Modelling C9orf72-linked frontotemporal dementia and amyotrophic lateral sclerosisRyan, Sarah January 2015 (has links)
Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative diseases with considerable clinical, genetic and pathological overlap. A GGGGCC hexanucleotide repeat expansion in a non-coding region of C9orf72 on chromosome 9 is the major cause of both FTLD and ALS. An understanding of the mechanisms through which the expansion leads to neurodegeneration will therefore be vital for development of novel therapeutics. There are 3 possible mechanisms through which the GGGGCC expansion may cause toxicity: (i) through haploinsufficiency of C9orf72, (ii) repetitive RNA transcripts arising from the expansion may be toxic or (iii) translation of the expansion may produce toxic peptides. Whilst the expansion is located in a non-coding region of the gene, long GC-rich RNA transcripts may be translated in the absence of an ATG start codon, through a process known as repeat-associated non-ATG translation (RAN-translation). 5 distinct dipeptide repeat proteins (DPRs) have been found to arise from the expansion through RAN-translation of the sense and antisense strands in all frames: poly-GA, -GR, -PR, -AP and –GP. All 5 DPRs have been shown to aggregate in patient tissue, indicating that they may play a role in C9FTLD/ALS pathogenesis. This project aimed to generate a series of models to investigate the mechanisms of neurodegeneration in C9FTLD/ALS, using C. elegans and cell culture. A transgenic worm strain which does not express the C. elegans orthologue of C9orf72 was first characterised. No impairments were observed in motility or life-span, demonstrating that loss of C9orf72 function does not cause an ALS-like phenotype in C. elegans. When considered alongside recent literature, this finding suggests that toxic gain of function mechanisms may be more important in C9FTLD/ALS pathogenesis. The impact of DPRs on cellular function was next investigated. Constructs were generated containing alternative codon sequences for each of the 5 DPRs, to express each peptide in the absence of the repetitive GGGGCC RNA sequences found in disease. A step-wise cloning strategy was employed to progressively increase repeat-length in these constructs until physiologically-relevant lengths of > 1000 repeats were obtained. DPRs were then expressed in HeLa cells, in order to assess the individual effects of each peptide on cellular function. Poly-GA formed large, star-shaped cytoplasmic inclusions which co-localised with ubiquilin-2 and p62, closely resembling the inclusions observed in patient tissue. This implies a potential role of proteasome dysfunction in C9FTLD/ALS. Conversely, the alanine-rich DPRs, poly-GR and –PR, translocated to the nucleolus, where poly-GR in particular caused nucleolar stress. Furthermore, nucleolar poly-GR caused loss of Cajal bodies from the nucleus, and a loss or mislocalisation of survival motor neuron protein (SMN). This is of particular interest to C9ALS, since loss of SMN is selectively toxic to motor neurons. Furthermore, loss of Cajal bodies and nucleolar stress are likely to cause defects in RNA processing, which may contribute to neurotoxicity.
Repute and remedy : psychiatric patients and their treatment at Bethlem Royal Hospital, 1930-1983Walke, J. January 2015 (has links)
Bethlem Royal Hospital is Britain’s oldest and arguably foremost mental hospital; a centre of psychiatric innovation notable for its early acceptance of voluntary, predominantly middle-class, patients. The study begins with the 1930 Mental Treatment Act, which endorsed voluntary and outpatient psychiatric treatment, and ends with the 1983 Mental Health Act, which placed legal controls on certain therapies, and introduced the Mental Health Act Commission. Although not wholly representative of other institutions, scrutiny of Bethlem can inform debates on the flux of ideas and methods within twentieth-century psychiatry, and further knowledge of the hospital in this era. The primary research aim is to analyse the changing nature of institutional care for psychiatric patients in mid twentieth-century Britain, through a case study of Bethlem Royal Hospital. Secondary objectives are to: * Explore the role of legislative frameworks in shaping the institution in terms of admissions and governance; * Investigate changing definitions of mental illness through analysis of the composition of patients with respect to their diagnosis and social factors; * Examine the impact of scientific developments in the theory and practice of psychiatry on service organisation and treatment. At the local level, combined admissions data and qualitative evidence provide a detailed, contextualised account of the Bethlem inpatient ‘journey’. Four national level themes emerged: first, a consumerist model of mental health was evidenced through hospital marketing materials, and, reciprocally, the preferences of patients and their families. A second key theme was a mid twentieth-century transition from aetiologic to diagnostic frameworks of mental illness. Thirdly, gender-specific attributions and treatment observed in interwar records were followed, in subsequent decades, by signs of a ‘meeting of minds’ in recorded experiences of male and female inpatients. Finally, this thesis addresses how changes in patients’ backgrounds, knowledge, and expectations, were intertwined with the development of psychotherapeutic approaches.
The role of amyloid precursor protein in neuronal and non-neuronal cell linesTse, Erin January 2015 (has links)
Models of Alzheimer’s disease (AD) have provided useful insights into the pathogenesis and mechanistic pathways that lead to its development. One emerging idea about AD is that it may be described as a hypometabolic disorder due to the reduction of glucose uptake in AD brains. Inappropriate processing of Amyloid Precursor Protein (APP) is considered central to the initiation and progression of the disease. Although the exact role of APP misprocessing is unclear, it may play a role in neuronal metabolism before the onset of neurodegeneration. To investigate the potential role of APP in neuronal metabolism, the SHSY5Y neuroblastoma cell line was used to generate cell lines that stably overexpress wild type APP695 or express Swedish mutated-APP observed in familial AD (FAD), both under the control of the neuronal promoter, Synapsin I. The effects of APP on glucose uptake, cellular stress and energy homeostasis were studied extensively. It was found that APP-overexpressing cells exhibited decreased glucose uptake with changes in basal oxygen consumption in comparison to control cell lines. Similar studies were also performed in fibroblasts taken from FAD patients compared with control fibroblasts. Previous studies found FAD-derived fibroblasts displayed altered metabolic profiles, calcium homeostasis and oxidative stress when compared to controls. As such, in this study fibroblasts were studied in terms of their ability to metabolise glucose and their mitochondrial function. Results show that FAD-derived fibroblasts demonstrate no differences in mitochondrial function, or response to oxidative stress compared to control fibroblasts. However, control fibroblasts treated with Aβ1-42 demonstrated changes in glucose uptake. This study highlights the importance of APP expression within non-neuronal cell lines, suggesting that whilst AD is considered a brain-associated disorder, peripheral effects in non-neuronal cell types should also be considered when studying the effects of Aβ on metabolism.
Behavioural investigation into whether L-DOPA, the current 'gold standard' pharmacotherapy for Parkinson's disease, can be improved by optimising its treatment strategiesTayarani-Binazir, Kayhan Ashley January 2014 (has links)
Long-term use of L-DOPA in Parkinson’s disease results in motor complications and a progressive reduction in clinical efficacy. No new pharmacologic agents and treatment options have been able to deliver a more effective treatment than L-DOPA and therefore L-DOPA treatment strategies could offer improved clinical outcomes. This led to the hypothesis that L-DOPA, the current 'gold standard' pharmacotherapy for Parkinson’s disease can be improved by optimising its treatment strategies. Using validated animal models of Parkinson’s disease, behavioural studies were performed to test this hypothesis to evaluate if we could: (1) potentiate the clinical response of L-DOPA by maximising the efficiency of peripheral decarboxylase (DDC) inhibition, (2) enhance the clinical response of L-DOPA through prodrug delivery and (3) optimise L-DOPA's clinical therapeutic window through combination therapy with dopamine agonists. Firstly, it was shown that the efficiency of DDC inhibition could improve the L-DOPA response particularly when L-alpha-methyl dopa (L-AMD) was utilised. Secondly, the novel L-DOPA prodrug PRX 1354, induced improvement in L-DOPA motor function in MPTP treated marmosets but did not have the same positive effect on dyskinesia expression. Lastly, in the MPTP-treated common marmosets, L-DOPA combined with the dopamine agonist pramipexole resulted in improved motor function and a reduction of dyskinesia. In conclusion, manipulating L-DOPA treatment strategies can improve motor function while reducing dyskinesia expression. These results suggest that optimizing the treatment of Parkinson’s disease by improving L-DOPA treatment strategies could reduce the impact of dyskinesia in Parkinson’s disease patients.
Musculoskeletal and spinal cord imaging in bilateral spastic cerebral palsyNoble, Jonathan James January 2014 (has links)
Periventricular leucomalacia (PVL) is the most common brain injury in bilateral spastic cerebral palsy (BSCP). Cerebral palsy is a group of conditions that affect the development of the motor system, that are attributed to non-progressive lesions in the developing brain. In PVL, damage is caused by a primary arterial ischemic injury to the white matter in the posterior limb of the internal capsule, although other regions of the brain can also be affected including the spinal cord. Alterations in spinal cord development may lead to many of the clinical problems observed in BSCP, including altered motor control, co-contraction of agonist and antagonist muscle groups, progressive musculoskeletal deformities and weakness. Further to this, a heightened fracture risk of the long bones of the skeleton may be related to poor muscle development subsequent to the original brain injury. In the work contributing to this thesis, the fat content of five muscles and the volume of nine major muscles of the lower limbs of ambulant adolescents and young adults with and without BSCP are investigated using MRI. The relationship between bony geometry and muscle volume are also studied. Studies of spinal cord white matter organisation are also performed using diffusion tensor imaging (DTI) MRI techniques to investigate whether there are associations between spinal cord organisation and gross functional development in BSCP. Lower limb muscle volumes in BSCP were found to be smaller with increased intramuscular fat compared to their typically developing peers. Bone strength estimated from bony geometry was found to be significantly dependent on muscle volume independent of diagnosis. No differences were observed in spinal cord white matter microstructure between the subject groups, although a reduced white matter crosssectional area was observed in the BSCP group. The clinical implications of this work are discussed in detail.
Investigating group III metabotropic glutamate receptors as novel therapeutic targets in Parkinson's disease and Levodopa-induced dyskinesiaFinlay, Clare Judith January 2014 (has links)
The loss of nigrostriatal dopamine neurones in Parkinson’s disease causes characteristic motor symptoms resulting from signalling alterations in the basal ganglia. An important consequence of this is increased firing of the glutamatergic subthalamic nucleus (STN). Since the STN innervates dopaminergic neurones in the substantia nigra pars compacta (SNc), any increased firing could perpetuate degeneration of these cells by promoting excitotoxicity. Activation of group III mGlu receptors reportedly reduces glutamatergic transmission at the subthalamonigral synapse suggesting activation of these receptors might provide neuroprotection in PD. The results reported in this thesis support site-directed targeting of the group III receptor subtype mGlu4 in the SNc as a neuroprotective approach in the 6-hydroxydopamine lesioned rat; however while one mGlu4 positive allosteric modulator tested was successful another was not, highlighting several future considerations for the use of these agents. Unexpectedly, activation of group III and mGlu4 receptors increased, rather than reduced glutamate release in the intact SNc. However, this effect was lost in the 6-hydroxydopamine lesioned SNc, reassuring us that in the parkinsonian state activation of these receptors should not exacerbate excitotoxicity. Further experiments are required to define the mechanisms by which the mGlu4-mediated protection is afforded. Additional studies are also required to shed light on why these protective effects were lost in a subsequent study following systemic injection of an mGlu4 PAM (LuAF21934); might this relate to severity of lesion, or a detrimental effect of activating mGlu4 receptors outside the SNc, for example? The lack of protective efficacy subsequently found with a systemically administered mGlu7 agonist (AMN082) against a severe 6-hydroxydopamine lesion also points towards partial lesion models for future testing. Finally, since other antiglutamatergic strategies have proven successful, we investigated mGlu4 activation as a means to inhibit L-DOPA-induced dyskinesia expression or development in rodents. While this approach was ineffective at reversing established dyskinesia there was a hint that it might be efficacious at delaying the onset of this complication that will be worth investigating further. In conclusion, targeting the mGlu4 receptor has shown some beneficial effects in relation to Parkinson’s disease but much remains to be discovered about the actions of these agents both within and outside of the basal ganglia before any clear potential is revealed.
The role of nicotinamide N-methyltransferase in Parkinson's diseaseThomas, Martin Geoffrey January 2015 (has links)
Parkinson’s disease (PD) is a progressive neurological movement disorder characterised by degenerating dopaminergic neurons in the midbrain’s substantia nigra pars compacta (SNpc). In the majority of cases, PD is thought to be caused by a plethora of overlapping factors that combine to cause toxicity in SNpc neurons. The enzyme nicotinamide N-methyltransferase (NNMT) is expressed at higher levels in the brain of PD patients. However, whether this association is indicative of a causative role in PD is unclear. The biochemical effects of NNMT expression were determined by comparing the NNMT-V5 expressing S.NNMT.LP cell line with its parent SH-SY5Y. Furthermore, the potential for NNMT to potentiate toxicity of β-carbolines (βC), a group of endogenous compounds with increased prevalence in PD, and 4-phenylpyridine (4PP), an environmental analogue of the neurotoxin 1-methyl-4-phenylpyridine, were determined by MTT and ATP assay. Finally, the ability of purified NNMT to metabolise the βC norharman (NH) via 2N-methylation was determined via enzyme assay and the toxicity of 2N-methylated NH (2-MeNH) was determined via MTT and ATP assay. S.NNMT.LP cells contained a significantly higher ATP content than SH-SY5Y cells (p < 0.01). No difference was seen between S.NNMT.LP and SH-SY5Y toxicity to the βC tetrahydronorharman or 4PP. However, 200 μM NH significantly lowered ATP but not cell viability in S.NNMT.LP cells compared with SH-SY5Y (p < 0.01). NNMT was found to be capable of producing 2-MeNH from NH and S.NNMT.LP cells were significantly protected from 2-MeNH toxicity (p < 0.001). Moreover, 2-MeNH was significantly less toxic to both cell lines than NH (p < 0.05). The metabolism of NH to 2-MeNH may, therefore, be a detoxification mechanism. Accordingly, the expression of NNMT in PD may be a protective response in PD by increasing ATP production and removing NH from the cytoplasm in favour of the less toxic 2-MeNH.
Pilot studies to develop and evaluate a muscle srengthening programme to reduce the risk of aspiration and improve outcome in stroke patientsKulnik, Stefan Tino January 2015 (has links)
Stroke can impair cough function. Respiratory muscle training (RMT) was investigated as an intervention for improving cough function in acute stroke; and as a potential strategy for preventing aspiration-related post-stroke pneumonia. Measures of cough function (volitional tests of cough flow and respiratory muscle strength, automated cough frequency measurement) required validation in the acute stroke population. Test-retest reliability was equally high in eleven healthy volunteers and six stroke survivors (ICCs >0.90). Minimal detectable difference was ≈7%. A calibrated pneumotachograph was found most appropriate for cough flow assessments, due to inaccuracy of portable flow meters (Bland-Altman 95% limits of agreement spanning ≈150 L/min). Automated cough frequency measurements (Leicester Cough Monitor) showed high accuracy (ICC >0.99). The effectiveness of RMT was investigated in a single-blind randomised placebo-controlled trial of 82 acute stroke survivors in three parallel groups (inspiratory, expiratory, and sham training). Mean group changes from baseline (SEM), respectively, were: 91 (42), 49 (27) and 84 (34) L/min for peak voluntary cough flow (p=0.46); -4 (28), 17 (19) and 32 (18) L/min for peak reflex cough flow (p=0.41); 20 (4), 12 (3) and 12 (4) cmH2O for maximal expiratory mouth pressure (p=0.35); and 18 (4), 10 (3) and 14 (3) cmH2O for maximal inspiratory mouth pressure (p=0.40). Pneumonia occurred in 13 (16%) participants with no difference between groups (p=0.65). Higher voluntary cough flow at baseline predicted lower pneumonia risk in patients with unsafe swallow (OR 0.73, 95%CI 0.51-0.95, p=0.012), but not in patients with safe swallow. In a sub-group of 21 patients, 24-hour cough frequency was abnormally high at baseline (median (range) 118 (4, 375)) and decreased to 56 (1, 186) at four weeks and 34 (6, 108) at twelve weeks (p=0.0003). RMT did not improve cough flow or respiratory muscle strength beyond natural recovery. Stronger cough was protective from aspiration-related post-stroke pneumonia.
Novel [¹¹C]CO₂ radiolabelling methodologies for PET neuroimagingHaji Dheere, Abdul Karim January 2015 (has links)
PET is a non-invasive molecular imaging technique that is increasingly being used for medical imaging and drug development. Carbon-11 (11C; half-life 20.4 min) is one of the most commonly used radionuclides for PET molecular imaging. 11C is usually produced in the form of [11C]CO2 and converted into more reactive secondary precursors such as [11C]methyl iodide and [11C]carbon monoxide for radiolabelling. Although such secondary precursors are undoubtedly useful, given the short half-life of 11C, it would be advantageous to use [11C]CO2 directly from the cyclotron without additional time-consuming processing. Therefore, the development of radiochemical methods to efficiently radiolabel compounds directly with [11C]CO2 for applications in PET neuroimaging is an important goal and is the focus of this thesis. This work includes the development of novel radiolabelling methodology utilising [11C]CO2 for the radiolabelling of molecules based on urea and carbamate scaffolds. These functional groups are found in a plethora of biologically active molecules and pharmaceuticals. As proof of concept, the utility of the developed radiochemistry methods were applied to the synthesis of novel GABA and glutamate radiotracers. GABA and glutamate are major excitatory and inhibitory neurotransmitters in the brain. Although implicated in many diseases, the in vivo function of these neurotransmitter system is poorly understood. Their dysfunction are implicated in pathologies such as addiction, Alzheimer’s disease, Parkinson’s disease and autism. Monitoring the expression of the receptors in vivo and in vitro would enable better understanding of these diseases, their progression and treatment. The research described in this thesis unveils new methods to radiolabel novel molecules for these targets with 11C thereby enabling more opportunities to study them in vitro using autoradiography and in vivo using PET molecular imaging.
Association of Alzheimer's disease progression with white matter hyperintensities and blood biomarkersMikula, Margit January 2015 (has links)
Alzheimer’s disease (AD) is the most common form of dementia affecting around 70% of dementia cases. The global prevalence of dementia is estimated to be 44.4 million people. The pathological hallmarks of AD are amyloid plaques and neurofibrillary tangles. AD impairs many cognitive functions including memory, language, abstract thinking, and spatial orientation and alters mood and behaviour. These symptoms become gradually more severe, affecting common daily activities until eventually full time care is required. There is currently no cure. A large number of promising drugs are being tested in clinical trials, but their effect on disease progression is difficult to assess using existing psychometric measures. New biomarkers, including cognitive-, MRI-, and blood-based measures could be combined to accurately predict disease progression. First, potential associations of 1-year cognitive decline with baseline volumes of MRI White Matter Hyperintensities (WMH) from the whole brain, were evaluated. Higher baseline WMH volumes were found to be significantly associated with greater AD-cognitive decline (number of subjects (n) =84, P < 0.05), with a median, yearly, cognitive change of -1.2 points in the Minimal-Mental-State-Exam, 1.5 points in ADAS-cog, and 1.3 points in Clinical-Dementia-Rating. Second, WMH volume was associated with an identified panel of mRNAs (trainingset: n=53, P < 0.001, R2=0.75; validationset: n=21, R2=0.11), and with proteins (trainingset: n=59, P < 0.001, R2=0.76; validationset: n=30, R2=0.03), in individuals with AD. Disease progression, measured by AD-cognitive decline in CDR-SOB points, was associated with an identified panel of mRNA transcripts (trainingset: n=46, P < 0.001, R2=0.86; validationset: n=16, R2=0.13). It can be suggested that WMH and cognitive disease progression affect mRNA expression and protein concentration in blood. A limitation was that there were only small independent validationsets. Future work could investigate the identified molecular candidates in larger subject groups, and if confirmed, they could contribute to defining a blood-based disease progression biomarker panel.
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