Ryanodine receptors (RyRs) are the largest known ion channels composed of four identical subunits. Interactions between structural/functional domains have been proposed to regulate channel activity and play an important role in the pathogenesis of RyR-associated disorders. RyR2 mediates the release of calcium form sarcoplasmic reticulum of cardiac myocytes and its dysfunction is associated with life-threatening arrhythmias. The principal aim of this study was to characterise the self-association of the RyR2 N-terminus biochemically and evaluate its impact on channel function. Moreover, its role in channel dysfunction observed in arrhythmia-susceptible individuals was tested together with dantrolene’s ability to rescue the disease phenotype. RyR2 N-terminus self-association is mediated by multiple sites with two critical oligomerisation determinants located in the loops connecting strands β8-β9 and β20-β21, predicted to reside at the inter-subunit interface. N-terminus self-association is further stabilised by disulphide bonds most likely involving multiple cysteine residues with cysteine 361 contributing to this process. Normal N-terminal inter-subunit interactions within the full-length RyR2 appear to prevent spontaneous activation of the channel at diastolic calcium. Channel hypersensitivity is a common feature of the arrhythmia-associated phenotype suggesting that abnormal N-terminus self-interaction might be involved in RyR2 pathology. Indeed the presence of arrhythmia-linked mutations (L433P and R176Q) compromises the ability of the RyR2 N-terminus to oligomerise. Defective N-terminus self-association appears to underlie the functional impairment of RyR2L433P. The mutated channel displays compromised [3H]ryanodine binding and reduced stability of tetrameric assembly, both of which can be rescued by dantrolene at clinically relevant concentrations. Notably, dantrolene’s primary mode of action appears to involve stabilisation of the N-terminal inter-subunit interactions. In summary, the work presented here provides important insights into a novel domain-domain interaction and its role in the regulation of RyR2 function.

In wound healing and tissue repair, the presence of α-smooth muscle actin (α-SMA) containing myofibroblasts leads to wound closure and collagen-rich scar formation. This thesis investigated mechanisms of transforming growth factor-β1 (TGF-β1)-mediated differentiation and the dysfunctions involved in age-associated loss of differentiation. The loss of epidermal growth factor receptor (EGFR) and hyaluronan (HA) production, and diminished interaction of HA with its receptor CD44 (compromising its function) were the principal contributors to aged fibroblast resistance to differentiation. In response to TGF-β1, CD44 relocated to EGFR held in cholesterol-rich membrane-bound lipid rafts. This was HA-dependent, as hyaluronidase or 4-methylumbelliferone treatments restricted CD44 motility and prevented CD44-EGFR co-localisation. Additionally the intracellular signalling cascade was found to be a sequential phosphorylation of extracellular signal regulated kinase 1 & 2 followed by Ca2+/calmodulin kinase II. The activation of both proteins was required for differentiation. Elevated microRNA-7 (miR-7) expression was found in aged fibroblasts. Overexpressing miR-7 in young fibroblasts attenuated the expression of EGFR and inhibited differentiation. When miR-7 was inhibited, EGFR and hyaluronan synthase 2 expression, CD44 membrane motility, and TGF-β1-mediated differentiation in aged fibroblasts were restored. Activation of EGFR drove miR-7 promoter activity and expression in a JAK/STAT1 dependent manner. Treatments of aged fibroblasts with 17β-estradiol (E2) resulted in decreased miR-7 expression and, when TGF-β1 was added, restored the α-SMA-positive phenotype. E2 treatments had no impact on STAT1 phosphorylation; leading to the hypothesis that E2 regulation of inflammatory mediators may be involved. The data demonstrated different points of intervention for the promotion or prevention of TGF-β1-regulated myofibroblast differentiation. The interactions between HA-CD44 and EGFR were crucial elements in the differentiation process and the importance of miR-7 was apparent. The mechanisms shown here may have direct implications for modifying the wound healing response, particularly for developing therapeutic strategies to improve healing in the elderly.

Hereditary haemophilia A (HA), an X-linked bleeding disorder, is caused by mutations in the coagulation factor VIII gene (FVIII abbreviates protein, gene symbol F8). The aim of this study was (1) to characterise F8 mutations in HA cohort from Pakistan, (2) to investigate whether in vitro thrombin generation (TG) differs according to mutation type, and (3) to compare haemophilia joint health score (HJHS) and Gilbert score with severity, age, TG and underlying mutation in HA cohort which had minimal access to haemostatic replacement therapy. Methods: One hundred HA individuals and 100 healthy controls were recruited; clinical details were recorded. Results: Phenotypic measurements were re-evaulated in Cardiff; the essential regions of F8 were screened. Ninty two individuals were diagnosed with HA, 7 with haemophilia B and 1 was normal. The F8 defects were characterised and comprised point mutations, inversions and frameshifts. Thirty novel variants were identified. No significant difference was observed in vitro TG between severes with null mutation and those with a missense change. HJHS was strongly correlated with Gilbert score (r =0.98), both were siginificantly higher in severe compared with nonsevere before the age of 12 years (P≤0.01) but not thereafter. According to developmental age (<12 years, 12-16 years and > 16 years), both scores were significantly lower in the youngest group (P≤0.001). In severes there was no correlation between in vitro TG and joint score, no significant difference was observed for either joint score according to the underlying mutation type. Whereas in nonseveres, negative correlation between in vitro TG and joint score was observed. Conclusions: F8 defects in Pakistan is heterogenous; in severe HA in in vitro TG are not influenced by underlying mutation.

The ryanodine receptor (RyR) is a large (563 kDa) tetrameric calcium channel that controls the efflux of calcium ions from the large internal stores within myofibrils called the sarcoplasmic reticulum. The cytoplasmic domain is connected to the pore via an "interacting-domain" (I domain) which plays an important role in the mechanism of channel regulation. Within the I domain is a calcium binding region which resembles a lobe of calmodulin and is thus called the calmodulin-like-domain (CaMLD). CaMLD has a binding partner region, the calmodulin-binding-domain (CaMBD) located in the cytoplasmic domain. Mutations in the cardiac isoform of RyR have been shown to result in the clinical presentation of a lethal disease condition called catecholaminergic polymorphic ventricular tachycardia (CPVT). Thus, mutations proximal to the CaMLD and CaMBD region are of particular interest, since if these mutations affect the binding of calcium or result in any conformational change of the protein, then the role of the mutation could be explained. Computer simulation methods were employed to investigate the role of CaMLD and CaMBD in the overall biology of RyR. The individual domains and also the entire section of RyR containing both domains were modelled. This latter model underwent a simulated folding process to generate a tool to further investigate the role of CPVT mutations within this region in the context of the structural changes brought about by the changes in amino acid residues of the protein. CaMLD and CaMBD were produced as recombinant proteins and selected CaMLD CPVT mutants generated. The calcium dependent interaction of these two domains was examined through a range of functional techniques, in particular circular dichroism. All the studied mutants displayed a calcium induced conformational change associated with the functional protein. However, one particular mutation was identified which appeared to significantly weaken the structural stability and have reduced calcium sensitivity when compared to the wild type recombinant protein.

This thesis presents the development and testing of an online lovingkindness meditation (LKM) intervention. Previous studies were systematically reviewed, showing complex but encouraging evidence that LKM can enhance the wellbeing of individuals and communities by promoting pleasant emotions and empathic attitudes. However, previous randomised controlled trials (RCTs) are small and have methodological limitations. An online RCT was conducted, which recruited 809 adult volunteers to test whether an LKM intervention offered to the general population improves wellbeing through pleasant emotions, psychological resources, empathy and altruism. LKM was compared to a light physical exercise course (LE). Participants followed prepiloted videobased instruction, wrote about their experiences in online diaries and interactive fora, and completed questionnaires and an objective measure of helping behaviour. The data were analysed using a mixed methods approach. Both courses led to greater wellbeing. LKM participants were significantly less anxious and more likely to donate money to charity than LE participants. Differences in other outcomes were not significant. Attrition was high but generally unrelated to the interventions’ content. The pathways to wellbeing differed. LKM was an emotionally intense experience, generating deep reflections and an increased connectedness with self and others. LE led to increases in relaxation and physical wellbeing which generated a sense of achievement. Some participants had early difficulties with LKM, in which personal factors played an important role. The study provides suggestive evidence that both LKM and LE enhance pleasant emotions, psychological resources and wellbeing, and that LKM specifically stimulates empathy and altruism. The LKM training platform used in this study is available for immediate largescale implementation as an inexpensive public health intervention. However, future research is needed to confirm present findings and devise LKM interventions that reduce the negative impact of initial training. Completion rates might be improved by nesting online RCTs within cohort studies.

Rhythmic contraction of cardiac myocytes is maintained by precisely controlled Ca2+ efflux from intracellular stores mediated by the cardiac ryanodine receptor (RyR2). Mutations in RyR2 result in perturbed Ca2+ release that can trigger arrhythmias. RyR2- dependent ventricular tachyarrhythmia is an important cause of sudden cardiac death, the mechanistic basis of which remains unclear. RyR2 dysfunction has also been implicated in other cardiovascular disorders such as heart failure and cardiomyopathy, thereby becoming an important target for putative drugs. The massive size of RyR2 (~2.2 MDa) along with its intracellular location poses considerable challenges to studies aimed at understanding the mechanisms underlying channel dysfunction. Single channel studies of reconstituted RyR2 in artificial lipid bilayers have provided important insights into channel behaviour in response to various physiological ligands, toxins, drugs and biochemical modifications. However, the precise mechanisms by which RyR2 is activated by its primary physiological trigger, cytosolic Ca2+, and the structural determinants of channel gating are yet unknown. In this study, I aim to understand the actual physical reality of RyR2 gating behaviour using novel experimental approaches and analytical procedures. I have examined in detail, single channel kinetics of wild type purified recombinant human RyR2 (hRyR2) when activated by cytosolic Ca2+ in a precisely regulated minimal environment where the modulatory influence of factors external to the channel were minimised. This mathematical modelling of hRyR2 single channel behaviour will serve as a future experimental platform upon which the effects of disease causing mutations can be studied, as well as the influence of physiological modulators and potentially therapeutic compounds capable of stabilising mutant channel function. Single channel studies of hRyR2 when modified by its archetypal ligand ryanodine in the absence of Ca2+ have uncovered an unusual voltage sensitive gating behaviour in this ligand-gated channel, providing further insights into the mechanisms underlying channel modification.

The metastatic spread of cancer cells to distant sites in the body accounts for the majority of cancer-related death and significantly decreases patient survival. Whilst cell migration is a physiologically important process, when uncontrolled, it can be a contributing factor to the metastatic phenotype. Actin polymerisation enables the dynamic restructuring of the cytoskeleton which is fundamental to cell migration and is stimulated by the Arp (actin-related protein) 2/3 protein complex which in turn is activated by members of the WAVE (WASP Verprolin homologous protein) family. WAVE1 and 3 expression was targeted separately in the PC-3 cell line utilising ribozyme transgene transfection. In vitro experiments revealed a reduction in cell growth and invasion following WAVE1 or 3 knockdown in PC-3 cells. These experiments were also repeated with small molecule inhibitors targeting the Arp2/3 complex, ROCK and N-WASP independently. This inhibitor work implicates Arp2/3 as a facilitator of cell proliferation through which WAVE regulates. Inhibition of Arp2/3, ROCK or N-WASP in WAVE1 knockdown cells increased cell invasion which may be attributed to the regulatory role of WAVE3 on MMP activity. Co-localisation of WAVE1 and 3 with ARP2 and ROCK-I was observed in PC-3 cells whilst this affect was abolished with WAVE1 or 3 knockdown. Furthermore, WAVE3 and WAVE1 knockdown affected ARP2 and ROCK-II tyrosine phosphorylation, respectively. These results suggest WAVE1 and 3 proteins are involved in several metastatic traits that characterise PC-3 cells. Furthermore, the contribution of WAVE in the networks that influence these traits may also involve association with Arp2/3 complex, ROCK-I and –II and N-WASP. Additionally, it sheds light on the similarities between these two related proteins and also highlights their subtle distinctions in PC-3 cells. The data outlined here provides justification to futher explore WAVE1 and 3 as potential contributors of prostate cancer progression.

Human cardiac ryanodine receptors (hRyR2) are major Ca2+ release channels in the heart, which form quaternary complexes with luminal proteins, calsequestrin (CSQ2), junctin (JUN) and triadin (TRD1). These proteins facilitate Ca2+ release during excitation-contraction coupling, modulating the response of hRyR2 to luminal Ca2+ changes. Catecholaminergic Polymorphic Ventricular Tachycardia is an arrhythmogenic disorder, caused by mutations in RyR2 and CSQ2 genes. Defective sensing of cytosolic/luminal Ca2+ by hRyR2 is a candidate mechanism underlying disease pathogenesis, likely caused by defective luminal protein regulation. Using a recombinant approach, this study aimed to evaluate if mutant (N4104K and A4556T) hRyR2 respond to or interact differently with, these accessory proteins. Expression constructs corresponding to CSQ2 and JUN were generated and expressed stably and transiently with wild-type (WT) or mutant hRyR2 in HEK293 cells. Immunofluorescent co-localisation confirmed successful co-expression and association of these luminal proteins with hRyR2 in situ. Ca2+ activation of wild-type/mutant hRyR2 in the absence/presence of CSQ2 and/or JUN was assessed by [3H]-ryanodine binding, while luminal Ca2+ effects were monitored using single-cell Ca2+ imaging - examining spontaneous Ca2+ release events. A4556T-hRyR2 displayed similar cytosolic and luminal Ca2+ dependence to wild-type channels, whilst N4104K-hRyR2 displayed a remarkably different Ca2+ activation profile, demonstrating functional heterogeneity between hRyR2 mutants. Ca2+ imaging revealed an inhibitory effect of CSQ2 on WT and N4104K-hRyR2 activity, both in the presence and absence of JUN. In line with this, CSQ2 was found to bind directly to hRyR2 by co-immunoprecipitation, an observation that has not been previously demonstrated in the literature. Immunofluorescence studies suggested reduced CSQ2 and JUN association with A4556T-hRyR2, but this could not be confirmed with co-immunoprecipitation. Ca2+ imaging investigations with this mutant however, suggested that CSQ2 wasn’t able to regulate these channels in the same way as WT, implying a change in functional effect.

Osteocytes are currently regarded as being pivotal in maintaining bone homeostasis. They differentiate from osteoblasts, are embedded in mineralised matrix and difficult to isolate. Current models for in vitro osteocyte studies are limited. Others have suggested that osteoblasts in 3-dimensional (3D) cultures differentiate to osteocytes. This study aimed to develop 3D cultures enabling differentiation of osteoblasts to osteocytes, which could be used for studies of osteocyte differentiation and responses to mechanical loading. Furthermore, the effects of external compounds on osteoblast differentiation in 3D were assessed. Mouse (MC-3T3, IDG-SW3) and human primary osteoblasts (hOBs) were maintained in type I collagen gels in either non-osteogenic or osteogenic media, and +/- compounds such as insulin-like growth factor-1 (IGF-1). Furthermore, mechanical loading (5 mins, 10 Hz, 2.5 N) was applied to 3D cultures and responses characterised. Cells were viable in collagen gels for 25 days, and expressed mRNA for mature osteocyte markers e.g. sclerostin in osteogenic medium. Furthermore, IGF-1 upregulated mRNA expression of osteocyte markers and other molecules (e.g. receptor activator of the nuclear factor kappa-β ligand - RANKL - 43-fold) in MC-3T3 cells, indicating modulation of cell differentiation and function. Osteocyte markers were expressed earlier in IDG-SW3 cells in 3D compared to published marker expression profiles in 2D monolayer cultures. Following mechanical loading, known mechanosensitive markers were modulated in IDG-SW3 cells in 3D, for example, RANKL and vascular endothelial growth factor (VEGF) up-regulated and sclerostin downregulated post-loading. This 3D model enables differentiation of osteoblasts to osteocytes in an environment akin to osteocytes in vivo. External compounds accelerated cell differentiation, and this was also accelerated in 3D compared to monolayer. Furthermore, the 3D model enabled osteocyte mechanical loading. This model can be used with human cells, will further our understanding of osteocyte differentiation, and inform on osteocyte function including their responses to mechanical loading.

Chronic Obstructive Pulmonary Disease (COPD) is one of the commonest long-term conditions worldwide. It is characterised by chronic airflow limitation, pathological changes in the lung and significant extra-pulmonary manifestations. The treatment of an acute exacerbation of COPD (AECOPD), involves glucocorticoids and bronchodilators supplemented by antibiotics if needed. In-hospital, oxygen, which has potential risks as well as benefits, and additional respiratory support can be given if the patient deteriorates. Clinicians need to decide which treatment to provide and who can be safely discharged. This has led to the advent of scoring systems to define severity in COPD This thesis examines the evidence base for the use of magnesium in airways disease and presents the results of the first randomised double-blind placebo-controlled trial using nebulised magnesium in the treatment of AECOPD. 116 patients were randomised, but after 3 nebulisations over 90 minutes, there was no significant difference in FEV1 compared to placebo (p=0.67). In a second study, the CRB65 score was retrospectively assigned to a cohort of patients presenting to the emergency department with AECOPD, using data collected from a previous audit. Patients with a CRB65 score of 0 or 1 had a low risk of in-hospital and 30- day mortality and could be considered for discharge, whereas those with scores between 2-4 required admission with mortality increasing with the score. The CRB65 score showed a similar utility in AECOPD as it does in pneumonia. Finally, 18 subjects with stable but severe COPD were randomised in a crossover study to two nebulisations with salbutamol and ipratropium over 15 minutes with a five minute interval between nebulisations, using air or oxygen as the driving gas. When oxygen was used there was a 3.1mmHg difference (p<0.001) at 35 minutes, compared to air, illustrating the potential risks of repeated nebulisations to those with severe COPD.