Objectives: To determine the effectiveness of lateral approach to subacromial injection compared to posterior approach for the treatment of subacromial impingement syndrome (SAIS); and to establish the experiences of SAIS patients receiving these injections associated with better clinical outcomes. Design: This study used a mixed methods approach that combines a pragmatic randomised control trial to investigate which injection approach is better and a semi-structured qualitative interview to investigate the experiences of SAIS patients receiving these injections. Settings: Out-patients community musculoskeletal service Sample: 80 patients with SAIS for the randomised control study and 20 participants for the semi-structured qualitative interview. Interventions: The Intervention group received a single subacromial injection with a 21-gauge Green needle with a 40 mg/ml of Kenalog and a 4 ml 1% of Lidocaine through a lateral approach. The Control group received an identical treatment except that the location was by a posterior approach. Outcome measures: Difference in improvements in the overall patient reported outcome measures (PROMs) and shoulder pain and disability index score (SPADI) at 8 and 12 weeks follow-up between the two groups. Results: A moderate but statistically and clinically significant difference in improvement in day-time pain (mean change score) occurred in favour of the lateral group (mean = 3.7) compared with the posterior group (mean = 2.3) between week 0 to 8 (1.4 points [95% CI 0.3 to 2.6, p = 0.018]). However, there were no statistically significant differences between the groups in night-time pain, shoulder function and SPADI scores. There was a statistically and clinically significant difference (p = 0.001) within the groups for all clinical outcomes between week 0 to 8 and between week 0 and 12. This was confirmed by participants from the semi-structured interviews which were conducted 12 weeks after the injection. Conclusion: There were no real significant differences in the treatments; however, both forms of treatment were associated with significant improvement in shoulder pain, function and disability. This was confirmed by participants from the semi-structured interviews, who felt that they improved not only because of the effect of the cortisone injection, but also because of other factors such as education about their treatment, exercise information, the experience and skills of the injecting clinicians, access to treatment as well as good customer service.

Background: The p38 Mitogen Activated Protein Kinase (MAPK) system is increasingly recognised as an important inflammatory pathway in systemic vascular disease but its role in pulmonary vascular disease is unclear. Indeed, Inflammation is becoming increasingly recognised as driving the process of pulmonary vascular remodelling. Previous in vitro studies suggest p38MAPKa is critical in the proliferation of pulmonary artery fibroblasts, an important step in the pathogenesis of pulmonary vascular remodelling. In this study the role of the p38MAPK pathway was investigated in both in vitro and in vivo models of pulmonary hypertension and human disease. Aims: To investigate the role that the pro-inflammatory pathway mediated by p38MAPK and the alpha isoform in particular, might have in pulmonary hypertension and whether manipulation might offer a mechanism for reversal of pulmonary vascular remodelling. Methods and results: Pharmacological inhibition of p38MAPKa in both chronic hypoxic and monocrotaline rodent models of pulmonary hypertension prevented and reversed the pulmonary hypertensive phenotype. Furthermore by using a novel and clinically available p38MAPKa antagonist, reversal of pulmonary hypertension was obtained in both experimental models. Increased expression of phosphorylated p38MAPK and p38MAPK was observed in the pulmonary vasculature from patients with idiopathic pulmonary arterial hypertension, suggesting a role for activation of this pathway in pulmonary vascular remodelling. A reduction of IL-6 levels in both serum and lung tissue was found in the drug treated animals, suggesting a link between p38MAPK and the inflammatory pathway in pulmonary hypertension. Furthermore a reduction in the amount of soluble collagen was also observed in the drug treated animals. In vitro work has shown that the pulmonary artery fibroblast is an important source of both inflammatory mediators and collagen, released through a p38MAPK dependent system, and that this cell may be essential in the propagation of vascular remodelling. Conclusions: This study suggests that the p38 MAPK pathway plays a pathogenic role in both human disease and rodent models of pulmonary hypertension potentially mediated through IL-6. Selective inhibition of this pathway may provide a novel therapeutic approach that targets both remodelling and inflammatory pathways in pulmonary vascular disease.

The idea behind this thesis developed during my employment at the Department of Gastroenterology, Glasgow Royal Infirmary University NHS Trust, as a Clinical Research Fellow. The department is one of the very few that has been established over the years as a centre for gastrointestinal research and investigations. The studies I performed aimed to take advantage of the availability of techniques for the measurement of protein synthesis using stable (i.e non-radioactive) labelling methods and mass spectrometric analysis to provide comparison of the rates of incorporation of labelled amino acids into mucosal protein sampled by gastrointestinal biopsy when presented intravenously and intragastrically. This project concerns itself with the potential role of the tracer amino acid infusion techniques in the investigation of gastrointestinal diseases particularly those associated with mucosal atrophy and hypertrophy.

GABA is one of the two main inhibitory neurotransmitters in the central nervous system, CNS (along with glycine, which has a major role in the brainstem and spinal cord). GABA regulates a number of functions in the CNS, and in the spinal cord, it is responsible for presynaptic inhibition of primary afferents and postsynaptic inhibition of neurons. GABA is produced by decarboxylation of L-glutamate by glutamate decarboxylase (GAD). Two GAD isoforms have been identified, GAD65 and GAD67. Antibodies raised against glutaraldehyde conjugates of GABA have been used to investigate the distribution of GABAergic cell bodies, whilst the distribution of GABAergic terminals has been examined with antibodies against GAD. Although GABAergic cell bodies are detected throughout the spinal grey matter, these are concentrated in laminae I-III of the spinal dorsal horn. GAD is present in axon terminals in all laminae of the rat spinal cord, but only a few immunoreactive cell bodies have been detected in the superficial dorsal horn. This differs from the situation in the brain, where many GAD-immunoreactive cell bodies can be found. Studies in the brain suggest that while most (if not all GABAergic neurons) synthesise both GAD isoforms, many have relatively high levels of one or other isoform. It is not known whether this is the case in the spinal cord. Until recently, most studies that have looked at the distribution of GAD have used antibodies that do not differentiate between the two isoforms, and such studies in the spinal cord have been qualitative and no attempt has been made to quantify GAD levels in individual laminae, or examine the co-localisation of GAD isoforms in individual boutons. The recent availability of antibodies that are directed against each isoform separately enables detailed studies to be performed that compare the distribution and co-localisation of the two isoforms, in this study, immunocytochemistry and confocal microscopy were used to examine the distribution and co-localisation of GAD65 and GAD67 in individual axonal boutons in each lamina of the rat spinal grey matter. The main finding of this part of the study was that although most GAD- immunoreactive boutons were labelled with both GAD65 and GAD67 antibodies, some showed similar intensities of both types of immunoreactivity whilst others appeared to have relatively higher levels of one or other of the GAD isoforms. This suggests that GAD-immunoreactive neurons are a heterogeneous population. Also, GAD-immunoreaetivity differed between each lamina of the spinal cord e.g. in the superficial dorsal horn, boutons that had relatively higher levels of either GAD65 or GAD67 were frequently found. In contrast, most boutons in the ventral horn displayed relatively high levels of GAD67, although discrete clusters of boutons that had high levels of GAD65 immunoreactivity were detected in lamina IX.

Glutamate is the principal excitatory neurotransmitter throughout the CNS, including the spinal cord. It acts on ionotropic (iGluR) and metabotropic glutamate receptors. Three iGluR families have been identified by the development of more-or-less selective agonists: N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate receptors. Both AMPA (GluR1-4) and NMDA (NR1, NR2A-D) receptors have been detected in the spinal cord and these play a major role in physiological processes such as fast excitatory transmission, synaptic plasticity and neuronal development. In addition, they have also been implicated in pathological conditions including neuropathic pain and neurodegenerative disorders. However, very little is known about the synaptic distribution of these receptors in the spinal gray matter. This is because conventional immunocytochemical techniques, generally used to investigate the location of proteins in the CNS, fail to detect these subunits at synapses due to the presence of an elaborate protein meshwork associated with the postsynaptic membrane, which masks synaptic receptors. Postembedding immunocytochemistry on freeze-substituted, Lowicryl-embedded material is a technique which has been used exclusively for the detection of synaptic AMPA and NMDA receptors. This project initially set out to use this method to examine these receptors on neurons of the adult rat spinal cord, with emphasis on their involvement in the sensory processing of the dorsal horn. With antibodies against the GluR1, GluR2/3, NR1, NR2A and NR2B subunits, heavy labelling was observed at many asymmetrical synapses and where the plane of section was perpendicular to the cleft, most of the immunogold particles were associated with the postsynaptic density. To examine the receptor expression pattern of selected cell populations a new method was developed which involved the combination of postembedding electron microscopy with immunofluorescence and confocal microscopy. However, during the course of this study heavy immunogold labelling of dense-cored vesicles (dcvs) inside axonal boutons was observed with all NMDA antibodies. Several studies have found iGluRs in primary afferent terminals in the spinal gray matter and these are thought to function as presynaptic receptor, in order to determine whether gold particles found over dcvs corresponded to presynaptic receptors in transit, immunogold reactions were carried out on transgenic mice which lacked the NR2A subunit. Surprisingly, not only did the dev labelling remain in these knock-out animals, but there was also a significant synaptic labelling. This suggested that the postembedding immunogold labelling observed with the NR2A antibody was non-specific. Since the labelling patterns were similar with other NMDA antibodies this cast doubts on the validity of the postembedding method for detecting NMDA receptors.

The corneal epithelium forms the smooth anterior surface of the cornea, a structure which is a highly refractive component of the optical system. The corneal epithelium has several unusual properties; it is avascular; it is capable of rapid regeneration; it is a non-keratinised epithelium exposed to the external environment. This epithelium contains a high concentration of acetylcholine (ACh) and high levels of choline acetyltransferase (ChAc) activity, this being the enzyme which is responsible for the synthesis of ACh. There are relatively low levels of cholinesterases (ChE), the enzymes involvea in the breakdown of ACh. These components of the cholinergic system do not appear Go be entirely associated with the sensory nerves of the cornea. Several theories have been propounded for the function of this ACh, but none has received universal acceptance. This research was an attempt to elucidate possible functions for the ACh of the corneal epithelium of the rabbit. The first possibility was that the ACh played a role within the epithelium, maybe a role in the regeneration of the tissue after abrasive injury. Removal of the epithelium followed by investigation of the levels of protein and ChAc in the regenerating tissue presented some evidence to suggest that the enzyme ChAc does not play a major role in this process of regrowth. Thereafter, experiments investigated the postulate that corneal epithelial ACh may act at a site outwith the epithelium. Firstly, the possibility was explored of an anterior release of ACh into the tear film, perhaps to act on one of the structures bathed by this fluid. It was found that only drugs and stimuli which caused damage to the epithelial cells could increase the release of ACh into the tears. It seemed unlikely that this was a physiological function of epithelial ACh. Secondly, experiments were designed to investigate posterior release of ACh to act at a site most probably located in the anterior segment of the eye. Levels of cholinesterases in various tissues were measured and it was found that these levels were low in the corneal stroma and the aqueous humour, which the epithelial ACh would probably have to traverse to reach a distant site of action. A second series of experiments traced the tissues to which radio-labelled ACh diffused after it had been injected into the corneal stroma. These results indicated that detectable amounts of ACh could pass through the aqueous humour to reach the iris and ciliary body althougn only 1.5% of the injected ACh reaches the iris intact and less tnan this reaches the ciliary body. When ACh was injected into the corneal stroma and its effect on the iris observed, it was found that an injected dose of around 2nmol could produce a significant miosis. This dose of ACh is of the same order as the total amount of endogenous ACh in the corneal epithelium. A total release of the complete store of epithelial ACh to have a physiological role in the iris seemed improbable. The effect of cholinomimetics on the outflow of aqueous humour is well documented. These drugs, especially pilocarpine, are used in the treatment of glaucoma and probably act by increasing the outflow of aqueous humour from the anterior chamber. The main outflow system from the rabbit eye is situated in the iridocorneal angle of the anterior chamber. It was found that small doses of ACh (40pmol) injected into the corneal stroma could significantly increase the apparent facility of outflow of aqueous humour. The ACh appeared to act on muscarinic receptors and act through a Ca2+-mediated mechanism. It was potentiated by anticholinesterases. The mechanism of action of ACh on the outflow mechanism is discussed. The possibility that ACh from the corneal epithelium might exert an influence on the outflow of aqueous humour is put forward.

Background: The consumption of plant based foods has demonstrated an inverse association with disease prevalence. Among the components of plant based foods, polyphenol and fibre are two of the main contenders for many health benefits. The majority of polyphenols and fibre pass through the small intestine unabsorbed, reaching the colon where they are subjected to the action of the colonic microbiota; resulting in the production of the potentially bioactive metabolites: phenolic acids and SCFA. These metabolites are potentially responsible for many of the health benefits exerted by polyphenols rich foods and fibre. Given the recent advances in understanding the role of colonic microbiota in metabolic and immune responses, factors, which may positively or negatively modify the composition of the colonic bacteria have also received much attention. Foods rich in dietary fibre and polyphenols have the potential to modify colonic bacteria through prebiotic and antibiotic action. The potential bacterial inhibition by polyphenolics and the stimulation of bacterial growth by fibre and polyphenols means potential for both sets of compounds to influence metabolite production from each other. Polyphenols and fibre are most often present in the same foods and may be found together in plant cell walls. Thus they most often enter the colon together. We aimed to explore how the presence of these two components in the diet may impact on the metabolite production from the other by the colonic microbiota. Methods: The food matrix interaction of fibres and polyphenols was assessed using the fibres: raftiline, pectin and ispaghula, having different physio-chemical properties (rate of fermentation and viscosity) and the polyphenols: rutin and catechin, epicatechin and other polyphenols present in cocoa in vitro models of phenolic acid and short chain fatty acid (SCFA) production. The impact of ispaghula on urinary phenolic acids after cocoa ingestion was then investigated in an acute human study. In Chapter-3 the impact of the fermentable fibres on phenolic acid production from isolated parent compound: rutin in-vitro using 24 hour batch cultures using human faecal samples from volunteers (n=10) after being on a 3-day low polyphenol diet was investigated. Using the same model the impact of rutin, quercetin and their metabolites on SCFA production from raftiline, ispaghula and pectin was then investigate. The SCFA were measured by GC-FID and phenolic acids by GCMS. pH and gas were also measured. Using the same methodology the matrix interaction between raftiline, ispaghula and pectin separately on phenolic acid production from their parent compounds within their food matrix was investigated using cocoa as a rich source of polyphenols, as well as the impact of cocoa polyphenols and their metabolites on SCFA production from the fermentable fibres (Chapter-4) In Chapter-5, 24-hour urinary polyphenolic acids were measured in 5 batches (0, 0-2, 2-5, 5-8, 8-24 hour) in 12 human volunteers after ingestion of 1g paracetamol with 20g cocoa (extra brute Cocoa-Cacao Barry, Barry Callebaut, Hardricourt, France) with water, 15g of ispaghula with water or the combination of the two. Urine samples were also used for total phenol and antioxidant capacity measurement. Plasma was collected over six hours (every half hour for 4 hours and at 6th hour) and used for the measurement of total phenols as well as paracetamol concentrations for the estimation of gastric emptying rate. Breath hydrogen was used for estimation of small bowel transit time and visual analogue scales (VAS) were used for the estimation of subjective appetite response to meals. Results: The faecal fermentation of rutin resulted in the production of the following phenolic acids: PAA, 4-HBA, 3-HPAA, 4-HPAA, 3,4-DHPAA, 3-HPPA and 4-HPPA. All of these phenolic acids were significantly reduced by at least one of the three fibres, with the exception of 3-HPPA and 4-HPPA. The extent of inhibition of total sum of phenolic acids from raftiline and pectin was similar (p < 0.01) and ispaghula demonstrated the least inhibitory effect (p=0.03). Rutin and quercetin had no impact on the SCFA production from the fermentable fibres. The phenolic acids identified from cocoa faecal incubations consisted of: of PAA, 3-HPAA, 4-HPAA, 3,4-DHPAA, 3-HPPA, 4-HPPA, 3,4-DHPPA, 4-HBA, 3,4-DHBA, hippuric acid and vanillic acid. Unlike the rutin study where majority of phenolic acids were significantly reduced, in this study only four of eleven phenolic acids were affected (PAA, 3-HPAA, 4-HPAA, 4-HBA: also inhibited in the rutin study).The extent of phenolic acid reduction was the highest for pectin (p < 0.01), followed by raftiline (p < 0.01) and ispaghula (p=0.03). These phenolic acids or their parent compounds had no impact on SCFA production from the fermentable fibres. The consumption of cocoa resulted in the urinary excretion of the following phenolic acids: 3-HPAA, 4-HPAA, 3,4-DHPAA, Hippuric, 4-HPA, 4-HBA, 3,4-DHBA, Vanillic, 4-HVA, Mandelic and 4-HMA. All of which, with the exception of vanillic acid and 3,4-DHPAA, were reduced by ispaghula (Table-I). Ispaghula accelerated gastric emptying rate but had no impact on small bowel transit time. The analysis of total phenol (TP assay) concentration (plasma and urine) and antioxidant capacity (urine) did not demonstrate any difference between cocoa and ispaghula, which were both high. However when they were ingested together there was a signification reduction in both total phenol and antioxidant capacity (p < 0.01). Given that urinary and plasma concentration of total phenols was no different for ispaghula and cocoa we analysed the free phenolic and bound phenolics in both ispaghula and cocoa, showing that cocoa has significantly higher free phenolics than ispaghula, whereas bound phenolics were higher in ispaghula. The sum of bound and free total phenols was higher in cocoa than ispaghula (approximately 10 fold). Urinary, faecal SCFA were not measured as they are not validated to represent in-vivo production. Conclusion: there is a strong inhibition of phenolic acid production from polyphenol by the fermentable fibres and their metabolites. This inhibition is stronger in-vivo than in-vitro for ispaghula, which may reflect the longer interaction time in the colon and potential small bowel interaction. The production of SCFA from fermentable fibres was not inhibited by the polyphenols or their metabolites. These interactions need to be considered when assessing the bioavailability of phenolic acid production and their potential health benefits.

This thesis describes the isolation, structure elucidation, anti-trypanosomal activity and molecular modelling of the aporphine alkaloids obtained from Enantia chlorantha. Phytochemical studies on the Enantia chlorantha stem bark yielded six dimeric and one monomeric aporphine alkaloid along with one protoberberine type alkaloid, palmatine. Three dimeric aporphine alkaloids were novel namely: 1,1',2,2',3-pentamethoxy-6-methyl-5,5',6,6'-tetrahydro-4H,4'H-7,7'-bidibenzo[de,g]quinoline, ECP-19 (89),8-(1,2,3-trimethoxy-5,6-dihydro-4H-dibenzo[de,g]quinolin-7-yl)-6,7-dihydro-5H-[1,3]dioxolo[4',5':4,5]benzo[1,2,3-de]benzo[g]quinoline, ECHE-45 (90), and 7-methyl-8-(1,2,3-trimethoxyl-5,6-dihydro-4H-dibenzo(de,g)quinolin-7-yl)-6,7-dihydro-5H-(1,3)dioxolo(4'5':4,5)benzo(1,2,3-de) benzo(g)quinoline ECH-56 (91). The structures of the alkaloids were determined using 2D NMR experiments and their masses confirmed using ESI Mass Spectrome-ter. Anti-trypanosomal screening of these alkaloids for activity against the non-virulent bloodstream form of T.brucei brucei ,carried out using a modified microplate Alamar blue™ assay revealed that these alkaloids had excellent anti-trypanosomal activity, with MICs 1.27 to 10.96 nanomolar compared to the positive control Suramin with MIC of 9.6 nanomolar. Of the three novel dimeric alkaloids, ECP-19 (89) was the most active with MIC of 1.27 nanomolar. Molecular modelling was carried out for all of these alkaloids as well as their derivatives monomeric aporphine alkaloids using the GRIP technique with Vlife molecular Design Suite (V life MDS 4.2) on seven validated Trypanosoma brucei protein targets from the Protein Data Bank (PDB). These protein targets were: T. brucei Glutathione Synthetase , Glutathione peroxidase-type tryparedoxin peroxidase, oxidized form ,Glutathione peroxidase-type tryparedoxin peroxidase, reduced form , Sterol 14-alpha demethylase (CYP51) from T. brucei in complex with the tipifarnib derivative 6-(4-chlorophenyl)(methoxy)(1-methyl-1H-imidazol-5-yl)methyl)-4-(2,6-difluorophenyl)-1-methylquinolin-2(1H)-one , T. brucei Ornithine Decarboxylase , Riboflavin kinase ,and Trypanothione reductase from T. brucei. The inhibition of T. brucei ornithine decarboxylase was the most significant, hence the possibility of it being a likely mechanism of action for these alkaloids. Further molecular modelling studies of the eight alkaloids whose structure were elucidated in this thesis, as well as six derivate monomeric alka-loids were carried out to pinpoint the 'best fit' alkaloids to Ornithine decarboxylase's active site Ly-sine 69 using the GOLD 5.5.2 software. This revealed the dimeric aporphine alkaloids isolated in this study had docking score as a function of GOLD.PLP.Fitness which ranged from -95.1384 to 27.8819 for dimers,which is not as good as the docking scores ranging from 26.5959 - 38.4616 for monomers isolated in this study, as well as monomeric derivatives of the dimers. Derivative monomer, Compound 95 had the best docking score of 38.4616. This set of results in terms of the novelty of the dimeric alkaloids, their excellent anti-trypanosomal activity in vitro, significant results in molecular models and the fact that anecdotal evidence of use of Enantia chlorantha extracts in vivo for treatment of ailments traditionally in rural West Africa for cen-turies may form a basis for further drug development studies. As it is the norm in drug discovery syn-thetic analogues developed from a natural product scaffold tend to provide a vast number of molecules to test and develop further, therefore future molecular modelling studies are currently being tailored to optimising the 'best fit' monomeric alkaloids to hybrid modelled synthetic analogues for further drug development studies beyond the scope of the PhD study.

Oligodendrocyte precursor cells (OPCs) are a major transplant population to promote myelin repair and central nervous system (CNS) regeneration in conditions such as Multiple Sclerosis and spinal cord injury. Magnetic particles (MPs) can offer a multifunctional platform for cell therapies, facilitating labelling for cell tracking (e.g. by MRI and histopathology); biomolecule delivery (including nonviral gene delivery, enhanceable by novel ‘magnetofection’ strategies); and magnetic cell targeting of transplant populations. However, MP-based applications for neural tissue engineering have received limited attention to date. This thesis demonstrates that ~60% of OPCs (derived from a primary source) can be safely labelled using two well-characterised MP formulations, including a novel multimodal MP with transfection plus cell labelling capabilities. A rapid, technically simple, high-throughput ultrastructural imaging technique, OTOTO SEM, has been developed to study the surface interactions of MPs with precursor cells. Safe MP-mediated transfection of OPCs was demonstrated, including with multiple and therapeutic genes. Transfection efficiency was enhanced by static/oscillating ‘magnetofection’ techniques (~21%; competitive with nonviral alternatives). Organotypic cerebellar slice cultures were developed as a model of ‘host’ neural tissue, and ‘magnetofected’ OPCs exhibited normal migration, proliferation and differentiation profiles following transplantation onto such slices. Safe labelling (~45%) and transfection (enhanced by static/oscillating magnetofection strategies: ~6%) of oligodendrocytes was achieved utilising identical protocols to those developed for OPCs. A comparative intralineage analysis demonstrated that MP-uptake and amenability to transfection were significantly lower in oligodendrocytes compared to OPCs. Inter-cellular comparisons of MP-handling by the four major CNS glial subtypes (viz. OPCs, oligodendrocytes, astrocytes, microglia; derived from the same primary source) revealed major differences in the rate/extent of MP uptake, amenability to transfection, optimal magnetofection frequency, and MP-associated toxicity. Finally, a stoichiometrically-defined glial co-culture model was developed and utilised to test the hypothesis that microglia represent an ‘extracellular barrier’ to MP uptake by other glia.

Acute pancreatitis is a human disease in which cytosolic Ca2+ overload and mitochondrial dysfunction have been implicated as the initial triggers. Sustained elevations in cytosolic Ca2+ result in intracellular activation of digestive enzymes, mitochondrial dysfunction and cellular necrosis. This leads to autodigestion of the pancreas and subsequent inflammation, which can prove fatal. Intracellular Ca2+ release from stores is coupled to the activation of store-operated Ca2+ entry (SOCE) channels such as the Ca2+ release activated Ca2+ (CRAC) channel or the canonical transient receptor potential (TRPC) 3 channels in the plasma membrane. These channels drive cytosolic Ca2+ overload. Targeting these channels is an appealing therapeutic strategy. One of aim of this study was to determine if there are any inhibitors, in addition to GSK-7975A, that can specifically block these channels and prevent cytosolic Ca2+ overload. Analogues of 2-APB, a well know but problematic inhibitor of SOCE channels, DPB163-AE and DPB162-AE were found to inhibit SOCE channels efficiently at a low micromolar concentration. This was similar to the effect seen with the inhibitor GSK-7975A. However, increasing concentrations of DPB163 resulted in impaired clearance of Ca2+ from the cytosol, whereas DPB162 inhibited SOCE further. RO2959, another reported CRAC channel inhibitor, was found to be less effective at inhibiting SOCE in pancreatic acinar cells than in other cell types. This phenomenon was true for the DPB compounds. RO2959 was effective at inhibiting SOCE in dose dependent manner when applied acutely or when pre-incubated with cells. In addition, there were observed effects of RO2959 on Ca2+ clearance from the cytosol. This likely contributed to the observed finding that RO2959 also increased levels of necrosis, due to impaired Ca2+ clearance. Targeting TRPC3 channels, the non-specific cation channels, has been difficult as it is lacking a specific inhibitor. Treatment with Pyr3, initially thought to be a specific TRPC3 inhibitor was particularly effective at inhibiting SOCE. However, during the time course of this work it came to light that TRPC3 at low micromolar concentration does not discriminate between TRPC3 and CRAC channels. Treatment with a reportedly specific inhibitor, Pyr10, resulted in a reduction in Ca2+ influx. However, in a similar manner to DPB163 and RO2959 there appeared to be an effect of Pyr10 on the Ca2+ clearance from the cell. A second aim of this thesis was to investigate the role of calmodulin in SOCE in pancreatic acinar cells. Calmodulin is thought to regulate CRAC channels by mediating Ca2+-dependent inactivation of the channel. Inhibitors of calmodulin: calmidazolium and W-7 were used. Both inhibitors resulted in an inhibition of SOCE. However, this inhibition was not as remarkable as CRAC channel inhibition. This could have been because the calmodulin inhibitors were not directly inhibiting the Orai1 protein of the CRAC channel rather were targeting one of the channels regulating proteins - calmodulin. A cell permeable activator of calmodulin was also utilised in this study - CALP3. CALP3 had previously been shown to protect acinar cells from cytosolic Ca2+ overload and also to block non-specific cation channels in T cells. At the concentration found to protect against Ca2+ overload, no effect on SOCE was observed. Increasing the concentration resulted in significant inhibition, but it was likely due to a non-specific effect of a supramaximal concentration. CRAC channel inhibition was the more effective as a target for reducing SOCE and subsequent Ca2+ overload than targeting calmodulin-regulation of CRAC channels or targeting TRPC3 channels. However, several of the 'specific' inhibitors available to target this channel, including DPB163 and RO2959 had a multitude of off-target effects including a significant effect on the Ca2+ extrusion mechanism of pancreatic acinar cells. GSK-7975A had the fewest undesired effects and also inhibited SOCE more than any other inhibitor. It is an ideal candidate for future drug development. When the work in this study is viewed in the context of the wider published work, the idea of focusing on CRAC channel inhibition as a potential therapeutic for acute pancreatitis is a promising future avenue.