There is an intimate spatial and functional relationship between cerebral microvasculature and the neuroglial tissues. It is known that both cerebrovascular and neuroglial alterations occur in multiple sclerosis (MS), but the pathophysiological relationships between these alterations, and the functional consequences, are not well characterised in vivo. Evidence from previous literature indicates that there are subtle changes in blood brain barrier (BBB) in normal appearing white matter (NAWM) of MS. However, lesion development due to subtle BBB leakage remains unclear. Additionally, the haemodynamic alteration and microstructural changes in grey matter (GM) is a well-known feature in MS. Nevertheless, the structural and functional relevance of GM perfusion and diffusion necessitate more exploration. The main objective of this study was further interpretation of lesion development in MS, using advanced MRI techniques. Moreover, the study also aimed to provide valuable understandings into the association of different MRI measures with structural changes and clinical performance in MS, using advanced image analysis methods. The study has focused on the following goals: to determine BBB permeability in NAWM of MS and its correlation with microstructural damage, the relationship of subtle BBB leakage to lesion development, the association of grey matter perfusion with structural changes and functional performance and finally, the cortical diffusion alteration and its relevance with functional performance. Therefore, the main hypothesises of this thesis are: firstly, MS patients have impaired BBB permeability in NAWM compared to healthy controls. Secondly, that mean diffusivity (MD) in NAWM will correlate with BBB permeability in MS. Thirdly, that subtle BBB leakage in NAWM may precede lesion progression in MS. Fourthly, that cortical and deep GM perfusion is reduced in MS patients compared to healthy controls. Fifthly, that cortical perfusion correlates inversely with cortical atrophy in MS and finally, pattern of regional cortical MD variability will explain performance in different functional domains in MS. All MS and healthy participants included in this study underwent an MRI scan at 3T. Functional and cognitive assessment was performed on MS. Dynamic contrast enhance-MRI was used to generate perfusion and permeability maps. The microstructural changes were measured using diffusion imaging. The cortical perfusion and diffusion changes were explored by Surface based analysis approach. Increased BBB permeability in NAWM of MS was detected when compared to healthy controls (p<0.05) and it revealed an association with MD in NAWM (r= 0.48, p = 0.01). The findings also revealed subtle BBB breakdown indicated by an increase in the permeability parameter (Ktrans) in prelesion NAWM. GM hypoperfusion was shown in MS when compared to healthy controls (P<0.05). However, surface based analysis revealed no correlation between cortical hypoperfusion with cortical atrophy and microstructural damage in MS. Furthermore, there was no association between GM perfusion and clinical scores. In addition, the cortical diffusion alteration revealed vertex-wise correlation with functional scores in MS (P<0.05). In conclusion, advanced MRI and advanced image analysis in this study has delivered novel insights on lesion development and other pathological changes affecting GM and WM in MS, which might have prognostic and therapeutic importance in MS.

The main aim of this thesis was to examine the role of metabotropic glutamate receptors (mGluRs) in the control of the excitability of sympathetic preganglionic neurones (SPNs). A review of the literature concerning SPNs and glutamate receptors has been performed. Experiments were made using whole-cell patch-clamp recordings from neurone in slices of spinal cord. During recording neurones were perfused with either Lucifer yellow or biocytin and following recording the slices were processed and the morphology of the neurones examined. SPNs were identified by their characteristic electrophysiological and morphological properties. The pharmacology of mGluR agonist-induced changes in the excitability of SPNs was examined. Almost all SPNs demonstrated a depolarisation with a pharmacological profile suggesting that it was mediated by a group I mGluR. A minority of SPNs also demonstrated hyperpolarising responses to agonists with activity at group II mGluRs, suggesting that activation of group II mGluRs may have inhibitory effects on some SPNs. Activation of mGluRs modulated the frequency of ongoing membrane potential oscillations and induced membrane potential oscillations in a subpopulation of previously silent SPNs. Responses involving induction of oscillations or increases in the frequency of spontaneous oscillations had agoinst profiles suggesting involvement of a group I mGluR, and responses involving decreases in the frequency of oscillations had agonists profiles suggesting that they were mediated by a group II mGluR. This study has demonstrated excitatory and inhibitory effects mediated by activation of different mGluRs on SPNs and has investigated some of the underlying mechanisms. Activation of mGluRs was also shown to modulate glutamatergic synaptic inputs to SPNs. Synaptically evoked responses mediated by mGluRs remain to be demonstrated.

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.

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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.

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.

Synaptic vesicles are the substrate of neurotransmission in most nerve terminals in the central nervous system. These small membrane spheres fuse with the synaptic membrane in an activity-dependent manner and release neurotransmitter into the synaptic cleft. Subsequently, vesicles are reclaimed through endocytosis prior to reuse. This recycling process is key to supporting ongoing signalling in the brain. While substantial effort has gone into defining basic characteristics of vesicle recycling, for example elucidating the timing of vesicle turnover, key questions remain unanswered. An important area with significant knowledge deficits relates to the relationship between vesicle function and ultrastructural organisation in the terminal. The aim of this thesis is to address this issue, exploiting new methodologies which provide novel insights into function-structure relationships of vesicle populations in acute brain slices. Specifically, this study considers organisational principles of three defined vesicle pools as well as examining the impact of an established plasticity protocol on pool properties. The first results chapter, Chapter 3, outlines and validates the novel protocol used for fluorescently labelling functional recycling vesicle populations in acute rat brain slices using the vesicle-labelling dye FM1-43 and new antibody based probes (syt1-Oyster, CypHer5E). Reporter-labelling and release properties are compared to similar approaches using cultured neurons. We conclude that this approach provides a more physiologically relevant method to study the functional properties of cells than used previously in cultured neurons. Chapter 4 outlines experiments utilising the capability of FM 1-43 to be photoconverted to an electron-dense form to allow a defined vesicle population, the readily releasable pool (RRP), to be characterised ultrastructurally. The RRP is arguably the most significant pool class, released first in response to an activity train. Functional assays and time-stamped electron microscopy are used to define basic properties of this pool, including its size, functional release kinetics, and temporal organisation. Specifically, the results demonstrate that retrieved vesicles are close to the active zone after stimulation, but mixed randomly in the terminal volume over 20 min. These findings address fundamental questions about vesicle reuse, the composition of future vesicle pools, and thus the mechanism of ongoing signalling in the brain. The same approach was used in Chapter 5 to examine the influence of Long Term Depression (LTD) on pool function and ultrastructure. LTD was induced in presynaptic terminals in CA1 via Schaffer collateral activation, and the following effects were observed: 1) a change in release kinetics; 2) a reduction in the total recycling pool size; and 3) no change in the composition of the docked pool. These findings demonstrate that there is a presynaptic component to LTD and that vesicle recruitment into the recycling pool appears to be an important possible substrate. However, the results suggest that such changes appear to be selective for specific pool subsets. Overall, work in this chapter offers new insights into fundamental principles supporting synaptic plasticity. Chapter 6 expands on previous studies which have demonstrated that recycling vesicles are constitutively shared between neighbours. This sharing of a ‘superpool' of vesicles has implications for the ability of synapses to adapt to changes in input weighting. In this chapter, the methods outlined above, as well as a new 3D EM technology, are used to define the size, positional organisation, and clustering properties of this pool in native hippocampal slice system. The findings in this chapter reveal that extrasynaptic vesicles appear to show a greater degree of motility than vesicles which remain in the intrasynaptic cluster, perhaps implying differential interactions with structural proteins in the synapse. Characterising the superpool is increasingly relevant, as it is now implicated in models of plasticity and disease. Taken together, these results show that the ultrastructural arrangement of recycling vesicles is highly activity-dependent, and that the cytoarchitecture plays a large role in determining the functionality of individual vesicles and synapses.

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.

Sustained neural activity critically relies on the ongoing function of small central synapses. In particular, activity-driven fusion and recycling of neurotransmitter-filled vesicles at presynaptic terminals are key processes responsible for information transfer. Despite the fact that vesicle exocytosis and endocytosis are of great interest, the mechanisms of their regulation are still poorly understood. Moreover, hippocampal synapses exhibit high levels of variability in their structure and function, but the basis for this remains unclear. The aim of this work was to investigate these fundamental properties and establish key rules of regulation. Specifically, we wanted to test whether the timing of endocytosis of single synaptic vesicles was characteristic at individual boutons, and to investigate structural and molecular properties of synapses that underlie their particular behaviour. To explore this, we used a variety of optical imaging techniques in rat hippocampal neurons based on acutely applied probes such as FM1-43 dye, fluorescently tagged antibodies and genetically encoded reporters of presynaptic function, as well as ultrastructural readouts using electron microscopy. We found that although the timing of vesicle retrieval, measured with the optical reporter sypHy2x, was highly variable across the population of synapses, individual boutons showed signature endocytic kinetics. We also uncovered the properties of synapses that determine this behaviour, and demonstrated that these could be modulated, leading to predictable changes in the timing of recycling. These findings offer new insights into the rules that govern the function of presynaptic terminals. A second related objective examined was whether amyloid beta, the misfolding protein implicated in Alzheimer's disease, causes changes that are detrimental for efficient vesicle recycling. We showed that oligomeric amyloid beta 1-42 impaired endocytosis and disrupted other related presynaptic processes. We suggest that vesicle recycling mechanisms are important target substrates in Alzheimer's disease providing potential new avenues for development of therapeutic approaches.

Functional brain networks are interconnected brain regions that flexibly coordinate their activity to support cognitive demands (Fair et al., 2009). Functional brain connectivity describes a statistical dependency between the activities recorded at spatially distinct brain regions (Friston, 2009). Changes in the pattern of connections and level of activation in functional brain networks are thought to occur across development (Taylor, Donner, & Pang, 2012) but the nature of these changes and their relationship to cognitive development have yet to be delineated clearly. This thesis seeks to deepen our understanding of the development of functional brain connectivity across the age range 9-25 years. We used magnetoencephalography in conjunction with canonical correlation analysis to explore functional connectivity via amplitude-amplitude envelope correlations in 110 datasets (39 working memory, 33 relevance modulation (attention processing) and 38 resting state). At the core of this thesis, we have presented novel findings that show non-linear functional connectivity changes across development, with an increase from childhood (age 9-12) to late adolescence (age 17-20) followed by a reduction into young adulthood (age 21-25), resembling an inverted-U-shaped trajectory at least in the females included in this study. Whilst there are subtle yet statistically significant differences in how the functional connectivity profile from 1-100 Hz is modulated by different factors, the overall pattern of functional connectivity development appears to be remarkably consistent across cognitive demands and networks. Critically, this work is the first example of such findings and suggests that functional brain networks supporting higher order cognitive function are not alone in undergoing functional development; sensory networks that reach structural maturity early on in life also undergo functional development from age 9 to 25.