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Amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are the commonest motor neuron diseases of adult- and childhood onset. Alterations of the RNA binding protein TDP-43 are associated with most cases of ALS, while SMA is caused by deletion of the Survival Motor Neuron (SMN1) gene. SMN has been well characterised in its role in the assembly of the cellular machinery that carries out splicing of pre-mRNA, but is thought to have other functions in RNA metabolism unrelated to pre-mRNA splicing. It is conceivable that specific aspects of RNA handling are disrupted in both SMA and ALS. A variety of genetic, molecular and neuropathological approaches were applied to investigate a potential common pathway in these diseases. The spectrum of genetic mutations underlying motor neuron disorders were explored by screening patient DNA. Cell culture and mouse models were used to test the hypothesis that altered pre-mRNA splicing causes motor neuron death. Human neuropathological specimens were examined for changes in proteins involved in RNA metabolism. The results indicate that altered pre-mRNA splicing is a late occurrence in disease and more likely to be a consequence rather than the cause of motor neuron degeneration. However, the notion that RNA metabolism is highly relevant to motor neuron diseases was strengthened by the discovery of mutations in another RNA binding protein, FUS, in cases of ALS without TDP-43 pathology. Overall the findings highlight the need to consider disruption of mRNA transport and regulation of mRNA translation in future motor neuron disease research.

Motor neuron disorders are a heterogeneous group of diseases characterized by the selective degeneration of motor neurons leading to muscle wasting and atrophy. Amyotrophic Lateral Sclerosis (ALS) is the most common amongst these disorders and is characterized by the selective loss of both upper and lower motor neurons in the brain and spinal cord. 20% of familial cases of ALS are caused by mutations in the Cu, Zn-superoxide dismutase gene (SOD1), a ubiquitously expressed enzyme responsible for scavenging superoxide radicals. The exact mechanisms underlying mutant SOD1-mediated neurotoxicity are unknown. Misfolded mutant SOD1 accumulates in the cytosol and mitochondrial intermembrane space (IMS) indicating the involvement of unfolded protein responses in ALS pathogenesis. Unfolded protein responses (UPRs) are complex signal transduction cascades which detect perturbations in protein folding and couple them to the expression of protein quality control machinery thereby allowing individual compartments to adapt to stress. In the cytosol, this study has shown that HspB8 was upregulated by SOD1 mutants, where it induced the clearance of aggregates by macroautophagy. This is a protective mechanism, as overexpression of HspB8 suppressed mutant-SOD1 mediated toxicity. In contrast, HspB8 mutants were impaired in macroautophagy and are toxic to NSC-34 cells. The mechanisms for the IMS-UPR have not been previously identified. To address this issue, a model for the accumulation of misfolded mutant SOD1 within the IMS was created and candidate proteins involved in protein quality control within the IMS were explored at the transcriptional level and at the level of protein expression. Preliminary results revealed some possible candidates that may have a role in the adaptation to mitochondrial stress. Interestingly, increased mitophagy was also found in IMS-G93A expressing cells, advocating the central role of macroautophagy in eliminating protein aggregates and damaged mitochondria in SOD1-FALS.

Made available in DSpace on 2015-03-26T13:47:26Z (GMT). No. of bitstreams: 1 texto completo.pdf: 330178 bytes, checksum: 0d7d63bcceac97aa987ceb95abd2345a (MD5) Previous issue date: 2003-12-15 / The aim of this study was to evaluate the effects of gallium arsenite (Ga-As) diode laser application on rabbits knees with ostheoarthritis induced by intrarticular injection of carrageenan. Clinical evaluation in vivo showed that there was reduction in inflammatory signs and pain, with treated animals presenting a better performance and greater amplitude of movements, if compared with untreated animals. The degenerative processes resulting from the administration of carrageenan were evidenced in both groups by post-mortem macroscopic and microscopic assessment in both treated and control groups, which allowed inferring that laser diminished the presence of chemical mediators, reducing the inflammatory response and local lesions, including the degradation of glycosaminoglycans. Histological analysis of the synovial membrane revealed the presence of inflammatory cells distributed diffusely or in foci, the intensity of which was higher in control groups. It was concluded therefore that the rate of progression of ostheoarthritis was slowed down by lasertherapy. / O objetivo deste estudo foi avaliar os efeitos do laser a diodo de Arseneto de Gálio (As-Ga) aplicado no joelho de coelhos portadores de osteoartrite induzida pela aplicação intrarticular de carragenina. A avaliação clínica in vivo demonstrou diminuição dos sinais inflamatórios e da dor, permitindo melhor desempenho e maior amplitude de movimento do joelho dos coelhos tratados em comparação aos dos animais não tratados. A avaliação macroscópica e microscópica post-mortem evidenciaram os processos degenerativos decorrentes da administração de carragenina em ambos os grupos, tratado e controle. Os resultados permitiram inferir que o laser diminuiu a presença de mediadores químicos reduzindo, assim, a resposta inflamatória e as lesões locais, inclusive a degradação dos glicosaminoglicanos. A análise histológica da membrana sinovial demonstrou a presença de células inflamatórias, distribuídas difusamente ou em focos, em maior intensidade nos grupos controle. Concluiu-se, portanto, que o caráter progressivo da osteoartrite foi retardado pela laserterapia.

De nombreuses méthodes ont été développées pour estimer l’âge au décès de l’adulte mature. De plus, l’observation de certaines lésions dégénératives amène parfois l’anthropologue à classer un sujet chez les individus âgés, plus spécifiquement lorsqu’elles atteignent la colonne vertébrale. Or si l’ostéoarthrose rachidienne a fait l’objet de nombreux articles en paléopathologie, l’exploitation de l’ensemble des articulations vertébrales au sein d’une méthodologie est quasi inexistante. Ainsi, afin de déterminer le rôle d’une étude paléopathologique dans le vieillissement osseux, nous avons mis au point une méthode originale d’enregistrement des données.Cette méthode, basée sur un découpage topographique du rachis et un système de cotation des lésions arthrosiques, permet d’étudier l’atteinte dégénérative grâce à l’obtention d’un score de sévérité pondéré à l’état de conservation de la vertèbre. Le matériel d’étude est constitué de 750 individus répartis équitablement sur 3 périodes historiques (médiévale, moderne et contemporaine) permettant ainsi d’effectuer une comparaison diachronique des résultats. Les 250 individus constituant l’échantillon contemporain proviennent de collections documentées (Schoten, Belgique ; Bologne, Italie ; Sassari, Sardaigne). L’âge des sujets ostéoarchéologiques a été estimé grâce aux méthodes utilisant la surface auriculaire de l’os coxal (Lovejoy, 1985 et Schmitt, 2005). La même estimation a été faite sur les collections âge/sexe connus afin de déterminer le pourcentage d’erreur commis sur nos échantillons médiéval et moderne. Pour chaque articulation rachidienne nous avons testé statistiquement la relation entre l’âge au décès et les scores de sévérité, ainsi que les dissemblances d’atteinte en fonction du sexe et de la latéralité. Des études qualitatives ont également été entreprises, permettant ainsi d’observer l’expansion des modifications dégénératives en fonction des différentes classes d’âge.Au terme de ce travail de recherche, nous avons constaté l’existence d’une relation entre l’âge et la sévérité des atteintes dégénératives vertébrales. Cependant ce lien est modéré par divers facteurs variant en fonction des articulations et des segments vertébraux. Parmi ces facteurs, nous avons relevé le sexe et la latéralité. Des informations concernant le mode de développement des lésions dégénératives rachidiennes ont également été acquises. Ainsi, nous avons observé que l’OA se développait dans un premier temps de manière progressive le long du rachis, puis, avec l’âge et selon le type d’articulation, les lésions se concentrent sur les mêmes vertèbres (celles étant le plus soumises aux forces biomécaniques). C’est également sur ces mêmes vertèbres que se manifestent les lésions les plus sévères. Ainsi, il semble indispensable, lors d’une étude paléoépidémiologique, d’avoir un nombre minimum de vertèbres et parmi celles-ci les vertèbres les plus sollicitées en pré requis. De plus, nous préconisons lors d’un examen paléopathologique, de considérer plus que la sévérité même de la lésion, le nombre de zone atteintes comme estimateur de l’âge au décès. / Various methods have been developed to estimate age at death of adults and anthropologists sometimes use degenerative vertebral lesions. In paleopathology contrary to vertebral degenerative disease (VDD) that has been extensively studied, the whole vertebral joints have almost never been investigated by standardized methodology that allows approximating the epidemiological performance of various vertebral joints in relation to articular degeneration. In order to better define the paleoepidemiological aspects of bone ageing we have developed a research program based on the recording of degenerative lesions in the vertebral joints.This approach is based on a topographic division of spine and a grading system of degenerative lesions, permits the study VDD using “a severity score” according to the state of vertebral preservation. We studied 750 spines provided equally from three samples (medieval, modern and contemporary) to compare periods. The 250 contemporary spines are from documented collections of Schoten (Belgium), Bologna (Italy) and Sassari (Sardinia). Age estimation was made for each archaeological sample using methods based on the observation of the sacro-iliac joint surface (Lovejoy, 1985 and Schmitt, 2005). The same estimation of age has been made with known sex and age collections in order to calculate error estimates. For each joint of the spine, statistical tests have been made to study relationship between the age at death and “the severity score” and to compare variability between sex and spine laterality. Quantitative studies have also been made to observe degenerative change resulting from advancing age.At the end of this work, we observed the relation between age and the severity of VDD. That link can vary according to joint type, spinal segment and various factors such as sex or laterality. Information concerning the evolution of degenerative lesions has been acquired. VDD appears initially to progressively affect the entire spine and then with aging, degenerative lesions focus on the same vertebrae (those that are the most used biomechanically). Those vertebrae always show the most severe lesions. Thus it is essential for paleoepidemiologic studies to include a minimal number of vertebrae and among them those that are the most used. We also suggest considering the number of affected zones to estimate age at death instead of the lesion severity.

No / Objectives This paper describes DCM 8 and reports on the initial validation study of DCM 8. Methods Between 2001-2003, a series of international expert working groups were established to examine various aspects of DCM with the intention of revising and refining it. During 2004-2005 the revised tool (DCM 8) was piloted in seven service settings in the UK and validated against DCM 7th edition. Results At a group score level, WIB scores and spread of Behavioural Category Codes were very similar, suggesting that group scores are comparable between DCM 7 and 8. Interviews with mappers and focus groups with staff teams suggested that DCM 8 was preferable to DCM 7th edition because of the clarification and simplification of codes; the addition of new codes relevant to person-centred care; and the replacement of Positive Events with a more structured recording of Personal Enhancers. Conclusions DCM 8 appears comparable with DCM 7th edition in terms of data produced and is well received by mappers and dementia care staff.

No / The maintenance of self in dementia is associated with socio-biographical factors. The theoretical literature suggests that interpersonal relationships, the social context, and the generation of stories are important in maintenance of self. Empirical research on self in dementia supports this but has been predominantly conducted with participants living in the community. Living in residential care brings additional threats to self. This paper presents a study examining the relevance of a socio-biographical theory of self to people with dementia living in residential care. Between 3 and 8 tape-recorded and transcribed unstructured interviews were conducted with 14 people with dementia who were living in 4 residential homes throughout England and Wales, over a 6-24-month period. They were analysed using an interpretive biographical methodology. The results provide evidence to support the relevance of a socio-biographical theory of self to this group. Relationships with family, other residents and care home staff were important for maintenance of self. Social roles related to work, being part of a family, caring for others and being cared for, were particularly significant for self in this group. The creation of a life story, stories of selected life events, and the telling of stories with possible metaphorical interpretations were also important for the maintenance of self. The results also suggest that psychological and embodied factors may be relevant to the self in dementia. The study suggests that staff working in residential homes should consider these elements if they are to provide care that supports maintenance of self for people with dementia. Implications for future research are discussed.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disorder characterized by loss of upper and lower motor neurons. TDP-43 was identified as a major protein component of the characteristic neuronal inclusions and it has been detected in 90% of ALS cases. Furthermore, pathogenic mutations in the gene encoding TDP-43, TARDBP, were found in both sporadic and familial ALS cases. The aim of this study is to investigate the molecular mechanisms of cellular dysfunction and ultimately death caused by TDP-43 mutations in human cells using established cell lines and human motor neurons derived from induced pluripotent stem cells (iPSCs). We generated a novel in vitro cellular model using a fluorescently tagged human genomic TARDBP locus carrying three ALS-specific mutations, A382T, M337V or Y374X. In site specific bacterial artificial chromosome (BAC) human stable cell lines, TDP M337V mislocalized to the cytoplasm more frequently than wild-type TDP-43 (TDP Ypet) and TDP-A382T, an effect potentiated by oxidative stress. Cytoplasmic mislocalization was significantly higher in TDP M337V cells compared to TDP-Ypet and correlated with cell death. Cells expressing the mislocalized TDP M337V mutant spontaneously developed cytoplasmic punctae, while for TDP-A382T punctae were only revealed after endoplasmic reticulum (ER) stress induced by the calcium-modifying drug thapsigargin (TG). Lowering Ca2+ concentration in the ER of TDP-Ypet cells partially recapitulated the effect of pathogenic mutations by increasing TDP-43 cytoplasmic mislocalization, suggesting Ca²⁺ dysregulation as a potential mediator of pathology. Ca²⁺ signaling from the ER was impaired in cells carrying TDP-43 mutations, with a 50% reduction in the levels of luminal ER Ca²⁺ stores content and delayed Ca²⁺ release induced by carbachol compared to TDP-Ypet cells. The deficits in Ca²⁺ release correlated with upregulation of Bcl-2 and siRNA-mediated knockdown of Bcl-2 restored amplitude of Ca²⁺ oscillations in TDP-M337V cells. These results suggest that TDP-43 pathogenic mutations elicit cytoplasmic mislocalization of TDP-43 through Bcl-2 regulation of ER Ca²⁺ signalling. Preliminary work in iPSC-derived motor neurons transduced with genomic DNA expression TDP-43 vectors using Herpes Simplex Virus type 1 (HSV-1) amplicons showed cytoplasmic redistribution of TDP-43 under high oxidative stress, without significant differences between mutations and wild-type. TDP-43 mutations delivered by HSV-1 amplicons also did not affect survival of iPSC-derived motor neurons. In ALS patient-derived motor neurons carrying C9orf72 expansions, TDP-43 pathology was not detected. However, preliminary data indicate that C9orf72 MNs present ER Ca²⁺ dysregulation with significantly high intracellular Ca²⁺ concentration, which correlates with high protein levels of ER stress markers and low levels of Bcl 2. This work highlights a potentially pathogenic role for TDP-43 mutations in the dysregulation of Ca²⁺ homeostasis and explores the use of iPS technology to investigate the effects of ALS-associated mutations in healthy and patient-derived motor neurons.

Aggregations of fused in sarcoma (FUS), a multifunctional RNA processing protein, define a pathological subtype of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), whilst mutations in the FUS gene are causative for ALS. To model the impact of FUS mutations, expression vectors containing the entire genomic sequence of FUS, up and downstream regions, and native promoter sequences have been generated. The constructs have been tagged with an mCherry fluorescent tag, and three separate pathological mutations (R244C, R521C, and P525L) have been separately inserted. Transgenic mice have been generated using the WT and P525L FUS vectors to provide a highly physiological model of FUS in disease. Within transfected HEK293 cells, insertion of the P525L and R521C FUS mutations leads to relocalisation of FUS from the nucleus to the cytoplasm. R521C and P525L mutant FUS incorporates into cytoplasmic aggregations of untranslated mRNA and RNA binding proteins known as stress granules. The strong relocalisation seen with P525L-FUS is associated with a gain of cytotoxicity. Reversal of this cytoplasmic relocalisation by demethylation of FUS rescues this cytotoxicity, suggesting a toxic gain of cytoplasmic function in the majority of FUS mutations. By contrast, insertion of the R244C mutation leads to neither relocalisation, stress granule association, nor cytotoxicity. Notably the R244C mutation, located away from the nuclear localization domain in which the majority of FUS mutations are found, leads to the presence of smaller FUS fragments in western blot analyses. These fragments appear not to be due to splicing defects in FUS but rather are due to post-translational modifications or aberrant protein cleavage. These data suggest an alternative pathway for FUS toxicity based upon a nuclear loss of function.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised clinically by rapidly progressive paralysis leading ultimately to death from respiratory failure. There is no cure for ALS and no definitive explanation for the onset and rapid progression of motor neuron degeneration. Genetics is a known risk factor for a portion of familial cases. However, the role of genetics in the commoner sporadic form of the disease is poorly understood, although numerous genes have been implicated. The primary aim of this thesis project is to uncover the genetic causes that underlie ALS. To accomplish this goal, the main focus of this thesis is to perform genome-wide association analysis of sporadic ALS using high density SNP arrays. This thesis describes the first and the largest genome-wide association studies of ALS to date. Results demonstrate that there is no single large effect susceptibility variant underlying a large proportion of ALS, such as ApoE in Alzheimer’s disease. However, the genotyping data has been made publically available and the digital nature of this data means that it is a resource that can grow with future studies. Beyond genome-wide association, this thesis describes work using linkage, haplotype and sequence analysis to investigate the genetic overlap between ALS and frontotemporal dementia. Lastly, this thesis presents a novel method for linkage analysis using high throughput SNP arrays. Ultimately, it is hoped that by uncovering the genes that cause ALS, such knowledge will shed light on the pathogenic mechanisms underlying motor neuron degeneration and potentially lead to new rational therapies effective in slowing or even halting disease progression.