p.175 / Chapter 3.3.6.1 --- Effect of TNF-a on IL-lα Expression --- p.177 / Chapter 3.3.6.2 --- Effect of A23187 on the IL-lα Expression --- p.177 / Chapter 3.3.6.3 --- Effect of Calcium Ionophore and TNF-α Combined Treatment on IL-1α Expression --- p.179 / Chapter 3.3.6.4 --- Effects of Calcium-inducing Agents on IL-lα Expression --- p.179 / Chapter 3.3.6.5 --- Effect of PKC Activator on the IL-1α Expression --- p.181 / Chapter CHAPTER 4 --- DISCUSSIONS AND CONCLUSIONS / Chapter 4.1 --- "Effects of Cytokines, Ca2+ and PKC and Anti-TNF-α Antibodies on C6 Glioma Cells Proliferation" --- p.184 / Chapter 4.2 --- The Role of Calcium in TNF-α-induced Signal Transduction Pathways --- p.186 / Chapter 4.3 --- Gene Expressions in C6 Cells after TNF-a Stimulation --- p.187 / Chapter 4.3.1 --- "Expression of TNF-α, TNF-Receptors and IL-1" --- p.187 / Chapter 4.3.2 --- CaSR Expression --- p.190 / Chapter 4.3.3 --- PKC Isoforms Expressions --- p.192 / Chapter 4.3.4 --- Expression of c-fos and c-jun --- p.193 / Chapter 4.4 --- Conclusion --- p.194 / REFERENCES --- p.200

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.

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.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Tumour necrosis factor alpha (TNFα) is a pleiotropic cytokine and has a wide variety of dose dependent cellular effects ranging from cell growth and differentiation, to inducing apoptosis. It has long been implicated in muscle and non-muscle inflammatory disorders, such as muscle wasting in chronic disease states, and rheumatoid arthritis. However, a physiological role for TNFα in muscle regeneration has been proposed as elevated levels of the cytokine are present when muscle regeneration processes are initiated: TNFα is secreted by infiltrating inflammatory cells, and by injured muscle fibres. Adult skeletal muscle contains a population of resident stem cell-like cells called satellite cells, which become activated, proliferate and differentiate following muscle injury to bring about repair of damaged muscle. Much research on the effects of TNFα on satellite cell differentiation has been conducted in recent years. It is however difficult to get a complete characterisation of the cytokine’s action as all models used slightly differ. We aimed therefore at providing comprehensive assessment of the effects of increasing doses of chronically supplemented TNFα on differentiating C2C12 cells. Cells were allowed to differentiate with or without TNFα supplementation for 7 days. Differentiation was induced at day 0. The effect on differentiation was assessed at days 1, 3, 5, and 7 by western blot analysis, and supplementary immunohistochemical analysis at days 1, 4, and 7 of markers of differentiation - muscle regulatory factors: MyoD and myogenin, markers of the cell cycle p21, PCNA, and the integral signalling molecule, p38MAPK. TNFα supplementation at day 1 tended to positively regulate early markers of differentiation. With continued supplementation however, markers of differentiation decreased dose dependently in treated cultures as the initial effect appeared to be reversed: A trend towards a dose dependent decrease in MyoD, myogenin and p21 protein existed in treated cultures at days 3, 5, and 7. These findings were significant at day 5 (p21, p<0.05), and day 7 (myogenin, p<0.05). A significant dose dependent decrease in p38 phosphorylation was evident at day 3 (p<0.05), while phospho-p38 was dose dependently increased at day 7 (p<0.05). Taken together, these data show that TNFα supplementation for 24 hours following the induction of differentiation in vitro, tends to increase levels of early markers of differentiation, and with continued TNFα supplementation decrease markers of differentiation in a dose dependent fashion. This study provides a comprehensive characterisation of the dose and time dependent effects of TNFα on satellite cell differentiaton in vitro. The model system used in the current study, allows us to make conclusions on more chronic disease states. / AFRIKAANSE OPSOMMING: Tumor nekrose faktor alfa (TNFα) is ‘n pleiotropiese sitokien wat ‘n wye verskeidenheid, dosis afhanklike, sellulêre effekte te weeg bring. Hierdie sellulêre effekte sluit sel groei en differensiasie tot sel dood in. TNFα is by beide spier en niespier inflammatoriese stoornisse soos spier tering in kroniese siektetoestande, en rumatiese artritis betrek. ‘n Fisiologiese rol vir TNFα is egter voorgestel aangesien verhoogde vlakke van die sitokien tydens inisiasie van spier herstel meganismes teenwoordig is: TNFα word deur infiltrerende inflammatoriese selle, asook deur beseerde spier vesels afgeskei. Volwasse skeletspier bevat ‘n populasie stamselagtige selle, sogenoemde satelliet selle. Laasgenoemde word geaktiveer, prolifereer en differensieër volgende spierbesering, om sodoende herstel van beskadigde spier te weeg te bring. Baie navorsing op die effekte van TNFα op satelliet sel differensiasie is onlangs uitgevoer. Dit is egter aansienlik moeilik om volgens hierdie navorsing‘n algehele beeld van TNFα se aksies te vorm aangesien alle modelle wat gebruik word verskil. Ons doel was daarom om ‘n omvangryke assessering van toenemende konsentrasies kronies gesupplementeerde TNFα op differensieërende C2C12 selle op ‘n enkele model uit te voer. Selle was vir 7 dae met of sonder TNFα supplementasie gedifferentieër. Differensiasie was by Dag 0 geïnduseer. TNFα se effek op differensiasie is op dae 1, 3, 5, en 7 deur middel van western blot analise geassesseer. Aanvullende immunohistochemiese bepalings op dae 1, 4, en 7 is verder deurgevoer. Merkers vir differensiasie het die spier regulatoriese faktore MyoD en miogenien, sel siklus merkers p21 en PCNA, asook die integrale sein transduksie molekule p38MAPK ingesluit. TNFα supplementasie by dag 1 het geneig om vroeë merkers van differensiasie positief te reguleer. Met voortdurende supplementasie is die vroeë positiewe effekte (op ‘n dosis afhanklike manier) egter omgekeer: ‘n neiging teenoor (‘n dosis afhanklike) vermindering in MyoD, miogenien en p21 proteïen het in behandelde kulture op dae 3, 5, en 7 bestaan. Hierdie bevindinge was beduidend by dag 5 (p21, p<0.05), en dag 7 (miogenien, p<0.05). A beduidende dosis afhanklike afname in p38 fosforilasie was duidelik by dag 3 (p<0.05), terwyl fosfo-p38 by dag 7 verhoog het met verhoogde konsentrasie TNFα (p<0.05). Bogenoemde saamgevat, dui aan dat TNFα supplementasie 24h volgende die induksie van differensiasie in vitro, verhoogde vlakke van vroeë differnsiasie merkers te weeg bring. Met voortdurende TNFα supplementasie, word differensiasie merkers egter met toenemende dosis verminder. Hierdie studie voorsien ‘n omvattende karakterisering van die dosis- en tyd afhanklike effekte van TNFα op satelliet sel differesiasie in vitro. Die model sisteem in hierdie studie gebruik, maak afleidings oor meer kroniese siektetoestande moontlik.

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.

Doctor of Philosophy / Department of Animal Sciences and Industry / Barry J. Bradford / The transition period in dairy cows is characterized by dramatic increases in nutrient requirements for lactation and substantial metabolic stress. The disturbed metabolic balance, coupled with suppressed immune function, contributes to markedly elevated incidence of health disorders. Several lines of evidence suggest that increased inflammation is common during the transition period. Unlike the classical inflammation associated with acute infection, the postpartum inflammatory state is low-grade and often of metabolic origin. This metabolic inflammation plays a key role in numerous disorders; an improved understanding of inflammatory pathways in transition cows may improve our ability to predict and prevent disorders. To mimic metabolic inflammation, in Experiment 1, we administered low amounts of recombinant bovine tumor necrosis factor-α (rbTNFα), a pro-inflammatory cytokine, to early lactation cows, and evaluated whether rbTNFα affects milk production, metabolism, and health. We found that rbTNFα administration increased systemic inflammation, decreased feed intake and milk yield, and increased incidence of disorders. Conversely, preventing excessive inflammation has the potential to improve productivity and health of dairy cows. To identify nutritional strategies that could enhance metabolism and immunity, we evaluated the efficacy of several feed additives. In Experiment 2, we evaluated effects of chromium propionate, rumen-protected lysine and methionine, or both on metabolism and immunity in lactating dairy cows, and found that supplementation of these nutrients may enhance neutrophil function. In Experiment 3, we determined whether supplementation of yeast product to transition cows could enhance production, metabolism, and immunity, and found that yeast product modulated feeding behavior, metabolism, immunity, and uterine inflammation. Overall, a greater understanding of the role of metabolic inflammation in the transition period and the nutritional strategies that could modulate these signals may improve the production and health of dairy cows.

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.