Background: Rheumatoid arthritis (RA) and psoriatic arthritis (PsA) are two distinct forms of chronic auto-immunity; understanding the transcriptomic profiles of key leukocyte subsets implicated in these arthritides could improve the diagnosis and treatment of patients. Current microarray analyses of samples derived from RA and PsA patients have examined the genetic profiles of whole blood or diseased tissue which, although informative, can mask the genetic contributions of individual cell types. Monocytes and macrophages are a cellular subset known to play a major role in PsA and RA through the production of pro-inflammatory chemokines, cytokines and destructive proteinases. Aim: To define the transcriptome in CD14+ cells separated from the blood and synovial fluid of PsA and RA patients, and to then compare and contrast that signature in health and disease. Thereafter to define the relevant activities of selected novel moieties described in the foregoing analysis. Methods & Results: The transcriptomic profiles of healthy, RA and PsA CD14+ blood cells were remarkably similar - few genes could distinguish diseased from healthy CD14+ cells. Comparison of the genetic signature of the RA and PsA synovial fluid CD14+ cells revealed that just over 50% of the differentially expressed genes were shared between the two disease groups. Furthermore, analysing the canonical pathways in the synovial fluid cells compared to the matched peripheral blood of both patient groups surprisingly revealed Liver X receptor (LXR) activation pathway as the most significantly upregulated pathway: this pathway has been previously shown by our group to play a pro-inflammatory role in arthritis. Examination of specific upregulated mRNAs in the synovial fluid CD14+ cells from both disease types revealed two novel genes that had not previously been associated with arthritis, the lysosomal enzyme legumain and the cell surface molecule plexin A1. Legumain was demonstrated to be present in RA and PsA CD14+ cells by RNA and protein analysis and was physiologically active. Incubation of CD14+ cells with patient synovial fluid under hypoxic conditions also potentiated legumain expression. Plexin A1 was confirmed to be expressed at the mRNA level within RA synovium. siRNA knockdown of plexin A1 suggested that it may play a pro-inflammatory role within macrophages since subsequent treatment of these macrophages with LPS resulted in decreased TNFα production. However, investigations into the identity of the specific ligands for plexin A1 in arthritis, known as semaphorins, were inconclusive. I finally generated microarray data to evaluate the transcriptome of macrophages activated via cel contact with activated T cells. Such cells shared only a small percentage of genes with those dysregulated in the RA and PsA synovial fluid derived CD14+ cells suggesting that this model at the time points chosen may not be an appropriate in vitro representation of articular macrophages. An imaging system of this in vitro model was also established to visualise the dynamic nature of the T cell – macrophage interactions and demonstrated that variables such as duration or method of T cell activation could alter the number and duration of interactions between the two cell types. Conclusions: These studies demonstrate that the CD14+ cells isolated from the blood are similar transcriptomically between healthy controls and RA and PsA patients. The synovial fluid CD14+ cells from RA and PsA patients exhibit substantial overlap in terms of their genetic profile. Two novel molecules expressed by diseased patients namely plexin A1 and legumain have been identified and their preliminary characteristics in the context of synovitis have been defined.

Kinesins are molecular motors that use energy from ATP hydrolysis to transport cargoes along microtubule tracks. There are at least 14 families of kinesins with different structural organisations but all kinesins have a motor domain that is the catalytic core for ATP hydrolysis and the binding site for microtubules. Most kinesins have a stalk domain, which facilitates oligomerisation, and a tail domain that is implicated in cargo binding and regulation. Depending on their structural organisation, each kinesin is suited for different functions. Some are involved in transporting vesicles and organelles in cells, while others are essential for axonal transport in neurons. Still others are involved in intraflagellar transport in cilia. Lastly, a group of kinesins participate in different steps of mitosis. One such kinesin is the human mitotic kinesin Eg5. It is a homotetrameric kinesin that is made up of a dimer of anti-parallel dimers. By cross-linking anti-parallel microtubules and moving towards their plus ends, Eg5 slides them apart and establishes the bipolar spindle. When Eg5 is inhibited by antibodies or siRNA, cells arrest in mitosis with non-separated centrosomes and monoastral spindles. Prolonged mitotic arrest eventually leads to apoptotic cell death. For that reason, Eg5 is a potential target for drug development in cancer chemotherapy with seven inhibitors in Phase I and II clinical trials. The first inhibitor of Eg5 was discovered in a phenotype-based screen and is called monastrol. Since then, several classes of inhibitors, such as ispinesib (a clinical trial candidate) and Strityl- L-cysteine (STLC), have been discovered. To develop more potent inhibitors, we employed a structure-based drug design approach. By determining crystal structures of the Eg5 motor domain in complex with various inhibitors, we can understand the interactions between the inhibitor and Eg5; thus, analysis of the structure-activity relationship (SAR) can help us to improve their potency. Consequently, these inhibitors could complement or act as alternatives to taxanes and vinca alkaloids, which are successful cancer chemotherapeutics currently used in the clinic, but have the tendency to cause neurotoxicities and develop resistance in patients. Here, I report the crystal structures of Eg5 in complex with three monastrol analogues, STLC, and four STLC analogues separately. Based on the crystal structures with monastrol analogues, I identified the preferential binding mode of each inhibitor and the main reasons for increased potency: namely the better fit of the ligand and the addition of two fluorine atoms. Next, the crystal structure of Eg5-STLC indicates that the three phenyl rings in STLC are buried in a mainly hydrophobic region, while the cysteine moiety of STLC is solventexposed. In addition, structures of Eg5 in complex with STLC analogues, which have meta- or para-substituents on one or more of the phenyl rings, reveal the positions of the substituents and provide valuable information for the SAR study. In short, these structures reveal important interactions in the inhibitor-binding pocket that will aid development of more potent inhibitors. To understand the molecular mechanism of inhibition, I examined the structure of the Eg5-STLC complex, which revealed an unprecedented intermediate state, whereby local changes at the inhibitor-binding pocket have not propagated to structural changes at the switch II cluster and neck linker. This provides structural evidence for the sequence of drug-induced conformational changes. In addition, I performed isothermal titration calorimetry to determine the thermodynamic parameters of the interaction between Eg5 and its inhibitors. The structural information and the thermodynamic parameters obtained help us to gain a better understanding of the molecular mechanism of inhibition by an Eg5 inhibitor. While there is a large amount of information about the motor domain of Eg5, less is known about the stalk domain, which facilitates oligomerisation. A prediction program showed that the first ~100 residues of the stalk domain have a high probability of forming a coiled-coil structure, while the middle ~150 residues have a low probability. Using analytical ultracentrifugation, I showed that the Eg5 stalk364-520 domain exists predominantly as a dimer with a sedimentation coefficient of 1.76 S. The purported coiled-coil quaternary structure is backed-up by circular dichroism data, which showed that Eg5 stalk364-520 domain contains about 52 % helical content. Finally, the low resolution solution structure of Eg5 stalk364-520 domain was determined by small angle X-ray scattering, which revealed an elongated structure that is ~165 Å in length. Together, these data give us a glimpse into the structural characteristics of the Eg5 stalk364-520 domain. Besides gaining a better understanding of Eg5, I decided to investigate the molecular mechanism of autoinhibition in conventional kinesin (later known as kinesin-1). As the founding member of kinesins, it was first discovered to be involved in axonal transport. When not transporting cargo, kinesin-1 is autoinhibited to prevent squandering of ATP. Although it is widely accepted that the tail binds to the motor domain to keep it in a folded autoinhibited state, the molecular mechanism remains unclear and several mechanisms have been proposed. Here, I report the crystal structures of the Drosophila melanogaster kinesin-1 motor domain dimer and the dimer-tail complex. The dimer, which exhibits ~180° rotational symmetry between the monomers, provides valuable structural information for modeling the motility of kinesins on microtubules. By comparing the free dimer with the dimer-tail complex, we observe that the motor domains have considerable freedom of movement in the absence of tail binding. However, in the dimer-tail complex, a ‘double lockdown’ at both the neck coil and the tail interface freezes out major movements. This could prevent conformational changes, such as neck linker undocking. Data from our collaborator (David Hackney) showed that a covalent cross-link, which mimics double lockdown of the dimer, prevents ADP release. Together, we propose a ‘double lockdown’ mechanism, in which cross-linking at both the coiled-coil and tail interface prevents the movement of the motor domains that is needed to undock the neck linker and release ADP. In short, the structures shed light on the autoinhibition mechanism, reveal important residues at the dimer-tail interface, invalidate other proposed mechanisms, and open up the possibility that other kinesins may be regulated by the same mechanism.

This is a general investigation of the SPECT imaging process. The primary aim is to determine the manner in which the SPECT studies should be performed in order to maximise the relevant clinical information given the characteristics and limitations of the particular gamma camera imaging system used. Thus the first part of this thesis is concerned with an assessment of the performance characteristics of the SPECT system itself. This involves the measurement of the fundamental planar imaging properties of the camera, their stability with rotation, the ability of the camera to rotate in a perfect circle and the accuracy of the transfer of the information from the camera to the computing system. Following this the performance of the SPECT system as a whole is optimised. This is achieved by examining the fundamental aspects of the SPECT imaging process and by optimising the selection of the parameters chosen for the acquisition and reconstruction of the data. As an aid to this a novel mathematical construct is introduced. By taking the logarithm of the power spectrum of the normalised projection profile data the relationship between the signal power and the noise power in the detected data can be visualised. From a theoretical consideration of the available options the Butterworth filter is chosen for use because it provides the best combination of spatial frequency transfer characteristics and flexibility. The flexibility of the Butterworth filter is an important feature because it means that the form of the actual function used in the reconstruction of a transaxial section can be chosen with regard to the relationship between the signal and the noise in the data. A novel method is developed to match the filter to the projection data. This consists of the construction of a mean angular power spectrum from the set of projection profiles required for the reconstruction of the particular transaxial section in question. From this the spatial frequency at which the the signal becomes dominated by the noise is identified. The value which the Butterworth filter should take at this point can then be determined with regard to the requirements of the particular clinical investigation to be performed. The filter matching procedure can be extended to two dimensions in a practical manner by operating on the projection data after it has been filtered in the y direction. The efficacy of several methods to correct for the effects of scatter, attenuation and camera non-uniformity are also investigated. Having developed the optimised methodology for the acquisition and reconstruction of the SPECT data the results which are obtained are applied in the investigation of some specific clinical problems. The assessment of intractable epilepsy using 99mTc-HMPAO is performed followed by the investigation of ischaemic heart disease using 99mTc-MIBI and finally, the diagnosis of avascular necrosis of the femoral head using 99mTc-MDP is studied. The SPECT studies described in this thesis make a significant contribution to patient management.

The lateral spinal nucleus (LSN), located in the dorsolateral funiculus, is an area that has been poorly understood, but has been implicated in nociception. To investigate the function of this nucleus, three broad areas were investigated: responses to nociceptive stimuli, neurochemical relations to the NK-1 receptor, and projections from this nucleus to several brain centres, to try to gain a greater understanding of the functions of this nucleus. The following conclusions can be drawn from the studies undertaken here: • A series of double-labelling experiments for confocal microscopy were carried out in the rat (Sprague-Dawley) to investigate the LSN responses to a variety of peripheral cutaneous noxious stimuli. It was found that the LSN responds to both thermal and chemical peripheral cutaneous noxious stimulation. However, unlike as previously thought, only a small number of neurons in the LSN are activated by a peripheral noxious stimulus, with hot water (55°C applied to the hind-paw) activating the most, as revealed by Fos immunoreactivity. Only 15% of LSN neurons showed response to this peripheral noxious stimulus. Interestingly, unlike the superficial dorsal horn (SDH), bilateral activation of LSN neurons after the application of a peripheral noxious stimulus was found in most of the experiments carried out. • Triple and quadruple-labelling experiments for confocal microscopy were carried out in the rat to investigate neurochemical relations at this site. It was found that although the LSN is abundant in staining for substance P, the number of LSN neurons showing immunoreactivity for the target of substance P (the NK-1 receptor) represented only one-third of all neurons at this site. However, substance P and nitric oxide synthase were associated with NK-1 neurons, and specifically nitric oxide synthase terminals were preferentially associated with NK-1 neuronal cell bodies. However, unlike the superficial dorsal horn, nitric oxide synthase terminals were not associated with inhibitory GABAergic neurons. • Using retrograde injection techniques (in the rat) combined with multiple immunolabelling for confocal microscopy, the LSN was shown to project to areas traditionally associated with nociception (caudal ventrolateral medulla and mediodorsal thalamus) but also projected to the hypothalamus and also the lateral globus pallidus. Indeed, the regions found to have the most projections from the LSN were the lateral and medial hypothalamus, with most of those neurons (>80%) possessing the NK-1 receptor. Interestingly, although numbers of retrogradely labelled neurons were low, they represented 30% of all labelled neurons that projected from the LSN to the lateral globus pallidus. In conclusion, the extent of involvement of the LSN in nociception is less than previously thought, but with projections to the hypothalamus, it could be postulated that the LSN functions as an integrative nucleus for autonomic and homeostatic functions, and related motivational and affective responses to autonomic function.

Membrane fusion in all eukaryotic cells is facilitated by the formation of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes; a process that is regulated by Sec1p/Munc18 (SM) proteins. Membrane fusion has been conserved through evolution and hence a lot or our knowledge about the molecular mechanism that regulates membrane traffic has come from experimentally tractable model organisms such as Saccharomyces cerevisiae. The work presented in this thesis demonstrates that the mammalian SNARE protein, syntaxin 16 (Sx16), is a functional homologue of the yeast SNARE protein, Tlg2p, as expression of Sx16 in tlg2Δ cells, fully complements trafficking defects displayed by these cells. This finding is supported by experiments demonstrating that Sx16 interacts both physically and functionally with the SM protein of Tlg2p, Vps45p, both in vivo and in vitro. Vps45p regulates Sx16 in a manner similar to the way that it regulates Tlg2p; controlling entry into functional SNARE complexes and regulating cellular levels. A model, in which Vps45p is required for regulating membrane fusion and regulating the cellular levels of Tlg2p, is also presented and discussed in this thesis.

Autoimmune inflammatory reactions occur in a number of disorders to a variety of self antigens but the precise cellular and molecular mechanisms resulting in pathology are largely unresolved. In some instances T cell mediated reactions are thought to be the main contributors to disease mechanisms but it is becoming increasingly evident that B cells, and their cognate antibodies, play a significant and contributory role in disease pathogenesis. Therefore establishing the occurrence of highly-specific B cell antigen-driven adaptive immune responses within autoimmune disorders would provide a valuable understanding into the roles of B cells within these disorders. The work of this study sought to determine the incidence of these B cell antigen-driven adaptive immune responses in the target tissues of the autoimmune disorders myositis and vasculitis, two autoimmune disorders characterised by a wide range of autoantibodies which have been implicated in the pathological mechanisms of these diseases. This study aimed to test the hypothesis that infiltrating B cells within the target tissue of myositis and vasculitis patients were being stimulated by antigen present within the tissue resulting in clonal diversification and affinity maturation mechanisms which would contribute to the pathological mechanisms of these disorders. Initially the cellular phenotypes and organisations of the inflammatory infiltrating cell population were characterised by immunohistochemical techniques, with particular interest on the infiltrating B cell and plasma cell populations. No typical ectopic germinal centre structures were observed in the target tissues from either disorder; cellular aggregations varied from loose aggregations to dense cellular follicles. Both B cell and plasma cell phenotypes were included in these infiltrating populations in correlation with varying numbers of cells positive for helper, cytotoxic and regulatory T cell, follicular dendritic cell, macrophage and proliferating cell markers. Double fluorescent labelling of B cells with the Ki67 proliferation marker indicated the possible expansion and clonal diversification of these cells within the target tissues. To address the main objective of this study and the possible antigen-driven mechanisms and active participation of these infiltrating B cells within the target tissue of these disorders, Ig gene repertoires, mutational characteristics, clonal diversification and affinity maturation were examined from infiltrating cells microdissected from areas of aggregation within the target tissues of both disorders. From the muscle infiltrating cells, Ig gene selections which were both patient and disease specific, mutational characteristics and oligoclonal expansion of B cells were observed establishing the involvement of muscle infiltrating B cells in the antigen-driven responses within inflamed muscle. Alternatively in the inflamed skin of vasculitis patients very few Ig gene rearrangements could be identified and no clonally related sequences or oligoclonal expansion of B cells were observed despite mutational characteristics indicating that antigen-driven diversification had occurred within these cells. Collectively the results indicate a role for B cells in the antigen-driven responses towards autoantigens within both disorders, either within an active antigen-driven response within the target tissue or at alternative sites resulting in cell migration into the target tissues. In the final part of this study recombinant antigens were used to identify antigen-specific B and plasma cells within the inflammatory infiltrating cell population in some cases of myositis. Pilot experiments were conducted to establish the sequence characteristics of Ig genes from these antigen-specific cells. Results of this study demonstrate the influence of B cell antigen-driven responses, either directly or indirectly, within the target tissues of myositis and vasculitis patients respectively, although the exact mechanisms leading to autoimmune reactions, and additional roles of B cells within these reactions, remain unresolved. The work presented from this study provides a foundation for further work to fully ascertain the role of B cells and antibodies within the target tissues of autoimmune disorders and in characterising the disease-associated antibodies, identifying stimulating antigens as well as assessing the effects of somatic hypermutation on the affinity and specificity of autoantigen specific antibodies. Increased understanding of B cells in these disorders will ultimately assist in the diagnosis and management of these diseases.

Actin polymerisation is a highly dynamic process which drives many cellular events, including endocytosis and cell motility. It is known that actin monomers are added to the filaments at the leading edge of a migrating cell and that this polymerisation is the driving force of protrusion. Much is known about the activation and regulation of this dynamic actin remodelling, but many questions about the exact nature of the interaction between the actin filaments and the plasma membrane remain. Weisswange et al (Weisswange et al., 2005) found that the leading edge of protruding fish keratocytes functions as a diffusion barrier for lipid dyes. The aim of the here presented thesis was to continue this project and to study the actin-membrane interaction at the leading edge, using the diffusion barrier as an initial read-out method. First it was investigated whether this diffusion barrier is present in other cell types and could therefore be seen as a general feature of protrusion. Using Fluorescence Recovery After Photo-bleaching (FRAP)in B16 F1 cells, it could be shown that the diffusion of membrane anchored GFP (GFP-F) is significantly inhibited at the leading edge compared to lamellar regions. No reduction in diffusion could be observed after destruction of the actin meshwork or at non-protruding sites of the lamellipodial periphery, showing that the diffusion barrier depends on active protrusion. The diffusion of cytoplasmic GFP was not altered near the leading edge compared to in the lamellipodium, indicating that only membrane bound proteins are affected. After showing that the diffusion barrier is a general feature of protrusion, the exact nature of the actin-membrane interaction causing this phenomenon was investigated. No direct interaction between actin and the membrane could be observed using FLIM-FRET, but FRAP experiments on fixed cells and a correlation between the strength of the diffusion barrier with the speed of protrusion indicate that the reason for the reduction in diffusion around the leading edge is the force created by the actin filaments pushing against the membrane. Further FRAP experiments indicate that actin regulating proteins such as IRSp53 are influenced by the restricted diffusion zone at the leading edge. We propose that the lipid diffusion barrier traps regulatory proteins at the leading edge and can therefore be seen as a positive feedback mechanism for actin polymerisation.

Depression and rheumatoid arthritis have an estimated co-morbidity of 13-20%. However, the mechanisms whereby peripheral inflammation might alter brain function are unknown. We hypothesised that pro-inflammatory cytokines released in the periphery will result in neurochemical, structural and functional changes related to depression. Therefore, the aim of this thesis was to investigate altered central nervous system function in a rodent model of rheumatoid arthritis, the murine model of collagen induced arthritis (CIA). The CIA model is an established model used to investigate novel anti-inflammatory agents and resembles rheumatoid arthritis as the model is chronic and involves an autoimmune response to type II collagen. To our knowledge brain function in the CIA model has not previously been examined. Therefore, we began by identifying key neurochemical, cellular and functional changes associated with depression which had the greatest likelihood of being influenced by pro-inflammatory cytokines. Serotonin and dopamine transporter densities. The serotonergic system is implicated in the pathology of depression and there is evidence that pro-inflammatory cytokines may influence the serotonin transporter (SERT) in vitro. In vitro autoradiography binding of [125I]-β-carbomethoxy-3-β-(4 iodophenyl)tropane ([125I]-β-CIT) in the presence of mazindol and [3H]-citalopram was used to determine SERT binding in mice with CIA and controls. Out of 15 regions of interest investigated a significant change in SERT binding was identified by [125I]-β-CIT binding, in the nucleus accumbens (58%), thalamus (62%), and dentate gyrus (-60%) in CIA mice compared to controls. However, no significant difference in SERT density was detected in any region by [3H]-citalopram binding. Dopamine transporter (DAT) binding sites were also examined using [125I]-βCIT in the presence of displacer fluoxetine and [3H]-WIN 35,428. Out of 14 regions investigated a significant difference in DAT binding was only observed in the caudate putamen (95%) in the CIA group in comparison to the control group. However, no significant difference in DAT binding was detected in any region by [3H]-WIN 35,428 binding. A limitation of this study was the small group sizes and the degree of clinical symptoms in the CIA group. The data suggest that SERT and DAT transporter densities are not altered by CIA. [14C]-2-Deoxyglucose autoradiographic study of local cerebral glucose utilisation. To investigate brain function the [14C]-2-deoxyglucose ([14C]-2-DG) autoradiographic technique and a challenge to the serotonergic system were employed to identify any abnormalities in regional cerebral glucose utilisation. Overall there was no significant difference in the index of cerebral glucose utilization (iLCMRglu) in mice with chronic clinical symptoms of CIA. To investigate altered serotonergic function in the CIA model fenfluramine, a drug which stimulates serotonin release and blocks serotonin re-uptake was employed. Fenfluramine challenge in the CIA group resulted in only 3 out of the 35 regions of interest examined being significantly different from fenfluramine challenged controls. The orbital cortex (-41%) and the molecular level of the hippocampus (-26%) demonstrated a significant difference in iLCMRglu Overall the data suggest minimal influence of CIA on brain function. Cell proliferation and cell survival in the hippocampus. Hippocampal atrophy is implicated in the pathology of depression and there is evidence to suggest that pro-inflammatory cytokines reduce cell proliferation in vitro. To investigate hippocampal cell proliferation mice were administered 5’ –bromo-2’-deoxyuridine (BrdU), a marker of proliferating cells, prior to and after developing chronic clinical symptoms of CIA. There was no significant difference in cell proliferation prior to the development of clinical symptoms. There was a statistically significant increase in cell proliferation after chronic clinical symptoms in the CIA model in one out of two separate experiments. The data has been interpreted cautiously due to the fact the significant increase in cell proliferation was not reproduced. Cell survival was also investigated during the onset of clinical symptoms and the data demonstrated no significant effect of CIA on cell survival. Conclusion. The data indicate minimal influence of peripheral inflammation on the central nervous system, at least in the murine CIA model. Two possible explanations are that the CIA murine model is not a suitable model to detect changes in brain function associated with rheumatoid arthritis or that uninvestigated neurochemical systems play a role. This thesis highlights our limited understanding of the CIA model and whether or not it represents the features associated with rheumatoid arthritis other then peripheral inflammation. Further characterisation of the brain and development of the CIA model is required to establish if it is a suitable model to investigate the association between depression and rheumatoid arthritis. This is important as understanding the cause of depression and how the cause influences the brain will allow for the development of more specific treatments.

The results obtained from the experiments presented in this study aimed to further explore the role of cyclin dependent kinases and cyclins in the protozoan parasite Leishmania major. Cdks in kinetoplastids, CRKs, are the key regulators that allow cells to progress through different cell cycle phases and promote parasite proliferation during infection. In chapter 3 of this study, the results presented showed that L. major CYCA is capable of activating CRK3 in an in vitro kinase assay using histone H1 as substrate. The CRK3/CYCA active complex was then used to analyse the effect of the phosphorylation at the CRK3 activation threonine using a kinase activating kinase (yeast CAK or Civ-1). Phosphorylated CRK3 activity was compared to non-phosphorylated CRK3 and it was found that the phosphorylation promotes a 5-fold increase in kinase activity of the complex. The accessory protein Cks1 was assayed in vitro with the active CRK3/CYCA complex and it was shown that Cks1 might have an inhibitory effect when histone H1 substrate is used. The IC50 for two different kinase inhibitors (Flavopiridol and Indirubin) was determined for the in vitro CRK3/CYCA complex and compared with the values found for the in vivo purified CRK3. Similar values were obtained suggesting that the in vivo complex is indeed represented by the recombinant complex. In the following chapter 4, yeast Civ-1 purified from E. coli, was used to try to phosphorylate, in a similar manner, the activation of threonine/serine residues from other L. major CRKs. The kinases assessed were CRK1, CRK2, CRK4, CRK6 and CRK7. None of these were phosphorylated by Civ-1 suggesting that the only CRK under this type of regulation is CRK3. L. major CRK1-4 and CRK6-8 were tested in kinase assays by mixing under described conditions with L. major CYC9 and kinase activities towards three different substrates were assessed. L. major CYC9 was not able to activate the above kinases and the kinase subunit that interacts with this cyclin could not be identified. In chapter 5, the L. major CYCA was used to elucidate the characteristics of this cyclin in vivo. A gene disruption strategy aimed to replace the two genomic alleles of this protein gene by homologous recombination. Plasmids were developed with flanking regions of this gene placed in association with two different drug resistance genes, one for each of the allele’s disruption. These constructs were not able to produce the first allele knock out suggesting that not only this gene might be essential but the levels of expression may also be important. Tagging L. major CYCA was also attempted in vivo using two different strategies (i.e. two different tagging systems). The first tag employed was the TAP tag syste. Although drug resistant transfected cell lines were obtained, no tag detection could be observed by western blot using different tag-specific antibodies (α-protein-A and α-calmodulin antibodies). The second tag employed was HA, the 9-amino acid sequence YPYDVPDYA, derived from the human influenza hemagglutinin (HA) protein. Plasmids that contained C and N-terminal HA tagged L. major CYCA were used to transfect WT cells and cells extracts of resistant cell lines analysed by western blot. Both C and N-terminal HA tagged CYCA were detected by the α-HA antibody. Following the confirmation of the presence of the tagged CYCA in the cell extracts an affinity purification using an HA affinity matrix was attempted and the matrix binding material was used in in vitro kinase assays. The presence of kinase activity towards Histone H1 confirmed that CYCA was being succesfully immunoprecipitated in complex with a kinase partner. The identity of the co-eluted CRK could be confirmed using specific α-CRK3 antibody that detected CRK3 in the eluted material.

Endothelial inflammation leading to vascular dysfunction is a major contributor to the development of atherosclerosis. The release of adenosine at sites of inflammation represents an endogenous mechanism for limiting excessive inflammation and tissue damage. The majority of the anti-inflammatory effects of adenosine are mediated by signalling through the A2AAR and activation of the A2AAR has been shown to be protective in numerous models of inflammatory disease. Little is known about the molecular mechanisms behind these effects. However, in vitro studies using cultured endothelial cells indicate that signalling through the A2AAR can suppress activation of the NF kappa B and JAK/STAT proinflammatory signalling pathways. NF kappa B appears to be primed for activation in atherosclerosis-prone regions of the aorta indicating that suppression of NF kappa B signalling may protect against the development of atherosclerosis. In this study, the role of the A2AAR in regulating NF kappa B and JAK/STAT signalling pathway activation in the aorta was studied using A2AAR-deficient mice subjected to an LPS-induced model of septic shock. In response to LPS treatment, these mice displayed markedly elevated plasma levels of the pro-inflammatory cytokines TNF-alpha, IL-6, IL-1 beta and GMCSF compared to wild-type mice. Consistent with this finding, heightened activation of the NF kappa B and JAK/STAT pathways was detected in aortic protein samples from A2AAR-deficient mice as demonstrated by increased levels of the phosphorylated forms of I kappa B alpha and STAT1. However, expression of the NF kappa B and STAT1-regulated genes ICAM-1, VCAM-1 and TAP-1 was unaffected indicating the involvement of compensatory negative feedback mechanisms. These findings confirm a role for the A2AAR in regulation of pro-inflammatory signalling in the aorta. Further analysis of mechanisms which mediate this response may reveal new targets for therapeutic intervention to suppress inflammation in inflammatory disorders such as atherosclerosis. While the wide range of anti-inflammatory and tissue-protective responses elicited by the A2AAR have been well documented, the molecular regulation of the A2AAR has been less well studied. The presence of several serine and threonine residues in the extended C-terminal tail of the A2AAR suggests that it may be regulated by phosphorylation events occurring in this region. Indeed, the canine A2AAR is phosphorylated in response to PKC activation. Interestingly, several proteins have recently been identified as being able to interact with the C-terminal tail of the A2AAR. However, how these interactions are regulated is not known. One of the aims of this study was to characterise phosphorylation of the human A2AAR and to determine whether this could provide a means for regulating the binding of C-terminal interacting proteins. This was examined using human umbilical vein endothelial cells infected with recombinant adenovirus encoding the human A2AAR. It was found that phosphorylation of the human A2AAR could be induced in HUVECs by treatment with PMA or by stimulation of endogenous histamine H1 receptors. This was due to activation of PKC, as phosphorylation was inhibited by the PKC inhibitor GF109203X and by depletion of PKC following chronic treatment with PMA. Treatment of cells with the calcium-chelating agent BAPTA/AM did not inhibit PMA-induced phosphorylation indicating that a calcium-insensitive isoform of PKC was responsible. Meanwhile an siRNA-mediated gene silencing approach confirmed that PKC epsilon was not responsible indicating the involvement of either PKC delta or PKC theta. Previously reported interactions between the A2AAR and TRAX and 14-3-3 tau were confirmed in vitro by GST pull-down assay. Binding of 14-3-3 tau to the A2AAR appeared to be unaffected by treatment of HUVECs with PMA. However, A2AAR complex formation with TRAX was significantly reduced in samples from PMA-stimulated cells. These findings indicate that PKC-mediated phosphorylation may represent a means of regulating which proteins can interact with the C-terminal tail of the A2AAR. This may allow the A2AAR to initiate distinct signalling pathways depending on the cellular context in order to achieve the appropriate response.