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Introduction: Executive function (EF) is impaired In up to 75% of patients following stroke and can hinder recovery. Despite this high incidence of executive dysfunction (EO), interventions to improve task performance focus on repetitive practice which does not address EF development. To explore further options for addressing ED following stroke, the work in this thesis includes three key studies: 1. Review of executive function research and development of the e){executive function task application model (EFTAM): An exploration of components within established definitions of EF suggested that EF can be explained by five core components: concept formation, planning, initiation, inhibition and flexibility. However, within existing EF models the task process was limited to one stage, while, in contrast, occupational performance models conceptualize tasks as multifactorial. This suggested the need for a new model which integrated models of EF and occupational performance. Hence the EFTAM was constructed with the key aim of providing a model to demonstrate how EF is applied at the various stages of task performance. 2. Cochrane systematic review of cognitive rehabilitation: This review found insufficient evidence to support the effectiveness of cognitive rehabilitation for improving EF after stroke and acquired brain injury. The review also identified limitations relating to assessment of EF leading to the hypothesis that instructing patients to verbalise their thoughts may be a method of determining how patients apply EF during tasks. Thus, a qualitative study to explore how participants expressed EF through their actions and talking was indicated. 3. Ethnomethodological study to explore how individuals express EF through talking and actions: 20 participants with stroke, upper limb injury and a healthy individual were Video-recorded during a semi-structured interview including an upper body dressing task. Data analysis, using a narrative analysis framework, and based around the EF core components and the EFTAM, explored how EF was expressed during application to the task. The participants demonstrated several patterns of EF expression and dressing ability from their combined actions and talking. Conclusion: This thesis provides a comprehensive synthesis of current evidence relating to interventions for EF and identifies the clear limitation within current literature as the lack of suitable assessment tools. A new model integrating EF and task performance theories has been generated with the potential to provide a useful tool for profiling EF during task performance. Innovative methods have been used to explore storytelling during task performance, providing new insights in relation to how EF is applied to the different stages of a task. Future research should include further exploration of the application of these methods to a range of different tasks to unlock the potential of storytelling during task performance. This could enhance our understanding of EF and lead to the development of a new standardised EF assessment and effective interventions to treat ED.

Acute stroke is known to lead to impairment of cerebral blood flow (CBF) regulation, but its natural history and techniques for its comprehensive assessment have not been previously reported. Noninvasive measurements of blood pressure (BP), end-tidal CO2 and CBF velocity (CBFv, using transcranial Doppler ultrasound) during active, passive and motor imagery paradigms were performed in healthy older controls (n=27) and in stroke patients (n=27). Two innovative analytical techniques were firstly used in stroke studies: subcomponent analysis and multivariate dynamic modeling. In controls, significant increase in CBFv during the paradigms with no significant difference in the response amplitude was found. A reproducibility study, not previously reported, was also performed. Following acute stroke, subcomponents analysis revealed a decrease of CBFv response to the passive paradigm and impairment of the myogenic pathways of CBF regulation. Multivariate dynamic modeling removed the influences of BP and PaCO[subscript 2] showing that the reduced CBFv response to neural activation was directly related and better expressed by the contribution of the stimulation component, instead of the CBFv raw change. The contribution of motor imagery in the CBFv increase was lower compared to the other two paradigms. Impairment of cerebrovascular reactivity to CO[subscript 2] was also detected by the model, without the need of performing specific tests for this purpose. The natural history of CBF regulation revealed a deterioration of control mechanisms in both the acute (< 72h) and subacute (2 weeks) phases, reaching the controls’ levels in the chronic phases (1 and 3 months). It has been demonstrated in this thesis that CBF regulation changes significantly over time after stroke (particularly in the first weeks after onset), having potential impact not only immediately post ictus but also during the subsequent rehabilitation phase.

Introduction: Options for imaging in acute stroke are expanding with the potential to select therapy based on imaging targets, as well as providing additional diagnostic and prognostic information. Multimodal CT has been used to image the ischemic penumbra, infarct core, and to detect leptomeningeal collateral flow although the optimum way to image these variables is not clear. Methods: In addition to a systematic literature review of imaging for leptomeningeal collaterals, Data from observational studies of acute stroke which employed multimodal CT imaging on admission and follow up was used to evaluate feasibility of acute stroke imaging with CT and MRI, Perfusion thresholds for core and ischemic penumbra, methods to quantify leptomeningeal collateral flow and sensitivity of non contrast CT for detecting infarct core pixels. Results: Advanced imaging in acute stroke and at follow up was more feasible with CT compared to MRI with the possible suggestion that imaging with MRI alone could introduce a bias regarding age and clinical severity for patients entered into clinical studies Heterogeneity in grading and detecting collateral flow was found in the literature providing an opportunity to devise a novel assessment method. Well developed collaterals were associated with imaging and clinical markers for good outcome as well as some potential biomarkers including atrial fibrillation and blood fibrinogen level. Relative cerebral blood flow and delay time were found to be the best predictors on infarct core and ischemic penumbra after derivation of optimum perfusion thresholds and subsequent validation in independent patient groups. Pixel based comparison of infarct core on CT perfusion and non contrast CT highlighted the lack of sensitivity of CT for detecting infarct core based on Hounsfield unit value alone. Conclusion: Multimodal CT for acute stroke assessment offers the potential for measuring infarct core, ischemic penumbra and leptomeningeal collateral flow status rapidly according to novel grading scales and thresholds and provides information on tissue viability which cannot be detected on non-contrast CT. Further evaluation on the impact additional imaging should have in clinical practice is needed.

Stroke affects more than 150,000 people every year and is the third major cause of adult disability in the UK. Stroke rehabilitation plays an important part in the motor skills recovery of the stroke patients. This thesis forms part of the development of an upper arm rehabilitation system which involves the use of Functional Electrical Stimulation (FES). Motivation for this use of stimulation to augment remaining voluntary effort in strokepatients is explained and the necessary components comprising the system are described. The task considered in this thesis is reaching, which involves elbow extension and shoulder elevation. FES is applied to two muscles, triceps and anterior deltoid respectively, to assist in these movements. A review of the literature has revealed possible control schemes which could be implemented with FES. Relatively few, however, have actually been implemented in clinical trials. This work, aims to apply selected controllers in clinical applications. A series of controllers are examined, starting from the simplest feedback controller going to more advanced model-based Iterative Learning Control (ILC) controllers. These include phase-lead ILC, input-output linearisation, and Newton-method based ILC. ILC algorithms are commonly used in industrial robots for precise control. The aim of this work is to transfer these algorithms to clinical settings. ILC algorithms are used to provide finely-controlled levels of FES assistance to patients during repetitive training tasks. To use a model-based controller, kinematic and dynamic models of the Armeo and human arm have been developed. The muscle model of the human arm has been derived using a Hill-type model while the Hammerstein model is used to model the stimulated muscle. The complete system has then been used in a clinical study involving five stroke patients. Improvements in clinical measured Fugl-Meyer Assessment (FMA) scores were seen in the stroke patients after the trials.

Progesterone has been shown to be neuroprotective in a number of central nervous system injury models, including cerebral ischaemia. There is still a lack of understanding behind progesterone’s neuropotective properties, and the purpose of this project is to clarify some of these issues. Osmotic mini-pump infusion was hypothesised to more effective in delivering progesterone to the target organ of the brain, when compared to a bolus intraperitoneal injection. Progesterone pharmacokinetic profiles were compared between different dosing regimes. Intraperitoneal progesterone injection had a short half-life in both plasma and brain, while osmotic mini-pumps delivered higher concentrations of progesterone in plasma and particularly in brain, over a longer period, which supports the hypothesis. It was hypothesised that progesterone will reduce NO production and cell death in in vitro. Progesterone reduced nitric oxide production after challenging microglia with LPS, which supports our hypothesis and the nuclear progesterone receptor was found not to have a major role in nitric oxide attenuation. Neither of the microglial cell lines, BV-2 and HAPI cells produced elevations in NO formation in ischaemic conditions. The in vitro oxygen and glucose deprived model of ischaemia, reduced viability in both microglial and neuronal cells. Also, high pharmacological concentrations of progesterone exacerbated ischaemic injury, which does not support the hypothesis of progesterone in reducing cell death. Progesterone administration, via osmotic mini-pump infusion, was hypothesised to have a better outcome compared to vehicle treatment. After the onset of experimental stroke, progesterone delivery via osmotic mini-pump with loading dose was found to be beneficial in terms of neurological deficit score in adult male mice, which supports the hypothesis. Also, we hypothesise that co-morbidity can affect the efficacy of progesterone treatment in outcomes. Aged animals have an increased sensitivity to experimentally induce stroke and did not display, in the outcomes measured, any benefit from progesterone treatment. NOD/ShiLtJ mice had severe symptoms, resulting in high mortality after surgery and are not recommended as a model of diabetes for experimental stroke. Hypertensive BPH/2 mice are a potential hypertensive model and had better functional outcomes after treatment with intraperitoneally administered progesterone, compared to non-treated hypertensive animals in our small preliminary study. This supports our hypothesis that co-morbidity can affect the efficacy of progesterone treatment in outcomes. The gold-standard for assessing intervention effects across studies within and between subgroups is to use meta-analysis based on individual animal data. We hypothesise meta-analysis would reveal progesterone to reduce lesion volume, but also discover other effects in different subgroups of animals. Progesterone significantly reduced lesion volume, it also appeared to increase the incidence of death following experimental stroke. Furthermore, this negative effect appears to be particularly apparent in young ovariectomised female animals. These findings support the hypothesis that progesterone reduces lesion volume and progesterone having other effects in different subgroups. This investigation has clarified some issues and expanded our understanding on the neuroprotective properties of progesterone. However, these findings indicate further investigation is still required before progesterone can be considered for use in clinical trials as a neuroprotectant in stroke.

Introduction: Oxygen-glucose deprivation (OGD)±reperfusion and hyperglycaemia exacerbate the ischaemic cerebral injuries during or after a stroke. The key biochemical events associated with these pathologies include excessive cytoskeletal remodelling, modulation of tight junction proteins and the induction of oxidative stress. Recently, the overactivities of protein kinase C (PKC), RhoA/Rho-kinase, and pro-oxidant NADPH oxidase have been shown to account for the development of these events and the consequent disruption of human blood-brain barrier (BBB) integrity. Objectives: This thesis focused on the putative roles of RhoA/Rho-kinase signalling in OGD and OGD+reperfusion-evoked modulation of cytoskeletal remodelling, tight junction proteins and oxidative stress in human brain microvascular endothelial cells (HBMEC). The effects of hyperglycaemia-mediated PKC overactivities in modulating the RhoA/Rho-kinase pathway with reference to the aforementioned parameters i.e. cytoskeletal remodelling and tight junction protein expression and localisation have also been the focus of this thesis. Methods: For the OGD studies, the HBMEC were exposed to normoxia (controls), OGD (4, 20 hours) alone and followed by reperfusion (20 hours). The HBMEC-human astrocyte (HA) cocultures were established to mimic human BBB before exposing them to the experimental conditions. The integrity and function of HBMEC-HA cocultures were measured by transendothelial electrical resistance (TEER) and flux of permeability markers sodium fluorescein (NaF) and Evan’s blue-labelled albumin (EBA), respectively. For the hyperglycaemia studies, the HBMEC monolayers and the cocultures were exposed to normoglycaemia (5.5 mM D-glucose), hyperglycaemia (25 mM D-glucose), and hyperglycaemia with inhibitors of Rho-kinase, PKC, PKC-α, PKC-β, PKC-βII, PKC-δ; and the BBB integrity and function were measured by the TEER and flux studies, respectively. Fold differences in the protein expression or activity of RhoA, Rho-kinase-2, mono- and di-phosphorylated myosin light chain-2 (MLC2), total MLC2, gp91-phox (a pivotal NADPH oxidase subunit), catalase, occludin, claudin-5, zonula occludens-1 (ZO-1), β-catenin, and vinculin were either measured by in-cell or ordinary Western analyses. Results from the OGD studies: OGD compromised the barrier integrity as observed by decreases in TEER values and concomitant increases in flux of EBA and NaF across the cocultures. Transfection of HBMEC with constitutively active RhoA also decreased the TEER and increased the NaF paracellular permeability, whereas inactivation of RhoA by anti-RhoA-IgG electroporation exerted barrier protective effects. Moreover, OGD alone and after constitutively active RhoA transfection introduced stress fibres in HBMEC, which were abrogated by inactivation of RhoA and the specific inhibition of its main effector Rho-kinase by Y-27632. In addition, dramatic increases in the protein expressions of RhoA-GTP, Rho-kinase-2, gp91-phox, and antioxidant catalase were observed in HBMEC exposed to OGD+reperfusion conditions. These along with increases in the NADPH oxidase activity and total superoxide anion levels confirmed the oxidative stress in HBMEC under these experimental conditions. A marked rise in the protein expressions of claudin-5 and β-catenin observed after OGD (20 hours) alone and followed by reperfusion may represent the effects of oxidative stress on tight and adherens junction proteins stability, respectively. These results also concurred with marked decreases in TEER and concomitant increases in the flux of EBA across the in vitro models of human BBB exposed to OGD±reperfusion conditions when compared with the controls. Cotreatment with Y-27632 under OGD±reperfusion normalised the protein expressions of RhoA, Rho-kinase-2, gp91-phox, claudin-5, catalase; activities of RhoA and NADPH oxidase; and total superoxide anions levels, alongside improving the expression of occludin and the coculture integrity under the OGD±reperfusion conditions. Results from the hyperglycaemia studies: Hyperglycaemia also increased RhoA-GTP, Rho-kinase-2, mono- and di-phosphorylated MLC2 protein levels and total PKC activity. These changes were consistent with the actin stress fibre formations, ZO-1 and occludin redistribution from HBMEC periphery. Hyperglycaemia-mediated endothelial-barrier dysfunction was further characterised by reduction in TEER and elevation in flux of EBA. Glucose normalisation, RhoA neutralisation by anti-RhoA-IgG electroporation and Rho-kinase-2 inhibition by Y-27632 normalised all abovementioned protein expressions, restored actin and tight junction protein localisations and barrier integrity. Cotreatment of HBMEC with hyperglycaemia and a general PKC inhibitor namely, bisindolylmaleimide-I normalised the Rho-kinase-2, mono- and di-phosphorylated MLC2 levels. Moreover, specific inhibitors of PKC-α (Ro-32-0432), PKC-β (LY333531), PKC-βII (CGP53353) attenuated the PKC overactivity, normalised all protein expressions, restored actin localisation and improved barrier integrity. In addition, the PKC-α and PKC-β siRNA transfections mimicked the effects of the specific inhibitors and attenuated the hyperglycaemia-evoked RhoA-GTP, mono- and di-phosphorylated MLC2 protein levels and stress fibre formations. Conclusions: The RhoA/Rho-kinase overactivities compromise the endothelial-barrier integrity, in part, by modulating the cytoskeletal remodelling and inducing the NADPH oxidase-evoked oxidative stress under OGD±reperfusion pathology. Moreover, hyperglycaemia-mediated increases in PKC-α and PKC-β activities exacerbate the endothelial-barrier dysfunction by modulating RhoA/Rho-kinase signalling pathway. Summary: These findings support the hypothesis that OGD±reperfusion and hyperglycaemia perturb BBB integrity through regulation of RhoA/Rho-kinase activity and modulation of cytoskeletal reorganisation, oxidative stress and tight junction protein expressions or localisations.

Stroke services provide patients with immediate assessment and treatment on a hyper acute stroke unit prior to being transferred for intense rehabilitation. Patients' experience of transfer from a hyper acute stroke unit has yet to be explored. A patient's beliefs regarding their illness are important factors that aid a patients' recovery yet these have not been fully explored in patients following a stroke. The aims of this study are to explore the patients' perspectives of transfer from a hyper acute stroke unit to a stroke unit and their itIness beliefs. Data were collected from a purposive sample of patients (n=6) on a stroke unit following transfer from a hyper acute stroke unit. Semi-structured interviews were carried out to explore patients' experience of transfer and their illness beliefs. The interview schedule was based on literature and interviews with a Clinical Nurse Specialist and a stroke patient. Data were analysed using Interpretative Phenomenological Analysis (IPA). The emergent super-ordinate themes included: disassociation from being in the world, search for understanding. strive for independence and acceptance of support, and hope and uncertainty. A chronic illness model of illness representation dimensions emerged; higher reporting of identity, consequences, and timel ine was associated with lower personal and treatment control and an emotional response. Self-regulatory coping strategies and health beliefs within patients following a stroke were identified as important constructs to include in healthcare assessments with the aim of improving psychological, physical and social outcomes.

Introduction: Elevated blood levels of Cardiac Troponin (cTnT) are the gold standard for diagnosis of acute coronary syndrome (ACS). However cTnT is not detectable for 3-4 hours after symptom onset; an early marker of myocardial ischaemia is therefore desirable. The peptide Calcitonin Gene-Related Peptide (CGRP) is released from nerve endings during myocardial ischaemia and has cardioprotective effects. Intermedin (IMD), from the same family, is elevated in myocardial infarction and oxidative stress in animal studies. Methods: I enrolled 81 patients with chest pain, suspected to have acute coronary syndrome to a chest pain study (1). Samples were taken on admission, at 12hours after pain, the next day and the following day. Samples were analysed for intermedin, & CGRP (radioimmunoassay) and High-Sensitivity troponin (HsTnT). Study 2 assessed 30 patients undergoing Percutaneous Coronary intervention (PCI) with 6 controls. Samples taken, pre, 1,5, 10,30, 120 or 240 minutes post balloon inflation and next day. Results: Study 1- Intermedin showed a non-significant trend towards elevation in ACS patients. In those patients who were (cTnT) negative «0.03ng/ml) on presentation, IMD & CGRP were significantly elevated. CGRP was significantly elevated at 12 hours after ACS onset and appeared to remain elevated for 48 hours. HsTnT identified more ACS on an admission sample than cTnT. Study 2 - IMD showed a small but significant rise at ten minutes after balloon inflation. HsTnT was significantly elevated the next day following PC!. Neither marker was related to number of stents/inflations, balloon pressure or length of inflation. Conclusions: The novel peptide IMD is elevated in ischaemia and infarction, but the rise is too small to be clinically useful. CGRP had a significant rise in ACS patients, but again of a small magnitude. The time course of their elevation is incompletely defined. HsTnT is more sensitive in ACS than cTnT on a presenting blood sample.

Electrophysiological stimulation has been proffered as having the potential to improve rehabilitation in stroke survivors suffering residual motor deficits. Interindividual variation exists in the response to these interventions, undoubtedly mediated at least in part by genetic factors. The thesis identified 3 candidate genes in neural plasticity, and determined their impact on the efficacy of paired associative stimulation (PAS) and transcranial direct current stimulation tDeS in forearm muscles, which are often the target of rehabilitation therapies . Three candidate genes in neural plasticity were examined- BDNF, COMT and ApoE. Genotype-mediated baseline differences in cortical excitability were found. Females who were homozygous for the BDNF val allele had significantly larger VO curves than those who carried a met allele. COMT val homozygotes showed a trend towards smaller VO curve area at baseline, which approached significance (p=O.0504). Baseline I/O curve area was undifferentiated across ApoE genotype groups.

Ischaemic stroke instigates a series of pathological mechanisms which contribute to injury. Despite significant research in this field successful clinical treatment is limited. This highlights the need to identify novel therapeutic targets in order to assess whether clinical investigation is warranted. The enzyme cyclooxygenase-2 (COX-2) is a key contributor to inflammatory injury following stroke. Upregulation of this enzyme results in increased prostanoid synthesis, which mediate many physiological and pathological functions. Prostaglandin E₂ (PGE₂) is a key mediator in inflammation and activation of its receptor subtypes is both neuroprotective and neurotoxic. COX-2 inhibition in animal models of stroke has demonstrated neuroprotection. However, long term arthritis trials have revealed detrimental effects of COX-2 inhibitors. This highlights the need to identify mediators of the detrimental effects of COX-2 and capitalise those that mediate the beneficial effects. The aim of this study was to investigate the role of the PGE₂ receptor EP₄ following in vitro and in vivo ischaemia. Organotypic hippocampal sliced cultures (OHSCs) were exposed to oxygen and glucose deprivation (OGD). Treatment with a selective EP₄ agonist following OGD significantly reduced cell death, whereas application of the EP₄ receptor antagonist exacerbated injury. C57/BL6 mice were subjected to focal cerebral ischaemia via middle cerebral artery occlusion (MCAO). Administration of the selective EP₄ agonist significantly reduced infarct volume and prevented the decline in neurological function. COX-2 inhibition and EP₄ receptor stimulation resulted in similar levels of protection both in vitro and in vivo ischaemia. EP₄ receptor expression was assessed using immunohistochemistry and real time-PCR. This study provides evidence that selective activation of the EP₄ receptor following ischaemic injury is as neuroprotective as COX-2 inhibition but possibly without the deleterious side effects of COX-2 inhibitors. This supports the concept of targeting protective prostaglandin receptor signalling as a potential therapeutic target for stroke.

The purpose of this thesis was to inform the development of a complex intervention for improving secondary stroke prevention in people who have experienced a Transient Ischaemic Attack (TIA). The work was guided by the Medical Research Council (MRC) framework for the development and evaluation of complex interventions. A mixed methods approach was taken, incorporating three inter-related studies. The first study was an audit investigating the quality of secondary stroke prevention in primary care following diagnosis of TIA in a specialist clinic. The second study was a systematic review of randomised controlled trials evaluating the effectiveness of stroke service interventions for secondary stroke prevention. The third study was a qualitative study, involving 20 interviews with TIA patients, using a discursive psychology approach to explore barriers and facilitators to secondary stroke prevention. Key findings: • Results of the audit demonstrated that monitoring and achievement of risk factor control in primary care was suboptimal; potential areas for quality improvement included blood pressure (BP) control, lipid control and provision of dietary and exercise advice. • Findings from the systematic review indicated that organisational interventions were associated with significant reductions in mean systolic BP, diastolic BP and body mass index (BMI). • The qualitative study, through an analysis of the ‘action-orientation’ of participants’ accounts, identified discursive features that functioned to justify adherence or non-adherence to recommendations for secondary stroke prevention. The key findings from these studies indicated that an organisational intervention should be developed based on the principles of integrated care. The qualitative study provided insights for understanding and optimising the intervention. Based on these findings, recommendations are made for further intervention development work. The findings also have relevance to the development and application of the MRC framework; efforts should be directed towards developing practical guidance for the integration of mixed methods research.

This thesis examined the application of psychological principles to stroke care. The literature investigating psychological adjustment post stroke was reviewed and the validity of the Distress Thermometer (DT) and Problem List was investigated in an empirical study. A systematic review focussed on the recently published literature using psychological theory to understand what cognitive factors are protective, or not, in the process of adjustment. Twenty papers were reviewed and nine theoretical models of psychological adjustment used. The most commonly referred to model was the Transactional Theory of Stress and Coping (Lazarus & Folkman, 1984). Factors found to be associated with positive mood included internal locus of control, finding meaning and satisfaction with treatment. The complex and dynamic nature of adjustment was highlighted by the role of time and individual differences. The results of the review provided support for a recently developed model of adjustment post stroke (Social Cognitive Transition Model for Stroke, SCoTs, Taylor, Todman & Broomfield, 2011). The DT and stroke specific Problem List offers a valuable tool for assessing and understanding distress post stroke. Forty-eight participants completed the DT, Problem List, Hospital Anxiety and Depression Scale (HADS, Zigmond & Snaith, 1983), Brief Assessment Schedule Depression Cards (BASDEC; Ashead et al, 1992) and the Visual Analogue Mood Scale Revised (Kontou et al., 2012) at one time point. Correlation coefficients were significant and positive between all measures, supporting concurrent validity. AUC analysis suggested a cut-off of 4.5 on the DT as suitable for the detection of anxiety. Cronbach's alpha found the Problem List to be most reliable when used as one whole scale, however this was most likely because of the large number of items in the overall scale, rather than the items being clearly associated to one another. Bladder and bowel problems were the most commonly reported distressing problem, with fatigue, worry and depression being frequently identified. These findings supported the used of the DT and Problem List in the early stages post stroke.

The molecular mechanism that leads to ischemic preconditioning and hence to ischemic tolerance, are not completely understood although it is clear that multiple effectors and pathways contribute to the instauration of this neuroprotective profile. To study the mechanism/pathway involved in the ischemic tolerance, brain proteins, plasma proteins and plasma metabolites were analyzed in preconditioning stimulus (7'Middle Cerebral Artery occlusion or 7'MCAo), in severe stroke and (permanent Middle Cerebral Artery occlusion or pMCAo) and in preconditioned (7'MCAo/pMCAo) mouse model. A conventional 2-DE approach was used to study technical replicates of pooled brain proteins revealing an involvement of energy metabolism, mitochondrial electron transport, synaptic vesicle transport and antioxidant processes; moreover network analysis suggested an involvement of the androgen receptor that was validated on technical replicates of pooled brain proteins by western blot analysis revealing an increased expression in preconditioned stimulus animals (7'MCAo). Plasma proteins were analyzed using a i-DE LC-MS/MS approach on technical replicates of pooled plasma proteins revealing decreased levels of epidermal growth factor receptor (EGFR) and increased levels of insuline like growth factor acid labile subunit (IGFALS), which expression was paralleled by increased insulin like growth factor 1 (IGFi) plasma concentration, as validated by ELISA on biological replicates, in preconditioning stimulus animals (7'MCAo). Finally an untarget metabolomics analysis was applied to technical replicates of pooled plasma proteins revealing fatty acid oxidation and branched-chain aminoacid metabolism as the main biological processes modulated in ischemic tolerance and highlighted an involvement of the aminoacid leucine, carnitine esters and adenosine. The results described in this thesis represents the first application of both proteomic and metabolomic approaches in cerebral ischemic sets, highlighting the androgen receptor as an important mediator between proteins and metabolites and adding new evidence to the current knowledge on ischemic preconditioning that may represent a starting point for future experiments on investigating candidate pathways that relate to the Androgen receptor.

Introduction: Sub-arachnoid haemorrhage (SAH) occurs when cerebral artery ruptures and blood leaks out into the sub-arachnoid space. This is often a catastrophic event for the individual and morbidity and mortality rates are significantly influenced by early intervention. This makes the role of the clinical biochemistry laboratory in early diagnosis vitally important, as delays in diagnosis can have a major clinical impact. The cerebrospinal fluid (CSF) of healthy individuals is optically clear. It has, however, been recognised for over a century that it can become coloured (xanthochromia) following a cerebrovascular incident such as a SAH. This has made the main role of the clinical biochemistry laboratory in SAH diagnosis that of detecting xanthochromia in the CSF. The majority of laboratories which offer a xanthochromia screening service use the national guidelines that are based upon ultra-violet scanning spectrophotometry (350 nm to 600 nm). This analytical technique is not without its problems: it is subjective, has a possibility of inter-operator variability and due to the specialised nature of the test can take many hours or even days for a result to be issued. This project aimed to improve the current laboratory service by investigating: turnaround times, users opinions of the current service and potential alternative analytical methods. Methods: An audit of the current analytical provision was used to assess its effectiveness and in order to elucidate the service users’ perception. This was effected by a questionnaire that was distributed to service users across three different NHS Trusts in England and Wales. In an attempt to improve the laboratory service, alternatives to scanning spectrophotometry were investigated. These were selected through consideration of the nature of SAH i.e. blood is released into the subarachnoid space and the brain is damaged. Laboratory analysis therefore needed to focus on detecting the presence of blood and/or its breakdown products, any change in CSF constituents that arise as a direct consequence of blood being introduced in to the subarachnoid space or a specific analyte which would only be present if brain damage occurred. Investigation of current research into subarachnoid haemorrhage identified the following analytes as potential alternatives: CSF diazo bilirubin, CSF Ferritin, CSF protein S100 and serum protein S100. Results: The audit revealed the average turnaround time for reporting xanthochromia results to be 26 hours, with almost 20% of samples being reported as equivocal. The service user’s questionnaire revealed a general lack of awareness of current United Kingdom National External Quality Assurance Scheme (UKNEQAS) guidelines for the ‘Analysis of cerebrospinal fluid for bilirubin in suspected Subarachnoid haemorrhage’ and a lack of understanding regarding the timing of lumbar punctures. Additionally, one third of users felt that the turnaround time for results was inadequate. CSF protein S100 was found to be unsuitable due to the difficulty in achieving a suitable balance between sensitivity and specificity; at a cut-off of 0.40 μg/l sensitivity is 80% and specificity is 4%, at a cut-off of 1.60 μg/l sensitivity is 40% and specificity is 94%. Serum protein S100 was found to be unsuitable due to the difficulty in achieving a suitable balance between sensitivity and specificity at appropriate cut-offs (66 % and 73%, respectively, at a cut-off of 0.09 μg/l). When the CSF diazo bilirubin and CSF ferritin were compared to current laboratory practises using pre-defined criteria then CSF diazo bilirubin was found to be the analyte of choice to base new guidelines upon. CSF diazo bilirubin was then used as an initial ‘rule-out’ step in a new set of guidelines for the determination of SAH utilising CSF analysis. Conclusion: The new guidelines employ CSF diazo bilirubin analysis as a ‘rule-out’ step with all samples that are above the cut-off (300 nmol/l) being processed through the UKNEQAS guidelines. In order for the guidelines to be introduced and accepted, local training and education programmes for laboratory and clinical staff will need to be developed and implemented and they will need to be disseminated through publication of articles in journals relevant to both the clinical biochemistry community and requesting clinicians.

Acquired brain injury (ABI) is an injury that is caused to the brain after birth. The impact of brain injury on the survivor may be to leave cognitive and behavioural difficulties that result in them requiring care. Many carers are the relatives of the individual and this can have a considerable impact on the carer’s well-being as well as their relationship with the injured person. The objective of this literature review is to evaluate recent literature around how family members cope with the process of caring for a relative with a brain injury. I start by introducing coping and the models that inform how coping mediates stress; I then go on to review some of the recent research that has looked at coping in carer relatives and finally I evaluate some of the challenges that are faced in interpreting the research and moving forward in the field. Although some of the evidence for particular coping strategies is mixed, one consistent finding is social support for carers appears to be a key factor in reducing distress and promoting good family functioning. The implications of this on clinical practice are then discussed.

Stroke is the principal cause of long-term disability. Hemiplegia affects up to 80% of people with stroke and a significant number will not recover use of the affected arm. People with profoundly-affected arm may experience pain, stiffness and difficulty with care activities. We cannot currently predict who is most at risk of these difficulties, and historically interventions have been designed without understanding the temporal evolution of impairment or disability. The International Classification of Functioning, Disability and Health (WHO, 2001) was used to develop a model of the consequences of the profoundly-affected arm on impairment, disability, and participation. A systematic review of thirty observational studies was undertaken and identified potential predictors of increased impairment in general populations of people with stroke. However, there was a paucity of evidence directed at people with profoundly-affected arm or regarding impact on passive care. The aim of this study was to test the feasibility of using an observational study design to develop a longitudinal profile of the profoundly-affected arm. Specific objectives of the feasibility study were to assess the processes of recruitment and follow-up, to review the sample characteristics, and to establish the acceptability and responsiveness of the predictor variables and outcome measures. Key tenets of the project were to involve people with cognitive and communication disability, and to use assessments that could be adopted by therapists working in a patient’s own home. Forty people with stroke and nine carers were recruited and followed up at three and six months post-stroke. Using enhanced communication techniques and personal consultees, it was possible to include people with severe cognitive and communication disability. The baseline demographic characteristics and the rate of loss to follow-up of participants reflect that expected in people more severely affected by stroke. Qualitative data suggest that participants affirmed the model of impairments and disabilities that had been developed. The predictor variables and outcome measures were considered acceptable to participants, and collected a range of data, generally performing in the manner expected. However, there were a number of exceptions. Cognitive and communication disability impacted on completion of the self-reported assessments, and may have affected performance on measures of mood and sensation/perception. In addition to this, measures of range of movement varied at each time point, in a manner not in accordance with expected change over time. The evidence from this thesis suggests the research design has potential to be used to develop a longitudinal profile of the profoundly-affected arm. Further work is required to improve carer recruitment, establish the best assessments for those with severest cognitive and communication disability, and review the method of measuring range of movement.

Cerebral small vessel disease (SVD) is a chronic disease of the small cerebral blood vessels and a common cause of dementia. Surrogate markers for cognitive impairment are required for future treatment trials. Patients with SVD have similar risk factors and MRI features as Alzheimer's disease. Blood biomarkers would be useful for diagnostic and prognostic purposes. St George's Cognition and Neuroimaging Study (SCANS) is a longitudinal study investigating the potential of MRI as a surrogate marker for cognitive impairment. This report examines the baseline data from SCANS. 121 patients with lacunar stroke and white matter hyperintensities had multi modal MRI, neuropsychological testing and had blood sampling for biomarkers of endothelial activation . Patient group performed worse in executive function and information processing speed compared to normative data. Patients had more white matter lesions, smaller whole brain volumes and lower fractional anisotropy and higher mean diffusivity on diffusion tensor imaging. Multiple regression, suggested brain volume was the best predictor of impaired information speed processing (P=0.001) and median white matter FA for executive dysfunction (p=0.004). High numbers (>9) of cerebral microbleeds were associated with impaired executive dysfunction. Blood biomarkers were tested in 106 patients. Patients had lower tissue factor (TF), tissue plasminogen activator (tPA) antigen and plasmin activation inhibitor-1 than controls. TF was associated with lesion volume and tPA was associated with the number of clinical strokes.

Stroke is a public health concern throughout the world due to its association with high levels of mortality and long-term morbidity. Stroke is a chronic condition. There is evidence that the use of self-management strategies can be associated with positive outcomes for people with chronic conditions. The evidence relating to stroke and self-management informs stroke policies in the UK. However, there is minimal understanding of self-management in the context of stroke. Rehabilitation interventions which can adequately address the longer term needs require an understanding of key factors which may influence self-management in stroke survivors. The overall aim of the research presented in this thesis was to explore the concept of self-management in the context of stroke. This thesis presents a mixed methods study that was designed to address three specific and connected research questions. This thesis described three studies. Firstly, through a systematic review of qualitative studies with thematic synthesis it systematically examined the current literature to determine stroke survivors' experiences of living with stroke and the biopsychosocial factors that may influence these experiences of post-stroke life as well as the components of these factors that might be relevant to self-management. Secondly, through a prospective quantitative cohort study, it investigated the biopsychosocial individual characteristics associated with self-management and self-efficacy in adults with stroke. Finally, through a qualitative interview study, it explored the meaning(s) ascribed to "self-management" by stroke survivors and healthcare professionals. The key findings taken together reveal new insights into the psychosocial phenomena that shape coping and adjustment after stroke; the biopsychosocial factors that influence selfmanagement and self-efficacy; and the conceptualisations of self-management. This thesis therefore develops new understandings of self-management in the context of stroke. Findings have implications for formulation, organization and delivery of better aftercare and the rehabilitation that follows.

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-PM). 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 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 of fractalkine receptor (CX3CR1) 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 CD11b, CD45, 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 CD11b morphology. M2 microglia/macrophage peaked at 24h after injury, whereas Ml 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. I finally investigated T-cells dynamism within the ischemic territory by in vivo 2-PM. I described two populations characterized by different track velocities and found that motile cells preferentially moved along the perivascular space, where they contacted astrocytes and perivascular macrophages. These data provide novel information on the inflammatory response after stroke and pave the way for developing strategies resulting in promotion of a protective inflammatory phenotype.

National guidelines for stroke recommend that all patients entering rehabilitation are screened for mood disturbance using a validated measure. The first half of this thesis presents a literature review of 25 self-report screening measures for the detection of post-stroke distress. A total of 26 studies were identified as meeting the search criteria. Fifteen self-report measures met recommended levels of sensitivity (≥0.80) and specificity (≥0.60) when screening for post-stroke depression. The Hospital Anxiety and Depression Scale (HADS) was the only measure to meet recommended levels of accuracy for post-stroke anxiety. At the commencement of this thesis, the Distress Thermometer (DT) had not been validated among stroke survivors despite being recommended by NICE (2009). The study presented in the second half of this thesis investigates the diagnostic accuracy and clinical utility of the DT and associated Problem List (PL), the Brief Assessment Schedule Cards (BASDEC), and the Yale. Relative to the HADS, the area under the curve (AUC) for the DT was significantly greater than an AUC of 0.50. Cut-off scores of at least 4 and 5 on the DT met recommended levels of sensitivity and specificity when screening for post-stroke depression and anxiety. The accuracy of the BASDEC and Yale was non-significant. Due to a small sample size, these results should be taken with caution. However, this study provides preliminary evidence to support the use of the DT and PL as a holistic and person-centred screening tool for the prevention and recognition of post-stroke distress.

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.

Lacunar strokes account for approximately a quarter of all ischaemic strokes and traditionally are thought to result from occlusion of a small deep perforating arteriole in the brain. Lacunar infarcts can be up to 2cm in diameter and are found in deep brain structures such as the thalamus and internal capsule. Despite their prevalence and specific accompanying clinical syndromes, the cause of lacunar stroke and its associated vascular pathology remain unclear. Many hypotheses as to the cause exist, which fall broadly into two categories; firstly, a direct occlusion via emboli or thrombus usually from a cardiac or large artery source, microatheroma (intrinsic lenticulostriate occlusion) or macroatheroma (parent artery occlusion) all operating primarily via ischaemia. Secondly, there could be an indirect occlusion resulting from vasospasm, endothelial dysfunction or other forms of endovascular damage (e.g. inflammation). Therefore the question of whether the resulting lesions are truly “ischaemic” or actually arise secondary to an alternative process is still under debate. To clarify the chain of pathological events ultimately resulting in lacunar stroke, in this thesis I firstly undertook a systematic assessment of human lacunar stroke pathology literature to determine the information currently available and the quality of these studies (including terminology). The majority of these studies were performed in patients who had died long after their stroke making it difficult to determine the early changes, and there were few patients with a clinically verified lacunar syndrome. Therefore I adopted alternative approaches. In this thesis, I systematically looked for all potential experimental models of lacunar stroke and identified what appears at present to be the most pertinent - the spontaneous pathology of the stroke-prone spontaneously hypertensive rat (SHRSP). However, the cerebral pathology described in this model to date is biased towards end stage pathology, with little information concerning the microvasculature (as opposed to the brain parenchyma) and confounding by use of salt to exacerbate pathology. Therefore, the aim of the experimental work in this thesis was to assess pathological changes within the cerebral vasculature and brain parenchyma of the SHRSP across a variety of ages (particularly young pre-hypertensive animals) and to look at the effects of salt loading on both the SHRSP and its parent strain (the Wistar Kyoto rat - WKY). Three related studies (qualitative and quantitative histology, immunohistochemistry and a microarray study of gene expression confirmed by quantitative PCR), revealed that the presence of inflammation (via significant changes in gene expression in the acute phase response pathway and increased immunostaining of activated microglia and astrocytes) plus alterations in vascular tone regulation, (via genetic alteration of the nitric oxide signaling pathway probably secondary to abnormal oxidative state), impaired structural integrity of the blood brain barrier (histological evidence of endothelial dysfunction and significantly decreased Claudin-5 staining) and reduced plasma oncotic potential (reduced albumin gene expression) are all present in the native SHRSP at 5 weeks of age, i.e. well before the onset of hypertension and without exposure to high levels of salt. We also confirmed previous findings of vessel remodelling at older ages likely as a secondary response to hypertension (thickened arteriolar smooth muscle, increased smooth muscle actin immunostaining). Furthermore, we found not only that salt exacerbated the changes see in the SHRSP at 21 weeks, but also that the control animals (WKY) exposed to a high salt intake developed features of cerebral microvascular pathology independently of hypertension (e.g. white matter vacuolation and significant changes in myelin basic protein expression). In conclusion, via the assessment of the most pertinent experimental model of lacunar stroke currently available, this thesis has provided two very important pieces of evidence: firstly that cerebral small vessel disease is primarily caused by a non-ischaemic mechanism and that any thrombotic vessel lesions occur as secondary end stage pathology; secondly that these features are not simply the consequence of exposure to raised blood pressure but occur secondary to abnormal endothelial integrity, inflammation, abnormal oxidative pathways influencing regulation of vascular tone and low plasma oncotic pressure. Patients with an innate susceptibility to increased blood brain barrier permeability and/or chronic inflammation could therefore have a higher risk of developing small vessel disease pathology and ultimately lacunar stroke and other features of small vessel disease. Research, addressing whether lacunar stroke patients should be treated differently to those with atherothromboembolic stroke is urgently needed.

Stroke is the single largest cause of disability and second highest cause of death globally. It is estimated that 10 million people a year are affected by stroke in the United Kingdom (UK). Of the 130,000 annual new stroke occurrences in the UK, one third will go on to have a further stroke. Recurrent stroke is more likely to be fatal than first stroke and survivors are more likely to be left with major disability. Many stroke patients do not adhere to secondary prevention strategies due to complex reasons, including lack of appreciation of their high risk of a secondary cardiovascular event. Long-term secondary prevention remains a desired goal in the management of stroke survivors, however, studies have shown that current strategies are not routinely and universally working. Hypothesis: Raising awareness of secondary stroke risk may improve stroke survivor’s adherence to secondary prevention strategies after stroke. Results: A survey of the general public (n=1019) and a population-based study of over 600 stroke survivors found that knowledge about Blood Pressure (BP) and stroke risk factors was poor in high risk populations. Only 55% of stroke survivors were able to cite any well-known vascular risk factors. However, those who were appropriately risk-aware significantly improved their health behaviour post-stroke by consuming less alcohol (P<0.001), less salt (P=0.05) and eating a healthy diet (P=0.02). Further, In a Randomised Controlled Trial setting an intervention to increase risk awareness was successful in increasing awareness (P=0.04) and resulted in a significant increase in knowledge of stroke sub-type (95% CI 0.72-0.677, P<0.001), risk factor control of systolic BP (95% CI 12.1-10.4, P=0.01) and increased the number of healthy lifestyle behaviour changes made at follow-up (P<0.001). Conclusions: Increasing risk awareness is potentially an important mechanism to improve health behaviour following stroke and may improve risk factor control as part of secondary stroke prevention.

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.

White matter injury often results in clinical neurodevelopmental deficits; however, molecular mechanisms remain partially understood and there is currently no clinically effective treatment. The purpose of this study was to elucidate the molecular and cellular mechanisms underlying developmental white matter ischaemia to aid the search for therapeutic interventions. In this thesis, a particular priority was to develop an ischaemia paradigm enabling study of the extended effects of ischaemia in a controlled environment. To achieve this, I sought to use organotypic slice cultures (OSC), whereby cerebellar slices could be generated from animals where the white matter is developing and maintain it whilst exposing it to a short oxygen-glucose deprivation (OGD) insult at some defined point in the culture cycle. Using this approach, I show that culture treatment at 7 days in vitro (DIV) with OGD for twenty-minutes triggered significant injury as judged by a 58.6% reduction in cell viability 3 days post-injury. TUNEL labelling showed about 60% of cell death was apoptotic in nature. Gene expression studies using Q-RT -PCR confirmed caspase-dependent cell death. OGD also produced marked oligodendrocyte loss and myelination disturbances as seen by immunocytochemistry. Post-OGD, astrocytes and microglia became activated, and cytokine and iNOS mRNA expression was upregulated. After a transient demyelinating insult with OGD, GluR-antagonists were effective against cellular damage and myelination disturbances. Myelin gene, oligodendroglial transcription factor, and BCL-2 mRNA expression were also maintained following administration of GluR-antagonists. In addition, there seemed to be some NG2+ OPCs differentiated into MBP+ oligodendrocytes during the recovery phase as seen by proliferation- marker BrdU. Western blotting showed that a mechanism by which GluR-antagonists confer protection was independent of the CREB transcriptional activity. The results also suggest ischaemia-induced NF-kB and p38MAPK signalling pathway activation may be attributable to ionotropic GluR stimulation. The cellular damage and myelination disturbances in this ex vivo model of white matter ischaemia provides a system to study remyelination following CNS injury- induced demyelination, and may serve as a valid pre-animal test-bed for examining potential pharmacotherapies.

Background and Purpose: Sleep problems are commonly reported by stroke patients. Poor sleep quality can detrimentally impact upon multiple clinical variables, including mood, physical health, cognition and the rehabilitation process itself. However, the relationship between sleep and stroke is complex and not fully understood. Pre-sleep cognitions and pre-sleep arousal have been proposed as contributing factors in sleep disturbance within the general population and this novel study investigates these variables as potential factors associated with sleep post-stroke. Methods: Stroke rehabilitation inpatients (N=21) were classified as good or poor sleepers using the Pittsburgh Sleep Quality Index (PSQI) and compared using measures of pre-sleep cognitions and pre-sleep arousal; relevant factors including daytime sleepiness, fatigue, mood and environmental disturbance were also explored. Results: Poor sleepers reported a significantly higher level of pre-sleep cognitions, pre-sleep cognitive arousal, fatigue and mood disturbance than good sleepers. The level of daytime sleepiness and perceptions of environmental disturbance did not differ significantly between groups. Conclusions: This study revealed a high level of poor sleep within the current sample (48%) based on the PSQI and pre-sleep cognitions and cognitive arousal appear potentially important factors in sleep quality post-stroke. Theoretical and practical implications and future directions for research are discussed.

In the UK, stroke is the third most common cause of death after heart disease and cancer. Importantly, most strokes are not fatal meaning stroke is the leading cause of adult disability, with one third of survivors still functionally dependent after one year. Post stroke hyperglycaemia (PSH) predicts poor outcome independent of age, stroke type or severity and occurs in over 60% of patients without a diagnosis of diabetes and in more than 90% of diabetic patients. Around 56% of hyperglycaemic acute stroke patients are subsequently diagnosed with insulin resistance, manifesting as impaired glucose tolerance, impaired fasting glucose or the “metabolic syndrome.” Clinical guidelines recommend routine blood glucose monitoring, and intervention with insulin for hyperglycaemia. However, there is currently no clinical evidence of benefit from insulin use, and recent findings raise concerns about the safety of insulin use in predominantly non-diabetic acute stroke populations. Hyperglycaemia in animal models of cerebral ischaemia is associated with increased infarct size. However, a recent systematic review highlighted uncertainties about the relevance of many previously published preclinical studies to the typical clinical picture of PSH with respect to whether clinically relevant elevations in blood glucose exacerbate ischaemic brain damage in models other than those of type I diabetes, or whether responses are different in animals with and without features of metabolic syndrome. The aim of this thesis was to investigate the differential effects of clinically relevant hyperglycaemia on the acute evolution of damage in animals with and without features of the metabolic syndrome and to investigate the potential mechanistic role of oxidative stress in glucose mediated ischaemic damage. I hypothesised that hyperglycaemia would exacerbate acute lesion evolution and final infarct volume in animals with and without features of the metabolic syndrome and that oxidative stress mechanisms would be involved. Developing suitable animal models for investigating post stroke hyperglycaemia and metabolic syndrome The first aim of this thesis was to develop a series of animal models for the investigation of clinically relevant PSH, metabolic syndrome in the presence and absence of PSH, and an appropriate focal cerebral ischaemia model. A previously described model of metabolic syndrome using the spontaneously hypertensive stroke-prone rat (SHRSP) fed on a 60% fructose diet for two weeks was set up in the laboratory and glucose tolerance, adiposity, plasma insulin, plasma triglycerides, cholesterol were measured and compared to the SHRSP reference strain, the Wistar-Kyoto (WKY). Fructose fed-SHRSP rats exhibited glucose intolerance, hypertriglyceridaemia, hyperinsulinaemia, increase adiposity and reduced HDL cholesterol compared to the WKY controls. This suitably modelled the features of the metabolic syndrome. Clinically relevant levels of PSH were achieved using a bolus intraperitoneal injection of 15% glucose (10ml/kg) 10 minutes prior to permanent middle cerebral artery occlusion (MCAO). Two methods of MCAO were compared; intraluminal filament (ILF) model and a distal diathermy MCAO model. The ILF method produced expansive lesions incorporating the majority of the ipsilateral hemisphere whereas the distal diathermy occlusion produced smaller cortical infarcts which were shown to produce a large volume of penumbra. The diathermy model was selected for all future experiments. Investigating the effects of hyperglycaemia on acute lesion growth after focal cerebral ischaemia in rats with and without components of the metabolic syndrome The second aim of this thesis was to determine the effects of hyperglycaemia on acute ischaemic lesion evolution and infarct volume in rats with and without features of the metabolic syndrome. Hyperglycaemia was induced in WKY or fructose-fed SHRSP rats 10 minutes prior to permanent MCAO. Magnetic resonance imaging (MRI) was used to quantify the expansion of the lesion using ADC maps calculated from diffusion weighted imaging (DWI) carried out over the first 4 hours after MCAO. Acute hyperglycaemia, at clinically relevant levels, exacerbated early ischaemic damage in both normal and metabolic syndrome rats. Hyperglycaemia worsened infarct volume at 24h in normal but not metabolic syndrome rats. These data suggest that management of hyperglycaemia may be most beneficial in the absence of an underlying dysglycaemia. Using a marker of oxidative stress to probe the tissue sections suggested that perhaps hyperglycaemic effects were mediated through oxidative stress mechanisms. Investigation of oxidative stress mechanisms in hyperglycaemia-dependant ischaemic damage. Based on the potential involvement of oxidative stress mechanisms in hyperglycaemia-associated ischaemic damage this was investigated in a proof of concept study using pre-treatment with a SOD/catalase mimetic, EUK-134. The rationale for this study was based on the involvement of increased superoxide production in chronic hyperglycaemia and diabetic complications. Sprague-Dawley rats received the same dose of glucose previously used 10 minutes prior to MCAO by distal diathermy. EUK-134 (2.5mg/kg) or vehicle (saline) was administered 20 minutes prior to MCAO. Infarct volume, calculated from RARE T2-weighted MR images and neurological scoring was measured 24 hours post MCAO. Arterial superoxide levels were measured along with the lipid peroxidation marker malondialdehyde in cerebral tissue. Hyperglycaemia increased infarct volume although no effect of high blood glucose on tissue lipid peroxidation was observed. EUK-134 failed to reduce infarct volume. EUK-134 reduced lipid peroxidation in ipsilateral cortex of normoglycaemic rats but not in hyperglycaemic rats. Thus, hyperglycaemia associated damage was not reduced by attenuating oxidative stress. Generating a threshold for quantitative T2 maps Using the data generated in the previous Chapter a study was carried out to determine if improvements could be made in the analysis of MRI data in pre-clinical rat studies. RARE T2-weighted images, where T2 relaxation time denotes contrast, have been used for infarct quantification 24 hours post MCAO. Generating a lesion volume from these images requires manual tracing of the visually defined infarct from the images. A degree of error is associated with this methodology either as inter-investigator reproducibility or in repeats conducted by the same investigator. The RARE T2 images are not quantifiable as arbitrary values denote contrast and this prevents direct comparison of T2 relaxation times for a particular structure between subjects. Using a MSME T2 sequence, quantitative T2 maps can be generated which calculate absolute T2 relaxation times for each voxel. Using quantitative maps an attempt was made to establish a threshold to discriminate between normal and ischaemically damaged tissue in animals that had undergone MCAO using the distal diathermy model. This threshold aimed to minimise inter- or intra-investigator error. A T2 threshold of abnormality for the quantitative T2 maps was established from the RARE T2 weighted images. Tissue that had a T2 relaxation time of 76 ms or more was deemed to be infarct according to the derived threshold. The suitability of the threshold was then assessed in a separate cohort and was found to produce similar infarct volumes to the manual delineation of RARE T2-weighted images. The 76ms threshold was also applied to animals that had undergone MCAO using the intraluminal filament model. However, the calculated threshold was not suitable for the intraluminal filament model due to interferences from cerebrospinal fluid signal. A more selective method for determining infarct volume in animals subjected to intraluminal filament MCAO needs to be established.

Stroke is a global health concern, with a significant impact on mortality and disability. Motor impairment, including upper limb impairment is particularly common following stroke. Upper limb impairment impacts on an individual’s ability to complete activities of daily living and quality of life. Effective interventions targeted at upper limb recovery are therefore important and further research, within this area, has been identified as necessary. However, challenges researching such complex interventions have been recognised. To attempt to overcome such difficulties the Medical Research Council (MRC) proposed a framework for the development and evaluation of RCTs for complex interventions. In this thesis the MRC framework has been used, focusing on the processes of developing and feasibility/piloting, to provide information for a phase III randomised controlled trial (RCT) of a novel intervention targeted at upper limb recovery following stroke. A systematic review and meta-analysis was undertaken to investigate and clarify any possible association between predictive variables and upper limb recovery. Observational studies of stroke patients investigating at least one predictive variable and its relationship with a defined measure of upper limb recovery at a future time point were included. For this review data analysis combined several approaches. Fifty eight studies were included and 41 predictor variables identified. Initial measures of upper limb function and impairment were found to be the most significant predictors of upper limb recovery; odds ratio (OR) 38.62 (95% confidence interval (CI) 8.40-177.55) and OR 14.84 (95% CI 9.08-24.25) respectively. Neurophysiological factors (motor evoked potentials and somatosensory evoked potentials) were also consistently identified as strongly associated with upper limb recovery; OR 11.76 (95% CI 2.73-69.05) and OR 13.73 (95% CI 2.73-69.05) respectively. Moderate evidence of association was found for global disability and lower limb impairment. Interpretation of results is complicated by methodological factors, particularly relating to the heterogeneous nature of the included studies. In order to identify interventions which show potential for reducing impairment and/or improving upper limb function after stroke, an overview of the available evidence was completed. This systematic review and meta-analysis included Cochrane systematic reviews, other reviews and, where necessary, additional RCTs of interventions to promote upper limb recovery. Thirteen relevant interventions were found, covered by nine Cochrane systematic reviews (bilateral training, constraint-induced movement therapy (CIMT), electromyograhphic (EMG) biofeedback, electrostimulation, hands-on therapy interventions, mental practice, repetitive task training (RTT), electromechanical/robotic devices and virtual reality) and four other reviews (neurophysiological approaches, high-intensity therapy, mirror therapy and splinting). A statistically significant result, in terms of arm recovery, was found in favour of eight of the interventions: CIMT (standardised mean difference (SMD) 0.74 95% CI 0.44-1.03), EMG biofeedback (SMD 0.41 95% CI 0.05-0.77), electrostimulation (SMD 0.40 95% CI 0.02-0.77), mental practice (SMD 1.37 95% CI 0.60-2.15), mirror therapy (SMD 0.41 95%CI 0.05-0.77), RTT (SMD 0.23 95% CI 0.06-0.41), electromechanical/robotic devices (SMD 0.30 95% CI 0.02-0.58) and virtual reality (SMD 0.52 95% CI 0.25-0.78). Two out of the eleven interventions, which investigated hand function outcomes found a positive result (CIMT SMD 0.39 95% CI 0.11-0.68 and repetitive task training SMD 0.27 95% CI 0.06-0.47). Analyses were limited by a relatively small number of RCTs, which were also generally small in size. Heterogeneity of the available data and methodological limitations further impacts on the conclusions. Despite these limitations this overview provided a concise and informative summary of the available evidence. The interventions found to be beneficial, or showing promise tend to include elements of intensive, repetitive, task-specific practice. To build the evidence base for upper limb interventions, two Cochrane systematic reviews were undertaken. These reviews investigated the effects of bilateral training and home therapy programmes on upper limb recovery. Both included RCTs of stroke patients. Eighteen trials were included in the bilateral review, of which 14 were included in the analyses. Most of the included trials were considered to be at high risk of bias and the evidence was further limited by heterogeneity. No statistically significant results were found for any of the primary outcomes. One study found a statistically significant result in favour of another upper limb intervention for performance in extended ADL. No statistically significant differences were found for any of the other secondary outcomes. Four RCTs were included in the home-based therapy programmes review. No statistically significant result was found for any of the outcomes. There is currently insufficient good quality evidence to determine the effects of both the interventions studied. Following the evidence gained from the overview of interventions elements of intensive, repetitive and task-specific practice were to be included in a novel upper limb intervention. Robotic interventions, which incorporate these principles, were also found to have a positive effect on upper limb outcomes. Therefore a pilot, feasibility and acceptability study of a novel device (Armeo®Spring) that included these elements was completed. Medically stable adults with a clinical diagnosis of stroke and arm deficits admitted to an acute stroke unit were recruited. Participants were randomly allocated to experimental intervention (high or low intensity training with the Armeo®Spring arm orthosis) or usual stroke unit care. Primary outcomes were feasibility and acceptability of the experimental device recorded at postintervention. Secondary outcomes were; safety and three efficacy outcomes recorded at post-intervention, and 3 month follow-up. Patient recruitment was challenging; over eight months 393 consecutive stroke admissions were screened and 12 participants recruited. This study demonstrated that per-protocol levels of intensity were not feasible to provide in an acute stroke unit. However, higher levels of intensity could be achieved and this novel intervention was found to be acceptable to patients. This pilot trial also found higher change scores on the three efficacy outcomes within both intervention groups, compared to the control group. Due to small sample size and other possible confounding factors, these findings must be interpreted with caution. Using the MRC complex intervention framework as a guide I completed development and feasibility/piloting work surrounding an upper limb intervention, following stroke. Following the results of this research further development, feasibility/piloting work is suggested for the ArmeoSpring device prior to the undertaking of a phase III RCT. The information gained from this research could be used to inform phase III RCTs of other upper limb interventions.

There is increasing evidence that inflammatory processes play a pivotal role in the pathophysiology of ischaemic brain injury. Cerebrovascular endothelial cells that form the blood-brain barrier are critical for maintaining brain homeostasis, however, during cerebral ischaemia they contribute to the post-ischaemic inflammatory responses. It is not yet fully understood how different cerebral cells interact during this inflammatory response. This study aimed to test the hypothesis that oxygen-glucose deprivation (OGD) induces the inflammatory activation of the cerebrovascular endothelium and glial cells in vitro and that intercommunication between these cells regulate their responses to OGD. Primary murine brain endothelial cells (MBECs) monocultures, murine mixed-glial monocultures and MBEC-glial co-cultures were exposed to OGD for up to 24 hours (h), then reperfused cultures were returned to normoxia for a further 24 hours. MBECs and glia remained viable over a 24 h OGD exposure and during reperfusion. OGD induced a time-dependent increase in MBEC glucose transporter 1 (GLUT-1) expression but a time-dependent decline in expression and secretion of monocyte chemoattractant protein-1 (MCP-1). A significant increase in keratinocyte-derived chemokine (KC) secretion by MBEC monocultures was observed during reperfusion after prolonged exposure (18-24 h) to OGD whereas, KC secretion by co-cultured MBECs was increased during reperfusion after short exposure (4 h) to OGD. Co-cultured MBECs displayed a significant increase in intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in response to a short or prolonged exposure to OGD with 24 h of reperfusion. Neither OGD nor reperfusion had any effect on permeability of the MBEC monolayer. OGD induced a time-dependent increase in nuclear stabilisation of hypoxia inducible factor-1 alpha (HIF-1α) in glial cells which correlated to vascular endothelial growth factor (VEGF) secretion during OGD and subsequent reperfusion. Nuclear stabilisation of the nuclear factor kappa B (NFκB)p65 subunit by glial cells was dependent upon the duration of OGD. Reperfusion induced a significant increase in KC secretion by co-cultured glial cells after short exposure to OGD. Inflammatory activation of co-cultured MBECs and glia after 4 or 24 h OGD caused a significant increase in neutrophil transendothelial migration which correlated with MBEC expression of ICAM-1 and VCAM-1. A combination of these cell adhesion molecules with neutrophil integrins and soluble glial-derived mediators contributed to neutrophil transendothelial migration. These studies provide evidence that combined hypoxia and glucose withdrawal induces the activation of MBECs and glial cells in vitro. Cross-talk between these two cell types may further regulate their activation. As a result of this inflammatory activation, soluble MBEC and glial-derived mediators may contribute to neutrophil transendothelial migration through the regulation of MBEC cell adhesion molecule expression.

Clinical manipulation of angiogenesis (the formation of new blood vessels from pre-existing vasculature) is of interest to treat diseases such as cancer and ischemic tissue where it is not properly regulated. Several treatments targeting vascular endothelial growth factor (VEGF) and its receptors - which are abundant at sites of angiogenesis - are currently in use to treat various types of cancer, however they have severe vascular side effects. Conversely, VEGF has been used clinically to promote angiogenesis to treat ischemic tissue. However, despite encouraging data from pre-clinical models, trials in humans have been disappointing. For further therapies to be developed, more information on how VEGF interacts with its receptors is required. Heparan sulfate (HS) is a ubiquitous glycosaminoglycan involved in a number of physiological processes including angiogenesis. HS facilitates the interaction of VEGF with its receptors, which is crucial for angiogenesis. Modification of this interaction via synthetic mimetics of HS may allow clinical intervention of angiogenesis. The current investigation aims first, to clarify the requirement for the interaction between VEGF and HS in angiogenesis; second to characterise the structure of HS that binds to VEGF so that mimetics can be developed; and third, to determine the effect of HS mimetics on angiogenesis in vivo. To determine the requirement for VEGF/HS interaction in angiogenesis, several mutants of VEGF165 that had lower affinities for HS were assayed for their ability to induce ectopic angiogenesis in the subintestinal baskets of zebrafish embryos. Wild type VEGF165 induced a 200-250% increase in ectopic vessels, which was matched only by a control mutant. Other mutants did not induce ectopic vessels, suggesting that this interaction is required for angiogenesis. To characterise the structure of HS that binds to VEGF, various HS mimetics were assayed against heparin in a VEGF competition assay using Biacore. Of these, the strongest inhibition (IC¬50 =~16nM) was with 2O10, an oligosaccharide that consisted of two highly sulfated octasaccharide domains (NS domains) that flanked an unsulfated dodecasaccharide region. To determine the type of sulfation required for this interaction, HS fragments were assayed for interaction with VEGF165 using the filter binding assay, and analysed by HPLC which indicated 6-O sulfation may be preferential for VEGF binding to HS.To investigate the ability of HS to affect angiogenesis, the effects of HS mimetics on zebrafish embryo subintestinal baskets were measured. The most interesting of these was with 2O10, which had a biphasic response whereby low doses (3ng) increased basket vasculature by 30% and high doses (30ng) decreased the endogenous vessels by 20%. As 2O10 had a high affinity for VEGF, its effects on the vasculature may be due to interaction with endogenous VEGF, which would indicate that HS mimetics can be used to control angiogenesis by modification of growth factor signalling. The investigation concludes that the interaction between VEGF and HS is critical for angiogenesis, and that this can be modulated by the application of HS mimetics that bind strongly to VEGF.

The role of CEA for stroke prevention in the presence of symptomatic carotid artery stenosis is well established. In order to maximize the benefit of surgery, several perioperative processes of care have been under scrutiny, of which one is the choice of anaesthetic method. The differing effects of GA vs. LA on the cerebral circulation after CEA may be of significance, since changes in the cerebral circulation post-CEA may give rise to cerebral hyperperfusion and intracerebral haemorrhage. This work assessed the effect of GA vs. LA on cerebral haemodynamic control after CEA using transcranial Doppler (TCD) techniques, and correlated these changes with serum markers of cerebral injury. Subjects undergoing CEA had perioperative TCD monitoring of middle cerebral artery blood flow velocity (MCAV). Pre- and postoperative (within 48 hours of surgery) testing of cerebral autoregulation [CA] (tilt-testing) and cerebral vasoreactivity to CO2 [CVR] (rebreathing expired air) was conducted. Cerebral haemodynamic parameters and clinical outcome were correlated with changes in jugular venous and peripheral levels of protein S100β and neurone-specific enolase (NSE).The change in CA and CVR was not different between GA (n=16) and LA (n=20). Overall, CA and CVR improved significantly within 48 hours of CEA for patients with preoperative impairment of these parameters, although some patients with normal baseline CA and CVR exhibited postoperative impairment. Increase of MCAV >100% from baseline after restoration of carotid blood flow was observed in patients with impaired CVR, but resolved by the first postoperative day. Transient elevation in jugular venous (but not peripheral) S100β during surgery was seen. Both jugular and peripheral NSE levels dropped during surgery. Neither anaesthetic method nor CA or CVR status had any effect on changes in serum S100β or NSE. Cerebral autoregulatory parameters thus improve rapidly after CEA, but appear unaffected by anaesthetic technique. This supports the concept that cerebral hyperperfusion is dependent on factors in addition to impaired CA or CVR. Changes in serum S100β or NSE do not reflect cerebral haemodynamic changes. However, the variability encountered between patients warrants further investigation. The implications for clinical practice and directions for further research are discussed.

The occlusion of a cerebral artery or stroke often results in neuronal deficit and/or patient death. A partial recovery often follows non-fatal stroke and this may be due to the activation of the progenitor cells in the Sub- Ventricular Zone (SVZ) naturally occurring after ischemia. In order to clarify the role of the SVZ neurogenesis in animal recovery, the effect of neurogenesis inhibition and boosting were studied in the mouse Middle Cerebral Artery occlusion model (MCAo). 6 to 10-week-old male mice were pre-treated with intracranial injections of lentiviral vector (LV) or integration deficient lentiviral vectors (IDLV), in order to target the SVZ. The IDLV carried an expression cassette encoding for a precursor Glial cell-derived neurotrophic factor (GDNF) or the tetanus toxin fragment C (TTC), which recently has been demonstrated to have growth factor like behaviour. Another group of animals received the LV carrying a double promoter expression cassette encoding for an eGFP, and in order to inhibit the cell cycle in targeted cells the shRNA_Cyclin D1. All vectors were co-injected with the LV_ pHR'SIN-cPPT-SEW, which contains an eGFP cassette. Two weeks later the animals received the MCAo, and for three weeks the sensorimotor behaviour was tested. Neurological assessment showed the sensory-motor debilitation was significant increased after the treatment with the LV_shRNA_CyclinD1 (* p<0.05); the IDLV_GDNF and IDLV_TTC groups showed a trend to improve neurological deficit in the subjects alive until day 5. In the IDLV_GDNF, the SVZ's derived green cells were positively correlated with the ischemic volume, *p<0.05 R=0.68, and the neurodegeneration, ***p<0.001 R=0.92. Moreover, while the SVZ neurogenesis inhibition reduced life expectancy, the boosting significantly improved it. Immunofluorescence analysis showed a migration extended to the striatum and cortex with a max distance of 1.87 mm from the SVZ.

Stroke is the third most common cause of death in the United Kingdom and the single greatest cause of severe disability. The effects of stroke are complex but the impact on emotional wellbeing is arguably one of the most problematic aspects of stroke rehabilitation to address. Nurses play a key role in stroke rehabilitation and the ways in which they interact with and respond to the emotional experience of stroke, may be crucial to the well-being of the patient and their relatives. Informed by an interactionist theory of emotion, the aims of this interpretive ethnographic study were to explore the emotional experiences of persons affected by a stroke (patients and relatives), nurses’ interpretations of these experiences and how they used them to inform and influence person-to-person interactions during the period of hospital-based rehabilitation. Taking a case study approach, a purposive sample of 10 cases was selected, with each ‘case’ comprising a patient, their closest relative and the nurses who provide their care. Data were constructed through participant observation, interviews and documentary review. Data analysis revealed that through a complex interplay of core beliefs, personal and professional attributes and interpersonal skills, nurses enabled patients and relatives to access and utilise their own personal attributes in order to recover from stroke. An emergent relationship model explains how the relationships built and sustained between nurses, patients and relatives during their encounters with each other on a stroke rehabilitation unit are central to creating a positive culture of caring which promotes emotional wellbeing and aids recovery. The findings have implications for policy, clinical practice, health care education and research.

Stroke is the main cause of disability worldwide. With over 110,000 first strokes occurring in the UK every year, the individual and societal burden of stroke will continue to increase. Consequently, there is a pressing need to develop therapies to both reduce the burden of stroke and the risk of stroke recurrence. Hyperglycaemia, dyslipidaemia, hypertension, reduced cardiorespiratory fitness and obesity are metabolic irregularities closely linked to stroke risk and represent possible therapeutic targets. In healthy individuals, a physically active lifestyle and structured exercise have been found to improve metabolic control. Research reporting the impact of physical activity and exercise on metabolic control, stroke risk and wellbeing is lacking in the study of stroke. This thesis aims to define and explore physical activity, exercise and metabolic control following stroke with a view to improving clinical care. A cross-sectional longitudinal study (n=31) demonstrates physical activity levels are low immediately following stroke and do not return to levels comparable to healthy individuals after six months, despite individuals having only mild impairment. A randomised controlled trial (n=40) of a community based exercise intervention post stroke investigated the impact of exercise primarily on metabolic risk factors. Significant improvements occurred in cardiorespiratory fitness (p<0.01); high density lipoprotein cholesterol (p<0.01); and diastolic blood pressure (p<0.04). The intervention also resulted in improvements in cognition, overall stroke recovery and mood, with the most marked changes occurring in walking endurance, walking speed and balance. This thesis delivers two clear messages: physical activity is reduced following stroke and does not fully recover after time; and the reversal of low levels of physical activity with a community based exercise therapy programme provides significant benefit to individuals after stroke. This information suggests that clinicians should consider both the importance of physical activity and the therapeutic potential of structured exercise therapy post stroke.

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

Posterior circulation ischemic stroke accounts for a quarter of ischemic strokes. Recent studies have shown that these patients have a high early risk of further ischaemic events especially if they have 2:50% vertebrobasilar (VB) stenosis; there is paucity of data on the diagnosis and management of these patients. The gold standard technique to diagnose VB stenosis is intra-arterial angiography (IAA) which carries a risk of stroke; this can be avoided by using non-invasive imaging techniques. I systematically reviewed the literature on studies which compared the accuracy of three commonly used non-invasive imaging techniques (contrast-enhanced magnetic resonance angiography (CE-MRA), computed tomographic angiography (CTA) and duplex ultrasonography) to IAA, the gold standard, in order to diagnose VB stenosis. There were 13 studies for vertebral stenosis, these showed CE-MRA and possibly CTA to be better than duplex. I then performed the first prospective study (40 patients) comparing CE-MRA, CTA and duplex to IAA in the same patient group demonstrating that CE-MRA had the highest sensitivity followed by CTA and then duplex. The two main studies which have shown that 2:50% symptomatic VB stenosis carries a high risk of further ischaemic events followed-up patients for 90 days. I examined my patient group with longer term follow-up (mean 14 months). This study showed a higher risk of recurrent ischaemic events (odds ratio (OR) 2.56(95%CI 1.11-5.9),p=0.023) and VB stroke or TIA (OR 2.8(95%CI 1.24-6.32),p=0.01 ). I reviewed data from patients who had endovascular vertebral intervention at St George's and systematically reviewed the literature. This study showed that vertebral artery stenting is a technically successful procedure (99.4%) with a periprocedural stroke risk of 0.5%; one year TIA, stroke and mortality rate was 3.9%, 5.0%, and 3.6% respectively. However, the included studies were retrospective studies of varying quality; large-scale prospective trials are needed to determine the benefits of stenting.

Carotid stenosis causes 15-20% of all ischaemic strokes, which can be preceded by TIA or minor stroke but usually occur without warning. Risk of stroke is 10-15% in symptomatic disease and optimal medical treatment is unknown in the acute period prior to carotid endarterectomy (CEA) or where there are contraindications to surgery. In contrast, risk is now <2% per annum in asymptomatic disease; due to improved medical treatment however optimal treatments are unknown. Methods are required to identify asymptomatic patients at high risk of stroke to improve treatment by targeting CEA and intensive therapies to those who may benefit. The mechanism of these strokes in carotid atherosclerosis is predominantly thought to be due to thrombo-embolism. Transcranial Doppler (TCD) embolic signal (ES) detection may therefore be a useful technique in investigating the prevention of ischemic stroke. Data in symptomatic and occlusive disease suggest a haemodynamic mechanism may also contribute to stroke risk; however there are limited data in asymptomatic disease. Methods: In symptomatic carotid stenosis a randomised controlled trial was used to compare two anti- platelet regimens. A novel ambulatory TCD ES detection protocol and concurrent platelet aggregometry were used as surrogate markers. In asymptomatic carotid stenosis secondary analysis of a large multicentre international trial (ACES) was performed, analysing optimal protocols for TCD ES detection. In a sub study, TCD cerebrovascular reactivity (CVR) was also investigated. The influence of improvements in treatments on ES over time and changes in treatments over a two year follow-up period were also analysed to investigate prevention of ischemic stroke in asymptomatic carotid stenosis. Results: In symptomatic stenosis, aspirin in combination with either dipyridamole or clopidogrel reduced embolism to a similar extent. ES had temporal variation in ambulatory recordings. In asymptomatic carotid stenosis, ES detection on 2 baseline recordings is an independent risk prediction tool for ipsilateral stroke which was better than shorter recordings or one recording only. CVR did not predict risk but this lack of association may reflect a low number of endpoints. A trend, but non-significant decrease, in ES over time was observed despite changes in treatments. Higher blood pressure and absence of anti-platelet therapy were associated with risk of ipsilateral strokelTlA and any stroke/cardiovascular death. Conclusion: ES are surrogate markers in both symptomatic and asymptomatic carotid stenosis. In acute symptomatic atherosclerosis, clopidogrel or dipyridamole in addition to aspirin reduce embolism to a similar extent and may be useful for prevention of embolic ischemic stroke. In asymptomatics, ES on either of 2 baseline recordings predict 2 year risk of ipsilateral stroke, however there is insufficient data for the use of CVR. Improvements in treatments at baseline did not prevent ES and the technique may be useful to identify patients at high risk who may benefit from carotid endarterectomy to prevent future ischaemic stroke. ES detection may also be important to identify those at low risk to target treatments appropriately. Consistent use of anti-platelet therapy and control of blood pressure are important in the prevention of ischemic stroke and cardiovascular death in asymptomatic carotid stenosis.

This research portfolio is divided into three parts:Part one is a systematic literature review of the literature titled ‘Expressed Emotion (EE) in long term health conditions including those with a neurological basis’. A great deal of research has been carried out looking at the role of EE in psychiatric conditions where EE is now seen as a well established strong predictor of relapse in schizophrenia. More recently, research has turned its focus onto the effects of EE within the domain of chronic health conditions, however, whether this maintains the same significance as shown in psychiatric illnesses remains unclear. This review examines the concept of EE in relation to adjustment, course of illness and functional outcomes in long term health conditions. Broadly it explores the individual components of EE (criticism, emotional over-involvement, hostility, warmth and positive remarks) to identify which have been most associated with outcome. Furthermore, this review has also focused upon how EE has been operationalized and measured in to relation long term health conditions. Clinical and research implications are discussed further in this review. Part two is an empirical research study titled ‘Post stroke depression and expressed emotion’. The causes of PSD remain controversial, particularly regarding the location of lesion that could be linked to depression. What remains clear, however, is that depression after a stroke injury is commonly experienced and has been evidenced as one of the key factors influencing adjustment and rehabilitation outcomes. Given the potential of the impact of EE in long term health conditions, particularly on psychological distress, understanding the causes of PSD in terms of how organic and psycho-social factors might relate to each other is vital for recovery. A cross-sectional design was used to investigate the extent to which EE might interact with lesion laterality to determine levels of post stroke depression (PSD) in stroke survivors. It was hypothesised that stroke survivors with a left lesion stroke injury living in a high EE climate would experience higher levels of PSD compared to those with a right lesion stroke injury living in a low EE climate. Secondary aims of this research explored the relationship between lesion laterality and levels of PSD; and levels of EE and PSD. Additional exploratory research was also carried out to examine the extent to which stroke survivors’ perceptions of EE may interact with lesion laterality to determine levels of PSD. Clinical implications and scope for further research are discussed further. Part three contains the appendices which provide further information in relation to the systematic literature review, empirical paper and a reflective statement of the process on this research.

Loss of upper-limb function is a problem following stroke. Recent research has led to the emergence of new treatments but progress is hampered by lack of reliable objective measures of impairment, and understanding of the underlying impairment mechanisms associated with loss and recovery of functional activity. The aim of this research was to identify, using neuromechanical measurement methods, inter-relationships between motor impairments, and correlates of motor impairments with functional activity limitation in the upper limb of acute and chronic stroke survivors. An instrumented rig has been developed to measure impairments: muscle weakness, active range of movement, motor control accuracy in rhythmic and discrete tracking tasks, spasticity, coactivation, contracture and non-neural stiffness. In pilot studies, signal processing and data analysis techniques have been used to generate novel, clinically and physiologically relevant indices to quantify impairments. In a Main Study, 13 older impaired participants in the acute phase post-stroke, 13 in the chronic phase 14 age-matched unimpaired participants underwent rig assessments and performed a test of upper limb activity. A sub-group of impaired participants were tested on two days for test-retest reliability evaluation. Statistical tests have confirmed the validity of the impairments to distinguish between acute and chronic patients and unimpaired individuals, except coactivation during discrete movements and non-neural stiffness. Repeatability coefficients for the active test indices have been presented as benchmark values for use in future trials. The muscle activation indices showed lower repeatability which highlights the challenge of using these to measure change over time. The impairments that contributed to lower motor control accuracy were reduced extensor weakness, delayed extensor onset timing, coactivation and smaller extension AROM and PROM; coactivation was more strongly associated with motor control accuracy than with spasticity or stiffness. The most important contributors to functional activity in the acute group was extensor weakness, and in the chronic group was motor control accuracy and coactivation (rhythmic task). Contracture was important contributor in both groups, and was associated with weakness and loss of active range of movement rather than spasticity. The findings support the notion that rehabilitation strategies should focus on increasing muscle strength and prevention of contracture. However, assessment of more complex impairments like motor control accuracy and coactivation may be crucial to better target therapy, especially in the later phases post-stroke.

Cerebral ischaemia, or stroke, is a leading cause of death and disability worldwide. Ischaemic stroke, as a result of arterial occlusion, and subarachnoid haemorrhage (SAH), as a consequence of arterial rupture in the subarachnoid space, are major subtypes of stroke. Treatment options for both are limited, and many therapeutic strategies have failed. In ischaemic stroke, lack of evidence of brain penetration of treatments has been cited as a major weakness and contributing factor to failed clinical trials. In SAH, animal models do not always mimic key pathophysiological hallmarks of the disease, hindering development of new therapeutics. Inflammation is strongly associated with brain injury after cerebral ischaemia and inhibition of the pro-inflammatory cytokine interleukin-1 (IL-1) represents apossible therapeutic target. Therefore, the key objectives of this thesis were; (1) to improve preclinical data on a promising stroke treatment, interleukin-1 receptor antagonist (IL-1Ra), by investigating its pharmacokinetic profile and brain penetration in a rat model of ischaemic stroke, (2) to investigate the endovascular perforation model of SAH in rat, as a tool for the investigation of neuroprotectants, and (3) to examine the role of the inflammatory response in the SAH model and the effects of IL-1Ra. The neuroprotective effect, pharmacokinetic profile and brain penetration of IL-1Ra were assessed after a single subcutaneous (s.c.) dose (100mg/kg) in rats, after transient (90 min) middle cerebral artery occlusion (MCAo). A single s.c. dose of IL-1Ra reduced neuronal damage, resulted in sustained, high concentrations of IL-1Ra in plasma and cerebrospinal fluid and also penetrated brain tissue exclusively in areas of blood brain-barrier (BBB) breakdown. An endovascular perforation model of SAH in rat was investigated and produced widespread multifocal infarcts. In this model, administration of IL-1Ra (s.c.) reduced BBB breakdown, which correlated with injury at 48 h. IL-1_ was expressed in the brain early after SAH in areas associated with haem oxygenase-1 (HO-1) expression, indicating the presence of free haem. Stimulation of primary mouse mixed glial cells in vitro with haem induced expression and release of IL-1 alpha but not IL-1 beta. These data, after MCAo in rat, are the first to show that a single s.c. dose of IL-1Ra rapidly reaches salvageable brain tissue and is neuroprotective. This allows confidence that IL-1Ra is able to confer its protective actions both peripherally and centrally. After experimental SAH, we suggest that haem, a breakdown product of haemoglobin, released from lysed red blood cells in the subarachnoid space, acts as a danger associated molecular pattern (DAMP) driving IL-1- dependent inflammation. These data provide new insights into inflammation after SAH-induced brain injury and suggest IL-1Ra as a candidate treatment for the disease. Overall, these findings strengthen preclinical data supporting IL-1Ra as a neuroprotective therapy for ischaemic stroke, and identify SAH as a new indication for treatment with IL-1Ra.

Following stroke, a patient may fail to report touch administered by another person but claim that s/he feels touch when it is self-administered. In Part One, the self-touch rubber hand paradigm was used to investigate different explanations for this phenomenon, termed self-touch enhancement. The most important finding was that patients reported touch based on feeling rather than by using proprioceptive information. Some patients have residual sensation that could be targeted in sensory rehabilitation. Part Two is a systematic investigation of the illusion of self-touch conducted with neurologically healthy participants. Participants used the right hand to administer touch to a prosthetic hand while the left (receptive) hand, positioned 15 cm from the prosthetic hand, received Examiner-administered touch. Proprioceptively perceived position of the administering and receptive hand was measured. Most participants experienced the single event of self-touch at the location of the receptive hand. Previous investigations have relied on measurement of only one hand and have concluded that participants experience self-touch at the location of the prosthetic hand. Our findings have implications for the role of ownership in this illusion. There is also a series of experiments in Part Two which test four potential constraints on the illusion of self-touch – violated expectations about the object that is administering touch, increased distance between the hands, alignment mismatch, and anatomical implausibility. For example, one study uses a novel paradigm to demonstrate that, although the subjective intensity of the illusion of self-touch is diminished by anatomical implausibility, most participants report the impossible experience of touching their left elbow with their own left index finger. Taken together, these experiments highlight the malleability of body representation, and provide a comprehensive framework for understanding the illusion of self-touch.

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.

Ischaemic stroke is a major cause of mortality and chronic disability for which there is no effective treatment. The subventricular zone (SVZ) is an adult neurogenic niche which mediates limited endogenous repair following stroke. To harness this phenomenon for therapy, it is important to understand how the SVZ niche is altered in stroke, and the processes that recruit neural precursors to the site of injury, which becomes a de facto neurogenic niche. Galectin-3 (Gal-3) is a β-galactoside binding protein involved in cellular adhesion, inflammation and tumour metastasis. Gal-3 is specifically expressed in the SVZ and maintains neuroblast migration to the olfactory bulb, although its role in post-stroke neurogenesis is not well-understood. Therefore, this project aimed to (1) characterise the cytoarchitecture of the SVZ in response to stroke, and (2) examine the role of Gal-3 in stroke outcome and tissue remodelling, and test the hypothesis that Gal-3 is required for neuroblast ectopic migration into the ischaemic striatum. Using the intraluminal filament model of middle cerebral artery occlusion (MCAO) in mice, and whole mounts of the lateral ventricular wall, significant SVZ reactive astrocytosis and increased vascular branching were observed, thereby disrupting the neuroblast migratory scaffold. Stroke increased SVZ cell proliferation without increase in cell death. Post-stroke ependymal cells were enlarged and non-proliferative, and assumed a reactive astroglial phenotype, expressing de novo high levels of glial fibrillary acidic protein. This was associated with focal planar cell polarity misalignment, and turbulent and decreased rate of cerebrospinal fluid flow. These findings demonstrate significant changes in multiple SVZ cell types which are positioned to influence post-stroke neurogenesis and regulation of the neural stem cell niche Gal-3 was up-regulated in the ischaemic brain and ipsilateral SVZ. To elucidate the role of Gal-3 after stroke, MCAO was performed in wildtype and Gal-3 null (Gal-3-/-) mice, and parameters of stroke outcome and post-stroke neurogenesis compared. The deletion of Gal-3 did not affect infarct volumes or neurological outcomes, although neuroblast migration into the ischaemic striatum was increased in Gal-3-/- brains. Gal-3-/- mice failed to mount an angiogenic response in the ischaemic striatum, and this was associated with lower levels of vascular endothelial growth factor (VEGF) and increased anti-angiogenic protein levels. Loss of Gal-3 further disrupted the pro-proliferative neural-vascular interaction at the basement membrane. The current data indicate that Gal-3 is a pleiotropic molecule which has distinct roles in both the SVZ and the post-stroke striatum as niches of adult neurogenesis.

Introduction. The glial cell water channel aquaporin-4 (AQP4) plays an important ro le in brain oedema, astrocyte migration and neuronal excitability. Current theories of AQP4 function are based largely on experiments using AQP4 -1- mice. These mice have only been partially characterized. I therefore undertook a detailed investigation of baseline brain properties in AQP4 -1- mice. In the second part of my experiments I investigated the role of AQP4 in brain oedema in a mouse model of subarachnoid haemorrhage. Method. Gross anatomical measurements included estimates of brain and ventricle size. Neurons, astrocytes and oligodendrocytes were assessed using the neuronal nuclear marker NeuN, the astrocyte marker GFAP, and the myelin stain Luxol Fast Blue. The blood brain barrier was studied by electron microscopy and the horseradish peroxidase extravasation technique. A mouse model in which 30~1 of autologous blood was injected into the basal cisterns was used to reproduce subarachnoid haemorrhage. Brain water content, intracranial pressure and neurological score were compared in wildtype and AQP4 -/- mice. I also measured blood brain barrier permeability and the osmotic permeability of the glia lim itans, one of the routes of oedema elimination.

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

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is one of the most common forms of hereditary vascular dementia (VaD). Characterised by early-onset strokes and cognitive impairment in the absence of vascular risk factors, CADASIL is an ideal model to understand the pathophysiology of VaD. Pathogenic mutations in the NOTCH3 gene, which encodes a single-pass transmembrane cell surface receptor expressed predominantly in vascular smooth muscle cells (VSMC), cause severe vascular alterations including VSMC degeneration, hyalinosis, deposition of CADASIL-specific granular osmiophilic material (GOM) and white matter (WM) changes. While these changes have been well-described, their causative mechanism or difference between sporadic VaD is poorly understood. The aim of the project was to quantitatively characterise various aspects of cerebral pathology of CADASIL in order to reveal the pathological basis of CADASIL phenotypes, especially of cognitive dysfunction. Firstly, we assessed vascular and perivascular changes in CADASIL brain areas and found significant vessel wall thickening and perivascular space enlargement, even compared to sporadic VaD. Secondly, by using immunogold electron microscopy, NOTCH3 extracellular domain (N3ECD) was located within GOM in the wall of cerebral arteries/arterioles/capillaries, establishing at least one component of GOM and its wide-spread existence in the vasculature. This study also suggested the possible existence of intracellular N3ECD accumulation and involvement of inflammatory response in the pathogenesis of CADASIL. Finally, we provide neuronal density data from the hippocampal formation in CADASIL brains to identify the neural substrates of VaD in CADASIL. Overall, the number of neurons in CA1, CA2 and entorhinal cortex was relatively spared in CADASIL while pyramidal neuronal subpopulation, as shown by SMI32 immunoreactivity, was slightly decreased. In addition, SMI32 staining revealed extensive chronic damage to WM tracts, especially those in the frontal-parietal area. These data suggest that vascular dysfunction and inflammation result in frontal disconnection, which could underlie cognitive impairment in CADASIL patients.

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.

Cognitive-based techniques have been proposed as instruments for use within stroke rehabilitation as a means of promoting recovery from hemiparesis. This thesis explores the theory and practice of using imagery and observation as rehabilitation therapies for individuals affected by stroke and explores the use of an observation-based intervention for stroke rehabilitation. The first study aimed to develop an appropriate tool with which to explore the processes of observation and, in particular, preferred observation perspective.