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Randomised controlled trials in vascular prevention typically use binary events as the primary outcome. However, this practice is statistically inefficient and gives no indication of the severity of the event. Hypothetically, outcomes could be polychotomised, with ordering determined by severity. The primary aim of this study was to identify the most efficient type of outcome and analysis technique using individual patient data from randomised controlled trials. Another aim was then to review the use of these approaches on other vascular event outcomes; done using data from the TARDIS trial, the first trial to use an ordinal event measure as the primary outcome. An additional consideration was that some trials use a composite outcome as the primary measure. Therefore, a third aim was to determine if severity data can be incorporated here as well and whether to analyse these novel outcomes using standard methods or global analysis techniques, which can analyse multiple outcome measures at once to determine an overall effect. Chief investigators of vascular prevention trials were asked to share individual participant data. Binary and ordinal extensions of vascular outcomes were then developed and analysed using methods including binary logistic regression, bootstrapping, Chi-Square, Cochran-Armitage trend test, Cox proportional hazards, Mann-Whitney U, median test, multiple linear regression, ordinal logistic regression, t-test and the Win Ratio test. Regressions were performed adjusted and unadjusted. Global analysis techniques included the Wei-Lachin test (for ordinal/continuous outcomes), the Wald test (for binary outcomes) and the Hotelling's T2 test (for continuous outcomes). Once the appropriate tests has been performed the resulting p-values were ordered within each trial set and given a rank, with the smallest p-value given the top rank. For the primary and global analyses the Freidman and Duncan's multiple range tests were then performed to identify significant differences between statistical methods. In the case of the TARDIS trial analysis, the ranks of the p-values were compared using heat maps. Data from 35 trials (257,749 patients) were shared, thirteen trials had multiple comparator arms, resulting in 59 datasets. The most commonly shared outcomes were stroke, myocardial infarction (MI) and bleeding. When compared against analysis methods performed on their binary counterparts (event/no-event), the analyses performed on the extended outcomes tended to be more efficient; e.g. Mann-Whitney U for stroke and MI outcomes (both p<0.0001) and multiple regression for bleeding event outcomes (P<0.0001). As for the TARDIS analysis, ordinal versions of vascular event outcomes were created in 3096 participants for stroke, MI, major cardiac events, bleeding events, serious adverse events and venous thromboembolism (VTE), with 32 outcomes being created overall (29 in the subgroup population due to the absence of VTE events). Overall, tests run on the ordinal outcome appeared more efficient for 20/32 (63%) of the analyses in comparison with binary tests. 764 (24.7%) participants were recruited within 24 hours of a mild stroke/TIA; again, tests run on ordinal outcome were superior in 27/29 (93%) of the comparisons. For the analysis including global analysis techniques, of the 35 trials shared, 24 (158,901 patients) had outcome data for both stroke and MI. From these 24 trials it was possible to develop 35 comparator datasets, as 5 of the trials had multiple intervention arms. With the introduction of further levels based on severity (that is, 4 or 5-level stroke with 3-level MI), the Wei-Lachin test had the smallest average rank and therefore appeared to be more efficient statistically than those that analysed the composite outcomes (p<0.0001). This study found that methods that are used when analysing polychotomised outcomes appear to be more efficient than those used for binary outcomes. This was shown for both the primary analysis and the secondary TARDIS analysis. Based on this, there could be a potential role for using ordinal outcomes in vascular prevention trials. Furthermore, the global analysis suggested that, where there are multiple outcomes of interest, there could also be benefits to basing the primary measure on global techniques, as they appeared to be more efficient statistically.

Stroke remains a major cause of death and disability worldwide, despite advances in prevention and treatment. Improvements in acute care have led to many surviving after an incident stroke event. However, the prognosis after surviving remains compromised. This is due to the high risk of recurrent adverse cardiovascular events, greatest during the first year but persisting over one's lifetime. Reducing long-term residual cardiovascular risk and improving quality of life are primary goals for clinical practice and research. Identifying patients at the greatest risk of subsequent major adverse cardiovascular events (MACE) could help clinicians and policymakers determine which patients need to be prioritised. This thesis research aimed to identify clinical phenotypes (that is, patient characteristics and distinct patient clusters) that correlate with subsequent MACE outcomes (defined as a diagnosis of either CHD, recurrent stroke, PVD, heart failure, or CVD-related mortality) in adults with an incident stroke diagnosis. Firstly, a systematic review was completed to identify and summarise the available evidence on prognostic models and assess their accuracy for predicting MACE outcomes in an adult with established stroke. Forty (40) full-text articles with 23 distinct prognostic models for predicting MACE outcomes in adults with established stroke were identified by the systematic review. There were 11 prognostic model developments and 77 external validations of models reported. Among the 23 models, the most frequently used predictors were age, sex, history of transient ischaemic attack, hypertension (blood pressure), and diabetes. Critical appraisal identified methodological limitations, in particular: inadequate sample size, improper handling of missing data, and incomplete evaluation of model performance. The Clinical Practice Research Datalink (CPRD GOLD), a longitudinal database of anonymised electronic health records (UK primary care data) linked to Hospital Episode Statistics (HES APC), national death registry, and social deprivation data was then used to undertake a series of data-related studies. Four cohort studies were completed using patients aged ³18 years with an incident stroke diagnosis between 1 January 1998 and 31 December 2017, and no prior history of either CHD, PVD or heart failure, to assess the risk of subsequent cardiovascular morbidity and mortality outcomes. In the analysis of 9,997,376 individual records in CPRD GOLD database, there were 82,774 non-fatal incident stroke events recorded in either primary care or hospital data - a stroke incident rate of 109.20 per 100,000 person-years (95% CI: 108.46 - 109.95). Of the 82,774 patients, 13,879 (16.8%) patients had a prior history of major adverse outcomes (CHD, PVD, and heart failure) and were excluded. Subsequent MACE was recorded in 47,500 (69.0%) of the remaining 68,877 patients. In the UK, the incidence of stroke and subsequent major adverse cardiovascular morbidity and mortality outcomes were higher in women, older populations, and people living in socially deprived areas. After excluding patients with stroke not-otherwise specified (n=36,551) and adjusting for potential confounders, patients with incident haemorrhagic stroke (n=6,535, 20.4%) had no significantly different risk of subsequent cardiovascular morbidity, compared with patients with incident ischaemic stroke (n=25,556, 79.6%) - CHD [HR 0.86, 95% CI 0.56 - 1.32], recurrent stroke [HR 0.92, 95% CI 0.83 - 1.02], PVD [HR 1.15, 95% CI 0.56 - 2.38], or heart failure [HR 1.03, 95% CI 0.61 - 1.74]. However, patients with incident haemorrhagic stroke had a significantly higher risk of subsequent CVD-related mortality [HR 2.35, 95% CI 2.04 - 2.72] and all-cause mortality [HR 2.16, 95% CI 1.94 - 2.41]. Propensityscore matched analysis of 1,039 patients with haemorrhagic stroke and 1,039 with ischaemic stroke showed similar risk in subsequent cardiovascular morbidity outcomes - CHD, recurrent stroke, PVD and heart failure. Obesity, a risk factor for stroke and is also a risk factor for hypertension and diabetes (known risk factors for CVD), is commonly measured using body mass index (BMI). In a multivariable analysis of a cohort of 30,702 patients with incident stroke and BMI record, individuals in higher BMI categories were associated with lower risk of subsequent: • MACE [overweight (BMI: 25.0-29.9 kg/m2): HR 0.96, 95% CI 0.93 - 0.99)], • PVD [overweight: HR 0.65, 95% CI 0.49 - 0.85; obesity class III (BMI: ³40 kg/m2): HR 0.19, 95% CI 0.50 - 0.77], • CVD-related mortality [overweight: HR 0.80, 95% CI 0.74 - 0.86; obesity class I (BMI: 30.0-34.9 kg/m2): HR 0.79, 95% 0.71 - 0.88; class II (BMI: 35.0-39.9 kg/m2): HR 0.80, 95% CI 0.67 - 0.96]; and • all-cause mortality [overweight: HR 0.75, 95% CI 0.71 - 0.79; obesity class I: HR 0.75, 95% CI 0.70 - 0.81; class II: HR 0.77, 95% CI 0.68 - 0.86] when compared with those with normal BMI. The results were similar irrespective of sex, smoking status, history of diabetes mellitus or cancer at the time of incident stroke. Using a combination of data-driven feature selection approaches and clinical expert opinion, 39 out of 336 characteristics (clinical features including sociodemographic, biochemical, comorbid conditions, and prescribed medications related to stroke or CVD) at the time of incident stroke were selected. An unsupervised machine learning approach [clustering algorithm for mixed (both categorical and continuous) data] was used to identify 4 phenotypic clusters for a cohort of 48,114 patients with incident stroke and subsequent outcomes occurring 30 days after incident stroke. Cluster 1 (n=5,201, 10.8%) was a cohort with high prevalence of CHD-related risk factors and prescribed medications; cluster 2 (n=18,655, 38.8%) a cohort with low prevalence of multiple long-term conditions (MLTC); cluster 3 (n=10,244, 21.3%) a cohort with high prevalence of MLTC; and cluster 4 (n=14,014, 29.1%), the oldest population cohort and predominantly female. The phenotypic clusters had different incidences and risks for subsequent cardiovascular morbidity and mortality outcomes. For instance, the incidence of the composite outcome of recurrent stroke and CVD-related mortality was lowest in cluster 1 and highest in cluster 4 (15.13 and 23.17 per 100 person-years, respectively). The risk of subsequent recurrent stroke + CVD-related mortality was significantly increased in cluster 2 (HR 1.07, 95% CI 1.02 - 1.12); cluster 3 (HR 1.20, 95% CI 1.14 - 1.26), and cluster 4 (HR 1.29, 95% CI 1.26 - 1.33), when compared with cluster 1. Findings from this thesis research indicate patients with incident stroke experience considerable heterogeneity in subsequent clinical outcomes. In particular, women, older patients, and those living in socially deprived areas are at greater risk of subsequent major adverse outcomes. Additionally, age at incident stroke, blood pressure, LDL cholesterol level, a diagnosis of hypertension and potency of prescribed statin were identified as key indicators of patients' phenotypic clusters and associated risk for subsequent clinical outcomes. The studies add to growing and wider evidence to identify those who may most benefit from, and be least likely to be harmed by, preventive treatment. Stratifying patients with stroke early, could lower the burden of subsequent adverse clinical outcomes, improve patients' long-term outcomes, and reduce the associated economic burden. This should, therefore, be a continuing research and public health priority.

Background: The biopsychosocial model suggests that chronic pain is the result of a dynamic interplay between biological, psychological, and social factors. Psychological variables, such as anxiety, are recognised as key contributors to chronic pain. However, their role, and particularly that of cognitive ability, in chronic pain from osteoarthritis (OA) ¬- the main cause of chronic pain worldwide - is less well understood. Social stressors, such as social deprivation may further explain the pain experience and may predict increased functional disability (limitations in activities of daily living), but their interaction with psychological factors has not been studied much, especially in the early stages of OA. Meanwhile, anxiety and social isolation extending over many months during the coronavirus pandemic may exacerbate pain in chronic pain patients, but this has not been investigated over time. Chronic pain in OA is underpinned by neuropathic and pain sensitization mechanisms that may predict resistance to therapy. Considering biological factors, such as host genetics, may enhance our understanding of these key mechanisms in OA. Lastly, little is known about the gut microbiome's role in chronic pain. Identifying the gut microbiome's link with anxiety may give us some understanding of how it might be linked to pain. Therefore, the aim of the current thesis was to explore how psychosocial factors (cognitive ability, anxiety and social deprivation/isolation), host genetics and the gut microbiome, interact in their contribution to chronic pain. Objectives: [1] to design a study aimed at investigating the specific neuro-cognitive domains that are impaired in OA pain, [2] to explore the dynamic interplay between key socioeconomic (i.e. social deprivation, education) and psychological factors (i.e. cognitive function, anxiety) and limitations in activities of daily living in OA pain; [3] to explore the potential impact of the first coronavirus lockdown on social isolation, anxiety, pain and overall quality of life in chronic pain patients; [4] to identify genetic markers associated with pain sensitisation and neuropathic-like pain in OA; and [5] to identify bacterial taxa reproducibly associated with anxiety and the role of serum levels of microbial metabolites and neuromodulators. Methods: [1] We recruited eight participants from the Nottinghamshire area to conduct a feasibility study. Distinct cognitive functions were measured using the Cambridge Neuropsychological Test Automated Battery (CANTAB). Pain measures included Quantitative Sensory Testing and pain questionnaires, such as painDETECT. Confounders were measured, including depression/anxiety, sleep quality, quality of life and physical function. Additional cognitive measures were taken, along with the National Adult Reading Test that served as a proxy of general premorbid IQ. Data on prosociality were also collected. For identifying molecular biomarkers, gut microbiome measures and genetic markers, blood, urine and faecal samples were collected. We assessed the feasibility of the study based on the experiences of the patients. Descriptive statistics were reported and correlational analysis was used to investigate the relationship between cognitive function, anxiety and pain. [2] We used data from waves 4, 5, 6 and 7 of the Survey of Health, Ageing and Retirement in Europe (SHARE) (n = 971) and selected a subsample of respondents who initially did not report a diagnosis of OA until wave 6. We used path models to test how social deprivation, education and anxiety, before diagnosis (waves 4 and 5), affect the relationship between cognitive ability, pain and limitations in activities of daily living following diagnosis (waves 6 and 7). [3] We analysed data from a sample of chronic pain patients (N = 305 of which 28.9% reported having OA) from Investigating Musculoskeletal Health and Wellbeing cohort thrice during the pandemic (May 2020, July 2020, September 2020). We employed repeated measures ANOVAs (and Kruskal-Wallis where appropriate) to compare measures of affect, social isolation, exercise changes due to COVID-19, quality of life and pain at the various time points. [4] We performed a genome-wide gene-based association study using pressure pain detection thresholds (PPTs) from distal pain-free site (anterior tibia), a measure of distal sensitisation, and from proximal pain-affected site (lateral joint line), a measure of local sensitisation, in 320 knee OA participants from the Knee Pain and related health in the Community (KPIC) cohort. We next performed gene-based fixed-effects meta-analysis of PPTs and a neuropathic-like pain phenotype (painDETECT) using genome-wide association study data from KPIC and from an independent cohort of 613 OA participants, respectively. [5] We meta-analysed data from two cohorts (WebEx: n = 37 and Omega 3/Fibre: n = 58) and published literature (n = 60) in order to identify bacterial taxa reproducibly associated with anxiety. We further investigated the role of serum levels of SCFAs and indoles on anxiety and the links between anxiety-associated operational taxonomic units (OTUs) and neuromodulators involved in anxiety. Gut microbiota was characterized using 16S rRNA sequencing in stool samples. OTU data were transformed and normalized. Serum metabolites were measured using tandem mass spectrometry. Results: [1] The study was feasible and all practical aspects were executed as planned, and the study was well received by the patients. Our preliminary findings suggested that anxiety, pain and cognitive ability are correlated and that global cognitive impairment and impairments in certain aspects of cognitive ability might be associated with poor physical function, increased neuropathic-like pain and increased central pain processing. [2] We showed that high social deprivation before diagnosis predicted greater limitations in activities of daily living after diagnosis, with this effect partly mediated by impaired cognitive ability. We also found that higher educational attainment before diagnosis may protect against limitations in activities of daily living after diagnosis via better cognitive ability and lower anxiety. [3] Covid-19 lockdown increased anxiety in May and September, whereas pain levels were unaffected. The highest levels of social isolation and depression occurred in the early stages of lockdown but declined thereafter. The at least a bit anxious group had heightened pain in May and September compared to the not at all anxious group. Doing at least an hour of exercise per day (on a typical day) was a protective factor against increased anxiety during lockdown, and quality of life improved by September when lockdown rules were eased. [4] The most significant genes associated with distal and local sensitisation were OR5B3 and BRDT, respectively. We also found previously identified neuropathic pain-associated genes - KCNA1, MTOR, ADORA1, and SCN3B - associated with PPT at the anterior tibia and an inflammatory pain gene - PTAFR - associated with PPT at the lateral joint line. Meta-analysis results of anterior tibia and neuropathic-like pain phenotypes revealed genes associated with bone morphogenesis, neuro-inflammation, obesity, type 2 diabetes, cardiovascular disease and cognitive function. [5] We identified reproducible microbiome associations of anxiety with Escherichia-Shigella (fixed-effect beta (95% CI) = 0.28 (0.11, 0.45), False Detection Rate (FDR)-adj. p = 3 x 10−3), Christensenellaceae R-7 group (fixed-effect beta (95% CI) = -0.32 (-0.48, -0.15), FDR-adj. p = 1 x 10−3) and Ruminococcaceae UCG-010 (fixed-effect beta (95% CI) = -0.24 (-0.40, -0.08), FDR- adj. p = 6 x 10−3). We found no significant associations between anxiety and gut-derived metabolites nor between anxiety-associated bacteria and anxiety-related markers after meta-analysis and correcting for multiple testing. Conclusion: This thesis showed that improving cognitive ability and reducing anxiety early in the disease course might protect against the negative impact of socioeconomic factors on subsequent functional disability in painful OA. Furthermore, regular exercise during lockdown and a balanced gut microbiome may contribute to reduced anxiety levels in chronic pain. Therefore, interventions like cognitive behavioural therapy, exercise, and probiotic supplementation might alleviate the influence of pain and anxiety on poor health outcomes in older adults with OA. Future research will investigate the role of inflammation, and the gut microbiome in the link between pain sensitisation and psychosocial factors, such as cognitive ability.

Multiple Sclerosis (MS) is the most common chronic neurological condition affecting young adults. Many people are diagnosed with MS while they are of working age, and many leave the workplace prematurely. Vocational Rehabilitation (VR) aims to support those with illness or disability to find new employment, remain in or return to work (RTW). The effectiveness of VR for people with MS is inconclusive. This thesis aimed to develop, implement, and evaluate a job retention VR intervention for employed people with MS. The VR intervention was developed following the Medical Research Council Framework for developing and evaluating complex interventions and the person-based approach (PBA). The first study was a systematic review to identify VR interventions implemented to support people with MS to find new employment, remain in or RTW. Fourteen studies met the inclusion criteria describing thirteen interventions. There was considerable variability across the interventions and no clear conclusion about the most effective intervention characteristics or components was reached due to the poor reporting of the interventions. The second study was a qualitative study to explore the experiences of people with MS who work, their needs for VR, and perceived barriers and facilitators to implementing the intervention. I conducted 20 semi-structured interviews with people with MS, healthcare professionals, and employers. Analysis was informed by the framework method and interviews were underpinned by theoretical frameworks. I identified nine themes reflecting the main MS symptoms (e.g., cognition, fatigue), difficulties at work, and support received (e.g., change of working hours). Providing tailored support and early intervention were seen as important attributes for the intervention. The main barrier identified to delivering VR support referred to lack of resources. Having flexibility in the intervention delivery was seen as a facilitator to receiving VR. The findings were combined following the PBA to develop the intervention's guiding principles, logic model, and a job retention intervention. The feasibility and acceptability of the intervention were tested using a mixed-methods single case study design. Secondary objectives included determining whether receiving this intervention was associated with changes in quality of life, fatigue, functional outcomes, and goal attainment. The intervention was tested between June 2020 and January 2021. I recruited 15 participants with MS, 3 employers and 4 healthcare professionals. On average the participants with MS received 8.36 (SD=4.48) hours of intervention and the employers received 1.94 (SD=0.38) hours. The most common topics addressed were managing cognitive problems, fatigue management, and reasonable accommodations. The intervention was only delivered remotely due to the Covid-19 pandemic. It was feasible to deliver the intervention, but it had no impact on quality of life, fatigue, and functional outcomes. However, it had a positive impact on goal attainment. Compared to baseline, the paired t-test showed a significant difference on goal attainment at the post-intervention assessment (t(14)=7.44, p=.0001, d=1.9), three (t(13)=4.81, p=.0001, d=1.28), and six (t(11)= 4.45, p=.001, d=1.28) months follow-up. Participants reported that the intervention was acceptable in the post-intervention interviews. Four themes were derived from the post-intervention interviews regarding the (1) context, (2) the employer, (3) empowerment, and (4) intervention components and attributes. Future research should focus on understanding how VR interventions can be embedded within existing healthcare services.

Background Strong evidence exists relating to the efficacy of Early Supported Discharge (ESD) which has been synthesised in several Cochrane systematic reviews. National clinical guidelines recommend that ESD be offered to all eligible stroke survivors, however service models vary significantly and there are still areas of England where ESD has not been commissioned. Additionally, some services, particularly in rural areas, report challenges in delivering evidence-based services. The organisational core components necessary for evidence-based service implementation have been identified, informed by international consensus and randomised controlled trials. It is unknown if or how these organisational components facilitate the delivery of evidence-based community stroke services in rural areas. Methods A theory building realist evaluation case study of community stroke services delivering ESD in rural areas of England was conducted. Three exemplar rural services were identified from the post-acute phase 2 stroke sentinel national audit programme as fulfilling key evidence-based criteria as identified in the national clinical guidelines for stroke. Incorporating evidence from the randomised controlled trials and a panel of expert advisors, five initial candidate programme theories were developed to explain how the core components underpin evidence-based service delivery in rural services. These programme theories were iteratively tested and refined during a 12-month data collection period taking place from June 2017 to May 2018. Evidence from realist interviews, observations, and several informal conversations, and documents were interrogated to identify hidden causal mechanisms and corresponding contextual features influencing evidence-based service delivery in line with national clinical guidelines. Data analysis incorporated inductive, deductive, abductive and retroductive inferencing to generate context-mechanism-outcome configurations for each programme theory under evaluation. Findings Programme Theory One: The multidisciplinary team and interdisciplinary working identified the importance of a comprehensive team composition including service leads, rehabilitation assistants and administrators with staffing levels adjusted for rurality. A supportive interdisciplinary team culture alongside service leads who empower skill development were also identified as important factors of teamworking. Working in an inter and transdisciplinary manner was identified as a key mechanism to facilitate the delivery on evidence-based services resulting in improved service efficiency and delivery of holistic inter and transdisciplinary care. The lack of psychologists within the stroke services was thought to impede the level of interdisciplinary working and resulting in unmet needs for patients. This negatively impacted upon the self-confidence of staff who did not possess sufficient knowledge or skills to appropriately treat patients with psychological needs. Programme Theory Two: Inter and transdisciplinary training was inextricably linked with programme theory one and identified the importance of providing formal and informal opportunities for all staff, including administrators, to develop inter and transdisciplinary skills. Training was provided in house and delivered by staff within the service. Comprehensive competency-based training for rehabilitation assistants equipped these members of staff with appropriate knowledge, skills and confidence to engage in transdisciplinary working. Training also facilitated interprofessional trust associated with the delegation of specialised rehabilitation tasks. Programme Theory Three: Multidisciplinary Meetings found that weekly MDMs facilitated shared decision-making, inter and transdisciplinary working and increased the likelihood of adhering to key Stroke Sentinel National Audit Programme governance criteria and national clinical guidelines. For services with high caseloads, meeting effectiveness was facilitated through effectively chaired and formally structured meetings. Given that rural community stroke services have reduced opportunities for communication, meetings provided a forum for interdisciplinary discussions which occurred in supportive team climates and enabled staff to develop a shared understanding of patient and service objectives. Programme Theory Four: Care Transitions identified that functional proximity between services, collaborative working and good interpersonal relationships between service leads facilitated smooth, timely and appropriate care transitions between services. Cross-service collaboration encouraged the development of trust, role clarity and shared understanding of patient and service needs. Conversely, hard organisational boundaries created barriers to cross-service collaborations and negatively impacted upon care transitions. Programme Theory Five: Visit Scheduling was identified during data collection and suggested that implementing structured visit scheduling facilitated the delivery of equitable and efficient services. This was particularly true for the services with high caseloads operating over large geographical areas. Conclusion This realist evaluation has provided a valuable insight into how stroke service organisation, coordination and teamworking facilitates the delivery of evidence-based services in line with national clinical guidelines in rural areas of England. The revised programme theories highlight the importance of supportive service leads, collaborative inter and transdisciplinary working from all staff including administrators, the use of highly trained transdisciplinary rehabilitation assistants to deliver rehabilitation and structured visit scheduling as essential components to the successful delivery of services with large caseloads. Given that some services in rural areas have found it difficult to deliver evidence-based services, it is hoped that these findings provide some meaningful detail for service leads and healthcare providers to adapt and implement within their own stroke services both in rural and urban areas.

Background It is estimated that as many as 86% of people with Multiple Sclerosis (MS) can experience cognitive impairment, which can impact quality of life. Although the value of screening for cognitive problems is recognised, it is not routinely undertaken. Methodology The research comprises five studies to identify acceptable and appropriate methods of assessing cognitive impairment in people with MS. A systematic review (Study I) was conducted to determine the most frequently used cognitive assessments in MS research. Study II focused on one test, the Symbol Digit Modalities Test (SDMT), and explored the variability in scoring and interpretation in MS research. In Study III the domain structure of an MS-specific self-report measure of cognitive problems, and how it relates to an objective measure of information processing speed, was explored. Finally, two qualitative interview studies were conducted. Study VI examined stakeholders' experience and opinions of cognitive impairment and assessments, and explored perceived barriers and facilitators to implementation in clinical practice. In Study V, people with MS completed a digital test and were asked to 'talk through' the process. Results The systematic review (Study I) extracted data from 1,526 studies, using 5,665 cognitive assessments. The SDMT emerged as the most frequently used individual test. Focusing on this, there was variability in how results of this test are interpreted for people with MS noted in Study II. Whilst the SDMT is a promising objective measure of information processing speed, the results of Study III suggest that a reduced, multidimensional questionnaire has more promise than its unidimensional version and could be used in place of an objective test. In Study IV, people with MS and clinicians reported that, despite being a prevalent symptom, cognitive impairment was not assessed routinely. Participants felt that symbol substitution tests and subjective self-reported questionnaires were acceptable and appropriate in assessment and could be completed digitally on personal mobile devices. This would also be feasible to implement in NHS clinical care. Based on these findings, a webapp was developed, and Study V identified usability problems, and barriers and facilitators to its use. Conclusions Future research is needed to assess the psychometric properties of the developed digital task. Normative data from non-MS control participants should also be collected to provide benchmarks for interpreting clinical data. Digitised, remote assessments can enable more people with MS to access screening, which can facilitate discussion and referral for cognitive rehabilitation. Overall, the results of this research indicate the potential of using digital, remote cognitive assessment with a symbol substitution task in MS clinical care.

Changes in cerebral perfusion or metabolism can occur as a result of healthy ageing, and in conditions of impaired ageing such as mild cognitive impairment (MCI) or Alzheimer's disease (AD). Overarchingly, this thesis aimed to explore physiological magnetic resonance imaging (MRI) measures to study both cerebral perfusion and metabolism in the healthy ageing brain. Specifically, arterial spin labelling (ASL) and functional magnetic resonance spectroscopy (1H-fMRS) were employed in the elucidation of healthy ageing. Investigation of cerebral functionality is clinically important, enabling understanding of healthy ageing and disease pathology beyond that provided by structural measures. Given the necessity for tightly-regulated tissue perfusion in the delivery of oxygen to the brain, assessment of brain perfusion can enable elucidation of related brain health. Firstly, this thesis focused on changes in brain perfusion within a cross-sectional retrospective cohort of healthy subjects. This study aimed to assess the utility of univariate and multivariate pattern analysis (MVPA) techniques, and determine whether spatial coefficient of variation (sCoV) measures - which provide a method for inferring spatial heterogeneity of blood flow from single post-label delay (PLD) ASL data - are more significantly associated with age than standard perfusion metrics (ml/100g/min values). The impact of data processing steps on quantification of perfusion was initially assessed. Particularly, the influence of partial volume effect (PVE) correction and how this affected quantification of cerebral perfusion was of interest. The relationship between measures of cerebral perfusion - in regions of interest, vascular territories, and grey matter - and age were assessed, before grey matter (GM) spatial covariance patterns were identified, with MVPA hypothesised to elucidate more subtle age-related change than univariate, voxel-wise methodology. The executive control network (ECN) was the only network exhibiting a significant decline in perfusion with age, after controlling for relevant covariates. Interestingly, whilst the PCA approach resulted in a pattern of both positive and negative associations with age across cerebral GM, the surviving clusters in voxel-wise approaches were deemed spurious. Five-fold cross validation of PCA findings was used to assess whether the resultant spatial covariance patterns were able to predict subject age. This prediction was successful, with related r2 values of between 0.5316 and 0.7297 (p < 0.001 for all), however validation of these findings in an unseen dataset is required. The utility of the sCoV metric was also compared with standard tissue perfusion values, finding that sCoV may be more closely associated with ageing than ml/100g/min in certain regions. Particularly, a significant increase in whole GM sCoV with age was notable, given the absence of significant changes in perfusion with age in the same region. Additionally, a MVPA approach was used to establish the complex unknown relationship between cerebral perfusion and the Montreal Cognitive Assessment (MoCA), before graph visualisation was used to further understand the regional relatedness of the spatial covariance pattern. PCA resulted in a model which provided a moderate explanation of the aforementioned relationship, but this may be improved by inclusion of additional covariates in subsequent work, such as those pertaining to genetic status, such as apolipoprotein E (APOE). This study also replicates an FDG PET cognitive resilience signature in an ASL cohort for the first time, with a trend towards declining perfusion with age found (p = .08). Lastly, as ageing is associated with metabolic failure in the brain, which is often investigated using methodology which employs ionising radiation, the final study was motivated to investigate possible metabolic markers of brain ageing which can be measured using MRI. Metabolic-functional coupling can be studied using functional stimulation, and functional magnetic resonance spectroscopy (fMRS) is perfectly poised to elucidate certain metabolic behaviour. Given the close relationship between glucose (Glc) - the key fuel for cerebral functionality - and lactate (Lac) metabolism, an optimised long echo time (TE) semi-localized by adiabatic selective refocusing (semi-LASER) sequence (TE=144ms) with optimised J-modulation selection at 7T was employed to assess the effects of age on the dynamic behaviour of Lac, and determine its absolute concentrations throughout the time course, whilst a visual stimulation paradigm was viewed. Successful quantification of metabolite concentrations - including Lac, tCr and tNAA - was achieved in both the young and old cohorts, and their Lac peaks clearly visually identifiable throughout the time course. A significant increase in Lac concentration was observed between rest and stimulation, but not stimulation and recovery, in the young cohort. No significant Lac time course changes were identified in the full old cohort. This thesis concluded by summarising and contextualising the key findings herein, and discussion of possible directions for further associated research. The findings of this thesis broaden the field of knowledge around healthy ageing, and therefore may contribute to subsequent translation efforts for both clinical diagnostics and treatment approaches.

Background: Due to the world's ever-growing and ageing population, dementia and cognitive impairments are major global health and socioeconomic burdens. Stroke is a leading cause of acquired cognitive impairment and dementia. Multiple sclerosis (MS) although less common than stroke, also causes cognitive impairment and in rare cases overt dementia. The risk factors for cognitive decline are still not fully understood and there is currently no recommended intervention for preventing cognitive decline. Therefore, we must determine which risk factors are associated with cognitive impairment and which interventions can help to delay or prevent the onset of cognitive impairment, to reduce the number of people who suffer from cognitive decline. Methods and results: The clinical and radiological predictors of post-stroke cognitive impairment (PSCI), following an ischaemic stroke (IS) or transient ischaemic attack (TIA) in previously independent adults, were investigated using data from the international multicentre randomised control trials, ENOS and TARDIS. Together (Chapter 3), this dataset of 4798 participants, is one of the largest international hospital-based studies of PSCI to date. The predictors of post-stroke cognitive decline in this cohort were older age, greater stroke severity (National Institute of Health Stroke Scale (NIHSS)), pre-stroke disability (modified Rankin Scale (mRS)), non-UK participants, history of atrial fibrillation, lower systolic blood pressure, and higher heart rate. The clinical and radiological predictors of PSCI, following an intracerebral haemorrhage (ICH) in previously independent adults, were investigated using data from the international multicentre randomised control trial, TICH-2 (Chapter 4). This study involved 693 participants and is one of the largest cohorts of ICH survivors that focused on cognitive impairment. The predictors of long-term SCI were older age, non-Caucasian ethnicity, a greater level of deprivation (Index of Multiple Deprivations (IMD)), reduced level of consciousness (Glasgow coma scale (GCS)) and lobar ICH. The effectiveness of intensive blood pressure and lipid-lowering on cognitive outcomes in patients with a recent stroke was investigated in the multicentre randomised trial, PODCAST (Chapter 5). Intensive BP and lipid-lowering did not alter cognition and did not have any long-lasting effects on cognition in participants approximately 5 years after stroke. Intensive lipid-lowering might still be partially effective at 33 months according to the global outcome and global cognition analysis. The effectiveness of live hookworm infection on cognition in multiple sclerosis (MS) patients, as well as the clinical and radiological associations of cognitive function in MS patients, was investigated in the WIRMS trial (Chapter 6). We showed that there were no effects of hookworm infection on cognitive function, quality of life or fatigue over the 6 months of active treatment. We also showed there was no significant change in the cognition and functional outcomes over time in all participants and across both treatment groups. Worse cognitive function was associated with a greater number of T2 lesions and a poorer quality of life. Discussion: The key predictors of cognitive impairment after IS and ICH were older age, a greater stroke severity, and non-UK/non-Caucasian ethnicity. Blood pressure and heart rate were identified as potential therapeutic targets for the prevention of cognitive decline after IS. An important predictor of cognitive decline after ICH was a greater level of social deprivation. Blood pressure and lipid interventions in stroke patients, and parasitic infection in MS patients, did not affect cognitive function, however, they are still feasible and require further study. A major limitation of cognition studies is the loss of participants to follow up due to disease severity therefore informant-based cognition questionnaires are essential. Future research should consider accounting for a participant's level of education, ethnicity and other race-related factors, socioeconomic status, and should perform MRI scans and genetic analysis (AD biomarkers) where possible. Conclusion: In conclusion, cognitive impairment due to stroke or multiple sclerosis is a socioeconomic burden that will continue to grow without further research into its risk factors and in turn potential interventions.

Background: Multiple Sclerosis (MS) is a chronic inflammatory disease of the central nervous system, characterised by an abnormal immune system response which causes axonal demyelination. MS presents a range of debilitating cognitive, sensory, motor and affective symptoms. Some of the symptoms of MS are classified as 'invisible', due to the fact that they are not easily observable to others and are 'hidden'. Common invisible symptoms include cognitive impairment, fatigue, pain, bladder and bowel dysfunction, sexual problems, and sensory dysfunction. Research shows that living with invisible symptoms causes distress for people with MS that differs from that of visible symptoms, not only because of their physical impacts, but because of their invisible nature. Studies highlight the issues associated with invisible symptoms, whereby people with MS do not feel understood or believed by others in relation to their invisible symptoms and attempt to navigate their difficulties brought about by this. There is a dearth of research offering in-depth exploration of lived experience of a cluster of invisible MS symptoms. Aims: This research aimed to explore how people with MS experience living with and managing invisible symptoms in daily life. Method: Photovoice is a participatory visual research method, in which people are asked to produce their own images in order to share their experiences of a particular aspect of health and illness. Photovoice was used in this study to explore people's lived experience of MS invisible symptoms. Twelve people with MS produced digital images over a two-week period to capture their experiences of living with and managing their invisible symptoms. Participants discussed their images and respective meanings in semi-structured interviews. The semi-structured interviews were analysed using an Inductive-Deductive Thematic Analysis. Results: Three main themes and eight subthemes were developed. The first main theme encompasses the difficulties that exist around conceptualising invisible symptoms, not only due to their invisibility, but because people find it difficult to use language to accurately communicate their abstract symptom experiences. The second theme describes the conflicts of legitimacy this presents for people with MS, whereby they struggle to validate the reality of their invisible symptoms and this reality is also invalidated by others. The third theme elucidated how participants navigate these issues in dynamic ways, choosing to fit their symptoms to their lives or make space for their symptoms depending upon the context, often influenced by their desire for their symptoms to 'stay invisible' or to 'be seen'. Choosing how to navigate invisible symptoms places a continuous burden on people with MS. Discussion: This research provides insight into the challenges faced by people living with invisible MS symptoms, and the ways they negotiate daily life. It is important that clinicians and healthcare professionals are aware of the conflicts created by symptom invisibility so that they can offer appropriate support to people with MS. Further research is suggested to explore determinants of particular navigation styles, and also to explore whether improved psychological flexibility could support people with MS to navigate their invisible symptoms and associated impacts.

The human brain is a highly complex organ that integrates functionally specialised subunits. Underpinning this complexity and functional specialisation is a network of structural connections, which may be probed using diffusion tractography, a unique, powerful and non-invasive MRI technique. Estimates of brain connectivity derived through diffusion tractography allow for explorations of how the brain's functional subunits are inter-linked to subsequently produce experiences and behaviour. This thesis develops new diffusion tractography methodology for mapping brain connectivity, both across individuals and also across species; and explores frameworks for discovering associations of such brain connectivity features with behavioural traits. We build upon the hypothesis that connectional patterns can probe regions of functional equivalence across brains. To test this hypothesis we develop standardised and automated frameworks for mapping these patterns in very diverse brains, such as from human and non-human primates. We develop protocols to extract homologous fibre bundles across two species (human and macaque monkeys). We demonstrate robustness and generalisability of these protocols, but also their ability to capture individual variability. We also present investigations into how structural connectivity profiles may be used to inform us of how functionally-related features can be linked across different brains. Further, we explore how fully data-driven tractography techniques may be utilised for similar purposes, opening the door for future work on data-driven connectivity mapping. Subsequently, we explore how such individual variability in features that probe brain organisation are associated with differences in human behaviour. One approach to performing such explorations is the use of powerful multivariate statisitical techniques, such as canonical correlation analysis (CCA). After identifying issues in out-of-sample replication using multi-modal connectivity information, we perform comprehensive explorations into the robustness of such techniques and devise a generative model for forward predictions, demonstrating significant challlenges and limitations in their current applications. Specifically, we predict that the stability and generalisability of these techniques requires an order of magnitude more subjects than typically used to avoid overfitting and mis-interpretation of results. Using population-level data from the UK Biobank and confirmations from independent imaging modalities from the Human Connectome Project, we validate this prediction and demonstrate the direct link of CCA stability and generalisability with the number of subjects used per considered feature.

Background: Multiple Sclerosis (MS) is a progressive neurodegenerative disease that impacts on a range of both proximal and distal outcomes. People with MS commonly experience cognitive problems that have been shown to affect important aspects of life such as employment, mood, and quality of life. Whilst the disease is progressive, and the damage widespread, recovery from damage through neuronal plasticity does occur. There is growing evidence base in cognitive rehabilitation offerings but limited in quality to draw firm recommendations and studies not reporting a clear theoretical underpinning of their interventions hinders development. Training (SMART: Strengthening Mental Abilities with Relational Training) of Derived Relational Responding, a fundamental component of a well-established behavioural theory of cognition - Relational Frame Theory - has been shown to improve cognitive performance as measured by Intelligence Quotient tests. Studies have begun initial applications of relational responding training in cognitive rehabilitation studies. Objectives: The primary objective was to explore the effects of SMART on both objective and subjective assessments of cognitive functioning for people with MS. Secondary objectives were to assess changes in secondary outcomes (mood, fatigue, quality of life), explore the relationship between relational responding and outcomes, and investigate the acceptability, practicality, and feasibility of the intervention. Method: The study has a multiple baseline design with a feedback interview. The study aimed to recruit nine participants. Participants completed a battery of neuropsychological assessments in addition to secondary outcome measures. Participants then completed their respective baselines. They then began a 12-week intervention of 45- minutes of training per week. Participants completed subjective assessments on a weekly basis and relational skills assessments every other week. The full battery was administered again at week six of the intervention, and again one week after the intervention had ended. A feedback interview, conducted by independent researchers, was delivered a week after the intervention ended. The full test battery was administered a final time at a three-month follow-up [1]. Results: A total of eight participants were enrolled into the study. Weekly measurements of subjective cognitive performance showed no phase contrasts. Objective measurements showed improvements. Processing speed outcomes improved in 87.5% of participants and this also represented a clinically significant improvement for 100% of participants who were in a clinically impaired range at intake. 50% of participants improved in the Repeatable Battery for the Assessment of Neuropsychological Status Total Score. Feedback interviews made recommendations to increase the intensity and duration of the intervention. Relational abilities improved in all but one participant. Secondary outcomes remained stable. Conclusions: These preliminary findings on objective assessments are promising though are questioned by the lack of subjectively reported improvements. The relationship between relational skills and improved outcomes was inconclusive. The intervention appears to have been acceptable to participants, providing support for further development.

Upwards of 50% of stroke survivors show symptoms of swallowing impairment (dysphagia) post-stroke. Dysphagia is clinically important as it results in poorer outcomes and affects quality of life. Despite this, there are few proven treatments. In addition, the act of swallowing is intricate and complex and may be best measured using multiple outcomes. This thesis had three principal aims: firstly, to evaluate the current evidence base for swallowing therapy by updating the Cochrane review into swallowing therapy in acute and subacute stroke. Secondly, to evaluate the use of multiple measures of swallowing (timing and clearance measures) to detect change following a swallowing treatment (Pharyngeal Electrical Stimulation - PES) as opposed to only using a single measure of safety, the Penetration Aspiration Scale (PAS). This was done using retrospective data analysis of videofluoroscopic data from the Swallowing Treatment using Electrical Pharyngeal Stimulation Trial (STEPS) which had already been evaluated for safety using only the PAS. And thirdly, to expand the range of outcome measures available for measuring dysphagia by validating the dysphagia severity rating scale (DSRS), a clinical outcome measure currently in use but not yet validated. The results of this thesis have confirmed three main findings with regards to clinical and radiological outcomes post-stroke. Firstly, the Cochrane review has highlighted that currently, swallowing therapy does show some positive benefits but this is based on evidence of variable quality. Recommendations for conducting more robust trials in the future are discussed. Secondly, with regards to using multiple measures, videofluoroscopic data analysis revealed that additional measures of timing and clearance did not result in the identification of any improvements that may have gone undetected using safety measures alone (PAS). However, final numbers were reduced due to data quality and lower frame rate acquisition and hence it would be premature to conclude that using the PAS alone is sufficient when measuring swallowing outcomes post-stroke. Finally, with regards to measuring dysphagia severity post-stroke, the DSRS was validated. The results showed that it appears to be a valid tool for grading the severity of swallowing impairment in patients with post-stroke dysphagia and is appropriate for use in clinical research and clinical service delivery.

Introduction: Recurrent/metastatic medulloblastoma is a devastating disease with an abysmal prognosis of less than 10% five-year survival. Whilst primary tumours can be classified based on epigenetic and transcriptomic features, there is very little information regarding molecular signatures of metastatic tumours. The secretion of extracellular vesicles (EVs) has emerged as a pivotal mediator for communication in the tumour microenvironment during metastasis. The most investigated EVs are exosomes, nanovesicles secreted by all cell types and able to cross the blood-brain-barrier. The role of exosomes as vehicles for cell-to-cell communication between a tumour and its microenvironment is a relatively new concept, with only limited study of this potential mechanism in medulloblastoma. This study aims to gain insight into the differences between exosomes of primary and metastatic medulloblastoma cell lines to better understand how exosomal cargo may account for differences seen between these phenotypes and to identify their applicability as a prognostic biomarker of increased metastatic potential. Methods and Results: Metastatic medulloblastoma cells were found to secrete markedly more exosomes compared to non-metastatic primary cells. Additionally, metastatic exosomes significantly enhanced the migration and invasiveness of primary and non-malignant cells in a transwell migration assay. Moreover, we demonstrated that the pro-migratory function of metastatic exosomes was, in part, due to tumour-promoting proteins EMMPRIN and MMP-2 enriched on their external surface. Furthermore, using zymography assay, metastatic exosomes were shown to potentiate medulloblastoma migration resulting in degradation of extracellular matrix components, via the active protease, MMP-2, on their surface. In support of this, stable genetic knockdown of MMP-2 or EMMPRIN antagonised the pro-migratory function of exosomes. Next generation sequencing was utilised to characterise exosomal RNA cargo (mRNA and miRNA). Hierarchical clustering, demonstrated that metastatic exosomes had distinct genetic cargoes compared to primary and normal exosomes. Further characterisation of the RNA delineated target pathways of importance for medulloblastoma progression and interaction with the surrounding microenvironment, in agreement with the pro-migratory phenotype conferred by metastatic exosomes. Differential gene expression analysis of mRNA sequencing data, identified PCAT1, PAPPA2 and POU5F1B as highly enriched in metastatic exosomes compared to their matched primary counterpart. These genes had previously been implicated in cancer progression in other cancer types, and in this study their high expression correlated with poor overall survival and tumour metastasis in medulloblastoma. A number of miRNAs were also identified which were differentially secreted in metastatic exosomes relative to primary and normal exosomes, yielding a candidate set of exosomal miRNAs as potential metastatic medulloblastoma biomarkers. Notably, several of the candidate miRNAs (miR-382-5p, miR-381-3p, miR-346, miR-628-5p) were detected at substantially higher levels in metastatic disease CSF samples, compared to matched CSF samples from their primary tumour. Conclusion: This study demonstrates the importance of exosomes in creating a favourable environment to drive medulloblastoma metastasis, either through extracellular matrix signalling, via surface-bound proteins, or through intracellular delivery of their RNA cargo to recipient cells. Our observations have also highlighted the possibility of CSF-derived exosomal miRNAs as biomarkers for metastatic medulloblastoma.

Introduction Medulloblastoma is the most common malignant paediatric brain tumour. Many patients present with metastatic disease and sadly this remains a powerful predictor for poor outcome. Metastasis is a complex multi-step process and the underlying molecular mechanisms behind medulloblastoma metastasis are poorly understood. One of the initial stages of this process involves the invasion of tumour cells through the basement membrane and into the leptomeningeal space. The transcription factor TWIST1 has been extensively studied in epithelial tumours and is considered as one of the master regulators initiating the first stage of this process. An in-depth analysis of the functional role of TWIST1 in initiating medulloblastoma metastasis remains to be explored. This project therefore aimed to assess the functional role of TWIST1 and whether it could play an essential part in driving medulloblastoma metastasis. Methods TWIST1 gene and protein expression was analysed in medulloblastoma patient samples to investigate if expression correlated with metastatic disease and patient outcome. Metastatic cell lines representing two out of the four medulloblastoma molecular subgroups (SHH and Group 3) were employed for in vitro analysis of TWIST1 function. A 3D transwell assay was used to quantify cellular migration and invasion through a simple basement membrane. Inhibition of TWIST1 using genetic (shRNA) and chemical (harmine, a beta-carboline alkaloid) methods was investigated. We then explored the presence of a TWIST1-ABCB1 axis, using chromatin immunoprecipitation (ChIP) and the specific ABCB1 inhibitor vardenafil. Finally, ChIP-Sequencing was carried out to define additional members of TWIST1's transcriptional regulatory network. Results TWIST1 was highly expressed across the four medulloblastoma molecular subgroups compared to the normal cerebellum. Analysis of patient tissue microarrays revealed that high expression was a strong indicator for worse event-free survival and for driving metastatic disease in medulloblastoma patients. Reduced TWIST1 expression significantly decreased medulloblastoma cell migration in the 3D transwell migration and invasion assay. ChIP analysis confirmed TWIST1 bound to the ABCB1 promoter and inhibition of ABCB1 with vardenafil reduced cell migration. ChIP-Sequencing identified many TWIST1 downstream target genes and pathways that are important in initiating the metastatic process. This included pathways and genes that are involved in interacting with cell adhesion molecules, focal adhesions and the extracellular matrix. Conclusions This thesis has shown that TWIST1 expression is crucial for initiating medulloblastoma metastasis through its ability to target pathways and genes that are essential in initiating the metastatic process. Therapeutically targeting TWIST1 in the primary tumour offers a novel approach to prevent primary tumour cells from invading the extracellular matrix and ultimately halt metastatic dissemination and, thereby greatly improving patient outcome.

Multiple Sclerosis (MS) is a frequently disabling neurological disease affecting young adults. The disease has been characterized by recurrent areas of focal inflammation (plaques) in the CNS giving rise to episodic neurological signs and symptoms. Helminth-associated immunoregulation has been investigated by the utilization of controlled hookworm infection in MS. Many studies have reported brain atrophy in patients with MS. This has been demonstrated as a major factor for physical and cognitive impairment in MS. In this thesis, I present our studies using immunomodulation, coordinate-Based Meta-Analysis (CBMA), Meta-Analysis of Networks (CBMAN), and Meta-Regression and voxel based morphometry (VBM) to study disease activity in MS. 1) For a Randomized Double-Blinded Placebo-Controlled Trial, we examined the effect of hookworm treatment on white matter (WM) disease activity. 2) Localised grey matter (GM) Atrophy in Multiple Sclerosis and Clinically Isolated Syndrome (CIS) was investigated by Coordinate-Based Meta-Analysis, Meta-Analysis of Networks, and Meta-Regression of Voxel-Based Morphometry Studies to reveal the significantly consistent regions and networks of GM atrophy in MS or CIS. 3) A further VBM was conducted in the revealed clusters to investigate the difference in GM atrophy between hookworm and placebo arms of the clinical trial, during the course of intervention.

Introduction: Metastatic medulloblastoma (MB) manifests in one third of paediatric patients at diagnosis, conferring a poor prognosis. Our limited capacity to detect dissemination at diagnosis and disease recurrence however, results in the erroneous risk stratification of paediatric patients with MB, with serious subsequent prognostic implications. Hypothesis: Significantly correlated with metastasis and poor prognosis in other solid cancers, tumour-derived enzymes that facilitate their dissemination known as matrix metalloproteinases (MMPs) and specifically MMP-2 and MMP-9, were postulated to be potential biomarkers of metastasis in MB. We hypothesised that differential levels of functionally active MMP-2 and MMP-9, as expressed and secreted by a panel of primary and metastatic sub-grouped MB cell lines and patient tumours, would correlate with their propensity to metastasise. Once biologically characterised, these findings would be translatable, using our locally synthesised 19Fluorine-MMP-MRI biosensor, which would demonstrate an increase in the fluorine signal proportional to the concentration of the MMP-2/MMP-9 within the sample, as its substrate linker is cleaved from a paramagnetic gadolinium III-DOTA complex over time. Methods: Protein microarrays, western blotting and qRT-PCR were performed to determine the levels of MMP-2 and MMP-9 protein and gene expression in nine sub-grouped primary and metastatic MB cell lines. Levels of secreted functionally active MMP-2 and MMP-9 from conditioned supernatants from the same MB cell lines and cerebrospinal fluid (CSF) from six paediatric patients with MB were assayed using gelatin zymography. Functional migration and invasion assays of 3-D cultured MB were performed to determine whether the detected levels of functionally active MMP-2 and MMP-9, would correlate to their migratory capacity and expected metastatic potential. The same samples were further examined using fluorescence resonance energy transfer (FRET) assays, to simulate and optimise the conditions for the biosensor experiments, prior to their imaging. Results: Our data strongly suggests that MMP-2 gene and protein is upregulated in metastatic MB cell lines. This was most evident in a matched pair of Group 4 MB where the recurrent cell line CHLA-01R-MED consistently expressed higher MMP-2 at all regulatory levels in comparison to its primary counterpart, CHLA-01-MED. This finding was also reflected in their increased migratory capacity in invasion assays. Zymography of MB patient CSF demonstrated significantly increased levels of functionally active MMP-2 in the recurrent samples; a finding resonated in our FRET assay experiments. 19Fluorine-MRI-MMP biosensor imaging demonstrated increased signal from the Fluorine moiety, following enzymatic degradation of the sensor by recombinant MMP-2 protein, demonstrating its capacity to detect the levels of functionally active MMP-2 and MMP-9 in MB patient cerebrospinal fluid and its potential as a clinically relevant imaging biomarker, once optimised. Conclusions: Levels of functionally active MMP-2 could prove to be a sensitive marker of metastasis in paediatric patients with medulloblastoma, at their initial diagnosis and ongoing disease surveillance. With further optimisation, we believe that our MMP-2 biosensor could become a useful adjunct as an intra-operative marker of residual metastatic cells, to enhance our limits of safe maximal resection.

The blood brain barrier (BBB) is central to the neurovascular unit (NVU) where it creates a semi-permeable barrier between neuronal tissue and the vascular networks that feed the brain. In neurodegenerative conditions and ischaemic stroke, the BBB becomes compromised and as a result its permeability increases. This not only exacerbates neuronal damage at the site of injury but also causes unwanted extravasation of peripheral immune cells into the brain, fuelling the overactivation of the immune response. Endocannabinoids and phytocannabinoids have both displayed neuroprotective effects, attenuating damage in a range of models including Parkinson's, Huntington's, amyloid lateral sclerosis and ischaemic stroke. The current study aimed to investigate the neuroprotective properties of emerging phytocannabinoids; specifically focusing on the BBB and NVU in the context of ischaemic stroke pathophysiology. A four-cell blood brain barrier model was constructed consisting of; human brain microvascular endothelial cells (HBMECs), astrocytes, pericytes and neurons. Cells were cultured on collagen coated transwell inserts and permeability was assessed using transepithelial resistance (TEER). A systematic review was conducted to examine work on the neuroprotective properties of minor phytocannabinoids, aside from cannabidiol (CBD) and delta 9-tetrahydrocannabinol (Δ9-THC). Following on from this, in vitro experiments were conducted using minor phytocannabinoids with the most neuroprotective potential; cannabidivarin (CBDV), cannabigerol (CBG) and cannabidiolic acid (CBDA). Inserts or monocultures (four cell model and pericyte, HBMECs and neuronal monolayers) were subjected to either a 4 h oxygen-glucose deprivation (OGD) protocol or an 8 h OGD (astrocyte monocultures), to model ischaemic stroke in vitro. Media was analysed for various chemokines and cytokines using enzyme-linked immunoassays or multiplex assays. From the systematic review, emerging phytocannabinoids cannabidivarin (CBDV) and cannabigerol (CBG) were found to display efficacy in various neurogenerative conditions and of the limited available mechanistic data, were found to mediate some of their effects through peroxisome proliferator-activated receptor gamma (PPARy). Data showed CBDV (300 nM-10 µM) attenuated MCP-1 levels in HBMEC monolayers, as well as reducing IL-6 (30 nM, 1 µM and 10 µM; p<0.05) and VEGF (10 nM- 10 µM; p<0.01) levels in astrocyte monocultures post OGD. CBG (10 nM-3 µM; p<0.0001) also reduced levels of IL-6 secreted by astrocytes and decreased levels of DNA damage response proteins including Chk1, Chk2, H2A.X and p53 post OGD. Neither CBG, nor CBDV reduced levels of IL-6, VEG or IL-8 in pericytes compared to the vehicle control post OGD. Cannabidiolic acid (CBDA) was also investigated and was found to decrease IL-6 in pericyte monocultures which was mediated, at least in part, by 5-HT1A activation. In a four-cell model of the BBB, CBDA offset increases in permeability vs the vehicle control and offered direct protection to neurons, as shown by a lack of propidium iodide (PI) staining in CBDA treated cells, indicating live cells are present. Data presented in this thesis show minor phytocannabinoids CBDV, CBG and CBDA provide protection against OGD mediated damage, with CBDA also offering protection against increases in permeability of the BBB post OGD. These novel data warrant further investigation into the neuroprotective properties of phytocannabinoids, particularly in ischaemic stroke.

Radiotherapy is often prescribed for brain cancer, particularly high-grade malignant gliomas. Efficacy is, however, often limited by radioresistance. The thioredoxin (Trx) system, a key antioxidant pathway in defence against oxidative stress, represents a potentially attractive target to overcome such resistance. Trx system alterations contribute to altered treatment response in a number of tumour types but information is lacking in brain tumours. The aim of the current study is to explore the potentially prognostic role of Trx system proteins in different types brain tumours; and investigate the chemotherapeutic and radiosensitising effects of a novel TrxR inhibitor IQ10 on brain cancer cells and the mechanisms underlying its action. Expression of Trx system proteins was evaluated in four different brain tumour types by immunohistochemistry. A panel of five different brain cancer cell lines were used in this study: SNB19 (adult glioblastoma), SF188 and KNS42 (paediatric glioblastoma), and DAOY and UW228-3 (medulloblastoma); and a normal cell type, MRC5 lung fibroblasts, was used for comparing drug cytotoxicity. Professor Chris Moody's group (School of Chemistry, University of Nottingham) developed a series of Indolequinone (IQ) compounds that potently inhibited TrxR activity in pancreatic cancer cells. One of the most potent IQs, IQ10, was assessed for its inhibition of TrxR activity and Trx family expression in brain cancer cells using the insulin reduction assay and Western blotting respectively. The cellular cytotoxicity of IQ10 was evaluated using growth curves, resazurin reduction and clonogenic survival assays under both normoxic and hypoxic (1% O2) conditions and in 2D vs. 3D spheroid cultures. Validation of TrxR as an important target for IQ10 in brain cancer cells was carried out using an siRNA approach. IQ10's role in regulating radioresponse of brain cancer cells was examined via clonogenic assays. In addition, intracellular ROS levels were determined by H2DCFDA flow cytometry and γH2AX foci by immunofluorescence staining. Expression of genes associated with epithelial-mesenchymal transition was examined by RT2 ProfilerTM PCR Array. The retrospective immunohistochemical study demonstrated that Trx system proteins were expressed to variable levels across a variety of brain tumour types, with high expression of cytoplasmic TrxR consistently correlating with a worse prognosis in all tumour types, suggesting that it is potentially a very important therapeutic target in brain cancers generally. In vitro results showed that IQ10 treatment significantly inhibited TrxR activity in a dose-dependent manner but did not affect the expression levels of Trx system proteins in brain cancer cells. IQ10 exhibited potent single agent anti-proliferative and cytotoxic effects against brain cancer cells under both normoxic and hypoxic conditions in 2D and 3D systems, with IC50s in the submicromolar/low micromolar range. IQ10 was up to ~1000 fold more potent than temozolomide, the agent used clinically to treat brain tumours and used as a comparator in the current study. IQ10 seemed to preferentially kill brain cancer cells but spare normal fibroblasts MRC5. More importantly, TrxR was found to be a promising radiobiological target in that inhibition of TrxR by IQ10 substantially sensitised both 2D and 3D cultured brain cancer cells to radiation (sensitiser enhancement ratio's ranging between 1.2 - 1.7), with such radiosensitising effect being due, in part, to functional inhibition of TrxR, which made cells less able to deal with oxidative stress, leading to oxidative DNA damage. In addition to its cytotoxic and radiosensitising effects, IQ10 showed potential to inhibit invasion/migration as treatment significantly downregulated EMT associated gene expression in brain cancer cells. Collectively, although further confirmation is required both in vitro and in vivo, the current study highlights TrxR as a potential therapeutic target in brain cancers and suggests that IQ10 could be a promising agent, either single or combined with radiation, to improve patient outcome.

The amygdala is most known for its role in fear, threat, relevance processing but also aversive conditioning, is part of nociceptive pathways and has extensive reciprocal connections to many pain-implicated brain areas yet less than 5% of human pain imaging studies report the amygdala. The aim of this thesis is therefore to evaluate the amygdala's role in pain and pain progression mechanisms. Thus chapter 1 describes this relationship from conceptual and preclinical perspectives and elaborates on possible reasons for its infrequent appearance in human pain literature whilst chapters 4 and 5 evaluate it empirically and try to mitigate some of the knowledge gaps. Specifically, chapter 4 examined amygdala morphometry in chronic knee Osteoarthritis pain patients with voxel-based morphometry, hypothesizing increased amygdala gray matter density in pain patients relative to controls in line with some preclinical reports on stress-induced amygdala hypertrophy. This hypothesis could not be confirmed or refuted because extracting gray matter probabilities from quality-controlled gray matter segmentations with an accurate amygdala mask revealed practically no variance in either patients or controls, suggesting that this parameter reflected segmentation quality rather than neurobiological qualities. It is therefore advised not to use this parameter for clinically motivated questions regarding the amygdala unless gray matter atrophy is expected. Chapter 5 investigated pain-related alterations to amygdalae functional networks in the same patient and control population. It was hypothesized that chronic pain would be linked with decreased amygdala-vmPFC connectivity because of proposed inhibitory mechanisms from animal pain work but increased amygdala-dmPFC connectivity because of human imaging studies linking this connection to aversive amplification. Furthermore, sex-effects were hypothesized. The first hypothesis could not be tested. Interestingly, findings supported the second hypothesis but in the opposite direction and future work is advised to investigate the discrepancy of this finding further as it has clinical relevance for serotonergic drugs. Unexpectedly, connectivity between the amygdala and the postcentral gyrus was also altered in chronic12 pain patients relative to controls; this results should be investigated further as the postcentral gyrus is often part of imaging results in human pain but hardly discussed yet it was recently linked to genetic predisposition for anxiety disorders. Results are also suggestive of sex-effects in that the amygdala dmPFC connectivity alteration was not found in post-hoc subsample analysis in males whilst subanalyses in females did not show alterations in amygdala postcentral gyrus connectivity. This should be followed up as it may help to understand why females are more prone to develop chronic pain. The last chapter reviews findings of this thesis and suggests further empirical avenues.

Introduction Medulloblastoma is the most common type of malignant paediatric brain tumour, accounting for 10% of cancer deaths in children. While survival rates have improved in recent years, metastatic spread poses a significant challenge to therapy success. Current therapies also cause irreparable damage to the surrounding brain leading to a decreased quality of life in surviving patients, consequently there is a need for new targeted therapy options. One of the routes for dissemination of metastatic cells is invasion into the leptomeninges, a process during which cells cross basement membrane barriers as they travel from the primary tumour niche into the metastatic niche in the cerebrospinal fluid (CSF). The pathways involved in dissemination and metastasis remain elusive, and a better understanding is crucial to developing targeted therapies. This project aimed to develop an in vitro model system to mimic key characteristics of cell migration from the brain to the CSF, and identify novel candidate genes driving medulloblastoma metastasis. Methods An in vitro culture model representing certain features of the basement membrane interface between the brain parenchyma and the CSF was developed in order to isolate cells for transcriptomic analysis from three compartments: (a) non-metastatic suspension cells, (b) early metastatic adherent cells, and (c) metastatic migrated cells. Differentially expressed genes in these three compartments were identified by Next Generation 3ˡ UPX RNA Seq. The prognostic role of candidates was assessed by analysis of a large medulloblastoma gene expression dataset (Cavalli et al., 2017) on the R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl). SORCS2 expression was validated in medulloblastoma cell lines by qRT-PCR and western blotting, and a possible function assessed in vitro after siRNA knockdown. Results A trans-well migration assay format was modified to represent certain features of the basement membrane, collagen IV and laminin 111 were used as a basement membrane-like coating, and cells were induced to migrate towards artificial CSF supplemented with FBS. The transcripts SORCS2, CORO2B and LMNB1-DT were identified as candidates that may drive the process of migration. The gene SORCS2 demonstrated a significant increase in expression as Group 3 medulloblastoma cells progressed through the stages of migration, from the suspension compartment to the adherent compartment and then to the migration compartment. The transcripts CORO2B and LMNB1-DT were upregulated in cells in the adherent compartment. The expression of SORCS2, CORO2B and LMNB1-DT correlated with poor patient outcome and metastatic recurrence in medulloblastoma patients. SORCS2 was selected for further in vitro investigation as it most significantly correlated with an early poor survival trend in patients with metastatic tumours. SORCS2 gene and protein expression was confirmed in Group 3 and Group 4 cell lines. The results from in vitro experiments did not allow significant conclusions relating to SORCS2 function in migration to be drawn, however they offer preliminary data encouraging further investigation. Conclusions This non-biased, model-led, target identification approach identified genes of interest SORCS2 and CORO2B, and lncRNA LMNB1-DT, in migrating medulloblastoma cells. SORCS2 encodes a brain-specific transmembrane receptor in the VPS10 family of sorting proteins previously linked to actin dynamics in neuronal growth cones, and was implicated in the migratory behaviour of basal-like breast carcinoma. The analyses in this project suggested a link between SORCS2, metastasis, and tumour recurrence in medulloblastoma. The other candidates identified by these analyses warrant further validation and may shed light on the metastatic process in medulloblastoma. A better understanding of the processes and pathways that drive malignancy is a starting point for a push towards improving efficacy of our treatment options while reducing toxicity.

DNA methylation (5-methylcytosine, 5mC) is the major epigenetic modification involved in transcriptional regulation. Ten-eleven translocation (TET) proteins can enzymatically oxidise 5mC producing 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). According to several reports, 5hmC levels are reduced in human tumours; however, the distribution and the exact biological role of TET-dependent oxidation to 5fC and 5caC in cancers is poorly studied. Here, using a range of techniques including, immunocytochemistry, 2 dimensional ultra-performance liquid chromatography coupled with tandem mass-spectrometry (2D-UPLC-MS/MS) and CRISPR/Cas9 gene editing we studied the presence and the distribution of 5caC/5hmC in brain tumours cell lines. Our ultimate goal was to examine the role of TET2 in the oxidation of 5mC to 5hmC/5caC in glioblastoma multiforme (GBM) pathogenesis and the role of 5caC potential readers in the interpretation of active DNA demethylation in medulloblastoma (MB) (paediatric brain tumour) cell lines. We found that, while GBM cell lines exhibit low levels of 5hmC, they are, rather unexpectedly, characterised by detectable 5caC levels. Remarkably, 5caC content in GBM corresponds to elevated levels of TET2 transcript. We next used CRISPR/Cas9 to knockout (KO) TET2 in the LN18 GBM cell line. 5hmC levels were significantly reduced in TET2 KO cell line. Moreover, we showed significantly reduced tumorigenic ability of TET2 KO cells in parallel with the reduction of the transcript levels of key glial cancer stem cell markers. We also showed that SMARCC2 and RCOR2 transcription factors with unique roles in neurogenesis, are highly expressed in paediatric brain tumours, while their transient knockdown resulted at impaired 5caC levels in MB cell lines; indicating their role as 5caC readers. Our compiled data show the unique epigenetic signatures of adult and paediatric brain tumours, the role of TET2 in active DNA demethylation and its potential contribution to tumorigenesis.

Introduction: Multiple Sclerosis (MS) is the most common neurological disease in young adults, and it contains different stages. Transitioning to Secondary Progressive Multiple Sclerosis (SPMS) is a difficult time for patients, fraught with psychological uncertainty and an increase in physical disability. In parallel, healthcare appointments become less frequent, disease modifying treatments are withdrawn, and social contacts typically become more difficult to maintain. The aim of this study was to assess whether providing a brief, Acceptance and Commitment Therapy (ACT) based telephone supported bibliotherapy intervention during transition to SPMS is feasible, effective and valued by participants. A separate questionnaire study was conducted as an adjunct to the project to determine the strength of relationship between psychological flexibility and distress in SPMS. Method: A single centre, mixed-methods, two-arm feasibility randomised controlled trial (RCT), comparing (i) Acceptance Based Support + treatment as usual (TAU) to (ii) TAU for those who had transitioned to SPMS was conducted. Feasibility, signal of efficacy and acceptability were assessed in both groups by self-report measures at 3 timepoints (Baseline, 8 weeks, 12 weeks), and feedback interviews analysed using framework analysis following the completion of the study. The questionnaire study recruited participants through the MS Register, a large existing database of MS patients. Those with SPMS were invited to complete a single psychological flexibility questionnaire, which was paired with a previously completed anxiety/depression questionnaire to observe correlation direction and strength. Results: The recruitment strategy was not feasible: 14 of 40 were recruited (35%) during the four-month time period. Mean sample age of 53, ten women. The data collection procedures and trial processes were feasible and acceptable to participants, reflected through all measures being completed, no attrition, and positive participant interview feedback. The intervention did not demonstrate efficacy, with no practically important differences between baseline and 8-week or 12-week follow up for anxiety (d = 0.00 [-1.60, 1.60]), depression (d = 0.00 [-1.60, 1.60]), or psychological flexibility (d = 0.42 [-1.20, 2.04]). The control group showed a significant reduction in anxiety at 12-week (d = 1.91, [-0.03, 3.83], p = .04), but not at 8-week but was due to one outlier. Comparisons between control and intervention groups showed no significant difference in anxiety (F = 3.21, p = .10), depression (F = 0.12, p = .89) or psychological flexibility (F = 0.36, p = .56) scores. Positive interview feedback suggested potential impact not being captured through self-report measures. The questionnaire study (n = 688) found psychological flexibility was negatively correlated with distress (r = -0.65), anxiety (r = -0.58) and depression (r = -0.56). Discussion: A future trial with those transitioning to SPMS using a similar ACT-based telephone supported bibliotherapy intervention and this methodology is not feasible, due to an unsuccessful recruitment strategy and a lack of evidence of efficacy. A more efficient recruitment strategy, or longer recruitment period is needed to recruit a large enough sample. Adapting the ACT intervention is also needed to ensure that it targets psychological flexibility, which could include changing the workbook or session delivery. The questionnaire study finding that psychological flexibility is negatively correlated with distress provides justification for using a psychological flexibility focussed intervention, such as ACT in SPMS.

Survival rates for children with brain cancer have dramatically increased in the recent decades, but evidence demonstrates long-term physical impairments, academic difficulties and neuropsychological deficits even among children with non-aggressive tumours and treatments. Children with optic pathway glioma (OPG) are at risk of neuropsychological difficulties due to the resulting visual impairment, the neural damage of the tumour itself and its treatment, and the comorbid neurodevelopmental disorder called Neurofibromatosis Type 1 (NF1). Yet, the literature on the sequelae of OPG beyond vision is scarce. This thesis is a comprehensive evaluation of long-term visual and neuropsychological outcomes in a group of 12 children who were diagnosed with a glioma in optic pathway, who received either chemotherapy or no treatment for their tumour. On the ophthalmic exam, children demonstrated a significant structural and functional damage to the optic pathway in terms of reduced thickness of the retinal nerve fiber layer and as well as poor visual acuity. Visuo-perceptual and visual-motor abilities, which reflect the ability to process of physical and asemantic stimuli, were significantly hindered and half of the sample performed below the level expected for their age in this domain (Chapter 3). On the cognitive assessment, children had preserved reasoning abilities (both verbal and visuospatial), but mild difficulties in working memory and processing speed, similarly to other brain tumour survivors. Core scholastic abilities of reading and maths comprehension were intact, but children had mild problems in writing and oral language. Only a minority of children (mostly with NF1) showed severe problems in these domains (Chapter 4). Significant associations were found among measures of vision and visual perception; cognitive and scholastic abilities were associated with each other, but not with visual perception. Among the risk factors, younger age at diagnosis was associated with poor visual outcomes in the best eye and poor binocular vision had a negative impact on visuoperceptual, cognitive and scholastic abilities that heavily relied on sight. In addition, children with NF1 tended to underperform, unlike children without NF1 (Chapter 5). Finally, participants were examined on a series of abilities (i.e., fine motor control, attention, short-term and working memory, mathematics and English comprehension), using parallel tasks that rely on either visual or auditory input. In comparison to a large group of typically developing children (N = 96), OPG survivors performed overall in line with the level expected for their age in both visual and auditory domains, although some children with NF1 exhibited problems in maths and English comprehension. In addition, strong significant correlations between the two ophthalmic measures and neuropsychological skills indicated that more severe structural and functional damage in the best eye was associated with faster responses on the auditory attention task. This result, in combination with the mild working memory difficulties reported with the standardised assessment, suggests that a compensatory mechanism in the auditory modality might take place in children with OPG, but this does not enable them to develop superior auditory abilities (Chapter 6). Overall, this study demonstrated that children with OPG experience significant visual and visuo-perceptual problems, as well as mild and specific cognitive and scholastic difficulties. About half of the children were at risk of great visuoperceptual problems, while only a minority was specifically at risk of severe underperformance in terms of intellectual and academic functioning. Children with OPG do not develop superior auditory skills to compensate for the loss of sight.

5-methylcytosine (5mC) is an epigenetic modification usually associated with transcriptional repression. The Ten–Eleven Translocation proteins (Tet1/2/3) can oxidise 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) in vertebrate DNA. These modifications have not been well characterised but have been detected during embryogenesis, in somatic adult healthy and malignant tissues. Transcription factor and tumour suppressor/ oncogene Wilm’s Tumour protein 1 (WT1), though not expressed in healthy brains displays over-expression in high grade neoplasms including Glioblastoma multiforme (GBM) and has been associated with poor patient prognosis. Interestingly, WT1 possesses preferential binding affinity for 5mC and 5caC over unmodified cytosines, 5hmC and 5fC in vitro. Thus we speculate that a WT1-5caC relationship may exist within a DNA demethylation associated and possible a transcriptional regulatory context in brain tumours. Utilising a range of techniques including genome editing and Next Generation Sequencing, we demonstrate that 5caC is enriched in brain tumour cell lines, is depleted upon WT1 mutagenesis and may share genomic occupancy on gene regulatory and intragenic sequences. Additionally, we demonstrate a compromised neurosphere formation phenotype evident in WT1 zinc finger mutant GBM cell lines which correlates with perturbed extracellular matrix and cell adhesion gene expression, and is correlated with reduced tumour formation in vivo.

Introduction: Medulloblastoma, the most common malignant paediatric brain tumour, is a heterogeneous disease with tumours classified based on their histological and molecular profiles. Studying medulloblastoma in vitro is challenging, as traditional two-dimensional (2D) cell culture models fail to recapitulate the multi-dimensional growth, cell-cell interactions, and complex microenvironment that exists in vivo and in patient tumours. This project therefore aimed to respond to the need for a robust, reliable, and optimised three-dimensional (3D) in vitro culture method for medulloblastoma, which could be applied to study drug response and metastatic dissemination. Methods: A panel of medulloblastoma cell lines representing three of the four molecular subgroups (SHH, Group 3, and Group 4) were tested for use in the 3D spheroid model. Standardised growth conditions, including spheroid size and culture medium, were adopted to allow direct comparisons across cell lines and treatments. Spheroids were characterised by imaging analysis, luminescence-based assays, and immunohistochemical staining. Drug response of 3D spheroids was compared to 2D monolayer culture, with additional long-term and dual-inhibitor studies conducted to exemplify the use of the 3D spheroid model in drug screening approaches. SHH medulloblastoma spheroids were applied to migration and invasion models using basement membrane matrices and hyaluronan (HA) hydrogels containing additional extracellular matrix (ECM) components. Next-generation sequencing (NGS) technologies were adopted to gain an insight into the genes and pathways involved in SHH medulloblastoma leptomeningeal metastatic dissemination using the HA hydrogel model. Results: Medulloblastoma cell lines formed spheroids of different morphologies. The SHH subgroup cell lines (DAOY, ONS76, and UW228-3) formed tight, highly reproducible spheroids, and were therefore chosen as suitable models for downstream analysis. SHH medulloblastoma spheroids could be maintained for up to 3 weeks in culture, forming physiological gradients within the 3D structure over time. 3D spheroid culture increased resistance to standard-of-care chemotherapeutic drugs compared to 2D monolayer culture and a subpopulation of viable cells was present after treatment. Dual-inhibitor studies with vardenafil, an inhibitor of the multidrug transporter ABCB1, showed synergistic effects which could facilitate the use of lower doses of chemotherapy. The HA hydrogels, containing ECM components highly expressed in SHH patient tumours, were more suitable, physiologically-relevant models of SHH medulloblastoma metastasis than commercially-available basement membrane matrices. NGS revealed the downregulation of cell cycle genes and upregulation of cell movement genes in DAOY and ONS76 spheroids migrating on the HA hydrogel. Overexpression of the insulin pathway, consistent with in vivo SHH medulloblastoma metastasis models and patient tumours, was also observed. Thus our model allows us to recapitulate known, and identify novel, mechanisms of cell dissemination from the primary tumour. Conclusion: The 3D spheroid model of medulloblastoma described in this study can be used to analyse drug response and model metastatic dissemination. It is a significant improvement over current in vitro techniques and represents an important breakthrough in medulloblastoma research.

Background: Aggression can often occur in people who have an Acquired Brain Injury (ABI), and the prevalence of ABI in forensic settings is high. This thesis aimed to explore aggression following ABI, how aggression is measured in various clinical settings, and then how aggression is defined, triggered and managed in a forensic hospital setting. Method: A systematic review and meta-analysis was conducted, identifying and evaluating measures used to assess aggression in adults with ABI. One of these measures, the OAS-MNR was critically evaluated. A questionnaire was then developed based on the OAS-MNR to quantitatively assess triggers to aggression in patients with ABI in a forensic hospital from the staff’s perspective. Qualitative data was then collected through interviews with staff to provide deeper exploration of these triggers. Results: The OAS-MNR was the only identified measure which assesses antecedents to aggression in people with ABI. Using a questionnaire based on this measure, staff frequently reported internal and external triggers that can lead to aggressive incidents, including restrictions in the secure environment. Frustration was identified as the main trigger, which they explained could lead to aggression if the patient is unable to control or effectively communicate their frustration. Impairment as a result of the brain injury can make this more difficult. Discussion: Incidents of aggression in a forensic ABI setting are frequent, complex and include multiple triggers to aggression. Incidents can be better managed by awareness of individual patients’ triggers, and distraction techniques. This can be helpful when training new staff or managing incidents.

Glioblastoma (GBM) is the most common type of adult brain tumour, representing 45.2% of all malignant brain and central nervous system tumours. It is also the most lethal, with a median post-therapy survival of 14 months, due to the high likelihood of tumour recurrence. Intratumour heterogeneity is thought to be a key contributor to GBM tumour recurrence, because treatments such as radiotherapy and chemotherapy typically only successfully target the core of the tumour. Due to its high visibility, this core is excised during surgery, but there is another more subtle region of the tumour, the invasive margin, which generally remains behind. This invasive margin is a mixture between normal brain and highly invasive tumour cells. If left over from surgery, these remaining tumour cells infiltrate the normal brain, and cause recurrence within 2cm of the original site. However, if surgeons are too vigorous in removing the invasive margin, they may cause unnecessary removal of healthy brain tissue, thereby causing other complications. This make developing specific methods to target the cancer cells in the invasive margin a priority area in GBM research. In order to specifically target the invasive margin, we need to have a way of differentiating it reliably from the other regions. In this thesis, I investigated the role that micro RNAs (miRNAs), a type of small non-coding RNA, play in this intratumour heterogeneity. miRNAs act as post-transcriptional regulators of gene expression and can simultaneously suppress multiple genes within the same pathway, which makes them potentially valuable routes for therapy development. I performed a miRNA microarray on tissue samples from three tumour regions (tumour core, rim, and invasive margin) and validated the results with qRT-PCR. I then performed computational predictions of gene targets of the differentially expressed miRNAs and validated them in cell lines and patient samples at both the mRNA (qRT-PCR) and protein (Western blot and immunohistochemistry) levels. I then performed a bi-phasic extraction of polar and non-polar metabolites and employed liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectroscopy techniques to profile the intracellular metabolome and lipidome of U87 cells overexpressing miR-619-5p and miR-4793-3p. Transwell invasion assays were performed to investigate the effect of each miRNA and its target on GBM cell invasion. In this work, I found that two miRNAs, miR-619-5p and miR-4793-3p, are significantly downregulated in the invasive margin. Moreover, these miRNAs control the expression of genes that are involved in regulating lipid metabolism pathways. A second key finding was that the knockdown or pharmacological inhibition of the miR-619-5p target, CPT2, also significantly reduced the invasion of the cells in the invasive margin. Because CPT2 lies in the lipid metabolism pathway, my findings indicate that lipid metabolism may present a possible vulnerability of the GBM invasive cell population, and the understanding the function of miRNAs in regulating lipid homeostasis in GBM may provide novel avenues for GBM therapy.

Neural stem cells (NSCs) in the adult mammalian brain have been identified in the sub-ventricular zone (SVZ) of the lateral ventricles (LV) and the sub-granular zone (SGZ) of the hippocampus. Recently, the Bergmann glia (BG) of the adult mammalian cerebellum have been proposed as a NSC population of the adult brain. However, their regenerative potential is largely unclear. This project aims to investigate the properties of LV-derived and cerebellum-derived NSCs from the adult mouse brain by developing and optimising protocols to (a) differentiate primary NSCs, and (b) characterise extracellular vesicles (EV) secreted by NSCs. The first results chapter focuses on testing a clinical drug called fasudil for neural differentiation in vitro. Fasudil is a a ROCK (Rho associated kinase) inhibitor which has been recently shown to differentiate C17.2 cell line. Here, the results show that fasudil can promote radial-like gliogenesis in primary LV-NSCs with increase in radial-like movement, radial glial markers GFAP and Sox2, and reduction in the neuronal marker βIII-tubulin. Moreover, this effect was consistent in multiple culture conditions including changes in serum concentration, substrate coating, and the batch of primary NSCs. Fasudil-based medium was thus used to study in vitro differentiation of primary NSCs. The second results chapter focuses on developing an innovative tool to characterise EVs secreted by stem cells by using direct stochastic optical reconstruction microscopy (d-STORM- a super resolution imaging technique), and a lipid-based dye called DiD. The result show that the d-STORM approach was a reliable and a rapid alternative to conventional techniques used for EV characterisation including electron microscopy (EM), nanoparticle tracking analysis (NTA), and tunable resistive pulse sensing (TRPS). d-STORM provided superior sensitivity (20 nm to 30 nm) and speed (< 1 hour) for analysing the EV size and count. The approach was further demonstrated to be useful to study intracellular uptake and trafficking of EVs in neural cells. The third results chapter focuses on using the tools developed in the previous results chapters, including the fasudil-based NSC differentiation protocol and the d-STORM-based EV characterising approach, to investigate the NSC properties of BG isolated from the adult mouse cerebellum. Results showed that BG-NSCs can multiply and differentiate into neurons (expressing βIII-tubulin) and glia (expressing GFAP, S100b), similar to LV-NSCs. Image analysis of immunostained cerebellum sections from pcd (Purkinje cell degeneration) mutant mice model showed that BG increase in number during late stages of neurodegeneration. It was found that BG-NSCs secreted EVs that promote neuronal growth in a 2-D neuronal culture system, similar to LV-NSCs. BG-NSCs also showed some distinctive features compared to LV-NSCs such as the difference in morphology at early passages during cell culture, longer doubling time, producing larger neuronal bodies, expressing Calbindin in neural extensions, and secretion of 10-times more EVs per cell. The fourth result chapter focuses on discovering the undesired effects of low-power sonication during EV analysis for future refinement of EV processing protocols. Results show that low-power sonication induces changes in size distribution, surface membrane properties and its functional effect including cellular uptake. Importantly, by comparing sonicated vs non-sonicated EVs in a 2-D neuronal culture system, it is shown that sonication of EVs is necessary for the neuronal outgrowth promoted by NSC derived EVs observed in the previous chapter. This chapter hence provides insights into the future considerations during EV research. Taken together, this project provides a deeper understanding into NSC properties of BG, which was investigated by developing protocols to study cellular differentiation and extracellular vesicle characterisation.

Central adjudication in randomised trials refers to the evaluation of outcome data by independent experts who are typically part of an event or outcome adjudication committee. Alternatively, local site investigators can assess outcomes, but these evaluations are often discarded in favour of the centrally adjudicated outcome. Central adjudication is thought to improve the precision of treatment effect estimates by reducing random error (non-differential misclassification), and in open-label studies adjudication has the potential to limit systematic error (differential misclassification) as adjudicators can always be blinded to treatment allocation. However, secondary analysis of trial data suggests that adjudication makes no meaningful difference to the endpoints of stroke and functional outcome. In addition, adjudication is a time-consuming and often expensive process. Given that central adjudication is common in trials investigating stroke, research is needed to establish what the benefits and costs are of adjudicating outcomes in stroke trials. In a systematic review, 15 randomised stroke trials (69,560 participants) were identified that had their primary outcome assessed by both central adjudicators and site investigators. The primary outcomes included were stroke (8 out of 15 trials, i.e. 8/15, 53%), a composite including stroke (6/15, 40%) and functional outcome after stroke (1/15, 7%). Overall, there was no evidence of any difference in treatment effect estimates based on data from central adjudicators and site investigators (pooled Ratio of Treatment Effects=1.02, 95% C.I:[0.95, 1.09]). This thesis also investigated whether two different approaches for contacting authors had any impact on the probability of receiving a response when trying to acquire data for a systematic review in a methodological trial. This nested randomised trial found that a short email with a protocol attached elicited a similar response compared to a longer email without a protocol. To explore circumstances where central adjudication would change the treatment effect estimate, five of the trials identified in the review were included in a further simulation study. Differential misclassification was introduced into each study via simulation until the treatment effect estimate was altered. For trials with a binary outcome, between 2.1% and 6% of participants needed to be differentially misclassified before this situation ensued. In addition, hypothetical trials were simulated with a binary outcome and varying sample size (1000-10000), overall event rate (10-50%), and treatment effect (0.67-0.90). Non-differential misclassification was introduced until the treatment effect was non-significant at 5% level. For these hypothetical trials, extensive non-differential misclassification was needed before the treatment effect became non-significant; trials with an overall event rate close to 50% and a larger sample size needed the highest proportion of random error before this occurred. Nine of the trials included in the review provided data on the cost of adjudication. These costs included adjudicators' time, direct payments to adjudicators and co-ordinating centre costs. The number of events corrected after adjudication was the measure of benefit used. The mean cost per event corrected by adjudication was £2295.10. To investigate whether these findings were similar for adjudication of safety data, a case study was carried out using data from the Efficacy of Nitric Oxide in Stroke Trial. Serious adverse events were reported by site investigators who were not blinded to treatment allocation. Central blinded adjudicators reviewed the investigators' report and used evidence available to confirm or re-categorise the classification of event. Repeating the main trial safety analysis with investigator reported events showed that adjudication had no effect on the main trial safety conclusions. To conclude, these studies have shown that central adjudication of the primary outcome in stroke trials does not alter treatment effect estimates. However, for studies without adequate blinding, a small amount of systematic error has the potential to alter the primary analysis and in this circumstance, adjudication is important. Given that the cost of central adjudication is not trivial, the potential advantages of adjudication may not outweigh cost and time disadvantages in stroke trials with blinded outcome assessment.

Nitric oxide donors (e.g. glyceryl trinitrate (GTN)) are candidate treatments for acute stroke with haemodynamic, reperfusion, and neuroprotective properties. In order to establish the safety and efficacy of NO donors in acute stroke a Cochrane systematic review and meta-analysis was performed. Transdermal GTN was assessed in 5 trials (n=4197) in acute stroke. Overall, GTN was safe and lowered blood pressure but did not influence clinical outcome. However, early treatment with GTN within 6 hours (n=312) improved clinical outcomes across a variety of domains (death and dependency, death, disability, cognition, mood, and quality of life). Using data from the large Efficacy of Nitric Oxide in Stroke (ENOS) trial, the haemodynamic properties of GTN were explored. GTN lowered BP and its derivatives including BP variability. Further, increased BP variability was associated with poor functional and cognitive outcomes and increased death at day 90. The safety and efficacy of transdermal GTN in important subgroups in acute stroke was assessed. GTN was safe in the context of blood markers of dehydration with no precipitous drops in BP seen in such patients. GTN in the context of ipsilateral or bilateral carotid stenosis was safe and may improve outcome in severe ipsilateral carotid stenosis. Although GTN was safe, it did not improve outcome in patients with lacunar syndromes either overall or within 6 hours of onset. Baseline imaging markers of small vessel disease and 'brain frailty' were associated with functional and cognitive outcomes 90 days after stroke. In summary, this thesis has confirmed the safety of transdermal GTN in acute stroke both overall and in important subgroups. Mechanistic data suggest that GTN may reduce BP variability, which seems to be more strongly associated with outcome than absolute BP or trend in BP. Transdermal GTN is safe to be administered in acute stroke patients with elevated BP prior to blood markers of dehydration or carotid stenosis status being known. Baseline imaging markers of SVD and 'brain frailty' predict clinical outcome and should be used as minimisation criteria in future acute stroke trials and may help guide future clinical decision-making.

People with multiple sclerosis frequently report cognitive problems, which can affect function and quality of life. Although the effectiveness of cognitive rehabilitation to address these problems is evaluated in research studies, inadequate reporting of the content of the interventions may hamper clinical implementation. This research aimed to explore avenues to increase the clinical impact of trials of cognitive rehabilitation through the development of a clinician­informed, evidence­based checklist to guide researchers to better report cognitive rehabilitation studies in MS. The overarching design was exploratory mixed-methods, involving three separate intertwining studies. Study one: A systematic review of descriptions of the content of cognitive interventions to document salient details and omissions in reporting. Ten electronic databases were searched, and 54 studies involving various types of MS and techniques to improve difficulties with memory, attention and executive function were included in the review. The results showed that important features of cognitive rehabilitation were not reported well. This was particularly in relation to the content of interventions (reported completely in 48% of the articles), how the interventions were delivered (reported completely in 44% of the articles) and the mechanism of action of the interventions (reported completely in 39% of the articles). Study two: A video-based observational study, using time-sampling and content analysis to examine the content of treatment sessions delivered within the Cognitive Rehabilitation of Attention and Memory in MS (CRAMMS) trial. A total of 252 completely recorded treatment session videos were observed and coded. Content analysis indicated that all components of the CRAMMS manual were delivered as planned (intervention fidelity). The most frequently discussed topic for both therapists and the people with MS related to memory and attention coping strategies (17% and 16% of observations, respectively). Findings from studies one and two were integrated and informed the initial questionnaire for study three. Study three: A modified Delphi consensus study of three rounds, with clinicians and researchers who deliver cognitive rehabilitation to people with MS. This was conducted to ascertain the key aspects of cognitive rehabilitation that research studies should report to enable implementation in clinical practice. 'Experts' were asked to rate the importance of a number of reporting statements for inclusion in a reporting checklist (from low importance to high importance). Fourteen experts completed all three rounds. Based on the pre-specified criteria, consensus was achieved for 29 reporting statements. These statements were subsequently grouped based on how the expert panel rated their importance, forming a Priority 1 and 2 list of reporting statements. The checklist with the reporting statements should be used by researchers to describe cognitive rehabilitation programmes in the published literature. The evidence-based, clinician-informed checklist developed in this research is important because it will enable researchers to report important aspects of complex cognitive rehabilitation interventions. This should be regarded as the first step towards the implementation of such research in clinical practice.

Spontaneous intracerebral haemorrhage is a devastating condition with high morbidity and mortality. Haematoma expansion and subsequent secondary brain injuries lead to worse outcomes in intracerebral haemorrhage. Tranexamic acid is a promising haemostatic agent that may prevent haematoma expansion and improve outcomes. The current thesis explores the effects of tranexamic acid in intracerebral haemorrhage from clinical, radiological and biochemical aspects, based on data from the now completed Tranexamic acid in IntraCerebral Haemorrhage-2 (TICH-2) trial. Two systematic reviews explore available evidence on efficacy and safety of tranexamic acid and other haemostatic therapies in intracerebral haemorrhage. There was insufficient evidence to support the routine use of tranexamic acid or blood clotting factors in treating intracerebral haemorrhage. Platelet transfusion had been found to be harmful and should be avoided in intracerebral haemorrhage. Predictors of neurological deterioration and seizures and the effects of tranexamic acid on both were explored. Older age, more severe stroke, larger haematoma volume, intraventricular and subarachnoid extension, previous intracerebral haemorrhage and antiplatelet therapy independently predicted neurological deterioration. Tranexamic acid reduced early neurological deterioration occurring within 48 hours from symptom onset through a reduction in haematoma expansion, but had no significant effect on late neurological deterioration (48 hours to 7 days). Lobar location of haematoma was the strongest predictor of early (≤ 7 days) and late seizures (> 7 days). The risk of seizures in intracerebral haemorrhage was not increased in patients treated with tranexamic acid. The role of several noncontrast CT signs, namely blend sign, black hole sign, hypodensities and island sign, in predicting haematoma expansion and poor functional outcome was assessed. Blend sign, black hole sign and hypodensities independently predicted haematoma expansion while black hole sign, hypodensities and island sign predicted poor functional outcome (modified Rankin Scale of 4 to 6). Tranexamic acid reduced haematoma expansion but there was no benefit on functional outcome. There was no significant interaction between the presence of noncontrast CT signs and benefit from tranexamic acid in terms of reduction in haematoma expansion or improvement in functional outcome From a biochemical aspect, a range of plasma biomarkers involved in fibrinolysis and haemostasis were examined in a single-centre substudy of the TICH-2 trial involving 22 patients and 9 healthy volunteers. Patients with intracerebral haemorrhage had higher D-dimer and plasmin-alpha-2-antiplasmin complex levels compared to healthy controls. Elevated baseline tissue-type plasminogen activator and urokinase-type plasminogen activator, lower baseline plasminogen and elevated day 2 matrix metalloproteinase-9 levels were associated with haematoma progression. Tranexamic acid did not have significant effects on plasma biomarker levels. In summary, there were clinical and radiological signals that tranexamic acid may be beneficial in patients with spontaneous intracerebral haemorrhage. Plasma biomarker results suggest fibrinolysis may be a pathophysiological mechanism in intracerebral haemorrhage. There is a role for further trials of tranexamic acid in intracerebral haemorrhage.

Providing appropriate rehabilitation for stroke survivors with severe disabilities can be challenging. This is due to the magnitude of neurological impairment early after stroke as well as the complexity of delivering long-term care post-hospital discharge. Severe stroke survivors have limited access to rehabilitation as a likely result of uncertainty related to their potential for functional gains. They are also likely to require greater health and social care resources for long-term care with significant cost implications. Rehabilitation following stroke has been proven beneficial after a severe stroke; however, recovery remains ambiguous. Several clinical and non-clinical factors are evidenced to influence the provision of rehabilitation interventions. However, it is not known how the decisions are made in practice for service provision, what service they receive and how they relate to the stroke survivors' recovery and rehabilitation. Aims This thesis aimed to explore the recovery and rehabilitation of severe stroke survivors by investigating the decision-making process for determining rehabilitation and service provided as a result. The study also evaluated the functional recovery made by severe stroke survivors in the first six months and investigated their caregivers' perspectives on rehabilitation services they received. Methods A prospective design with a fixed convergent mixed method approach was chosen to investigate the research questions. Three independent studies were conducted concurrently in two distinctive phases with independent sets of results. A qualitative approach was taken in the first phase to explore the decision-making process using semi-structured interviews with 22 hospital staff responsible for determining rehabilitation for severe stroke survivors. Functional recovery was investigated in the second phase using an observational cohort study. Patient outcomes were longitudinal measured using validated tools in 52 severe stroke survivors (NIHSS ≥ 10 and mRS ≥4 at admission). Information was also obtained on rehabilitation services accessed post-hospital discharge in the first six months post-stroke. The second part of the study also used semi-structured interviews with 18 caregivers to investigate their experience of the rehabilitation received post-stroke. All data were collected from a single hospital site, covering a selected region in the East Midlands. The data were analysed separately and integrated using a structured triangulation method in the interpretation stage complement, validate and strengthen the overall findings. Results Results established a complex, dynamic, temporal process of decision-making for post-stroke rehabilitation of people with severe functional disability. This required multiple stages of corroborations amongst key decision makers to evaluate the impacts of several social and external factors, additional to severity of stroke. Severity of stroke was defined for rehabilitation purposes and a clinically meaningful change in functional abilities was evidenced in 69% of stroke survivors with severe disabilities with no causal relationship with the initial severity. A noticeable involvement of healthcare professionals during this period suggested the possible contribution of rehabilitation in the early phase post stroke. Rehabilitation was valued for recovery; however, an integrated, client-centred approach was identified as lacking for managing severe disabilities after stroke. This, along with limited shared-planning for rehabilitation led to a low confidence of caregivers in the current healthcare system. Conclusions The overall findings of the thesis established a complex, dynamic, cognitive process of clinical reasoning amongst hospital staff for deciding rehabilitation of severe stroke survivors. The study quantified the recovery made in the first six months and suggested that a significant functional improvement is possible in the severely disabled patients that could influence their rehabilitation plan. Thesis findings also highlighted the potential inadequacies in rehabilitation services provided and emphasized the need for incorporating the service users' expert knowledge in shaping future models of care for severe stroke survivors and their caregivers.

Introduction: Up to 70% of people with multiple sclerosis (MS) experience mild to moderate cognitive deficits in attention, memory, information processing speed, and executive functioning. Cognitive rehabilitation to address such deficits has emerged as a potential treatment approach, but the evidence regarding its effectiveness is mixed. It is also unclear how cognitive rehabilitation is currently delivered in the UK. Aim: To examine the perceived effectiveness and provision of cognitive rehabilitation services for people with MS. Methods: This mixed methods research comprised three studies. Study One was a meta-synthesis summarising findings of qualitative studies examining patient perspectives of the effectiveness of cognitive rehabilitation programmes. Study Two was a UK national survey examining the provision of cognitive treatment for people with MS from the perspective of healthcare professionals. Study Three used semi-structured interviews to investigate patient perspectives on the services they received for their cognitive problems: this study was embedded within a large, multi-centre randomised controlled trial (RCT). Participants from both the control and intervention groups of the RCT were interviewed to compare experiences. Results: (1) In the meta-synthesis, findings from seven individual studies highlighted the perceived benefits of cognitive rehabilitation for people with MS. Participants reported benefits in cognitive function, improved mood and quality of their relationships, and felt the programmes helped them change their perceptions of having MS. The group component was specifically referred to as beneficial as it helped participants experience a sense of community and support. Participants reported cognitive, behavioural, emotional and social improvements, and felt more optimistic. Overall, these changes had a positive impact on participants' quality of life. (2) Survey findings indicated that clinical pathways for assessing and managing cognitive problems varied greatly across the UK and were dependent on the individual healthcare professional's expertise, available resources, and access to specialist services. Of 109 healthcare professionals who responded, fewer than 50% reported that they developed and implemented a cognitive rehabilitation plan and only 3% followed a manual. The Montreal Cognitive Assessment was the most widely used cognitive assessment tool. (3) In the interview study, five main themes were identified through analysis. Participants reported on the services they received for their cognitive problems before the trial and on their perceived cognitive changes. Participants in the intervention group reported on the perceived mechanism of change of cognitive function after the trial and highlighted possible improvements to the treatment. Participants from both the intervention and control groups stated additional reasons for adherence to the treatment and trial. Participants in the intervention group perceived having better cognitive functioning than the participants in the control group. Results suggested that people adopted habits and coping behaviours after participating in a group-based rehabilitation programme, which had a positive impact on daily functioning. Conclusion: There is evidence that people with MS perceive cognitive rehabilitation programmes to have a positive impact on their wellbeing, daily activities, and cognitive functioning. In addition, all participants in the interview study recognised the importance of clinical services focusing on cognitive deficits in MS (i.e., offering cognitive rehabilitation). However, there were no UK-wide standard clinical pathways for the assessment and management of cognitive problems in people with MS. Cognitive rehabilitation was not routinely offered in practice. There is a gap between patient needs and current clinical practice. This is a concern for the management of people with MS and for the access to training for healthcare professionals to improve services, which will need to be addressed in future research.

Over one million people live with the long-term consequences of acquired brain injury (ABI). Individuals with ABI may find it difficult to self-manage the effects of injury and it is important to provide them with the skills to live independently. Many smart technologies exist to aid rehabilitation, but there is limited technology available to support self-management. Brain-in-Hand (BiH) is a smartphone application that was specifically engineered to support the management of persistent emotional and behavioural problems, which are commonly seen following ABI. However, it has not been systematically evaluated in this population. The aim of this thesis was to understand the perspectives and experiences of people with ABI and healthcare professionals after using BiH. A systematic review was conducted to highlight the level of evidence available for smart technologies in the ABI population, and determine the effectiveness of technologies compared to usual care/other technologies on independence and functional outcomes. The review included six randomised controlled trials that utilised a range of technology interventions (e.g., smartphone, iPad, Neuropage). All included a measure of goal attainment/function, however none measured independence or fatigue. Only one measured quality of life and participation. The review highlighted a lack of studies focusing on some of the hidden consequences of ABI such as lack of independence, fatigue, anxiety and poor participation. An engagement study was conducted with stakeholders (n=50) (including people with ABI, carers and professionals), to explore their views and opinions of BiH, and identify some of the barriers and facilitators to its use. A mixed methods approach was used (focus groups, questionnaires, and presentations) to obtain feedback. Stakeholders identified areas that BiH could potentially target such a memory and managing routine. They also highlighted some of the barriers to use such as smartphone competency, lack of interest in technology, and not seeing a personal use for BiH. These findings informed the design of the subsequent case studies. An n-of-1 mixed method case study design was used. All ABI participants were provided with BiH for 12 months. Data were collected using questionnaires (at baseline, 6 and 12 months post-intervention) and semi-structured interviews (at six months post-intervention) with ABI participants and healthcare professionals. Questionnaire data were used to determine whether BiH had any effect on functional outcomes, mood, fatigue, participation, quality of life and cognition of ABI participants. The interviews aimed to explore the barriers and facilitators associated with the use of BiH. These were thematically analysed using a framework informed by the Behaviour Change Wheel (BCW) and International Classification of Functioning, Disability and Health (ICF). A feasibility health economics evaluation was also conducted. The quantitative findings showed that BiH had no effect on mood, functional independence, participation, quality of life, fatigue, or quality of life. However, there was a significant increase in goal attainment over the first six months. There was also a slight improvement in cognitive function. An overarching theme of context (personal/environmental factors) was identified as a key factor influencing the use and effectiveness of BiH. The four subthemes were: 1) insight and self-awareness following injury and its impact on BiH use; 2) patient and therapist support/training to use BiH; 3) motivation to use BiH and achieve goals; 4) technology specific barriers/facilitators. Having a sufficient level of insight, appropriate support (set up, training, monitoring etc.), and motivation appeared to facilitate BiH use. The health economics evaluation identified potential cost drivers as therapist appointments and the cost of BiH. It was feasible to collect this data from the ABI population. Contextual factors need to be considered when implementing BiH, or similar technology interventions in the ABI population. Personal and environmental factors play a key role in the use and effectiveness of BiH. It is important to identify important barriers and facilitators early in the implementation process, to guide the development of BiH for ABI, and its wider applicability to other long-term neurological conditions. Additional research is required to determine its effectiveness in the ABI population. BiH has the potential to be a valuable tool in community rehabilitation by facilitating independent living for people with ABI and providing a way to self-monitor and manage their fatigue/anxiety.

Both impulsivity and empathy are considered crucial in clinical, in particular forensic, populations. A neuroscientific technique, namely repetitive transcranial magnetic stimulation (rTMS), has proven its therapeutic utility in a variety of neuropsychiatric disorders and may be effective in modulating empathy and impulsivity. This thesis aimed to examine the effects of rTMS on empathy and impulsivity in healthy adults via two systematic reviews with meta-analyses and two empirical rTMS studies, thereby contributing to the relatively small pool of knowledge in the field. The first systematic review and meta-analysis reviewed the literature on the effects of rTMS on empathy in healthy adults, and included 18 studies contributing to 24 effect sizes. The findings revealed an overall small effect favouring active rTMS over sham stimulation on cognitive empathy in healthy individuals. Differential effects across the domains of empathy, namely affective and cognitive empathy (also referred to as theory of mind (ToM)), were evident. Meta-regression revealed no significant between-study heterogeneity in respect of sex ratio, mean age, number of pulses, and stimulation intensity. In conclusion, the study found that rTMS may have discernible effects on different components of cognitive empathy. The second systematic review and meta-analysis which examined the effects of rTMS on impulsivity in healthy adults included 27 articles consisting of 50 effect sizes. Results indicate that rTMS has distinct effects on different impulsivity subdomains. A small and significant effect was found in respect of modulating motor impulsivity and a moderate effect was found in regards to temporal impulsivity. However, insufficient data was available to ascertain the effects of rTMS on reflection impulsivity. The first empirical study investigated the effects of excitatory rTMS at the rIFG of 20 healthy male adults in modulating motor and reflection impulsivity. It employed a single-blind randomised crossover design, and it also examined the relationship between trait and behavioural measures of impulsivity. Unexpectedly, no significant effect was found on either motor or reflection impulsivity, after including scores on trait impulsivity as covariates in the analyses. In accord with the extant literature, there were no significant associations between self-report (trait) and behavioural measures of impulsivity. The final study applied intermittent theta burst stimulation (iTBS) at the left dorsolateral prefrontal cortex (LDLPFC) to modulate cognitive empathy and both temporal and reflection impulsivity in 23 healthy adults, using a single-blind randomised crossover design. Non-significant post-iTBS effects on both cognitive empathy and impulsivity were found. Baseline trait measures of empathy and impulsivity were included in the analysis as covariates in order to control for the impact of pre-stimulation individual differences on the iTBS modulation. Despite the mixed results, I argue that rTMS can potentially be used to modulate empathy and impulsivity. Future studies in the field should address some of the limitations highlighted in this project, for instance, in terms of localisation of stimulation sites, stimulation parameters and sample sizes, to determine the utility of TMS.

Introduction: Ependymoma is the second most common malignant brain tumour of childhood. 50% of children with primary disease recur; three-quarters of these do not achieve long term survival. In the ‘molecular era’ of cancer research, diagnosis combines advanced molecular profiling with histopathological assessment. Whilst primary ependymomas can be classified based on epigenetic and transcriptomic features, there is little information on molecular signatures at recurrence. However, some small studies have implicated cancer immunity. Trials of novel therapies at recurrence have been disappointing. This study undertook molecular profiling of recurrent ependymoma, combined with contemporary clinical data, to better understand recurrence biology and potential therapy options. Methods: Clinical outcomes for 188 children with recurrent ependymoma were analysed. Cases with primary and matched recurrent samples were included in DNA methylation (n=56), RNA sequencing (n=52) and immunohistochemical (IHC) (n=56) analyses. RNA sequencing from FFPE tissue was validated to expand the cohort. Results: Recurrence was the strongest predictor of long term survival. Treatment approach at primary diagnosis was not associated with survival, but radiotherapy at first recurrence was associated with better short-term outcomes. Children with the commonest DNA methylation based diagnoses, EPN_PFA and EPN_RELA, had equally poor outcomes. RNA sequencing from FFPE tissue was effective, therefore tumours sequenced from FFPE and FF tissue were included in paired gene expression analyses. Transcriptomic and DNA methylation analyses identified three similar subgroups in FFPE and FF cohorts (PF1, PF2 and ST). At first recurrence, PF1 was associated with downregulated immune and inflammatory ontologies, which may indicate tumour immune escape. PF2 and ST subgroups demonstrated upregulation of ontologies associated with adaptive immunity. Despite this, there was little evidence of change in either immunogenicity or T-cell effector activity at first recurrence. IHC analysis identified a fall in inflammatory cells in posterior fossa tumours at recurrence and indicated that ependymoma is an immune excluded tumour. Conclusions: This study highlights both the abysmal prognosis for this disease, and the need for a better understanding of tumour biology to improve outcomes. This study has contributed novel data on changes at recurrence across molecular subgroups, and identified the immune excluded nature of ependymoma, which may be important in guiding therapy. The validation of RNA-seq from FFPE in childhood brain tumours has facilitated access to a large set of previously uninvestigated samples.

The UK has been at the forefront of young carer research internationally, creating a sound evidence base, influencing policy and developing support for young carers. Studies have consistently found young carers can gain positive attributes and skills from their caring role but that is not always the case. Where there are unmet needs, young carers can experience detriment to their health, well-being and future life chances. Some of the detrimental causes are condition-specific. Young carers of stroke survivors are not included in national stroke guidance. No UK studies were found of young carers of stroke survivors. There is therefore a significant knowledge gap about existing unmet needs of young carers of stroke survivors and consequent potential to develop support services to mitigate any detrimental effects of caring. This thesis reports on a series of three studies taking a sequential exploratory mixed design which aimed to begin to address the identified knowledge gap. The first study comprised a cross-sectional survey of stroke survivors newly discharged home from hospital. 100% of (n=75) respondents identified a need for support with Activities of Daily Living and 75% did not have any paid carers, suggesting a reliance on family and friends for support. Twelve stroke survivors had young carers (n=24), four were primary young carers and three were providing over 20hrs a week of emotional and over 20hrs of practical care. Eight families expressed an interest in participating in the second study. The second study explored the lived experience of young carers of stroke survivors through semi-structured interviews. Seven stroke survivors and their 11 young carers were interviewed. Thematic analysis revealed five themes with 14 sub-themes: Impact of stroke, Insulating the family, Children and young carers of stroke survivors, Knowledge information and learning about stroke and Statutory and voluntary sector services for young carers. Findings showed that young carers were providing a range of support including personal care and rehabilitation. The majority did not recognise themselves as young carers and none had been assessed or were in receipt of support. All young carers were happy providing care and most reported enhancing attributes and gaining positive skills from their caring role. Families were working together to protect and support each other but did not know how to talk to each other about their fears and worries, potentially increasing anxiety. Knowledge and understanding of stroke, and access to information for young carers was poor. This was compounded by a lack of engagement with young carers by health and social care professionals and their apparent lack of awareness of young carers' need for information and support. The greatest unmet need for all young carers was for information about stroke and how they could care more effectively. Experts (commissioners and providers of young carer services) (n=12) were also interviewed to explore the context and challenges for the provision of young carer services. No stroke-specific support services were identified for young carers of stroke survivors. The study found a large variation in the young carer's assessment tools in use and the way they were being implemented to triage young carers for support. This was contributing to inequity in access to services for some young carers. Experts identified challenges in identifying young carers compounded by many factors including the young carers and families not engaging with statutory services either because they were afraid of unwanted interference or were unaware of their rights to assessments and support; complex and variable pathways to assessment and support and a lack of awareness of the existence of young carers by front line health and social care professionals. This was particularly so for stroke. The final study reviewed and analysed printed and online information for children and young carers of stroke survivors which was compared and synthesized with information for brain injury, Multiple Sclerosis and Parkinson's disease as comparable neurological conditions. An Appreciative Inquiry approach was taken to identify and build upon best practice in information provision for young carers identified from condition specific information for Multiple Sclerosis and Parkinson's disease. A gold standard core information set was generated from the information synthesis. Conclusion This PhD provides the first insight into the lives of young carers of stroke survivors. It has started to address the existing knowledge gap by identifying the unmet needs of young carers of stroke survivors. A gold standard core information set has been identified as a potential support mechanism. Health and social care professionals could play a key role in supporting young carers of stroke survivors, but require support to maximise opportunities to adopt a more robust whole family approach by disseminating information, teaching families about stroke and encouraging openness. This could enhance outcomes for young carers by mitigating potential damaging effects of caring and optimise beneficial aspects of caring.

Recovery from a stroke is affected by a wide range of factors. A multitude of comorbidities, including old age and diabetes, all increase the risk of death or disability as a result of a stroke. In addition, medical complications can affect stroke outcome. Infections are known to increase the risk of death or disability when contracted in the first week after stroke onset. However, by analysing data from a clinical trial, we have found that infections are actually detrimental for a lot longer than one week. Any infection acquired in the first 76 days after stroke increases the risk of death in comparison to patients with other post-stroke medical complications. Additionally, patients who contract infections and survive up to 90 days post-stroke have more disabilities and are more reliant on assistance compared to stroke patients who did not acquire medical complications. Inflammation has been identified as a possible cause for the effect of infection and requires further investigation. Neuroinflammation is a firmly established response to stroke and inflammatory processes within the stroke infarct are well characterised. But inflammation in other brain regions, not directly affect by the stroke, receives less attention. Using immunohistochemistry techniques, we looked at microglia number and activation over 28 days post-stroke in nine different regions across the rodent brain, ranging from the prefrontal cortex to the substantia nigra. Microglia activation was present in all assessed brain regions at least once between 1 and 7 days post-stroke, showing that the neuroinflammatory response to a stroke is not limited to the stroke infarct, but instead encompasses the whole brain. In light of this, it is feasible that changes in neuroinflammation will contribute to the detrimental effects of post-stroke infection. We developed an animal model of infection at a chronic time point post-stroke by administering lipopolysaccharide (LPS) intraperitoneally at 15 days post-stroke. Not only did this initiate a peripheral inflammatory response and increased in symptoms of sickness, but in stroke animals the administration of LPS increased microglia activation in the stroke-affected striatum and decreased microglia activation in the prefrontal cortex compared to controls. This supports a possible role of neuroinflammation in propagating disability after infection. While the LPS model would be useful for investigating the role of neuroinflammation further this model does not account for other comorbidities which may also have an effect. Animal models incorporating multiple comorbidities common in stroke patients are rarely used. We aimed to develop a comorbid animal model which could be used in stroke research by administering 12 month old rats a high fat diet. Although these aged obese animals successfully underwent several procedures common in stroke research, they did not develop additional desired comorbidities such as insulin resistance, diabetes and hypertension. This limits their usefulness in stroke research. Overall, a range of work has been completed highlighting a number of areas in stroke research. While some of the work has limited potential, the detrimental effect of infections acquired post-stroke requires further attention with research needed to investigate its mechanisms and possible therapeutics. The LPS model of chronic post-stroke infection is an ideal model for this work.

G protein coupled receptor agonists can be described by two parameters; affinity (ability to bind a receptor) and efficacy (ability to activate a receptor once bound). While affinity is now an accessible parameter through binding studies, agonist efficacies have historically been difficult to determine. This is due to the significant system dependence of agonist responses and is particularly true of traditional maximal response and potency measurements. The Black and Leff operational model determines agonist affinity and efficacy estimations directly from functional data. Its measure of efficacy (τ) offers improvement over maximal response determinations but remains a system dependent parameter. More recently, the Slack and Hall operational model has been described. This model uses receptor constitutive activity to estimate the system contribution to agonist responses to derive system independent measures of intrinsic agonist efficacy. This thesis explores the use of operational models in the analysis of agonist functional responses at the dopamine D2 receptor. This receptor is an important therapeutic target in the treatment of schizophrenia and Parkinson’s disease, and has well documented receptor constitutive activity. Maximal responses and potencies in two representative functional assays, CRE-SPAP reporter gene and receptor internalisation, were determined for a panel of ligands at the D2L receptor. In the receptor internalisation assay, agonist maximal responses and potencies were decreased, consistent with an assay of reduced receptor reserve. Applying the Black and Leff operational model to the responses of four representative agonists, in the absence and presence of the irreversible antagonist phenoxybenzamine, yielded affinity and efficacy estimations consistent with the known pharmacology of the compounds. However, experiments did not provide evidence for the receptor constitutive activity necessary to apply the Slack and Hall operational model. As such, site-directed mutagenesis was performed to generate a constitutively active D2L receptor. T6.34R mutation increased agonist potencies and relative maximal responses consistent with a receptor more likely to adopt the active state; however, potencies of the ergo-derived compounds were selectively decreased. Mutagenesis of three key binding site serine residues examined the mode of agonist binding in both wild type (WT) and T6.34R backgrounds. Each serine mutation had differential effects on the responses of structurally distinct agonists, suggesting agonists engage specific serine residues; while S5.42 and S5.46 were required for catechol agonist responses, S5.46 was essential for the responses of the ergo-derived compounds, bromocriptine and dihydroergocristine. It was unknown whether these specific modes of binding would generate functionally similar active conformations. Generally, the differences between the effects on CRE-SPAP reporter gene and receptor internalisation assay responses were sufficiently explained by differences in receptor reserve. However, there were differential effects of serine mutations on WT and T6.34R D2L receptors, particularly the opposing effect of S5.42A on quinpirole potencies in the CRE-SPAP reporter gene assay. This suggests that WT and T6.34R D2L receptors adopt different conformations. Finally, we have described a method to determine agonist affinity and efficacy from [35S]-GTPγS binding assays using operational models, which does not rely on irreversible antagonist treatments. Buffer Na+ substitution revealed consistent constitutive activation of the D2S receptor, but not of the D2L or D2L T6.34R receptors. Agonist responses at the D2S receptor, in the presence and absence of buffer Na+, were analysed with the Black and Leff and Slack and Hall operational models respectively, generating agonist affinity and efficacy estimations. A novel operational model, written specifically for the analysis of [35S]-GTPγS binding assays, was also examined. This model provided similar affinity derivations to the Black and Leff operational model but provided a τ value that may have reduced system dependency. Although there may be compromises in the associated experimental conditions and which receptors can be investigated, the Slack and Hall operational model can be applied to constitutively active systems to provide system independent measures of agonist intrinsic efficacy.

Primary Intracerebral haemorrhage is a severe form of stroke with poor prognosis attributed to haematoma characteristics. High blood pressure is present during the acute phase of intracerebral haemorrhage and associated with poor outcome in part through expansion of haematoma. Data from the ‘Efficacy of Nitric Oxide in Stroke trial’ (ENOS) was used to analyse the performance characteristics of qualitative and quantitative descriptors of intracerebral haematoma. The results showed that formal measurement of haemorrhage characteristics and visual estimates are reproducible. Intracerebral haemorrhage volumes measured using the modified ABC/2 formula were significantly lower compared to standard ABC/2 and computer assisted semi-automatic segmentation. In 629 patients with intracerebral haemorrhage presenting within 48 hours, the effect of blood pressure lowering with transdermal glyceryl trinitrate was assessed. Glyceryl trinitrate lowered blood pressure, was safe but did not improve functional outcome. In a small group of patients treated within 6 hours, glyceryl trinitrate improved functional outcome. Analysis of 246 patients with acute intracerebral haemorrhage from ENOS was undertaken to assess whether there were any differences in functional outcome among those who continued prior antihypertensive drugs during the immediate stroke period compared to those assigned to stop temporarily for 7 days. The results were neutral indicating that there was no benefit in those who continued treatment. Data of 1,011 patients with intracerebral haemorrhage in hyperacute trials from the VISTA collaboration showed differences in baseline characteristics and functional outcomes among patients from various ethnic backgrounds. A systematic review was updated to assess the effect of 26 randomised controlled trials that aimed to alter blood pressure within one week of acute stroke. The results showed that blood pressure reduction did not improve functional outcome irrespective of stroke type. When examined by time, treatment within 6 hours appeared to benefit but the number of patients were small and more studies are needed. The analysis also showed that continuing prestroke antihypertensive drugs in the immediate period after stroke did not benefit and might be harmful. In summary, this thesis provides new information on parameters used to estimate intracerebral haematoma, relationship between management of blood pressure and outcomes after haemorrhagic stroke. The work supports testing of whether very early blood pressure lowering after ictus is beneficial as is being undertaken in ongoing randomised controlled trials. Adjusting for ethnic differences may further identify patients in whom treatment may confer measurable advantage.

Background: Central Nervous System (CNS) inflammatory syndromes represent a spectrum of diseases caused by infiltration of inflammatory cells into the tissue of the brain and spinal cord. They comprise a group of diseases that can be acute or chronic, including optic neuritis, transverse myelitis, acute disseminated encephalomyelitis (ADEM), neuromyelitis optica (NMO) and its spectrum disorders (NMOSD), clinically isolated syndrome (CIS) and multiple sclerosis (MS). MS is the most common one while NMO/NMOSD is relatively rare although the exact population-based epidemiological parameters are lacking. The distinction of MS and NMO/NMOSD in adult patients and of MS and ADEM in paediatric patients is important for considerations of prognosis and treatment. This thesis aims to differentiate NMO/NMOSD from MS using epidemiology analyses and quantitative magnetic resonance imaging at 7 Tesla. The hypothesis of the thesis is that NMO/NMOSD can be differentiated from MS by disease incidence, prevalence, severity, and the quantitative characteristics of the lesion and non-lesion white matter of the brain. Methods: The thesis firstly employed a comparative epidemiological study to estimate incidence and prevalence of MS and NMO/NMOSD, and to estimate risks for associated comorbidities and mortality between NMO/NMOSD and MS. The thesis then applied two quantitative techniques to compare the white matter lesions and non-lesion white matter (so-called normal-appearing white matter [NAWM]) of both NMO/NMOSD and MS. Results: The results are presented in seven chapters. Chapter 1 provides a general review of CNS inflammatory syndromes. Chapter 2 shows the estimated incidence and prevalence of CNS inflammatory diseases in 2012 using the Clinical Practice Research Datalink (CPRD). MS is estimated to be the most prevalent and the NMO/NMOSD the rarest. Chapter 3 shows the case-controlled analytic results on the impact of the burden of comorbidity in MS. The burden is cumulative after MS diagnosis and impacts on patient survival compared to non-MS patients. With an interest of new-onset epilepsy in the course of MS compared to non-MS controls, chapter 4 shows that MS patients have a higher risk of having the first medical recorded epilepsy at and after MS diagnosis. Chapter 5 shows the comorbidity burden in NMO/NMOSD, and estimates the risk for mortality between NMO/NMOSD and age- and gender-matched MS populations. Chapter 6 compares in vivo white matter lesions and NAWM of the brain between adult patients with NMO/NMOSD or MS and healthy subjects using quantitative T1 relaxation time and magnetisation transfer ratio (MTR) magnetic resonance imaging at 7 Tesla. The results show that myelin content of white matter lesions is relatively better-preserved in NMO/NMOSD than that in MS; and they also show some changes in the NAWM of NMO/NMOSD, despite the fact that the metrics of the histogram of both sequences are not different to those in MS. Conclusions: In the UK, NMO/NMOSD is rare with a higher disease severity compared to MS, which is the most prevalent inflammatory disease. In vivo, using T1 relaxation time and MTR imaging at 7 Tesla, the white matter lesions of the brain have more preserved myelin in patients with NMO/NMOSD than in those with MS. There is at least a small proportion of intracranial NAWM having subtle changes in patients with NMO/NMOSD that is detectable, but the changes could not be differentiated from MS in the current study. This thesis helps to better understand the epidemiology of NMO/NMOSD and MS, and MRI detectable changes of lesions and NAWM in NMO/NMOSD.

Introduction: Carotid artery stenosis is associated with stroke and large randomised controlled trials (RCTs) have shown that carotid endarterectomy (CEA) can reduce subsequent stroke risk in those with symptomatic 50-99% carotid stenosis. However, not all patients benefit from CEA equally and therefore other methods of risk stratification are needed. Non-invasive plaque imaging with ultrasound (US) and Magnetic Resonance Imaging (MRI) have shown potential in this regard, but ultrasound is much cheaper and more widely available. Both are associated with plaque instability features on histology and relate to symptom status. There a number of studies in asymptomatic patient cohorts, but only one that shows echolucent plaques on US are a risk predictor for stroke and TIA. Further, there is limited longitudinal data on the predictive value of plaque echolucency or other US features in recently symptomatic patients. We have previously shown that the presence of hyperintense signal in the carotid artery on MRI relates to plaque haemorrhage (MIR-PH) and is a strong predictor of recurrent stroke. However, the temporal stability of the MIR-PH signal is not known for beyond 12 months and how it is linked stenosis progression. The aims of this thesis are to investigate US features for carotid plaque instability and if they can predict stroke in a cohort study of symptomatic patients. These were also compared to MRI carotid plaque imaging and a clinical risk score. Methods: Patients mild to moderate carotid stenosis, not planned for CEA, were recruited into a prospective cohort study. They had ultrasound plaque imaging was performed and five features were compared to risk markers for stroke in a cross-sectional design. The presence of plaque haemorrhage on MRI (MRI-PH) was determined and the Carotid Artery Risk (CAR) score calculated. Patients were followed up until the end of the study or event. In another cohort of patients, serial MRI and US scans were performed to determine the temporal stability of the MRI-PH signal and its effect on stenosis progression. Results: In a cross-sectional cohort study (91 symptomatic and 85 asymptomatic contralateral carotids) I showed that although the US plaque features were associated with symptom status, MRI-PH and a higher CAR score only one (Juxtaluminal Black Area - JBA) was predictive (OR =2.3 and 2.9 and 12.7, respectively). In the longitudinal study of 89 patients with a median of 905 days follow-up, the Greyscale Median (GSM) (log rank test: P=0.995) and JBA (log rank test: P=0.248) were not predictive of future cerebrovascular events. I found that the MRI-PH signal is generally stable over 24 months and MRI-PH negative showed in increase in hyperintense signal (P=0.020) with 7% becoming MRI-PH positive. In a cohort of 88 patients with > 50% carotid stenosis, there were 16 ipsilateral cerebrovascular events over a median follow-up of 1038 days. MRI-PH status predicted stroke (HR = 4.49 (95% CI 1.35-14.94, P=0.014). However the CAR score was not significantly predictive (HR = 1.272, 95% CI 0.410 – 3.951, P=0.677). Conclusions: This thesis shows that JBAs hold promise in being able to predict unstable plaques, but that in the small cohort study it was not predictive of cerebrovascular events. This is probably due to the small effect size and a low number of events recorded. Larger studies are required to properly test this finding. The MRI-PH is stable over a 2-year period and it may be associated with faster rates of stenosis progression. I found that MRI-PH is predictive of stroke in patients with >50% stenosis, but the CAR score is not.

Convulsive status epilepticus (CSE) is an emergency condition associated with mortality and morbidity. It is commonly treated with antiepileptic drugs (AEDs), but these may cause serious adverse events and even death in children. Research on their effectiveness for CSE, and related adverse events in children remains limited. The primary aim of this research was thus to evaluate the effectiveness and safety of AEDs in treating acute tonic-clonic seizure including convulsive status epilepticus (CSE). Two systematic reviews and meta-analyses were conducted to address these aims. The first evaluated the effectiveness of AEDs in children with acute tonic-clonic seizures including (CSE). The second evaluated the safety of AEDs in this population. The systematic review of AED effectiveness identified 20 studies published between 1946 and April 2015. It showed that buccal midazolam was more effective than rectal diazepam for treating acute tonic-clonic seizures including CSE in children, and was associated with a lower recurrence rate. Lorazepam and diazepam were equally effective in terminating seizures, but for lorazepam, intravenous administration was more effective than the buccal, sublingual or intranasal routes. Intravenous valproate appeared to be more effective than intravenous phenytoin and phenobarbital; however, the difference was not significant. The systematic review of AED safety for children with acute tonic-clonic seizures identified 25 studies, published between 1946 and April 2015. These studies were predominantly randomised controlled trials and of these 19 studies reported more than one adverse event, while 6 reported none. A total of 203 adverse events were documented, most commonly respiratory depression (101 children), mainly after treatment with diazepam (46 children). The rates of respiratory depression with buccal midazolam and rectal diazepam were similar (3.0% and 3.3%, respectively). Compared to intravenous diazepam, intravenous lorazepam was associated with less respiratory depression. No child suffered respiratory depression associated with intravenous valproate treatment, compared to one child with intravenous phenobarbital. When looking at all adverse events, intravenous valproate was significantly safer than intravenous phenobarbital. Respiratory depression was not noted in children who received intravenous levetiracetam; however, all levetiracetam studies identified in this review were cohort and non-comparative. In conclusion, in the treatment of acute tonic-clonic seizures (including CSE), buccal was the best administration route for children admitted to the emergency department. Intravenous lorazepam treatment was associated with less respiratory depression than intravenous diazepam. Where IV access was practicable, intravenous lorazepam was the drug of choice. More randomised control trials are needed to evaluate the effectiveness and safety of AEDs as a second-line treatment.

Stroke is a highly prevalent condition. It is a principal cause of adult disability and a leading cause of death in the United Kingdom (UK). The lifetime risk of stroke recurrence is also high, particularly for those with specific risk factors (e.g., hypertension). The long-term management of stroke is multi-faceted and complex. Stroke survivors are often responsible for self-managing their secondary preventive treatment (e.g., oral medication(s)) alongside rehabilitation, which is most beneficial in the first six-months. However, patients undergo a period of psychological adjustment after stroke. This is particularly evident in the early days where mood problems, such as depression, are highly common and affect how well people engage with, and adhere to, their rehabilitation and treatment. The present PhD research was interested in further understanding adjustment after stroke, and in particular, by utilising key evidence from the field of Health Psychology (more specifically, the Common Sense Model (CSM)), examining whether survivors’ perceptions (or beliefs) about their stroke are influential in determining how well people recover. The overall aims of this work were to: • Examine and synthesise the current evidence, through a systematic review and meta-analysis on the CSM in relation to physical illnesses to determine whether individual illness belief domains were important predictors of adherence to self-management in adults with acute or chronic conditions; • Elicit views regarding an assessment method (the Stroke Illness Perception Questionnaire-Revised (Stroke IPQ-R)) that would be suitable and acceptable to a stroke population for accurately measuring peoples’ beliefs in the acute phase (≤three-months post-stroke); • Apply the CSM (utilising the Stroke IPQ-R) to a population within the acute phase of stroke, to determine whether the model provides a useful framework for understanding survivors’ recovery after stroke (e.g., Health-Related Quality of Life (HRQL), disability, and mood). This research incorporated three standalone though inter-related studies, the first of which was a systematic review and meta-analysis. The key findings from this meta-analysis indicated that the majority of illness belief domains outlined by the CSM significantly predicted (albeit weakly) adherence to a range of self-management behaviours (e.g., attendance; medication adherence; diet; physical activity; and other disease-specific behaviours (e.g., blood glucose self-monitoring)). Pooled effect sizes ranged from 0.04 and 0.13. None of the relationships varied according to acute and chronic illnesses; the type of self-management behaviour; or the duration of follow-up. A qualitative study was subsequently undertaken. A variety of approaches were employed (including, expert consultation and Think-Aloud interviews) to develop a stroke-specific version of the IPQ-R. Interview findings showed very few problems with completion of the instrument. Where there were problems, these related to the wording of items, specifically abstract, complex and negative wording; the response format for the identity sub-scale; and questions on the timeline-cyclical and treatment control sub-scales. The revised scale was used in the third and final study. The final study examined the relationship of illness beliefs, assessed by the Stroke IPQ-R developed in study 2, and measures of post-stroke recovery, including HRQL, disability and mood. This was a longitudinal observational study involving 50 survivors (average age=66.9 years, standard deviation =14.5 years) within three-months of their stroke. The primary outcome for this study, HRQL, was measured using the EQ-5D-5L instrument. The secondary outcomes were: disability, which was assessed using the Barthel Index, Modified Rankin Scale, and Nottingham Extended Activities of Daily Living Scale; and mood, measured using the Patient Health Questionnaire-9. Various statistical approaches were employed in the analyses of these data, the results of which are as follows. Spearman’s correlations indicated that participants who perceived their stroke to have fluctuating effects and considerable distress at baseline ( < eight-weeks post-stroke) also reported worse mood one-month later. Multiple mediation analyses were conducted to examine whether baseline mood and coping (follow-up medication adherence, measured using the Medication Adherence Report Scale) mediated any of these relationships. The results indicated that baseline mood rather than coping was a significant mediator in the prediction of worse mood three-months post-stroke. Multiple linear regression analyses were carried out to determine whether baseline illness belief domains explained the additional variance in three-month HRQL, disability and mood, over and above that explained by socio-demographic (e.g., age, gender, and deprivation) and clinical variables (e.g., baseline co-morbidities, stroke severity, and indices of recovery). The findings indicated that baseline illness belief domains significantly added between 7.4 and 29.9% to the overall variance in models for the majority of the abovementioned outcomes. Last, the results from the study demonstrated that baseline illness belief domains were highly significantly inter-correlated at baseline, and with follow-up illness belief domains. In light of this, cluster analysis was carried out to explore whether stroke survivors in the acute phase could be grouped according to their illness belief schema. Three clusters were identified: ‘Low Adjusters;’ ‘Moderate Adjusters;’ and ‘High Adjusters.’ In contrast to ‘High Adjusters,’ Low Adjusters’ were participants who perceived their stroke to be chronic, with fluctuating effects, associated with a lot of symptoms, serious consequences and considerable distress. While baseline cluster membership was not significantly related to follow-up markers of post-stroke recovery, there were trends to suggest that ‘Low Adjusters’ had worse HRQL and mood, and greater disability one-month later compared to ‘High Adjusters.’ The present research had several important implications. The principal theoretical implication concerns treatment beliefs. The findings from all three studies highlighted that people’s beliefs about the effectiveness of their treatment in managing their condition are examined in the CSM in a limited way. There is currently a substantial focus on medication-taking behaviours despite treatments often being more complex (e.g., lifestyle behaviour or surgery). Therefore, the findings indicated that there is scope to further elaborate this domain, above and beyond what has already been undertaken in the Necessity and Concerns Framework. This examines the cost versus benefits decision-making that patients undergo when taking their medication(s) (Horne and Weinman 1999). In addition, suitable instruments need to be further adapted and validated to accurately measure these treatment beliefs. There were also several clinical implications of this work. First, it was emphasised that mood assessments should be carried out in patients immediately after stroke. It was shown in this research that even mild depressive symptomatology affected peoples’ beliefs about their stroke, and thus how well they psychologically adjust in the acute phase. Second, there is a potential to develop brief belief-based interventions for modifying maladaptive beliefs about stroke. For instance, addressing perceptions around the fluctuating effects of stroke and the considerable distress caused by stroke could form the basis for such interventions. In addition, interventions can be targeted to a particular type of stroke survivor (i.e., ‘Low Adjusters’), who are likely to be the people to benefit most from receiving them. However, these findings need to be borne out in a larger sample beforehand. The primary methodological consideration in relation to this work was the logistics of recruiting stroke survivors in the acute phase, which led to issues including: a small sample size; sample non-representativeness; and statistical constraints (e.g., factor analysis).

Cognitive impairment and fatigue are prevalent and impactful symptoms of multiple sclerosis (MS). Effective markers are required by clinical studies to accurately test the efficacy of treatments for these symptoms. Graph analysis of brain networks based on magnetic resonance imaging (MRI) data can feasibly provide useful candidate markers of cognitive impairment and fatigability in MS which may be more objective, reliable and specific than existing markers. My original contribution to knowledge is therefore an exposition of the following hypothesis: “summary graph-theoretic descriptors of brain network organisation are good candidate markers of cognition or fatigue in MS”. To achieve this, network metrics were assessed based on three main criteria: reliability (“are the measurements the same across time and settings?”), validity (“do they measure what they are supposed to measure?”) and responsiveness (“are they altered when a change in cognitive state is induced?”). The applicability of the graph-theoretic approach was first established with a spatial meta-analysis of tract integrity and its relevance to cognition and disability. Reliability over time in healthy subjects was assessed by systematic review and reliability between different scanners and between MS and control groups was assessed in two longitudinal datasets by measuring intra-class correlation (ICC) of graph metrics. The validity criterion was assessed in an analysis of covariance and linear regression of cognitive and fatigue measures with brain network metrics in people with MS. Finally, an exploration of network dynamics during a sustained attention task with a sliding-window approach was performed to test the immediate responsiveness of the measures to alterations in cognitive state. Spatial meta-analysis of white matter tract degradation was performed using the Signed Differential Mapping method. Statistical maps were gathered from the original authors of studies which performed voxelwise correlations between fractional anisotropy (a measure of white matter integrity based on diffusion tensor imaging data) and measures of either cognitive impairment or physical disability. The combined sample included 495 people with MS and 253 controls from 12 studies. MS diagnosis was significantly associated with widespread lower tract fractional anisotropy. Distributions of voxels with significantly lower fractional anisotropy in relation to cognition and physical disability were only minimally overlapping. The number of and effect sizes for significant clusters in the cognition comparison were greater than those for the physical disability comparison, suggesting a greater relevance of cerebral white matter damage to cognition. The main results remained significant when using a stringent p-value threshold of 0.00001 to control for false positives. The next analysis was a systematic review of the reproducibility of graph metrics over time in healthy people. Online databases were searched for articles reporting ICCs for graph metrics based on imaging data and information was recorded on the sample size, acquisition method, inter-scan interval and reported ICCs. Twenty-six articles were included, with a combined sample size of 676. Overall, reproducibility over time was rated as “good”, but heterogeneity of methods precluded in-depth quantitative analysis. A qualitative synthesis of results highlighted the main methodologic factors affecting reproducibility, which included: ICC type, retest interval, fibre tracking algorithm, graph metric type, image processing strategy, region of interest size, graph threshold and acquisition method. Reliability of brain network metrics between scanners was tested using a travelling-subjects dataset in which 5 subjects each underwent a resting-state functional MRI scan at 10 sites. Graph metrics were calculated for each scan and then tested for ICC across sites. Reproducibility was “poor” for most metrics (characteristic path length ICC=0.23, global efficiency ICC=0.18, modularity ICC=0.24) and “fair” for two (clustering coefficient ICC=0.43, small-worldness ICC=0.42). There was limited evidence that some subjects tended to produce less reliable results and that magnets with higher field strengths did not produce more reliable results. The main implication is that multi-site studies using graph analysis of brain MRI data should investigate inter-site reproducibility beforehand. To investigate the validity of graph metrics as markers of cognitive impairment and fatigue, MRI and neurocognitive data were first gathered from 37 people with MS and 23 matched controls. The sample was characterised in detail and comprised a range of cognitive abilities. Data quality was investigated and the small-world structure of the data was confirmed by comparison to random and lattice graphs. Analysis of covariance controlling for age, sex and education showed significant group differences for all but one graph metric. Linear regression models predicted the main measures of cognitive impairment in the MS group, but not in the control group. Measures of fatigue were not well-explained by graph metrics. The direction of the relationships indicated that greater levels of cognitive impairment were related to increased network clustering and modularity, longer average path lengths, lower small-worldness, lower levels of education, old age and sleep disturbance. Finally, responsiveness of graph metrics was investigated in an analysis of functional network dynamics during performance of a sustained attention task. A “sliding-window” approach was taken, in which network metrics were calculated for 84 100-second windows at increments along the fMRI timeseries. Reaction times in the task showed a learning effect for both groups, but were consistently slower for the MS group. Plots of graph metrics over time showed differing responses to the task and to the transition between task and rest periods between groups. The small-worldness and clustering coefficient metrics were correlated with reaction times for both MS (small-worldness: r=0.623, < 0.001; clustering coefficient: r=0.554, p= < 0.001) and control (small-worldness: r=0.586, < 0.001; clustering coefficient: r=0.627, p= < 0.001) groups, but the characteristic path length metric was not (MS: r=-0.154, p=0.313; control: r=0.343, p=0.021). Disconnection of cortical areas by degradation of white matter is a viable explanation for cognitive symptoms in MS. There is some evidence that increased network segregation and decreased network integration may explain cognitive symptomatology. Graph theoretic summary brain network metrics do have potential for use as complimentary information to existing markers of cognitive impairment in clinical studies.

This thesis focuses on the use of the Magnetic Resonance Imaging (MRI) white matter lesion (WML) central vein as a biomarker for multiple sclerosis (MS). MS remains a clinical diagnosis, with reliance on MRI to support the diagnosis. Misinterpretation of the MRI can lead to misdiagnoses of diseases that mimic MS. With the increase in disease modifying treatments, accurate and timely diagnosis is needed now more than ever. Using T2* weighted imaging at 3 Tesla (T) MRI, I explored different aspects of WML central veins in patients with relapsing-remitting (RRMS), primary progressive MS (PPMS), and ischaemic small vessel disease (SVD) including: (1) the effect of using different T2* weighted sequences; (2) how T2* and susceptibility weighted imaging (SWI) and fused imaging techniques such as fluid attenuated inversion recovery (FLAIR)-SWI affected the proportion of WML central veins and; (3) determining if WML central veins were as prevalent in patients with PPMS. Further objectives included: (4) attempting to determine if vascular risk factors altered the proportion of WML central veins in patients with MS and; (5) using statistical modelling to calculate a simple diagnostic rule using WML central veins to differentiate MS from SVD. The proportion of WMLs with central veins differed significantly between patients with MS and SVD. Variations of the T2* sequence altered the proportion of WMLs with central veins, but the difference between MS and SVD remained statistically significant. T2* and SWI allowed a higher proportion of WMLs with central veins to be detected, with T2* being just as accurate as FLAIR-SWI in allowing the diagnosis of MS or SVD. Patients with PPMS and RRMS have a similarly high proportion of WMLs with central veins. High sensitivity and specificity for the diagnosis of MS versus SVD can be achieved by identifying a subset of WMLs with central veins. WML central veins could be used as an MRI biomarker using T2* imaging at 3T to differentiate cases of diagnostic uncertainty with RRMS, PPMS and SVD. Application of this imaging technique to patients with diagnostic uncertainty in prospective studies needs to be studied along with refining a clinical diagnostic rule.

Introduction: Medulloblastoma is an aggressive malignant neuro-ectodermal tumour of the cerebellum which accounts for 15-20% of childhood central nervous system tumours and frequently disseminates to the leptomeningeal spaces of the brain and spinal cord. Patients with disseminated disease respond poorly to post-surgical multimodal treatment which is thought to be explained by the intrinsic drug-resistant nature of these tumours. Here, we hypothesized that cells gain migratory and invasive capabilities by undergoing an Epithelial-Mesenchymal Transition (EMT)-like process whereby cells alter phenotypically and acquire stem cell-like properties during tumour dissemination. In this study, metastatic genes were identified and tested using 3D in vitro model systems which incorporated important components of the extracellular matrix. Using these markers, evidence for an EMT-like process and the role of the multi-drug transporter, ABCB1, was investigated in metastatic medulloblastomas. Small molecule inhibitors were also used to investigate whether metastatic processes could be targeted and drug resistance mechanisms could be circumvented in both in vitro and/or in vivo settings. Materials and Methods: Growth, morphology and biological processes (e.g. cell migration) were assessed in a panel of non-metastatic and metastatic medulloblastoma cell lines, as well as in non-tumourigenic neural stem cells cultured in a 3D basement membrane extract (BME) using the alamar blue assay (measure of metabolic activity) and time-lapse imaging. Putative metastatic markers were identified through literature review analysis of gene expression datasets or immunohistochemistry of tissue micro-arrays (TMA). These markers were then assessed in samples obtained from medulloblastoma cell lines cultured as 2D monolayers and grown in BME (for 3 and 6 days) by QRT-PCR analysis. Protein expression of selected markers were also assessed in mouse orthotopic xenograft samples by immunohistochemistry or in cell lines using immunofluorescence analysis. Finally, small molecule inhibitors (WIP1 and ABCB1 inhibitors) were used in 3D culture systems (3D spheroid and 3D BME assays) and in an orthotopic metastatic mouse model. Results: Medulloblastoma cell lines demonstrated different growth patterns in 3D. Metastatic cell lines formed metabolically active aggregates which sustained continual cell migration for at least 6 days; whilst non-metastatic and non-tumourigenic cells showed low metabolic activity and rapidly differentiated. Metastatic cell lines which were sustained longest in BME (D283 Med and MED1) demonstrated upregulation of the EMT transcription factor, TWIST1, along with several other EMT and TWIST1-related factors. Further analysis included overexpressing TWIST1 in a non-metastatic cell line (MED6 TWIST1) which induced a dispersed phenotype in 2D and cell aggregation in the 3D BME model which phenotypically resembled metastatic cell lines. TWIST1 and ABCB1 expression correlated with metastasis in patients and was upregulated in the invasive edge of primary tumours and in spinal metastases in an orthotopic metastatic mouse model. Small molecule inhibitors targeting WIP1 (a published metastatic marker) and ABCB1 inhibited cell migration of metastatic cell lines grown in 3D including the MED6 TWIST1 cell line. ABCB1 inhibition also increased sensitivity to etoposide treatment in 3D spheroid models and an orthotopic metastatic in vivo model. Conclusion: The 3D BME model utilised in this study can be used to distinguish metastatic capacity and transcriptional changes of medulloblastoma cell lines. Furthermore, data from this study supports a role for a TWIST1 driven EMT-like process in metastatic medulloblastoma and supports the use of ABCB1 inhibition to overcome chemoresistance.

Introduction Patients with symptomatic carotid artery disease have increased risk of cognitive impairment. Multiple mechanisms of cognitive impairment have been proposed. However, the contribution of each mechanism to cognitive impairment and the interrelation between these mechanisms remain unclear. Lesional and non-lesional damage of brain tissues may lead to cognitive impairment in patients with symptomatic carotid artery disease. The research presented aims to use whole brain statistical brain mapping and quantitative neuroimaging techniques to understand vascular cognitive impairment (VCI) in a group of patients with symptomatic carotid artery disease. Methods Studies presented in this thesis were based on available multimodal MRI and cognitive performance data from a study in patients with symptomatic carotid artery disease. Totally 108 subjects had FLAIR and diffusion MRI scans and 62 of them had resting-state fMRI scans in a 3T MRI scanner. All participants (n=108) underwent cognitive assessments using Addrenbrooke’s cognitive examination-revised (ACE-R). Voxel-based lesion symptom mapping (VLSM) technique was used to identify the contribution of ischaemic lesion presence to cognitive performance. Tract-based spatial statistics (TBSS) approach was used to investigate the association between microscopic damage of white matter and cognitive performance. Independent component analysis (ICA) and seed-based analysis approaches were used to investigate functional disconnection of large-scale cognitive networks and its contribution on cognitive impairment. The association between cognitive performance and ischaemic lesions volumes and microscopic damage of brain tissues were also assessed. Results The presence of chronic subcortical ischaemic lesions located in strategic white matter tracts including anterior thalamic radiation, forceps minor and forceps major contributed to global cognitive impairment in patients with symptomatic carotid artery disease. Ischaemic lesion volumes and microscopic damage of brain tissues including strategic brain areas, strategic white matter tracts, normal appearing white matter and white matter tract skeleton were related to cognitive impairment. Functional disconnection of large-scale cognitive networks including default mode network (DMN), dorsal attention network (DAN), left fronto-parietal network and salience network was found in patients with probable vascular mild cognitive impairment (VaMCI). Functional disconnection beyond the large-scale cognitive networks, showing as decreased functional connectivity between bilateral hippocampi, bilateral dorsal lateral prefrontal cortex (DLPFC), anterior cingulate cortex (ACC) and posterior cingulate cortex (PCC) with the rest of the brain, was also found in patients with probable VaMCI. Also, the thesis presented preliminary results of the role of lesional and non-lesion brain damage in fluency performance in patients with symptomatic carotid artery disease. Conclusions This thesis provides further evidence for the role and to the nature of disconnection in VCI and proposes several disconnection related imaging biomarkers.