KATP channels are ubiquitously expressed and link metabolic state to electrical excitability. In heart, in response to ischaemic stress, they play a protective role and in vascular smooth muscle regulation of vascular tone (vasorelaxation). Functional KATP channels are hetero-octamers composed of two subunits, a pore forming Kir6, which is a member of the inwardly rectifying potassium channels family and a regulatory sulphonylurea receptor (SUR). In response to nucleotides and pharmacological agents, SUR allosterically regulate KATP channel gating. Multidisciplinary techniques (molecular biology, biochemistry, electrophysiology, pharmacology) were used to study the allosteric regulation between these two heterologous subunits in KATP channels. This project was divided into three major sub-projects: 1) Application of site directed mutagenesis and biochemical techniques to identify the cognate interaction domain on Kir6.2 for SUR2A-NBD2 (nucleotide binding domain 2). 2) Electrophysiological techniques to investigate the allosteric information transfer between heterologous subunits Kir6 and SUR2A. 3) Recombinant fusion protein to express and purify the cytoplasmic domains of Kir6.2 for structural analysis of the interaction between the two subunits. This study reports on the identification of three cytoplasmic electrostatic interfaces between Kir6 and SUR2A involved in determining the sensitivity of KATP channel agonist, pinacidil, and antagonist, glibenclamide, from SUR2A to the Kir6 channel pore. For structural study of cytoplasmic domains of Kir6.2, bacterial TM1070 was used as fusion partner with Kir6.2. A TM1070-Kir6.2 NC (CT-His6 tag) fusion construct expressed in Arctic Express competent cells permitted successful expression of folded cytoplasmic domains of Kir6.2 in near native form. Immobilized metal ion affinity chromatography, IMAC (Ni2+), and gel filtration chromatography (GFC) column as second purification step were performed to purify this recombinant protein. The purification was confirmed by CBS and Western blot analysis. Possibly, this new information on channel structure-function relationships may contribute to the design of novel and more effective drugs.

Object. The primary objective of this thesis was to establish the pattern of change in haemostatic systems in patients following a subarachnoid haemorrhage (SAH). I hypothesise that following a SAH there is an undefined period of increasing hypercoagulability, which if present would predispose to ischaemic stroke. Methods. This was a prospective, observational study on 67 consecutive patients admitted with a primary diagnosis of SAH. There were 24 males, median age 47.5 years (25-75) and 43 females, median age 53 years (23- 80). Blood was taken at 4 time periods (<48hours, 4-5, 9-10 and 15-16 days) following the ictus depending on the day of hospital admission, and on regular intervals during the hospital stay. In addition, a sample was taken at 3 months from the ictus. A Thromboelastograph (TEG) profile performed at 37 C, and the routine coagulation studies, International Normalised Ratio (INR) and Activated Partial Thromboplastin Time Ratio (APTR) were obtained at each of these time points. In addition a full blood count, biochemical profile, and plasma for coagulation and fibrinolytic assays was also taken. Results. The results demonstrated that SAH patients were hypercoagulable immediately following the ictus, when compared with the blood sample taken 3 months later. In addition we observed the development of an increasingly hypercoagulable state for the first 21 days following the ictus. This increase in coagulation was demonstrated against a background of haemodilution during this time. Conclusions. This highly significant data demonstrates that SAH patients become increasingly hypercoagulable over time (maximum 21 days) following the ictus. This prothrombotic tendency has reversed by 3 months. This may provide a new direction in the treatment of symptomatic vasospasm. In addition, an in-vitro study using TEG has been performed in 20 volunteer subjects to assess whether haemodilution 'per se' has an intrinsic affect on coagulation specific to the dilutent itself. This study demonstrates that haemodilution does alter coagulation profiles measured using TEG. Different crystalloid and colloid fluids used to achieve haemodilution produce qualitatively consistent but quantitatively very different effects on coagulation in-vitro.

Prehension is the act of reaching towards, grasping, and manipulating an object with one's hand. It is an extraordinarily complex behaviour that requires the assimilation and processing of information from multiple sensory modalities, and generation of precise, but flexible actions to achieve desired outcomes. At the neocortical level, this behaviour is controlled by functional network interactions between several parietal and frontal regions. The experiments composing this thesis contribute to ongoing research investigating how these regions interact with one another during the planning, execution, and reprogramming of prehension behaviour. Here, these interactions are studied in both the healthy brain, and following an injury resulting from stroke. First, functional interactions between the ventral premotor cortex (PMv) and the primary motor cortex (Ml) were investigated during a naturalistic prehension task. Results suggest that PMv can exert either a facilitatory or inhibitory influence over Ml corticospinal output depending on whether the action must be reprogrammed. In a follow-up experiment, we used a novel approach with transcranial magnetic stimulation (TMS) to induce plasticity in the PMv-Ml pathway. The observed effect was consistent with Hebbian-based associative plasticity induction, and showed different forms of expression when measured either at rest or during the performance of a prehension task. Finally, we conducted a longitudinal training study in a group of chronic stroke patients suffering from severe hand plegia, in which they used prehension- related motor imagery to control a mechanical hand orthosis through a brain- computer interface. While most patients did show significant improvements in task performance during training, these improvements varied across the group. We attempted to explain this inter-patient variability by relating performance to graph theoretical measures of functional and structural network architecture. These results highlighted specific ipsilesional and contralesional parietofrontal regions and related white-matter connections that appear to have a crucial role in BCl training task performance.

Stroke is the second leading cause of death worldwide, accounting for about 9% of all deaths. It is the leading cause of neurological disability and in developed countries accounts for more than 4% of direct health care expenditure. The burden of stroke is predicted to increase during the next 20 years because of the ageing population. Whilst effective primary prevention is essential about 30% of strokes occur in individuals with a previous transient ischaemic attack: (TIA) or stroke. Recent prospective studies have shown high early risk of recurrent stroke in the days after TIA or minor stroke. Accurate identification and early treatment of these high risk patients is likely to have substantial benefits for stroke prevention. In this thesis, I aimed to study risk of recurrent stroke after TIA in the hyper-acute phase and the role of current clinical scoring systems for use in the hyperacute phase after TIA, in minor stroke, in posterior circulation TIAs and also for predicting the severity of recurrent events. I have also studied patient behaviour immediately after TIA and minor stroke to determine factors associated with delays to calling for medical attention. I have used data from a large population based study; the Oxford Vascular Study (OXVASC). OXVASC is a prospective, population-based incidence study of vascular disease in all territories in Oxfordshire, UK, which started in 2002 and is ongoing. The study population comprises all 91,106 individuals registered with nine general practices and uses multiple overlapping methods of "hot" and ' cold" pursuit to identify all patients with acute vascular events. The research described in this thesis has resulted in several clinically useful findings. Firstly, I have shown that about half of all recurrent strokes during the seven days after a TIA occur in the first 24 hours, with 6-h, 12-h and 24-h stroke risks of 1.2%, 2.1 % and 5.1 % respectively, and that the 24 hour risk was strongly related to the ABC02 score highlighting this as a reliable risk prediction tool in the hyperacute phase. Second ly, I showed that the ABC02 score was highly predictive of major recurrent stroke and inversely related to risk of recurrent TIA. These findings have implications for policies on hospital admission in patients with high scores and for the advice given to patients with low scores. Thirdly, I demonstrated that the predictive power of the ABC02 score is relatively modest in patients with minor stroke, and neither the Essen Stroke Risk Score (ESRS) nor the Stroke Prognostic Indicator II (SPI-II) predict 9O-day recurrence. Fourthly, I was able to show that the risk of stroke was as high after posterior circulation TIA as carotid TIA and that the ABC02 score was predictive in those patients presenting with posterior circulation TIA. Fifthly, I contributed to a study that showed that in patients presenting with TIA or minor stroke irrespective of age, early initiation of existing treatments in those referred to a daily clinic was associated with an 80% reduction in early recurrent strokes. Finally, I highlighted that about 70% of patients do not correctly recognise their TIA or minor stroke and about 30% delay seeking medical attention for over 24 hours. Higher risk patients lend to contact health services most quickly, but 30% of early recurrent strokes still occur prior to any attempt to do so. ii

Background: CBT for insomnia (CBTI) has been successfully applied to those with long term medical illness as an alternative to sleep medication. Such treatments have not yet been experimentally trialled in with stroke, nor has attention been paid to tailoring CBTI for those living with the challenges of stroke. The present study aimed to modify the standard CBTI protocol for post-stroke insomnia and to test the efficacy. Method: The first phase of the study comprised development of the protocol and consultation with a service user feedback group. The second phase involved a Single Case Experimental Design whereby 5 community dwelling persons with stroke, who met the DSM-IV criteria for insomnia, underwent the modified CBTI protocol. Efficacy of the protocol was determined by measuring sleep, beliefs about sleep, daytime functioning, mood, quality of life and whether participants met the criteria for insomnia. A content analysis was also carried out to elicit qualitative findings from participant feedback and therapist clinical notes. Results: The modified CBTI protocol showed both subjective and objective improvements on one or more sleep parameter for all participants. Three participants no longer met the criteria for insomnia at post-treatment. CBTI was associated with improvements in daytime sleepiness, quality of life and belief change about sleep. The content analysis suggested that behavioural techniques were preferred by participants over cognitive strategies. 3 I I ~ Conclusion: This study has shown that the modified CBTI protocol was beneficial for reversing insomnia symptoms in those with stroke. 4

Cognitive deficits in the domains of working memory (WM) and executive function are well documented following Childhood Arterial Ischaemic Stroke (AIS), though there are currently no evidence based cognitive interventions for this population. Computerised implicit WM training has been demonstrated to generate generalised cognitive gains for children with WM and attention deficits and for adults following stroke. This study investigated the long-term cognitive outcomes of childhood AIS and evaluated the efficacy and feasibility of an implicit WM training program for this population. A pre-post pilot design was used and both quantitative and qualitative outcomes were considered. Results suggested that, following AIS, the cognitive profiles of young people remains relatively stable over time. All partic.ipants who completed the intervention (N= 7) demonstrated improvements on untrained WM tasks and functional gains were reported by participants. In contrast to similar studies, no consistent improvements on measures of attention or executive function were observed following the intervention. The intervention was acceptable and feasible for most participants, though barriers to training, such as lack of parental support, were identified. This study concluded that following AIS, children's brains maintain the potential of plasticity. Implicit WM training is deemed a feasible intervention, which can produce improved working memory skills, for some children following AIS. The study highlights the potential role of technology in rehabilitation, as well as the importance of research to determine how interventions can be most effectively targeted. Follow-up data collection is needed to establish the impact of the intervention on academic attainment for this sample. The findings support further research, including randomised, controlled trials, to investigate cognitive interventions for this population.

Mechanisms contributing to the inflammatory cascade, including vascular modifications and immune cell recruitment/activation, act with high dynamism within a three-dimensional space, thus being ideally investigated by in vivo two-photon microscopy (2-P:M). In this, thesis I applied in vivo 2-PM and quantitative bright field and confocal microscopy to explore cerebrovascular remodelling, immune cell dynamism and phenotype in two models of ischemia in mice achieved by transient or permanent middle cerebral artery occlusion. To specifically visualize microglia and T -cells, I used cx3crl_eGFP and hCD2_eGFP mice, respectively. I imaged animals before and at different time points after ischemia, and I quantified blood flow velocity and extravasation in individual vessels. After reperfusion, blood flow exhibited >80% drop in most vessels and extravasation established as early as 20min after ischemia onset. In this ischemic territory, I analyzed motility and morphology of GFP+ microglia. Microglia were stationary and became ameboid at 24h after injury. The absence offractalkine receptor (CX3CRl) prevented the ameboid switch and favoured a protective M2 microglia polarization, characterized by decreased CD68 and iNOS expression and increased Yml expression. To assess the temporal evolution of microglia/macrophage polarization in the ischemic brain, I investigated the expression and coexpression pattern of CDllb, CD4S, CD68, Yml, CD206 and iNOS after ischemia by conventional immunohistochemistry. Microglia/macrophages showed multiple polarization states, with a specific pattern of distribution and association with globular or ramified CDII b morphology. M2 microglia/macrophage peaked at 24h after injury, whereas MI cells peaked at 48h. The phagocytic activity (CD68). mainly confined at the lesion borders at 6h-48h, dramatically increased and occupied all the ischemic territory at 7d.

Nitric oxide (NO) may modulate mitochondrial O2 consumption in the heart, and provide another level of respiratory control under normal or hypoxic conditions. NO and NO donors have also been proposed to have both beneficial and deleterious effects during ischaemia-reperfusion (IR) injury, and both of these maybe mediated by interactions of NO with mitochondria. The aims of the research in this thesis were to investigate (i) whether there is a specific form of mitochondrial nitric oxide synthase (NOS), (ii) which NO donors release NO spontaneously and which require bioactivation, (iii) whether different NO donors are protective or deleterious during IR injury, (iv) the effect of NO donors on mitochondrial membrane permeability transition pore (mPTP) opening. In this study, using 3 different techniques (Western blotting, immunoprecipitation and DAF fluorescence), no evidence was found to suggest the existence mitochondrial NOS in highly purified heart and liver mitochondria. However, it was possible to detect all forms of NOS in crude heart and liver mitochondria, and the same applied for the ryanodine receptor and caveolin, in "mitochondrial" fractions. Despite the fact that NO donors are commonly used in medicine and in research, in many cases it is still unclear as to how NO is released from the NO donors; whether it is spontaneously released or whether bioactivation is required. Using the NO- sensitive fluorescent dyes DAF-2 and DAF-2 DA, the results of this study showed that the NO donors DEA and SIN-l spontaneously released NO. SNAP, a donor that has been assumed to be spontaneous, showed no release of NO. Sodium nitroprusside did show significant spontaneous release of NO, but only at 5 and 10 mM. The novel NO donor NCX2057 was the only NO donor that showed release of NO by mitochondrial metabolism. The nitrates BDMN and ISMN both released NO in post-mitochondrial supernatant, indicating the involvement of cytosolic enzymes in NO donor metabolism. Previous studies have shown that NO can have both protective and deleterious effects in whole heart IR injury. In this study it was shown that the administration of the NO donors, SNAP and DEA before the onset of global ischaemia and throughout reperfusion, resulted in a concentration-dependent protection against IR injury. Beyond a concentration threshold level (40 ~ SNAP and 2 ~ DEA), the protection was lost. Since IR injury is critically regulated by the opening and closing of the mPTP during reperfusion, the effects of DEA on mPTP opening, mitochondrial Ca2+ uptake and membrane potential were investigated using isolated mitochondria. The lower concentrations of DEA (2-25 flM) caused partial mitochondrial membrane depolarisation, sensitised the mPTP to Ca2+ and promoted mPTP opening. By contrast, the higher concentrations of DEA depolarised the mitochondrial membrane potential, prevented Ca2+ uptake and inhibited mPTP opening. This biphasic effect observed could be attributed to the inhibition of cytochrome c oxidase, as potassium cyanide had similar effects on membrane potential, Ca2+ uptake and mPTP opening as DEA. DEA also inhibited mPTP opening under de-energised conditions, indicating that in addition to modulating mPTP opening via inhibition of cytochrome c oxidase, DEA can also directly interact and modify mPTP components, possibly via nitration.

The HSP70 family is the most highly conserved of the high molecular weight HSP's in both non-neuronal and neuronal systems. Recent studies have also shown that the overexpression of HSP70 is neuroprotective for stroke and the polyglutamine (PolyQ) disorders. Hence understanding the mechanisms that regulate HSP70 may allow new therapeutic strategies to be developed. MicroRNAs are a subset of small non-coding RNAs that have been shown to mediate an entirely new level of post-transcriptional gene regulation by inhibiting the translation of target mRNAs. MiRNAs bind complementary sequences within the 3 'UTR of specific mRNAs and mediate their translational repression. An aim of this study was to identify the miRNAs that regulate HSP70 and investigate their involvement in regulating HSP70 during a heat shock response. To achieve this I initially used the online algorithms designed to predict miRNA targets. The predictions made by the different algorithms showed considerable variation and we hence also carried out an empirical analysis. MiR-206 and miR-21 were found to down regulate luciferase expression when linked to the HSP70 3 'UTR. In order to study the role these two miRNAs play in controlling HSP70 expression further, a lentivirus expressing miR-206 and miR-21 was used. Transduction of HeLa cells with this virus and an HSP70 3'UTR luciferase reporter found the virus mediated a dose dependent decrease in translational activity. HeLa and C2C12 cells were used to study function and preliminary experiments showed that HSP70 mRNA expression was increased 1-3 hours after the induction of a heat shock (HS). HSP70 protein levels were also increased following a HS. Following heat shock miR-206 levels were found to fall significantly within the first hour after heat shock (in C2C12 cells) and then start to recover twenty-fours later. The fall in miR-206 levels correlated with increased HSP70 levels, though this trend did not reach statistical significance. Transduction with viruses expressing miR-206 and miR-21 were shown to significantly decrease HSP70 protein but not mRNA expression following a HS. Furthermore, miR-206 specific antagornirs mediated a decrease in endogenous miR-206 levels and a trend towards increased basal levels of HSP70 protein was also seen. These results suggest that miR-206 may be one of a number of miRNAs that help ensure conditions within a cell are permissive or non-permissive for the expression of heat shock proteins following a stress or consitutively.

Stroke is a condition associated with a number of cognitive, physical and emotional sequelae and as such is the leading cause of significant long-term disability (The Stroke Association, 2013). It is also changes a individual's sense of self, an experience of which several models of adjustment to stroke have attempted to capture (i.e, Gracey et aI., 2009; Taylor et al., 2011), The purpose of this study was investigate the internal psychological processes that have an effect on adjustment. In particular, the traits of self-criticism and perfectionism are examined in relation to their impact on outcomes post-stroke. This is investigated using two studies. Study 1 is a longitudinal study (n = 8) that aimed to examine stroke survivors within 8 weeks of their discharge from acute care (time 1) and follow their progress six months later (time 2). Unfortunately this study was limited by a poor response rate and consequent small sample size therefore any conclusions are drawn with extreme caution. Study 2 attempted to address the research question using a cross-sectional method. 69 stroke survivors completed a series of questionnaires relating to brain injury sequelae, perfectionism, self-criticism, mood and quality of life. Self-criticism was found to moderate the relationship between brain injury sequelae and quality of life and also the relationship between self-discrepancy and quality of life. Perfectionism was not found to have any effect on psychological well-being post stroke and the possible reasons for this result are discussed. The clinical implications of this study include an increased focus on building compassion and self-reassuring skills in stroke survivors.