Introdução: A Esclerose Lateral Amiotrófica (ELA) é uma neuronopatia de curso progressivo, caracterizada pela morte dos neurônios motores superiores e inferiores. Devido a rápida progressão da doença e ao aparecimento dos sintomas de incapacidade funcional os indivíduos com ELA buscam uma forma alternativa de comunicação e interação. Com isso, o desenvolvimento tecnológico utilizando programas de realidade virtual com ajuda de dispositivos de interação pode viabilizar mais função e auxiliar indivíduos com ELA a obter autonomia, independência, melhor qualidade de vida e inclusão. Objetivo: Identificar qual dispositivo de interação virtual é melhor para propiciar desempenho e funcionalidade em uma tarefa de realidade virtual para indivíduos com ELA. Método: Participaram do estudo 30 indivíduos que formaram o grupo ELA e 30 indivíduos com desenvolvimento típico que formaram o grupo controle, com idade entre 44 a 74 anos, pareados por idade e sexo. A tarefa utilizada, foi um jogo no computador, que consiste em estourar o maior número de bolhas possíveis durante 30 segundos. Os indivíduos foram separados em 3 grupos, cada qual utilizando uma interface diferente (Kinect, Leap Motion Controller ou Touchscreen) na fase de aquisição e retenção da tarefa. Após essas fases, foi realizada a fase de transferência com a troca de dispositivos e assim todos os grupos tiveram contato com todas as interfaces. Para análise estatística utilizou-se o número de bolhas alcançadas para cada participante, durante as fases de aquisição, retenção e transferências. Resultados: Todos os participantes, tanto do grupo ELA como do grupo controle, apresentaram melhor performance motora na utilização do dispositivo Touchscreen, porém o grupo ELA apresentou desempenho inferior com a prática de todos os dispositivos. A prática com o dispositivo Touchscreen não permitiu a transferência para os dispositivos Leap Motion Controller e Kinect, isso significa que a prática com dispositivo de característica mais real (Touchscreen) não permitiu a transferência para os dispositivos com características mais virtuais (Kinect® e Leap Motion Controller®), porém considerando a prática com os dispositivos virtuais essa transferência ocorre. Conclusão: O trabalho apresenta um avanço na compreensão de dispositivos apropriados para a utilização na reabilitação da funcionalidade de indivíduos com ELA. O dispositivo Touchscreen foi o que apresentou melhor desempenho funcional para essa população, podendo oferecer mais funcionalidades para os indivíduos na execução de tarefas virtuais / Introdution: Amyotrophic Lateral Sclerosis (ALS) is a progressive course of neuronopathy, characterized by the motor neurons death (MN) upper and lower. Due to rapid disease progression and the onset of symptoms of functional disability individuals with ALS seek an alternative form of communication and interaction. Technological development using virtual reality programs with the help of interaction devices can offer more function and assist individuals with ALS to obtain autonomy, independence, quality of life and inclusion. Objective: to identify which low-cost non-immersive interaction device, using a virtual task, is better for providing performance and functionality for individuals with ALS. Method This is an analytical cross-sectional study. A total of 60 people participated in this study, 30 individuals with ALS (18 men and 12 women, mean age = 59 years, range 44-74 years), while 30 people with normal development that were matched for age and gender with individuals with ALS formed the control group. The task used was a computer game, which consists of blowing the largest possible number of bubbles for 30 seconds. The subjects were divided into 3 groups, each using a different interface (Kinect®, Leap Motion Controller® or Touchscreen) in the task acquisition and retention stage. After these phases was carried out the transfer phase with the switching devices, then all groups had contact with all interfaces. For statistical analysis we used the number of bubbles achieved for each participant during the phases of acquisition, retention and transfer. Results: All participants, both the ALS group, both the control group showed better motor performance in the use of the Touchscreen device, but the ALS group had underperformed the practice of all devices. Practice with the touchscreen device did not allow the transfer to the Leap Motion Controller® and Kinect® devices, this means that the practice more real feature device (Touchscreen) did not allow the transfer to devices with more virtual features (Kinect® and Leap Motion controller®), but considering the practice with virtual devices that transfer occurs. Conclusion: This work presents a breakthrough in the understanding of appropriate devices for use in the rehabilitation of people with ALS functionality. The Touchscreen device showed the best functional performance for this population and can offer more features for individuals in executing virtual tasks

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron death and subsequent muscle atrophy. Approximately 15% of ALS cases are inheritable, and mutations in the Fused in Sarcoma (FUS) gene contribute to approximately 5% of these cases, as well as about 2% of sporadic cases. FUS performs a diverse set of cellular functions, including being a major regulator of RNA metabolism. FUS undergoes liquid- liquid phase transition in vitro, allowing for its participation in stress granules and RNA transport granules. Phase transition also contributes to the formation of cytoplasmic inclusions found in the cell bodies of FUS ALS patients motor neurons. The nature of these inclusions has remained elusive, as the proteins localized to them have not been identified. Additionally, the functional consequence of the accumulation of cytoplasmic FUS inclusions has not been established, nor is it understood how they contribute to selective motor neuron death. We carried out two related, but independent studies to characterize the proteins that may be included in FUS-positive inclusions. In this first study, we utilized immunoprecipitation of wild-type and mutant FUS in the presence and absence of RNase, followed by LC MS/MS. The identified proteins represent those that directly or indirectly interact with FUS, with relatively high affinity that can be pulled down with immunoprecipitation. A wide variety of interacting proteins were identified and they are involved in a multitude of pathways including: chromosomal organization, transcription, RNA splicing, RNA transport, localized translation, and stress response. Their interaction with FUS varied greatly in their requirements for RNA. Most notably, FUS interacted with hnRNPA1 and Matrin-3, proteins also known to cause familial ALS. Immunofluorescent staining of proteins interacting with mutant FUS were localized to cytoplasmic inclusions. We concluded that mis-localization of these proteins potentially lead to their dysregulation or loss of function, thus contributing to FUS pathogenesis. In the second study, we developed a protocol to isolate dynamic FUS inclusions and employed LC MS/MS to identify all proteins associated with FUS inclusions. We identified a cohort of proteins involved in translation, splicing, and RNA export to be associated with the FUS inclusions. Further pathway and disease association analysis suggested that proteins associated with translation and RNA quality control pathways may be the most significant. Protein translation assays using both N2A and ALS patient fibroblasts demonstrated suppression of protein biosynthesis in mutant FUS expressing cells. However, translation initiation was not impaired. To understand how protein synthesis is suppressed by mutant FUS mediated defects in RNA metabolism, we examined changes in a well conserved RNA turnover pathway namely: nonsense mediated decay (NMD). We found that NMD is hyperactivated in cells expressing mutant FUS, likely due to chronic suppression of protein translation shifting the pathways autoregulatory circuit to allow for hyperactivation. We concluded that mutant FUS suppresses protein biosynthesis and disrupts NMD regulation. These defects together likely contribute to motor neuron death.

There is little research studying the relationship between caregiver quality of life and gastrostomy, a palliative intervention recommended to manage dysphagia and malnutrition in patients diagnosed with Amyotrophic Lateral Sclerosis (ALS). To facilitate a more comprehensive assessment of treatment effectiveness and to better guide patients and their families, this study investigated the relationship between gastrostomy, caregiver strain, and patient disease-related factors. Patients with bulbar-onset ALS and their caregivers were recruited regardless of their decision to accept or decline future gastrostomy. Caregivers completed the Modified Caregiver Strain Index (MCSI) to assess levels of caregiver strain as an index of quality of life. Surveys were completed at 3-month intervals prior to gastrostomy and at a single time point following gastrostomy. Of 13 patient-caregiver dyads recruited, 1 dyad completed both phases of the study as of yet. This caregiver reported increased caregiver strain following gastrostomy. Medical interventions aimed at managing dysphagia, such as gastrostomy, may not have a predictable impact on caregiver strain, as indexed by the MCSI, or changes in caregiver strain may reflect characterological differences among patient-caregiver dyads. Other psychosocial factors within a given patient-caregiver dyad may be stronger predictors of caregiver strain, burden, and quality of life in caregivers.

La sclérose latérale amyotrophique (SLA) est une pathologie neurodégénerative progressive se déclarant vers 50-60 ans. Elle est majoritairement de nature sporadique son incidence est estimée à 1 :1000. La SLA mène à une paralysie progressive et entraine généralement à la mort des patients de 2 à 5 ans suivant le diagnostic aux suite d’une fonte musculaire importante liée à la perte des neurones moteurs. Au cours des années, plusieurs mutations ont été identifiées autant chez les patients atteints de SLA sporadique que de SLA familiale. Ces mutations interfèrent avec la fonction de gènes variés, tels que DCTN1, codant pour la protéine dynactine1, sous-unité du complexe multimoléculaire dynactine. Ce complexe sert d’adaptateur au moteur moléculaire dynéine, chargé du transport axonal rétrograde, où sa fonction permettrait de régir l’activité du complexe moteur et sa capacité à lier divers cargos. Nous avons donc entrepris la caractérisation d’une lignée de poissons zèbre mutants pour dynactin1a (nommés mikre okom632, mokm632), plus particulière en terme du développement d’un type de neurone moteur primaire (les CaPs), afin de déterminer l’effet de la perte de fonction de ce gène sur l’axonogenèse, la formation et la stabilisation de la jonction neuromusculaire, sur le comportement de l’embryon, ainsi que sur le transport axonal.Nous suggérons que dynactin1 favorise la stabilité synaptique, où une perte de fonction de ce gène entraine des défauts de croissance, des anomalies éléctrophysiologiques et un comportement anormal. Ce rôle semble être indépendant des fonctions connues de régulateur du moteur dynéine. / Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease, which is mainly sporadic in nature. This progressive pathology has an estimated incidence of 1:1000 and generally leads to death within 2-5 years of diagnosis due to muscle wasting and severe motor neuron loss. Over the last years, mutations have been identified in both sporadic and familial ALS patients, interfering with the function of many genes, including DCTN1, which encodes for a subunit of the motor protein complex subunit dynactin. The dynactin complex serves as an adaptor for the dynein motor complex, responsible for retrograde axonal transport, and it is believed to regulate dynein activity and the binding capacity for cargos. We set out to characterize a mutant zebrafish line for dynactn1a (named mikre okom632, mokm632), looking specifically at caudal primary motor neurons (CaPs), with regard to axonal development, formation and stability of the neuromuscular junction (NMJ) and the behavioral phenotype produced in embryos, as well as axonal transport metrics. We suggest a role for dynactin1 in synapse stability, where the loss-of-function of this gene leads to growth defects, electrophysiological abnormalities and behavioral deficits. This role appears to be independent of its known function as a regulator of dynein, its implication in axonal transport, or its regulation of microtubule dynamics. With this study, we hope to elucidate key molecular mechanisms in ALS etiology by revealing the role of dynactin1 in NMJ development and maintenance.

Amyotrophic lateral sclerosis (ALS), also known as Charcot’s disease, motor neuron disease (MND) and Lou Gehrig’s disease, is a deadly, adult-onset neurodegenerative disorder characterized by progressive loss of upper and lower motor neurons, resulting in evolving paresis of the linked muscles. ALS is defined by classical features of the disease, but may present as a wide spectrum of phenotypes. About 10% of all ALS cases have been reported as familial, of which about 20% have been associated with mutations in the gene encoding for CuZn superoxide dismutase (SOD1). The remaining cases are regarded as sporadic. Research has advanced our understanding of the disease, but the cause is still unknown, no reliable diagnostic test exists, no cure has been found and the current therapies are unsatisfactory. Riluzole (Rilutek®) is the only registered drug for the treatment of ALS. The drug has shown only a modest effect in prolonging life and the mechanism of action of riluzole is not yet fully understood. ALS is diagnosed by excluding diseases with similar symptoms. At an early stage, there are numerous possible diseases that may present with similar symptoms, thereby making the diagnostic procedure cumbersome, extensive and time consuming with a significant risk of misdiagnosis. Biomarkers that can be developed into diagnostic test of ALS are therefore needed. The high number of unsuccessful attempts at finding a single diseasespecific marker, in combination with the complexity of the disease, indicates that a pattern of several markers is perhaps more likely to provide a diagnostic signature for ALS. Metabolomics, in combination with chemometrics, can be a useful tool with which to study human disease. Metabolomics can screen for small molecules in biofluids such as cerebrospinal fluid (CSF) and chemometrics can provide structure and tools in order to handle the types of data generated from metabolomics. In this thesis, ALS has been studied using a combination of metabolomics and chemometrics. Collection and storage of CSF in relation to metabolite stability have been extensively evaluated. Protocols for metabolomics on CSF samples have been proposed, used and evaluated. In addition, a new feature of data processing allowing new samples to be predicted into existing models has been tested, evaluated and used for metabolomics on blood and CSF. A panel of potential biomarkers has been generated for ALS and subtypes of ALS. An overall decrease in metabolite concentration was found for subjects with ALS compared to their matched controls. Glutamic acid was one of the metabolites found to be decreased in patients with ALS. A larger metabolic heterogeneity was detected among SALS cases compared to FALS. This was also reflected in models of SALS and FALS against their respective matched controls, where no significant difference from control was found for SALS while the FALS samples significantly differed from their matched controls. Significant deviating metabolic patterns were also found between ALS subjects carrying different mutations in the gene encoding SOD1.

Introduction: Amyotrophic lateral sclerosis (ALS) is a deadly, progressive neuromuscular disease that affects individuals all over the world. About 10% of the patients have a familial predisposition (FALS) while the remainder of cases are isolated or sporadic (SALS) and of unknown cause. To date, the principal recognized risk factors for ALS are higher age, male gender, slim figure (BMI<23) and a family history of ALS. In 1993, Rosen et al. observed that some FALS cases were associated with mutations in the gene encoding the CuZn superoxide dismutase enzyme (SOD1). Since then, several mutations in the SOD1 gene have been discovered, and mutations in more than 18 other genes have been associated with causing ALS. The aim of this thesis was to identify new mutations associated with ALS pathogenesis, and by comparing patients from different countries, were we also able to identify population-specific genetic variations. The studies are referred to as I–V. Methods: With written informed consent and adhering to the tenets of the Declaration of Helsinki, through a national network of ALS clinicians´, venous blood samples were collected from ALS patients and healthy subjects in Europe and the USA. The patients were diagnosed according to the El Escorial criteria, and as having FALS according to the criteria of Byrne et al. (2011). The DNA variations were amplified by various PCR techniques. (I, III and IV) The amplicons of ataxin 2 (ATXN2), profilin 1 (PFN1), and vesicle-associated membrane protein type B (VAPB) were characterised by direct sequencing. (II) After quantitative PCR, a genotype-phenotype correlation was performed to assess whether the survival motor neuron gene (SMN) modulates the phenotype of ALS. (V) The amplicons of the 50 base pair deletion in the SOD1 promotor (50 bp) were separated by electrophoresis on agarose. Results: (I) We observed a significant association between CAG expansions in the ATXN2 gene and ALS in a European cohort. (II) Abnormal copy number of the SMN1 gene was identified as a risk factor in France, but not in Sweden. Homozygosity of the SMN2 deletion prolonged survival among Swedish ALS patients, compared to French patients. (III) We identified two mutations in the PFN1 gene, the novel p.Thr109Met mutation and the p.Gln117Gly mutation, in two unrelated FALS patients. (IV) In our cohort, we identified five VAPB mutations p.Asp130Glu, p.Ser160del, p.Asp162Glu, p.Met170Ile, and p.Arg184Trp, two of which are novel. (V) The 50 bp deletion upstream of the SOD1 gene was found in equal frequencies in both the patient and control cohorts. The 50 bp deletion did not affect SOD1 enzymatic activity. Furthermore, we found no differences in age of onset or disease duration in relation to the 50 bp deletion genotype.VI Conclusions: (I) Our findings indicate that ATXN2 plays an important role in the pathogenesis of ALS, and that CAG expansions in ATXN2 are a significant risk factor for the disease. (II) We suggest that abnormal SMN1 gene copynumber cannot be considered a universal genetic susceptibility factor for ALS. We also propose that the effect of abnormal SMN2 gene copy number on ALS phenotype may differ between populations. (III) This work provides evidence that PFN1 mutations can cause ALS as a Mendelian dominant trait. The novel p.Thr109Met mutation also shows that disturbance of actin dynamics can cause motor neuron degeneration. (IV) We find it unlikely that the VAPB mutations cause ALS in our cohorts. (V) We find it unlikely that the 50 bp region contains important regulatory elements for SOD1 expression. This thesis supports the theory that ALS is a multigenetic disease, but there appears to be great genetic variation among apparently identical populations. These studies emphasise the importance of continuous genetic screening, to identify further mutations and genes involved in ALS disease, but it also highlights the importance of cooperation and comparison between countries. / On the aetiology of ALS: A comprehensive genetic study

While great strides have been made in treating many classes of human disease, the late-onset neurodegenerative diseases continue to elude modern medicine. These diseases, which include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), the transmissible spongiform encephalopathies (TSEs), and amyotrophic lateral sclerosis (ALS), involve accumulation of insoluble aggregates of one or more causative proteins, leading to progressive loss of central nervous system neurons, progressively worsening neurological symptoms, and eventual patient death. All of these diseases are currently incurable and fatal. In the case of ALS, progressive death of upper and lower motor neurons leads to full-body paralysis, respiratory difficulty, and patient death. Of the subset of ALS cases showing familial inheritance, approximately 20% are caused by mutations in the SOD1 gene, encoding the Cu, Zn superoxide dismutase (SOD1). These mutations do not have the common property of impairing SOD1's normal function as a free radical scavenger. Instead, they are thought to increase the protein's likelihood of misfolding and aggregating via a poorly-understood aggregation cascade. It is believed that species populated along the misfolding and aggregation pathway may prove to be good targets for therapies designed to block accumulation of downstream toxic species, or to prevent aberrant protein-protein interactions responsible for neurotoxicity. In this thesis, several new techniques are developed to enable detailed elucidation of the SOD1 unfolding and misfolding pathways. Time-resolved measurements collected during SOD1 unfolding or misfolding of release of bound Cu and Zn, of changes in intrinsic fluorescence, of exposure of hydrophobic surface area, and of alterations in the chemical environment of histidine residues, are presented. A new mathematical analysis technique named the Analytical Laplace Inversion Algorithm is developed for rapid extraction of mechanistic information from these time-resolved signals. These tools are applied to the construction of the most detailed models to date of the unfolding and misfolding mechanisms of WT and ALS-causing mutant SOD1. The models presented identify several well-populated unfolding and misfolding intermediates that could serve as good targets for therapies designed to address the fundamental molecular mechanisms underlying SOD1-associated ALS, and to treat what is currently a devastating and incurable disease.

Amyotrophic lateral sclerosis (ALS) is a devastating disease. Currently, there is no cure for this disease, and effective treatment strategies are greatly needed. Calpain activation plays a major role in the motor neuron degeneration that causes ALS. Therefore, therapeutic strategies can inhibit calpain activity in the central nervous system (CNS) have great clinical potential. The calpain inhibitors AK295 and MDL-28170 have been demonstrated to be neuroprotective in animal models of neurological injury, and should have great potential to treat ALS; however delivery problems have hindered their clinical success. Therefore, development of a new strategy that can locally deliver the calpain inhibitors to the central nervous system could significantly improve the treatment of ALS. The objectives of my thesis research were (1) to develop high molecular weight polyketals that provide sustained release properties for hydrophobic molecules, (2) to formulate calpain inhibitor-encapsulated polyketal microparticles which have a release half life of one month in vitro, (3) and to evaluate the performance of polyketal microparticles for delivering calpain inhibitors to the spinal cord in vivo. In completing these specific aims, we have developed biodegradable polymeric microparticles for the delivery of calpain inhibitors, AK295 and MDL-28170 to treat ALS. The results of calpain assays showed that both AK-PKMs and MDL-PKMs maintained most of their inhibitory activities even after the robust emulsion process. The in vitro release profile of MDL-28170 in MDL-PKMs showed that 50 % of the drug was released in the first 30 days. Experiments using dye-encapsulated microparticles showed that polyketal microparticles (1-2 ìm) are not easily cleared in the neutral physiological environment and can have potential to continuously release drug from the injection sites in the spinal cord. The efficacy of calpain inhibitor-encapsulated PKMs were studied by evaluation the behavior and survival of SOD1G93A rats, a genetic rat model for ALS. We observed the trend toward improvements in grip strength and rotarod performance in the first two months from the AK-PKMs treated group, however, further improvements are needed to enhance their in vivo efficacy.

No description available.

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease which currently lacks rapid and definitive diagnostic tests. Recently identified neuron specific splice variant molecules, Per28 and NFL-60, have been shown to contain unique epitopes and to have altered levels of expression in ALS patients. It is believed that these factors make Per28 and NFL-60 excellent candidate biomarkers for the ALS disease state. In this study, we attempted to develop ELISA assays directed against Per28 and NFL-60, as well as a generalized guideline for splice variant ELISA development, which could be used in a clinical setting. Limitations in currently identified antibodies to the splice variants allowed only for the completion of a Per28 ELISA, which lacked the sensitivity for clinical relevance. This assay creation process, however, did produce a guideline for similar ELISA development, which should allow for the more expeditious creation future ELISA.