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Background: Previously, Vestibular Schwannomas (VS) were thought to arise as a consequence of the clonal expansion of neoplastic Schwann cells, but more recently there has been increasing interest in the role immune cell infiltration may play in their growth. The focus of this thesis was to investigate the link between immune cell infiltration and tumour growth, as well as to interrogate the relationship between inflammation and angiogenesis in VS. Methods: 101 growing and 19 static/shrinking sporadic VS were subjected to immunostaining for Iba1, CD31 and fibrinogen. Plasma samples from 50 patients with growing VS and 25 patients with static VS were analysed to assess the circulating concentrations of 47 cytokines, chemokines and growth factors. Finally, immunostaining for proteins associated with Angiopoietin/Tie2 angiogenesis pathway was performed on sporadic NF2-Schwannomatosis associated VS and this was further validated with quantification of angiopoeitin 1 and angiopoeitin 2 expression. Results: Growing VS were characterised by significantly higher infiltration of Iba1+ macrophages and increased microvessel surface area when compared to static tumours. There was a strong positive correlation between macrophage infiltration and VS growth rate, and this effect persisted following correction for tumour size. Significantly increased concentrations of IL-1α, IL-6, IL12p40 and IL-18 were observed in the plasma of patients with growing VS, alongside CCL22, CXCL9 and CCL4 when compared to plasma obtained from patients with static VS. Conclusions: This thesis provides clear evidence of a relationship between macrophage infiltration and VS growth. This macrophage infiltration may be driven by tumoral secretion of chemokines into the bloodstream and targeting these processes may yield therapeutic benefits in patients with sporadic and NF2-Schwannomatosis associated VS.

Parkinson's disease (PD) is the second most common neurodegenerative disorder following Alzheimer's disease (AD). In PD, extensive loss of dopamine neurons, mainly in the substantia nigra, is found in the brain alongside proteinaceous deposits of α-synuclein known as Lewy bodies. Although characterised primarily by motor dysfunction, up to 80% of individuals with PD go on to develop cognitive dysfunction within 20 years of initial diagnosis, in a condition referred to as Parkinson's disease dementia (PDD). PDD shares many characteristics with other neurodegenerative diseases, such as AD and Huntington's disease (HD), including progressive dementia symptoms, neuronal loss which is initially region-specific but eventually spreads throughout the brain, and metabolic perturbations-including mitochondrial dysfunction, oxidative stress, and energy pathway disruption. Previous studies have identified specific metabolic and metallic dysregulations shared in AD and HD brains, including marked widespread increases in glucose and urea, and decreases in copper. This raised the question as to whether similar perturbations may be present in PDD. Using mass spectrometric methods, this thesis aimed to perform parallel metabolomic and metallomic analyses of PDD brain tissues, the results of which could then be compared to those obtained in AD and HD. Firstly, investigations into the effects of tissue collection variables such as post mortem delay were carried out to ascertain the suitability of different donor tissues for metallomic and metabolomic analysis. Using the results of this preliminary study, suitable tissues were selected for investigations of metals and metabolites in PDD brain tissue obtained from both the UK and the US. These investigations revealed strikingly similar findings to those observed in AD and HD, including widespread decreases in copper, increases in urea, and perturbations in metabolic pathways involved in energy production and purine metabolism, as well as regional-selective decreases in pantothenic acid. These results indicate, for the first time, a shared metabolic basis for three neurodegenerative diseases featuring dementia-raising the possibility not only of shared pathogenic mechanisms, but shared therapeutic targets for these conditions.

The translocator protein (TSPO) is a mitochondrial protein with five transmembrane domains. It is involved in many biological functions including cholesterol transport, cell proliferation and apoptosis. TSPO is expressed at a low level in normal brain tissues but its expression is up-regulated in brain tumours and many inflammatory diseases. A positive correlation has been identified between TSPO and proliferation in brain tumours suggesting a possible role for TSPO in tumour progression. Vascular endothelial cells and glial cells have been shown to express TSPO in many neuropathological conditions, including tumours of the central nervous system (CNS). Therefore, studying the contribution of neurovascular unit (NVU) cellular components to TSPO expression warrants further research. Here, TSPO expression was examined for several cell types of the NVU via immunohistochemistry (IHC) and immunofluorescence (IF). TSPO was found to be expressed by astrocytes, macrophages and endothelial cells in normal condition. Additionally, TSPO expression levels in glioma associated macrophages/microglia (GAMMs) and vascular tissues in oligodendroglioma has not been previously studied. This perhaps represents a missed opportunity to utilize TSPO expression as a diagnostic marker or predictor of tumour behaviour. Therefore, tissue microarray (TMA) and Mass Cytometry have been used to examine the vascular, GAMMs and neoplastic TSPO expression in oligodendrogliomas. It was noted in TMA analysis that neoplastic cell contribution to overall TSPO expression levels was greater compared to the vascular (CD31) and inflammatory cell (GAMMs) components. Next, tissue from fifty oligodendrogliomas was examined by IHC to discern whether TSPO expression varied significantly between low (WHO II) and high grade (WHO III) tumours. TSPO expression was significantly increased in high-grade oligodendrogliomas compared to low-grade. Furthermore, TSPO was found to be a predictor of prognosis for oligodendroglioma patients. High TSPO expression levels correlated with poor outcome in terms of disease progression and mean survival time. Furthermore, the regulation of TSPO expression is very poorly understood. This thesis has studied the epigenetic regulatory mechanism that might control TSPO expression in oligodendroglioma. For that purpose, DNA was isolated from thirty-eight cases of oligodendrogliomas and the methylation status of the TSPO gene and its promoter examined by using the Infinium methylation EPIC beadChip. No difference within the promoter methylation state was seen between samples showing high or low TSPO expression. As an alternative mechanism, RNA sequencing for oligodendroglioma frozen tissues RNA samples was done to identify possible candidate genes which might regulate TSPO expression. In conclusion, this thesis has identified TSPO as a possible biomarker of oligodendroglioma progression and patient's survival. It provided a wide coverage of the possible cellular components that contributed to TSPO expression in NVU and oligodendrogliomas. Moreover, TSPO expression was not found to be regulated by promoter methylation suggesting other alternative mechanism which might be involved.

Introduction: Brain tumours continue to present specific imaging challenges, in particular related to grading and progression, from initial diagnosis, through surveillance and treatment planning, to response assessment and prognostication. In this project we explored the role of advanced MR biomarkers and PET imaging with two different tracers in glioma, [¹¹C]-Methionine and TSPO-binding ligand [¹¹C]-(R)-PK11195. Both PET tracers are ubiquitously low in the normal brain and high in the pathological tissue. Methods: In the first experimental chapter we investigated the role of [¹¹C]-(R)-PK11195 and relative cerebral blood volume (rCBV) in prediction of transformation in a cohort of 26 low grade gliomas (LGGs). Results were further validated on tissue derived from a consecutive cohort of 30 oligodendroglial tumours. The second step of our study extended to a larger cohort of 41 low and high grade gliomas, where we compared [¹¹C]-Methionine uptake to rCBV and diffusion tensor imaging (DTI) derived metrics in correlation to strongest signal areas of the tumour. Finally, in the last chapter we investigated the extent and potential causes of neuro-inflammation in the normal appearing brain of 66 patients with glioma, comparing to a cohort of 19 healthy controls and 27 patients with other types of intracranial tumours. Results: Progressively higher uptake of [¹¹C]-(R)PK11195 was seen in anaplastic gliomas compared to the lower grade (p=0.001), whilst rCBV failed to show a significant difference (p=0.23). Tissue data from TSPO antibody staining showed a very good correlation to the imaging results and were further validated in a cohort of 30 oligodendroglial tumours (sensitivity 92.85% and specificity 93.75%). In the second study, significant [¹¹C]-Methionine uptake was seen with increasing histological grade (p<0.001). Significant rank correlations were identified between peak methionine uptake ratios and peak values for both relative blood volume (p=0.004) and flow (p=0.036). Correlation of peak methionine values was additionally noted to inversely correlate with trough values of mean diffusivity (p=0.001). Tumours with vivid [¹¹C]-Methionine uptake appeared to have a poorer prognosis with shorter time-to-progression interval (p=0.015). Lastly, we found increased microglia activation, in both cerebral hemispheres in glioma patients compared to controls. This was most prominent in the tumour-bearing hemisphere (p<0.0001) but was also evident in the controlateral hemisphere (p=0.0078). Patients presenting with seizures showed a positive correlation between extra-tumoural microglial activation in the cerebrum and the duration of epilepsy. Conclusions: TSPO PET imaging can offer good accuracy in prediction of transformation which can be also translated in tissue analysis via antibody staining. Identifying tumour most malignant regions can be trivial using only MR derived data and PET imaging with [¹¹C]-Methionine can contribute to better surgical planning and understanding of tumour microenvironment. Increased neuro-inflammation in patients with glioma is linked to seizure activity, although it is difficult to ascertain cause and effect.

Idiopathic Inflammatory Myopathies (IIMs) are a heterogeneous group of acquired diseases characterised by inflammation of the skeletal muscle. The most common forms are sporadic inclusion body myositis (sIBM), dermatomyositis, polymyositis, overlap syndormes and necrotising myopathy. Sporadic IBM is the most common acquired myopathy over the age of 50 years, however its pathogenesis remains largely unsolved and there is still debate on whether it is a primary inflammatory or a degenerative condition with secondary inflammatory component. I used an in silico approach to identify molecules of interest that might shed light on the pathogenesis of sIBM. Emerin, a nuclear envelope protein, resulted as one of the candidate molecules. To verify in silico analysis results I reviewed a cohort of patients with sIBM phenotype identified at Charing Cross Hospital. This review allowed me to select 10 sIBM patients and identify 3 further patients with sIBM-like phenotype and good response to steroids. The selected patients have been then tested through immunohistochemical, immunoblotting and sequencing studies. Immunohistochemistry studies using anti-emerin antibodies showed abnormal distribution of emerin in the sIBM patients but not in the 3 patients with sIBM-like phenotype. Interestingly, sequencing of the emerin gene identified missense mutations in the 5'-untranslated region. The three IBM-like patients were extensively studied and serum analysis allowed me to identify a novel autoantibody that proved to target the sarcoglycans. Sequencing of the sarcoglycans genes in the three patients identified a missense mutation in the β-sarcoglycan affecting the 3D-structure of the extracellular component. The identification of this novel autoantibody prompted a further pilot study to develop a diagnostic test to identify novel autoantibodies to sarcolemmal proteins. Variable positive reactivity against sarcolemmal proteins, suggestive of presence of autoantibodies against sarcolemmal proteins was identified through immunoblotting in the sera of 10 further IIM patients, but not in 9 controls. This study highlights the complexity behind the pathogenesis of IIM and importance of unified classification criteria. Although further studies on a larger scale will be needed I suggest that emerin immunolabeling could be used as diagnostic biomarker in patients with sIBM. In addition, a pilot study using of skeletal muscle protein extract revealed novel autoantibodies in IIM whose pathogenicity remains to be established.

There is growing evidence that maximal surgical resection of malignant gliomas is beneficial, both by increasing the progression free survival (Stummer, Pichlmeier et al. 2006) and also by facilitating postoperative chemotherapy and radiotherapy (Stupp, Mason et al. 2005). In this context there has been an increased need for real time intraoperative imaging in brain tumour surgery. The complex theatre arrangements, prohibitive cost and prolonged theatre time has restricted the wide application of intraoperative MRI (iMRI). Modern intraoperative ultrasound, which in the past has been relatively underused in oncological neurosurgery, is affordable, and easy to use (Unsgaard, Ommedal et al. 2002), but has not been robustly validated. This study attempts to histologically validate the accuracy of the intraoperative US and to explore its potential as an intraoperative navigation tool, which can accurately guide biopsies and resections of intrinsic brain tumours. The digital data extracted from US images obtained during ultrasound guided biopsies was correlated with the histology of the relevant specimens. Image analysis of selected regions of interest (ROI) was employed to extract quantitative parameters from the digital ultrasound images. The mean pixel brightness (MPB) and the standard deviation (SD) were correlated with histological parameters. The pattern of histograms from the selected ROIs was observed and correlated with the histological findings. A close correlation was observed between mean pixel brightness (MPB), an objective measure of echogenicity, and cellularity and an equally close correlation between the standard deviation (SD) and the intrinsic cellular diversity of the analysed areas. These two together, despite not being specific, can indirectly suggest the nature of the tumour and also reflect the sensitivity of intraoperative US to detect the presence and the extent of intrinsic brain tumours. Our findings could have translational potential as an intraoperative guidance tool.

The translocator protein (TSPO) is an 18 kDa molecule spanning the outer mitochondrial membrane. Structural predictions, reported physical interactions and experimental data from diverse models suggest that TSPO is involved in apoptosis, proliferation and steroidogenesis. TSPO is up-regulated in several human cancers and its high levels are frequently associated with advanced disease and poor outcome. TSPO was shown to be up-regulated in astrocytomas and this was correlated with high tumour grade and shorter patient survival. Here it is confirmed that TSPO is highly expressed in astrocytomas and this is correlated with tumour grade. TSPO expression is significantly higher in astrocytic tumours than in oligodendrogliomas and this differential expression is epigenetically driven. Low TSPO expression in oligodendrogliomas is associated with frequent promoter methylation, whereas in astrocytomas, where TSPO is expressed at high levels, the promoter typically remains unmethylated. As oligodendrogliomas and astrocytomas have very different biology and prognosis, and several TSPO ligands, including those enabling positron emission tomography (PET) scanning, are in development, we propose pre-operative PET imaging to differentiate these two histotypes. TSPO is not an independent prognostic factor in our patient population. The main cell population expressing TSPO within the tumour bulk are neoplastic cells. Consistent with no evidence for TSPO as a strong prognostic factor in gliomas, no alteration in proliferation, cell cycle progression and baseline or induced apoptosis following TSPO siRNA knock-down in an in vitro astrocytoma model is demonstrated. Furthermore, there was no evidence of cortisol production by several astrocytoma cell lines in vitro despite TSPO protein expression. Out of the examined MPTP-related binding and functional partners of TSPO (VDAC1, ANT, HK I and II) only HKII up-regulation appears be reproducibly related to patient outcome in gliomas. Consistently, targeting hexokinases in vitro in a glioma model inhibited proliferation and increased apoptosis.