Parkinson's disease (PD) is a devastating neurodegenerative disorder with cardinal motor symptoms linked to death of nigrostriatal dopaminergic neurons. Several findings place the protein cc-synuclein firmly at the centre of PD research. First, co-synuclein is the main component of Lewy bodies, the defining inclusion in PD. Second, missense muta- tions and duplication or triplication of the SNCA locus cause autosomal dominant PD. Finally, recent genome-wide association studies revealed the involvement of SNCA vari- ants in sporadic PD, providing a central link between genetics and neurodegeneration in PD. The aim of this study is to elucidate the role of cc-synuclein on dopamine homeos tasis. To this end, two approaches to modulate cc-synuclein gene expression have been developed in a relevant human dopaminergic cell line: (i) RNAi-mediated knockdown of cc-synuclein and (ii) expression from a BAC vector carrying the entire human SNCA genomic locus under the control of endogenous promoter sequences. Modulation of «-synuclein expression caused several functional changes. cc-Synuclein was shown to influence cellular dopamine levels, specifically acting on dopamine biosyn- the tic enzymes TH and AADC. Suppression of cc-synuclein increased dopamine content, whereas modest overexpression of cc-synuclein was sufficient to dramatically reduce to- tal dopamine content. At the same time, the presence of A53T cc-synuclein mutation reduced TH activity. Changes in DAT function were also observed. Consistent with pre- vious findings, suppression of cc-synuclein led to a 50% decrease in dopamine uptake. A53T-mutated cc-synuclein also altered DAT activity, causing a reduction in dopamine uptake velocity coupled with a decrease in dopamine affinity for the transporter. This could be due to altered DAT post-translational modifications and consequently changes in DAT trafficking. This work highlights important roles for cc-synuclein in maintaining dopamine home- ostasis and demonstrates that modulation of cc-synuclein expression has complex effects on dopaminergic neuronal function.

In this thesis I explore the ethical debate over the use of 'sham surgery' control groups in the context of neurosurgical research for Parkinson's Disease, with specific reference to brain surgery techniques involving fetal cell transplantation. I use a 'dual mode' approach involving both theoretical ethical analysis and qualitative empirical research conducted with people with Parkinson's Disease and their relatives. A theoretical and organisational structure for this thesis is provided by the 'three approaches' framework for ethical analysis of clinical research developed by Clare Foster. This framework addresses three broad areas of moral concern in research: i) use of a scientifically appropriate methodology, ii) the duty to protect patients' best interests and iii) consideration of research participants' autonomy. Drawing on both theoretical analysis and the empirical data generated I conclude that the use of sham surgery control groups in the PD cell transplantation trials under discussion was problematic from all three of these ethical perspectives. A prohibition against all forms of sham surgery is not supported, however, and features which might make a sham-controlled surgical trial less ethically contentious are discussed.

MicroRNAs are a newly described class of short endogenous non-coding RNAs, which bind to the 3' untranslated region of a target mRNA molecule and result in either their degradation or inhibition of their translation. Recently microRNAs have been shown to play a role in neurogenesis in the adult brain and with neuronal deterioration in neurodegenerative disorders. In particular, microRNA-7 has been shown to bind to both alpha synuclein and the epidermal growth factor receptor (EGFR). Alpha synuclein is known to play a key role in the pathogenesis of Parkinson's disease (PD), which is a common neurodegenerative disorder, whilst the EGFR has been shown to be decreased in PD patients in the subventricular zone (SVZ). The SVZ is one of two areas of the adult brain thought to be involved in neurogenesis. Therefore the aim of this thesis has been to investigate the role of miRNA-7 in the regulation of alpha synuclein and the EGFR further. Two lentiviruses were produced, one to cause an overexpression of miRNA-7 and another to cause a loss of miRNA-7 by acting as a target/sponge sequence for miRNA-7 (miRNA-7T). These lentiviruses were firstly transduced into HEK293T cells where miRNA-7 was found to bind to the 3 'UTR of the SNCA gene and inhibit translation causing a decrease in alpha synuclein expression. The miRNA-7T lentivirus was found to effectively bind to endogenous miRNA-7 causing an increase in alpha synuclein expression in HEK293T cells. To investigate this further, the miRNA-7T lentivirus was injected into the SNpc of mice. Data showed that 2 injections of the virus were sufficient to cause an overexpression of the virus in the SNpc. This resulted in an upregulation of alpha synuclein 24 weeks after surgery and a significant loss of dopaminergic neurons. However, the animals did not show any motor impairment and the effects on striatal DA was only reduced to 30% at 8 weeks post surgery. There was therefore, a disparity between the loss of dopaminergic neurons and the levels of striatal DA. To investigate the effect of manipulating miRNA-7 levels on dopaminergic neurons the viruses were also transduced into iPSCs. Unfortunately, the viruses had no effect on alpha synuclein expression in these cells, which may have been due to limitations of the experiment.

The cause of impulse control behaviours (ICBs) in Parkinson's disease (PD) has recently become the focus of research attention. However, 'this research is limited to a biomedical model, centring on the role of dopamine replacement therapy in causing these behaviours. Furthermore, gaps remain in our understanding of ICBs. The first section of this work presents a narrative review of current evidence regarding the etiology of ICBs in people with PD. The review highlights areas of methodological and theoretical discrepancy in the current biomedical understanding. It is argued that a biopsychosocial model may provide a more adequate explanation of these behaviours within PD and thereby inform individual, formulation based approaches to assessment and treatment. Furthermore, research to date has failed to consider perceptions of cause in people with PD who have experienced these behaviours. Therefore, the second part of this work concerns a qualitative investigation which explored how people with PD perceive the cause and controllability of their ICBs. Interpretative phenomenological analysis (IPA) was carried out on data gathered via semi-structured interviews with 10 participants. The resulting themes were 1) 'It does seem to open a whole Pandora 's Box of who we are and why we do what we do; Conflicting views on causality.'; 2) 'Better to live like a tiger for a day than like a lamb for a year; Impulse control behaviours as a coping strategy.'; and 3) 'Just a thing I couldn't control, like a greater power than me; Relationship between causal attribution and perceived controllability.' Causal attributions were found to be fundamental to the perceived controllability of ICBs. In addition, ICBs were linked to coping with PD. Implications for clinical practice and future research are discussed. The final section of this work reflects on methodological, professional and ethical issues which arose throughout the research process.

Mitochondria are the powerhouses of eukaryotic cells and they must remain healthy in order to generate sufficient ATP for cellular function. Dysfunctional mitochondria pose a grave threat to high-energy demanding tissues and are associated with an array of human diseases. Mitochondria exist in a dynamic organelle network that is essential for their intracellular distribution and quality control. A damaged mitochondrion must first be exiled from the network by mitochondrial fission and next be neutralized by a process termed mitophagy. A number of mitophagy pathways exist to specifically target damaged or redundant mitochondria for engulfment by double-membrane autophagosomes in order to deliver them to the acidic lysosome for degradation. This dissertation explores the regulation and molecular mechanisms of the PINK1/Parkin mitophagy pathway. Mutated in several forms of Parkinson's disease, the PINK1 kinase and Parkin E3-ubiquitin ligase govern the selective degradation of dysfunctional mitochondria and they have been demonstrated to play key neuroprotective roles in vitro and in vivo. Here, the role of mitochondrial bioenergetics in regulating mitophagy is investigated. By employing a range of biochemical and imaging techniques in a cell-based model of Parkin-mediated mitophagy, the following data demonstrate how cells dependent on mitochondrial respiration can avoid mitophagy via intricate control of mitochondrial dynamics. In order to maintain the energy supply, respiring cells can resist mitophagy by preserving an interconnected mitochondrial network via inhibition of Drp1 and impaired OMA1-dependent OPA1 cleavage. This dissertation also questions the importance of close contact between the mitochondria and endoplasmic reticulum (ER) for the progression of Parkin-mediated mitophagy. A focused siRNA screen of ER-mitochondrial communication factors highlights a novel role for ER-mitochondrial Ca2+ signa ling during Parkin-mediated mitophagy. Together, the data presented in this dissertation place mitochondrial bioenergetic demand and Ca2+ flux as key players in the regulation of mitophagy. Further research will be required to identify whether these two regulatory arms are linked and will strengthen the therapeutic potential for positively modulating mitochondrial homeostasis in order to promote cell protection.

In this thesis results are presented which relate to using artificial neural networks to predict the onset of Parkinson's disease tremors in human subjects. Data for the networks was obtained from implanted deep brain electrodes in human subjects. A tuned artificial neural network was shown to be able to identify the pattern of the onset tremor from these real time recordings. Parkinson's disease (PO) is one disease in a group of conditions called movement disorders. One of the primary symptoms of Parkinson's disease is tremor, and in the extreme case, the patient can suffer loss of physical movement. There are two major types of treatment for PO currently available, namely chemical treatment (Levodopa) and surgical implants (Deep Brain Stimulation). Deep Brain Stimulation (DBS) has been widely accepted as an efficient treatment for PO over the past decade. Despite the high cost of surgical operation, deep brain stimulation has become a widely accepted alternative choice (if not the only) to medical treatment such as Levodopa for patients. In this work, number of methods have been applied on exploring the possibility of determining PO tremor onset from patient's brain signal, in particular using combination of artificial neural networks (ANN) and advanced signal processing algorithms. The result of this work could eventually lead to design a deep brain stimulation device with the ability to react on different brain activities, for example, start stimulation just before Parkinson's disease tremor onset. The benefits of such smart device are pre-Iong DBS battery life and reduce stimulation interference on normal brain functions.

Freezing of Gait (FOG) is a neuro-motor symptom associated with Parkinson's disease (PD), which is suitably managed by Rhythmic Auditory Stimulation (RAS) and music therapy, if applied upon or prior to symptom onset. This stipulates an objective measurement of gait to automatically detect FOG. This research has improved on existing methods for automatic detection of freeze states using vertical acceleration of the leg. Accordingly, a method was devised, implemented and evaluated with the DAPHNet Freezing of Gait dataset. The proposed method is based on Discrete Wavelet Transform (DWT) for feature extraction and Support Vector Machine (SVM) with a Radial Basis Function (RBF) kernel to distinguish freezing of gait from normal locomotion in a binary classification problem. The method was evaluated on the DAPHNet dataset containing over 8 hours of recorded data from PD patients with a history of FOG. The performance of the method was examined in user-dependent and user-independent experimental scenarios with respect to the analysis of feature combinations and sliding window size. The evaluated method exceeded the state-of-the-art performance results in user-independent settings giving an average sensitivity of 76.37% and an average specificity of 85.15% with a maximum detection latency of 2 seconds.

Growth factor receptor bound (Grb) protein 7 family and Proline-rich EVHl ligand (Prel) family proteins are adaptor proteins that share Pleckstrin homology (PH) and Ras-associating (RA) domain. Grb7 family members have been shown to play important roles in growth and signal transduction while members of the Prel family have been reported to function in cell adhesion and migration. Grb 10 interacting GYF domain (GIGYF) proteins were shown to interact with GrblO proline-rich moFif and have been implicated in familial Parkinson's Disease.

Non motor symptoms (NMS) have emerged as one of the key determinants of quality of life in people with Parkinson's and fatigue is a common specific and distinctive NMS in PD but is often under diagnosed. Aims: In this body of work, I have attempted to explore firstly, the clinical correlates of fatigue, which may confound the characterisation of fatigue. Thereafter, the work has attempted to explore possible patho-physiological basis of fatigue in PD, addressing peripheral mechanisms such as cardiac sympathetic dysfunction within the spectrum of dysautonomia or centrally mediated mechanisms via striatal and limbic dopaminergic or serotoninergic pathways. Methods: In the first study, 135 non-depressed PD patients with an age range of 50-75 years and a clinical diagnosis of idiopathic Parkinson's disease were studied using clinically validated scales and specifically the fatigue visual analogue scale initially to identify patients with central fatigue and those without. Collateral assessment of other confounders of fatigue chiefly depression and excessive daytime sleepiness were assessed by Non motor assessment sale (NMSS), Parkinson's Disease Sleep Scale (PDSS), Epworth Sleepiness Scale (ESS) and Hospital anxiety and Depression Scale (HADS) In the following study, 20 patients from the above cohort with significant fatigue were further corroborated using the Parkinson fatigue Scale (PFS-16). Then 10 patients with high fatigue scores, fatigue +v patients (PFS 16 score-> 8) and 10 patients with no fatigue, fatigue -ve cases ( PFS-16 < 8) were selected to undergo cardiac 123I_meta_ iodobenzylguanidine (MIBG Scanning) (using validated local protocol as well as cardiac 2- methoxy isobutyl isonitrile (MIBI scans) to study the integrity of cardiac sympathetic innervation, a sensitive marker of autonomic function. Peripheral and central mechanisms have been investigated by using combination of clinical assessments with imaging parameters of SPECT and PET scans in selected subjects. Findings were correlated with clinical measures. In the final assessment study, 40 patients were selected from the original cohort for a Positron emission tomography (PET) scan sub study (20 fatigue +v cases (PFS-16 > 8) and 20 fatigue - ve cases (PFS-16 < 8)) Patients were matched for motor severity ofPD and cases with significant depression or excessive daytime sleepiness were excluded. PET imaging was performed with l8Fluoro-dopa (dopaminergic) and C-amino-4-(2-dimethylaminomethylphenylsulfanyl) benzonitrile (11 )C-DASB) (serotoninergic) ligands. Results: In the first study, fatigue correlated with disease severity as measured by Hoehn and Yahr (HY) staging which stratified the condition into three categories (HY l-2.5=Mild; HY 3=Moderate; HY 4+5 = Severe; Kruskal-Wallis test, p=0.004). There were no differences in fatigue levels between different subtypes of PD while anxiety, depression and sleepiness emerged as key clinical associations of fatigue. In the second study, a pilot exploratory work, MIBG data from 20 non-depressed PD patients (53% male, mean age (mean ± SD) of 68.75 ± 9.7 years (range: 41-88 years), mean disease duration 7.65 ± 5.5 years (range: 1-35 years) were analysed based on fatigue positive and negative cases (10 in each group) after a total assessment of 30 patients where scan was only possible in 20. The majority (51 %) was at BY stage 2. Cardiac MIBG uptake was expressed as mediastinum to heart ratios at 15 min and 3 hrs (RI and R2) and showed no difference between the fatigue versus non fatigue cases (Mean RI of Fatigue Positive (1.6 ± 0.53) vs Mean RI of Fatigue -ves (1.5 ± 1.37) and mean R2 of Fatigue Positives ( 1.58 ± 0.48 ) vs Mean R2 of Fatigue -ves ( 1.48 ± 0.23 )). In the third stage PET data was analysed and Fatigue + cases showed, a significantly depressed uptake of lIC-DASB binding in comparison to PD-Fatigue - cases, in caudate, putamen, ventral striatum and thalamus (p<0.001, p<0.05, p<O.OI, p<O.OI; Mann-Whitney-Test) and fatigue severity was inversely correlated with lIC-DASB binding. This is a novel finding never reported before. 18F -dopa uptake in the same structures was similar in the two groups using a region of interest approach, however, voxel-based statistical parametric mapping detected relatively reduced 18F-dopa uptake in caudate, thalamus and the insula in the PD-F group (p<0.001). Conclusions: Our preliminary data suggest, fatigue in PD is associated with anxiety, depression and sleepiness and appears to increase with disease severity although also evident in early-untreated phase of PD. The underlying mechanism is likely to be independent of peripheral sympathetic dysfunction as judged by cardiac sympathetic function but is associated with a severe loss of serotoninergic and dopaminergic innervation in the basal ganglia and limbic system (ventral striatum and thalamus) while sparing the raphe serotoninergic innervations. This suggests a dominant role of central serotonergic and in part, dopaminergic dysfunction in the origin of fatigue in PD.

Awareness of the negative effect of non-motor symptoms (NMS) on quality of life in idiopathic Parkinson's disease (PD) has increased in recent years. The greatest degree of variation in the symptomatology in PD involves NMS, but the source of this susceptibility remains undetermined. The aetiology of NMS is likely to be multifactoria1 and involve neurochemical~ neuroanatomical, pharmacological and psychological explanations. Several symptoms may have a genetic basis - revealed within, or exposed by, the neurodegenerated milieu of the Parkinson's brain, akin to the processes that appear to operate in Alzheimer's disease. The comparatively unexplored area of epigenetics is also considered to be a potential cause of both PD and the neuropsychiatric symptoms that can accompany it. This study helps explain the biological mechanisms underlying PO development by exploring genetic and epigenetic profiles using a case-control approach. Elucidating the (epi)genetic determinants and consequences for common diseases such as PO can help inform personal and public strategies for minimising disease and optimising treatment.