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No description available.

Historically various CJD syndromes have been described with a spectrum of clinical presentations. Sporadic CJD (sCJD) is the most common form of human transmissible spongiform encephalopathy (TSE) and is characterised by accumulation of pathological prion protein (PrP) in the central nervous tissue. Classification criteria have changed with the advent of novel diagnostic tests over the last decade. The subclassification according to the codon 129 genotype and prion protein (PrP) type in the brain has led to the identification of six sCJD subtypes, but whether different phenotypes are related to distinct infectious agent strains remains unclear. In order to assess clinical phenotype in detail, four hundred and fourteen cases of sCJD referred to the NCJDSU and classified using the Rotterdam Criteria (1998) were retrospectively analysed (2000-2006). Seventy-four non-cases (1998-2008) were included for comparative analyses. The number of referrals seen has remained relatively uniform with necropsy performed on 70% of suspect cases. The clinical phenotype is less well defined than previously postulated with marked overlap of clinical symptoms and signs independent of PrP[superscript sc] subtype, PRNP genotype and age of onset. In particular the MM1 and MV1 subtypes are not as clinically matched as previously proposed, with the suggestion that these are two distinct forms. The early development of symptoms and signs however favours a diagnosis of sCJD compared with non-cases and may be a potential diagnostic indicator for sCJD. The utility of investigative tests in sCJD, including the EEG and CSF 14-3-3 have been validated. The additional role of the brain MRI, particularly in atypical subtypes, is also demonstrated. This thesis demonstrates that the current WHO diagnostic criteria for sCJD are of value. However, the inclusion of the results of brain MRI and the time to onset of neurological signs and symptoms may improve diagnostic accuracy and allow identification of atypical phenotypes.

Background: Previous studies have reported that there is both objective and subjective evidence that individuals with Fibromyalgia (FM) suffer from memory problems (Landro et al 1997; Katz et al 2004; Munoz et al 2005). The current literature has not examined how individuals cope with these difficulties. Aims of the study: The three aims were to: 1) measure the perceptions of managing memory problems for a FM population; 2) examine their coping response/strategies to establish if the responses can be explained by Models of Stress, Coping and Adjustment (The SRM and Transactional Model of Stress and Coping). 3) To explore the relationships between memory problems, mood, employment, health and pain. Methods: A mixed methodology design was adopted, within an internet survey. 100 participants (91 female, 4 males and 5 preferred to not answer) with FM were recruited from two FM Charity Websites. The modal age of the sample was 51-55 years old. 61 % of participants were from 'other' countries and 38% from the UK. Eight subjective questions (with comments) and psychometric tests were employed (Hospital Anxiety Depression Scale, Pain Catastrophizing Scale, Memory Aids Checklist and the Memory Complaint Questionnaire; and, demographic questions including employment). The questions were analysed using thematic analysis and inductive/deductive application of two theoretical models (Self Regulatory Model (Leventhal et al 2003) and Transactional Model of Stress and Coping (Lazarus et al 1984b). The data from the psychometric tests were analysed using SPSS (multiple regression and ANOVA). Results: Six themes emerged from the qualitative data, these were: "external memory aids"," impact upon daily life", "barriers that impact upon using strategies", "emotional well being", "other strategies and "denial of memory problems". The evidence from the themes implies that memory problems had a global impact upon a person's daily life: well being, home life, employment and coping behaviour. The SRM provided a framework for explaining the coping process. The Regression data proposed that depression significantly influenced perceptions of memory problems but not pain or anxiety. Conclusion: The outcome of this study suggests that both FM and memory problems impact upon well being by causing difficulties in the home and employment. The six themes suggest that memory problems can be managed by using external aids, family members/professionals and hobbies and interests. Recommendations from this research are that psycho-educational interventions would help to reduce memory problems and improve well being in this population.

Hepatic encephalopathy (HE) is a neuropsychiatric syndrome which develops commonly in liver cirrhosis following the build up of toxic substances in the blood that cross the blood-brain barrier and affect normal brain function. The diagnosis of HE is difficult due to only subtle impairments of cognitive function at early stages of the disease and the lack of a gold standard test that specifically and reliably detects the condition. HE is associated with a poor prognosis and effective treatment largely depends on early diagnosis. Thus the aim of this work was to investigate the use of breath analysis as a non-invasive and simpler means of diagnosing HE in cirrhotic patients. This was based on the hypothesis that toxins accumulating in the blood may, if sufficiently volatile, undergo alveolar gas exchange in the lungs to be excreted in the breath. Bespoke breath testing devices were utilised for the collection of breath onto Solid-Phase Micro-Extraction (SPME) fibres and adsorbent packed Automated Thermal Desorption (A TD) tubes from cirrhotic patients with and without HE, patients with early alcohol-related health problems, patients with respiratory disease, and healthy controls. Analysis of the breath samples collected was undertaken using Gas Chromatography Mass Spectrometry. In total, 237 different compounds were identified from all samples collected using the SPME breath analyser system and 385 using the ATD breath sampling device. Multivariate discriminant analysis was used to identify Volatile Organic Compounds (VOCs) that will discriminate patients according to disease status. More compounds were associated with the presence of HE compared to the absence of HE in alcoholic cirrhotic patients. Classification rules based on the presence or absence of volatiles correctly classified the presence of HE in 86% of patients tested with the SPME technique and 88% of patients tested with the A TD technique. Breath tests based on the presence or absence of discriminatory volatiles, correctly predicted the presence of cirrhosis in 93% and 96% of alcoholic patients tested using the SPME and ATD techniques, respectively. The presence or absence of four key volatiles on the breath also helped discriminate patients with early alcohol related health problems (ARHP) from healthy cases, correctly predicting the presence of ARHP in 78% and 91 % of alcoholic patients tested using the SPME and ATD systems, respectively. The use of a targeted sensor-array device found that, on average, HE patients exhaled higher concentrations of hydrogen, alcohol, and total VOCs compared to alcoholic cirrhotic patients without clinical signs of HE, especially in the non- smoking cohort of subjects studied. This finding shows that the use of gas sensor technologies in clinical practice can provide useful diagnostic information for clinical conditions at the bedside of patients in real-time. Not all breath volatiles come from the alveolar-blood interface; many can also be produced in the oral cavity by the action of bacterial or salivary enzymes on a range of substrates. Thus gases were sampled from tongue biofilm models in vitro and this identified 32 compounds commonly detected on breath. This highlights the need for further research to determine the major source of breath volatiles in order that suitable markers of systemic disease or metabolic disorders can be identified. Overall, the results reported in this thesis suggest that breath analysis is a useful tool for the non-invasive diagnosis of a range of conditions associated with the liver such as HE, cirrhosis, and, most importantly the presence of early alcohol related health problems before any significant damage to the liver has occurred.

The neuronal ceroid lipofuscinoses (NCLs, Batten Disease) are a group of lysosomal storage disorders caused by mutations in known and unknown proteins with different cellular locations. Mutations in the CLN6 gene cause a type of variant late infantile NCL (vLINCL). The CLN6 protein is located in the ER and how mutations in CLN6 cause accumulation of storage material in the lysosome is unclear. The aims of this thesis were to establish the tools and techniques necessary to analyse the CLN6 protein and to utilise these to investigate its subcellular location, to identify proteins that interact with CLN6 and to elucidate the early cellular responses to loss of functional vLINCL proteins, CLN5, CLN6 and CLN8. It was shown that CLN6 can be detected by Western blotting, indirect immunofluorescence and immunoprecipitation. A construct of CLN6 fused to horseradish peroxidase was cloned for localisation studies by indirect immunofluorescence and electron microscopy. CLN6 protein and mRNA levels could be depleted using RNA interference and this depletion assessed by Western blotting, indirect immunofluorescence and Q-PCR. It was not possible to establish whether CLN6 is located within a sub-domain of the ER using indirect immunofluorescence or electron microscopy. There was no effect on the localisation or stabilisation of wild-type or mutant CLN6 when proteasomal or lysosomal inhibitors were used, indicating that CLN6 does not traffic to the lysosome and that wild-type and mutant CLN6 were not degraded by ER-associated degradation. Endogenous CLN6 was identified in co-immunoprecipitation experiments, confirming that it may bind to itself. No other proteins were identified that bind to CLN6. Depletion of CLN5, CLN6 and CLN8 resulted in an increase in the size and a redistribution to perinuclear areas of late endosomes and lysosomes. An increase in long cis-Go g structures was also observed in response to siRNA against CLN6 and CLN8 but not CLN5.

Motor Neurone Disease (MND) is a life threatening, neurodegenerative disease for which there is no known cure. As the patient becomes increasingly disabled and subsequently dependent on others, responsibility often falls to close friends and relatives to care for them at home. The health of the carer is often at risk due to long hours of caring and the nature of the work involved.

Mitochondrial diseases affect the core energy generating pathways and can result in significant cognitive impairment and neurodegeneration. The mechanisms underlying the neurological and pathological consequences of mitochondrial disease are not understood and current treatment of mitochondrial diseases is limited in scope and efficacy. pyruvate dehydrogenase (PDH) and Complex I are key mitochondrial enzymes pivotal for cellular energy metabolism, and mutations in genes coding for PDH and Complex I proteins are common causes of mitochondrial disease. This thesis describes the use oftwo techniques, gene targeting, and RNA interference, to generate in vitro models of Complex I and PDH deficiency in pluripotent cell lines. Gene targeting of the ndufal gene was unsuccessful, however, a number of cell lines were isolated with significant reductions in PDH activity mediated by RNAi targeted against the Pdhal transcript. These cells lines were neurodifferentiated in vitro and used to characterise the developmental and degenerative consequences of PDH deficiency on neural cultures. The cultures were found to successfully differentiate into neural cells, but were developmentally abnormal with defective neuronal migration and neurite extension, and neuritic varicosities, an indication of neuritic degeneration in many neurological disease models. These features were activity-dependent with the most severe phenotype found in the cell line with the least residual PDH activity. These cultures were used to explore the nature of energy metabolism in PDH deficient neurons, and therapeutic strategies were successfully tested. This research has successfully established a reproducible and practical model to assess neurodegeneration in PDH deficiency, to test t reatments and to model theories of disease mechanisms. As such it provides promise for the improvement of the understanding of and treatment of disorders of brain energy metabolism in the future.

Mucopolysaccharidosis type IIIA (MPS-IIIA) is a severe neurodegenerative lysosomal storage disorder (LSD) inherited as an autosomal recessive trait and caused by sulfamidase deficiency. Using somatic gene transfer, we demonstrated therapeutic efficacy of a novel low-invasive gene therapy approach to treat the brain pathology in MPS-IIIA. The therapeutic strategy is based on a chimeric sulfamidase engineered with both the signal peptide (sp) from the highly secreted iduronate-2-sulfatase (IDS) linked to its N-terminal end and the blood-brain barrier (BBB)-binding domain of apolipoproteinB (ApoB-BO) linked to its C-terminal end. These modifications allow the enzyme (i) to be highly secreted from the liver and (ii) to efficiently cross the BBB. A single intravascular administration of vectors, based on adena-associated virus (AAV) serotype 8, was performed in one-month old MPS-IIIA mice to efficiently target the liver and convert it in a factory organ for sustained systemic release of high levels of the modified sulfamidase. We show that while the 10Ssp replacement results in higher enzyme secretion, the addition of the ApoB-BO allows efficient BBB transcytosis and restoration of sulfamidase activity in the brain of treated MPS-IIIA mice to ~ 12-15% of the normal levels. This, in turn, results in reduction of pathological vacuolization,

Amyotrophic lateral sclerosis (ALS) is a clinically heterogeneous disease with considerable variability in the disease course even in familial cases associated with autosomal dominant SOD1 gene mutations. Previously, we have shown that SOD1G93A mice, the most widely used model of ALS, exhibit remarkable variability in disease onset and progression according to the mouse genetic background used (Pizzasegola et al 2009). The overall aim of this thesis was to explain the phenotype diversity between two SODIG93A mouse strains on C57BLl6J and 129S2ISv backgrounds since they carry the same number of human SODl transgene copies and express the same amount of mutant SODl mRNA and protein. We found that the levels of insoluble proteins greatly differ between the two SODIG93A mouse strains. In the lumbar spinal cord, the mice with fast disease progression (129SvG93A) exhibited an earlier and higher accumulation of ubiquitinated protein aggregates, mutant-SOD! and several chaperone proteins in comparison with the slowly progressing mice (C57G93A). In contrast, the levels of insoluble TDP43 and its phosphorylated form were higher in the slow than in the fast progressing mice. This led us to perform an in depth investigation of the protein quality control pathways during the course of the disease in these two mouse strains. In the lumbar spinal cord, the fast progressing mice showed lower expression of the chaperone αBcrystallin and an earlier reduction 0[' all the catalytic subunits of the proteasome compared to the slow progressing mice. However, a later induced expression of proteins involved in the misfolded protein recoguition complex and autophagic degradation, such as HspBS, Bag3 and the soluble Hsc70 was observed in the 129SvG93A mice. These data emphasize the involvement of impaired protein quality control mechanisms, in particular the chaperones and proteasome, in determining a more aggressive disease course in in vivo models of ALS.