High-mobility group box 1 protein (HMGBl) is a nuclear component, but extracellularly it serves as a signaling molecule involved in acute and chronic inflammation, for example in sepsis and arthritis. The identification of HMGBl inhibitors is therefore of significant experimental and clinical interest. We show that glycyrrhizin, a natural anti-inflammatory and antiviral triterpene in clinical use, inhibits HMGBl chemoattractant and mitogenic activities, and has a weak inhibitory effect on its intranuclear DNA binding function.

Microglia are the resident immunocompetent cells of the central nervous system (CNS). In response to insult or injury, microglia become more amoeboid, migrate to the site of injury, proliferate, phagocytose pathological stimuli and release growth factors and cytokines, thus initiating the inflammatory response. Due to these immune functions, much research has been invested in characterising their membrane physiology to investigate potential therapeutic targets. Despite being a non-excitable cell, microglia express an array of ion channels, which shape their physiological functions. One aim of this project was to characterise the ion channels functionally present in the human microglial cell line, C13-NJ, through cell attached and whole-cell voltage-clamp techniques. These experiments revealed that C 13-NJ cells express functional Na + and large conductance Ca2+ activated K+ channels, a well as a channel mediating a TRPM7 -like current. Further to the characterisation of ion channels, Gq-protein-coupled receptor related Ca2+ signalling was investigated using standard Ca2+ imaging techniques. Numerous GPCRs were identified and mechanisms of Ca2+- mobilisation and influx were determined. The functional consequences of histamine receptor 1 activation were also fully investigated, which was found to be pro-inflammatory due to the resultant increased migration, proliferation and cytokine release upon application of histamine. In addition to the well established role microglia have in the inflammatory response, recent research has revealed a role for microglia in non-pathological neuronal and synaptic function. Chemokines tethered to neuronal membrane, such as fractalkine and CD200 bind to receptors that are primarily expressed on ~ cells of myeloid lineage (such as microglia) and are prime candidates for a mechanism of this interaction. Extracellular field recordings and a range of intracellular recording techniques in acute rat hippocampal brain slices demonstrated that exogenous application of fractalkine or CD200 evokes depression of basal synaptic transmission. Mechanisms of these actions were further elucidated and discussed.

Temporal rhythms are an important aspect of physiological and behavioural systems. A picture is now developing indicating the importance of biological rhythms in both health and disease. This is particularly evident in inflammatory diseases, such as rheumatoid arthritis (RA), which demonstrates a temporal aspect to disease severity. hi order to fully exploit the potential of timing pharmacological drug treatment to endogenous rhythms (chronotherapy), a greater understanding of temporal control of mediators involved in diseases, as well as the kinetics of the therapeutics, is required.

Interleukins ILIRA, 1L6R and 1L18 are important cytokines, implicated in ageing processes. Some of these interleukins or their receptors or antagonists already provide targets for treatments. We hypothesized that interleukin related genotypes alter serum concentrations and thereby alter inflammatory profiles in individuals from the general population.

Mitogen-actWated protein kinase phosphatases (MKPs) are a family of dualspecificity phosphatases known to regulate the activity of the mitogen-activated proteins kinases (MAP kinases). MKP-2 was one of the earliest MKPs to be identified and characterised as a nuclear MKP. It is abundantly expressed in most tissues and induced in response to different range of stimuli.

Background:. Neutrophils play a central role in inflammatory disease. Since neutrophil apoptosis is essential for the resolution of inflammation, understanding the mechanisms regulating this process in vivo are important, as is the potential to drive apoptosis using appropriate pro-apoptotic stimuli. Our group previously showed that a death receptor ligand, TRAIL (TNF-related apoptosis inducing ligand), accelerates neutrophil apoptosis in vitro without associated cell activation. This thesis examines the role of TRAIL in inflammation in vivo.

Inflammation is vital for host defence against infections and tissue injury. However, dysregulated inflammation contributes to the pathogenesis of major peripheral and central nervous system (CNS) diseases. lnterleukin-1 (IL-1) is a key proinflammatory cytokine, and is implicated in the pathogenesis of many of these diseases.

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Background: Patients with chronic inflammatory diseases including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) demonstrate endothelial dysfunction. The inflammatory response triggered by endothelial injury leads to reduced NO biosynthesis, while increasing superoxide generation, monocyte adhesion and endothelial cell (EC) apoptosis. Apoptosis occurs preferentially at atherosclerosis prone sites, where endothelial erosion increases the risk of thrombosis and plaque development. Thus, chronic endothelial injury is a significant factor in the accelerated atherosclerosis associated with SLE and RA. Identifying the mechanisms underlying vascular injury and understanding innate cytoprotective pathways are essential for novel therapy development. Results: Known to play a role in cardiomyocyte ischemic preconditioning, protein kinase C (PKC) ε co-precipitates with stress-activated proteins and induces anti-apoptotic genes. We have now identified PKCε as an important regulator of cytoprotective responses in the vascular endothelium. We have shown that activation of PKCε induces expression of the cytoprotective enzyme HO-1 in human umbilical vein EC, using qRT-PCR and immunoblotting. This response is dependent upon gene transcription and de novo protein synthesis. A combined siRNA and immunohistochemical approach revealed a role for CREB and Nrf2 in PKCε-mediated HO-1 induction via a novel mechanism, which may involve positive feedback mechanisms and the dimerization of these two transcription factors. PKCε- dependent HO-1 expression is inducible by angiotensin II and able to protect against apoptosis and TNF-α- induced ICAM-1 upregulation. Furthermore, analysis of cardiac EC isolated from PKCε-/- and wild-type mice demonstrated altered expression of HO-1 in PKCε-/- EC and increased generation of reactive oxygen species. Conclusion: We have demonstrated a regulatory role for PKCε in the expression of the cytoprotective enzyme HO-1 in the vascular endothelium, and unearthed a novel signalling pathway involving CREB and Nrf2. The ultimate aim of this work is to identify novel therapeutic targets for treatment of endothelial dysfunction in systemic inflammatory diseases.