TDP-43 est une protéine multifonctionnelle possédant des rôles dans la transcription, l'épissage des pré-ARNm, la stabilité et le transport des ARNm. TDP-43 interagit avec d'autres hnRNP, incluant hnRNP A2, via son extrémité C-terminale. Plusieurs membres de la famille des hnRNP étant impliqués dans la réponse au stress cellulaire, alors nous avons émis l’hypothèse que TDP-43 pouvait y participer aussi. Nos résultats démontrent que TDP-43 et hnRNP A2 sont localisés au niveau des granules de stress, à la suite d’un stress oxydatif, d’un choc thermique, et lors de l’exposition à la thapsigargine. TDP-43 contribue à la fois à l'assemblage et au maintien des granules de stress en réponse au stress oxydatif. TDP-43 régule aussi de façon différentielle les composants clés des granules de stress, notamment TIA-1 et G3BP. L'agrégation contrôlée de TIA-1 est perturbée en l'absence de TDP-43. En outre, TDP-43 régule le niveau d`ARNm de G3BP, un facteur de granule de stress de nucléation. La mutation associée à la sclérose latérale amyotrophique, TDP-43R361S, compromet la formation de granules de stress. Ainsi, la fonction cellulaire de TDP-43 s'étend au-delà de l’épissage; TDP-43 est aussi un composant de la réponse cellulaire au stress central et un acteur actif dans le stockage des ARNs. / TDP-43 is a multifunctional protein with roles in transcription, pre-mRNA splicing, mRNA stability and transport. TDP-43 interacts with other hnRNPs, including hnRNP A2, via its C-terminus and several hnRNP family members are involved in the cellular stress response. This relationship led us to investigate the role of TDP-43 in cellular stress. Our results demonstrate that TDP-43 and hnRNP A2 are localized to stress granules, following oxidative stress, heat shock, and exposure to thapsigargin. TDP-43 contributes to both the assembly and maintenance of stress granules in response to oxidative stress and differentially regulates key stress granules components including TIA-1 and G3BP. The controlled aggregation of TIA-1 is disrupted in the absence of TDP-43. In addition, TDP-43 regulates G3BP mRNA levels, a stress granule nucleating factor. A mutation associated with amyotrophic lateral sclerosis, TDP-43R361S, compromises stress granule formation. Thus, the cellular function of TDP-43 extends beyond splicing and places TDP-43 as a participant of the central cellular response to stress and an active player in RNA storage.

La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative due à une dégénérescence des motoneurones. Plus de 40 mutations du gène TARDBP ont été identifiées chez des patients SLA. Les défauts biochimiques de ces mutations étant encore inconnus, les modèles animaux sont présentement la seule mesure possible d’un phénotype. Pour étudier les conséquences physiopathologiques d’une de ces mutations, nous avons développé deux lignées transgéniques de poisson zébré, exprimant le gène humain TARDBP soit de type sauvage, soit avec la mutation G348C liée à la SLA, sous le contrôle d’un promoteur de choc thermique. Ces lignées ont été étudiées sur trois générations, après avoir établi un protocole de choc thermique induisant une expression ubiquitaire du transgène. Les embryons transgéniques de la génération F2 de la lignée exprimant la mutation développent un phénotype moteur suite à un choc thermique de 38.5°C pendant 30 minutes lorsque les embryons sont à 18 heures post-fertilisation. 60% des embryons ont une réponse anormale au toucher. De plus, une réduction de 28% de la longueur de pré-branchement des axones des motoneurones est observée. Ces résultats indiquent que notre lignée exprimant la protéine mutante TDP-43 est un modèle génétique de la SLA prometteur, qui ouvre des perspectives pour la compréhension de la physiopathologie de la maladie et la découverte de molécules thérapeutiques. / Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease due to motoneurons degeneration. More than 40 mutations of the gene TARDBP, coding for the protein TDP-43 have been found in ALS patients. As the biochemical defects of these mutations are not known, in vivo models are currently the only windows onto the pathology. To study the pathophysiological consequences of one of these mutations, we have generated two stable zebrafish transgenic lines, expressing the human gene TARDBP, either the wild-type version, or the G348C mutated version linked to ALS, under the control of a heat shock promotor. These lines were studied for three generation, after establishing a heat shock protocol sufficient to induce a ubiquitous expression of the transgene. The transgenic embryos of the F2 generation of the line expressing the mutant protein develop a motor phenotype after a 38.5°C heat shock for 30 minutes when the embryos are 18 hours post-fertilization. 60% of these embryos have an abnormal touch escaped evoked response, and a 28% reduction of the pre-branching axonal length of the motoneurons axons. These results indicate that our line expressing the mutant TDP-43 protein is a promising genetic model of ALS, opening perspectives for the pathophysiological understanding of the disease, and the discovery of new therapeutics.

La sclérose latérale amyotrophique est une maladie neurodégénérative fatale caractérisée par la dégénérescence progressive des neurones moteurs centraux et périphériques. L’un des premiers signes de la maladie est la dénervation de la jonction neuromusculaire (JNM). Les diverses unités motrices (UM) ne présentent toutefois pas la même vulnérabilité à la dénervation dans la SLA: les UM rapide fatigables sont en fait les plus vulnérables et les UM lentes sont les plus résistantes. Alors que des études précédentes ont démontré dans plusieurs modèles animaux de la SLA de nombreuses variations synaptiques, les découvertes ont été contradictoires. Par ailleurs, le type d’UM n’a pas été tenu en compte dans ces divers travaux. Nous avons donc émis l’hypothèse que la présence de la mutation SOD1 pourrait affecter différemment la transmission synaptique des UM, en accord avec leur vulnérabilité sélective. En effectuant des enregistrements électrophysiologiques et de l’immunohistochimie, nous avons étudié la transmission synaptique des différents types d’UM du muscle à contraction rapide Extensor Digitorum Longus (EDL; rapide fatigable (FF) MU) et du muscle à contraction lente Soleus (SOL; lente (S) and rapide fatigue-résistante (FR) MU) de la souris SOD1G37R et leur congénères WT. Pour identifier le type d’UM, un marquage par immunohistochimie des chaînes de myosine a été effectué. Un triple marquage de la JNM a également été effectué pour vérifier son intégrité aux différents stades de la maladie. À P160, dans la période asymptomatique de la maladie, alors qu’aucune altération morphologique n’était présente, l’activité évoquée était déjà altérée différemment en fonction des UM. Les JNMs FF mutantes ont démontré une diminution de l’amplitude des potentiels de plaque motrice (PPM) et du contenu quantique, alors que les JNMs lentes démontraient pratiquement le contraire. Les JNMs FR montraient quant à elles une force synaptique semblable au WT. À P380, dans la période présymtomatique, de nombreuses altérations morphologiques ont été observées dans le muscle EDL, incluant la dénervation complète, l’innervation partielle et les extensions du nerf. La transmission synaptique évoquée des UM FF étaient toujours réduites, de même que la fréquence des potentiels de plaque motrice miniatures. À P425, à l’apparition des premiers symptômes, l’activité synaptique des JNMs S était redevenue normale alors que les JNMs FR ont montré à ce moment une diminution du contenu quantique par rapport au contrôle. De manière surprenante, aucun changement du ratio de facilitation n’a été observé malgré les changements flagrants de la force synaptique. Ces résultats révèlent que la fonction de la JNM est modifiée différemment en fonction de la susceptibilité des UM dans l’ALS. Cette étude fournit des pistes pour une meilleure compréhension de la physiologie de la JNM durant la pathologie qui est cruciale au développement d’une thérapie adéquate ciblant la JNM dans la SLA. / Amyotrophic lateral sclerosis (ALS) is a fatal late-onset neurodegenerative disease characterized by the progressive loss of upper and lower motor neurons. Denervation of the neuromuscular junction (NMJ) is an early pathological event in various ALS models. Motor units (MU) appear unequally susceptible to denervation, the fast fatigable (FF) MU being the most vulnerable and the slow (S) MU the most resistant. While previous studies in several ALS models have consistently reported alterations in synaptic transmission, their findings have been contradictory. Interestingly, the MU types were not taken into account in these studies, which could explain these discrepancies. We hypothesized that the MU selective vulnerabilities observed in ALS will be associated with MU-specific NMJ alterations throughout the disease course. Using electrophysiology, we studied synaptic transmission of different types of MU in the fast-twitch Extensor Digitorum Longus (EDL; fast fatigable (FF) MU) and the slow-twitch Soleus (SOL; slow (S) and fast fatigue resistant (FR) MU) of the SOD1 mice and their WT littermates. MU types were identified using immunohistochemical labelling of the respective myosine heavy chains. Immunohistochemistry was also performed to assess NMJ integrity by using antibodies against main NMJ components. At a presymptomatic stage (P160), while no morphological alterations of NMJs were seen in both muscles, evoked activity was altered in a MU-specific manner in SOD1 mice. FF MU from SOD1 mice showed a decrease in EPP amplitude and quantal content whereas S MU showed the opposite. Mutant FR MU showed no difference in evoked activity compared to WT. At presymptomatic stage (P380), various morphological alterations were seen in the SOD1 EDL, including denervation, partial innervation and nerve sprouting. Evoked activity was still reduced in FF MU, as well as mEPP frequency. In contrast, at disease onset (P425), synaptic strength of the S MU was now similar to WT MU, whereas FR NMJs showed a decrease in EPP amplitude and quantal content. Surprisingly, paired-pulse facilitation was not altered in any MU type and at any age despite changes in synaptic strength. Taken together, these results reveal that NMJ function is differentially modified according to MU susceptibility in ALS. This study provides insights for a better understanding of NMJ physiology during the illness that is crucial to the development of a proper NMJ-targeted treatment in ALS.

La Sclérose Latérale Amyotrophique (SLA) est une maladie neurodégénérative qui affecte les neurones moteurs. 10% des cas sont des cas familiaux et l’étude de ces familles a mené à la découverte de plusieurs gènes pouvant causer la SLA, incluant SOD1, TARDBP et FUS. L’expansion de la répétition GGGGCC dans le gène C9orf72 est, à ce jour, la cause la plus connue de SLA. L’impact de cette expansion est encore méconnu et il reste à déterminer si la toxicité est causée par un gain de fonction, une perte de fonction ou les deux. Plusieurs gènes impliqués dans la SLA sont conservés entre le nématode Caenorhabditis elegans et l’humain. C. elegans est un vers transparent fréquemment utilisé pour des études anatomiques, comportementales et génétiques. Il possède une lignée cellulaire invariable qui inclue 302 neurones. Aussi, les mécanismes de réponse au stress ainsi que les mécanismes de vieillissement sont très bien conservés entre ce nématode et l’humain. Donc, notre groupe, et plusieurs autres, ont utilisé C. elegans pour étudier plusieurs aspects de la SLA. Pour mieux comprendre la toxicité causée par l’expansion GGGGCC de C9orf72, nous avons développé deux modèles de vers pour étudier l’impact d’une perte de fonction ainsi que d’un gain de toxicité de l’ARN. Pour voir les conséquences d’une perte de fonction, nous avons étudié l’orthologue de C9orf72 dans C. elegans, alfa-1 (ALS/FTD associated gene homolog). Les vers mutants alfa-1(ok3062) développent des problèmes moteurs causant une paralysie et une dégénérescence spécifique des neurones moteurs GABAergiques. De plus, les mutants sont sensibles au stress osmotique qui provoque une dégénérescence. D’autre part, l’expression de la séquence d’ARN contenant une répétition pathogénique GGGGCC cause aussi des problèmes moteurs et de la dégénérescence affectant les neurones moteurs. Nos résultats suggèrent donc qu’un gain de toxicité de l’ARN ainsi qu’une perte de fonction de C9orf72 sont donc toxiques pour les neurones. Puisque le mouvement du vers peut être rapidement évalué en cultivant les vers dans un milieu liquide, nous avons développé un criblage de molécules pouvant affecter le mouvement des vers mutants alfa-1 en culture liquide. Plus de 4 000 composés ont été évalués et 80 ameliore la mobilité des vers alfa-1. Onze molécules ont aussi été testées dans les vers exprimant l’expansion GGGGCC et huit diminuent aussi le phénotype moteur de ces vers. Finalement, des huit molécules qui diminent la toxicité causée par la perte de fonction de C9orf72 et la toxicité de l’ARN, deux restaurent aussi l’expression anormale de plusieurs transcrits d’ARN observée dans des cellules dérivées de patient C9orf72. Avec ce projet, nous voulons identifier des molécules pouvant affecter tous les modes de toxicité de C9orf72 et possiblement ouvrir de nouvelles avenues thérapeutiques / Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting the motor neurons. 10% of the cases are familial and using those families, many genes were shown to be involved in ALS pathogenesis, including SOD1, TARDBP and FUS. The GGGGCC repeat found in the first intron of C9orf72 is, to this day, the most common genetic cause of ALS. Many hypotheses have been speculated to explain the toxicity of the pathogenic GGGGCC repeat, including loss and gain of function mechanisms. Many proteins involved in amyotrophic lateral sclerosis (ALS) are evolutionarily conserved in the worm Caenorhabditis elegans. C. elegans is a transparent nematode widely used for anatomical, behavioural and genetic studies. It possesses an invariant cell lineage that includes 302 neurons in the adult nematode. Also, cellular stress responses and survival mechanisms are genetically regulated and conserved from the nematode and human. Therefore, our group, and others, have used C. elegans to model different aspects of neurodegenerative diseases including ALS. To better understand the toxicity caused by the GGGGCC repeat expansion in C9orf72, we have developed two C. elegans models to understand either the impact of the loss of function of C9orf72 or the gain of toxicity of the RNA containing the GGGGCC repeat. To understand the loss of function, we have characterized the orthologue of C9orf72 in C. elegans, alfa-1 (ALS/FTD associated gene homolog). Mutant alfa-1 worms exhibit motor impairments leading to paralysis and neurodegenereation of the GABAergic neurons. Mutant worms are also sensitive to osmotic stress which can lead to increased neurodegeneration. On the other part, exposure of C. elegans neurons to the RNA containing the GGGGCC repeat causes also motor problem and degeneration affecting the motor neurons. Therefore, our data suggest that both loss of function of C9orf72 and toxic gain of function are detrimental to neurons. Since motor dysfunctions in worms can be easily accessed in liquid culture, we have screened more than 4,000 FDA approved compounds in the alfa-1(ok3062) worms. 80 molecules were shown to improve alfa-1 impaired function and eleven of those were also tested for their effect to reduce the neurotoxicity caused by the GGGGCC repeat RNA. Eight molecules were shown to affect both types of neurotoxicity. Finally, from these eight molecules that can improveboth types of toxicity, two were shown to restore the abnormal RNA expression observed in C9orf72 patient-derive cells. With this project, we aimed to identify molecules that can affect the loss of C9orf72 toxicity and the toxic gain of RNA function containing the GGGGCC repeat to hopefully open new therapeutic avenues for ALS patients.

Mesenchymal stem cells (MSC) possess a multilineage differentiation potential and have the ability to regulate reactivity of the immune system. They are usually isolated and expanded from the bone marrow, adipose tissue or umbilical cord. MSC represent promising cell population for the treatment of some severe diseases, such as amyotrofic lateral sclerosis (ALS), due to the combination of regenerative and immunomodulatory properties. The aim of this study is to compare MSC from ALS patients and healthy donors in their phenotype, proliferative activity and mainly their immunomodulatory properties. The assessment of impact of the disease on the properties of MSC is important for their autologous use in clinical trials. In this study we used MSC isolated from bone marrow of 14 ALS patients and 15 patients undergoing mostly orthopedic surgery as control group. We also used MSC stimulated for 24 hours by poinflammatory cytokines. Cells were compared in terms of immunophenotype, differentiation in adipocytes and osteoblasts, metabolic activity, expression of selected genes for immunomodulatory molecules and for inhibition of lymphocyte proliferation. Further experiments were focused on evaluation of immunomodulatory properties of MSC. The effect of MSC on peripheral blood mononuclear cells stimulated...

LIGHT, un membre de la famille du TNF, déclenche la mort des motoneurones et contribue à la progression de la dégénérescence des motoneurones chez les souris modèles de la sclérose latérale amyotrophique. Étonnement, nous montrons que LIGHT peut également stimuler la croissance axonale in vitro et est nécessaire à la régénération axonale des motoneurones in vivo lors d'une lésion nerveuse périphérique. La compréhension des mécanismes dictant la bivalence de son récepteur mène à l'étude des effets de la régionalisation du signal LIGHT. Les chambres microfluidiques en isolant les axones des corps cellulaires, nous ont permis d'étudier la résultante fonctionnelle d'une stimulation axonale et somatique par LIGHT. Nous montrons que LIGHT active différentiellement la mort, quand il agit au niveau somatique, ou la régénération axonale, quand il stimule l'axone. L'ensemble de ces résultats mène à de nouvelles approches où l'administration de LIGHT au niveau du nerf lésé et du muscle correspondant nous renseignera sur le potentiel thérapeutique de LIGHT lors d'une lésion du système nerveux. / LIGHT, a member of the TNF superfamily, triggers death of motoneurons and contribute to the progression of motoneuron degeneration in an ALS mouse model. Interestingly, we demonstrate that LIGHT can elicit neurite outgrowth in vitro and contribute to axon regeneration in vivo following nerve injury. To better understand mechanisms governing the opposite effects of LIGHT on motoneurons we asked whether a regional activation of LIGHT pathway could trigger these opposite responses. Toward this goal we used microfluidic chambers to isolate the soma from the axon of motoneurons. We shows that LIGHT activates differentially motoneurons death when it acts at the soma and axon outgrowth when it acts at the level of the axon. Together, these results lead us to evaluate new therapeutic approaches to nerve injury.

A Esclerose Lateral Amiotrófica (ELA) é uma doença neurodegenerativa progressiva de evolução rápida, caracterizada pela perda seletiva dos neurônios motores (NM) superiores e inferiores. Recentemente, as células gliais centrais (astrócito, microglia e oligodendrócito) mostraram-se tóxicas aos NM, porém os detalhes moleculares não estão completamente elucidados. Em relação às células gliais periféricas, alterações eletrofisiológicas no nervo ciático do modelo animal da ELA na idade pré-sintomática foram reportadas pelo nosso grupo e os achados de denervação precoce tanto no modelo animal quanto em pacientes sugerem a participação das células de Schwann (CS) na morte neuronal retrógrada na ELA, teoria conhecida como dying back. Nesse contexto, as CS mostraram-se capazes de induzir a retração axonal e a denervação das junções neuromusculares, eventos precoces na doença, ocorrendo possivelmente na fase présintomática. O objetivo deste trabalho foi verificar a influência das CS do modelo experimental na fase pré-sintomática e do paciente com evolução recente da forma esporádica da ELA, na sobrevida e no tamanho dos prolongamentos dos NM in vitro e entender a natureza molecular do fenômeno. Culturas de CS altamente purificadas foram obtidas a partir do nervo ciático do camundongo modelo animal e do nervo periférico de pacientes com ELA. Os NM da medula espinal de camundongos neonatos foram co-cultivados com as CS. A neurodegeneração foi avaliada pela presença do marcador Fluoro-Jade C (FJC). Os NM também foram tratados com o meio condicionado das culturas de CS do modelo animal ou dos pacientes com ELA. Os motoneurônios tiveram os seus prolongamentos contados e a morte neuronal foi identificada pela presença do FJC. Diversos fatores neurotróficos foram quantificados no meio condicionado das culturas de CS pela técnica de ELISA. A reação em cadeia da polimerase quantitativa (do inglês, quantitative polymerase chain reaction - qPCR) foi realizada para detectar alterações nas CS e no nervo periférico que pudessem estar relacionadas com disfunção na unidade CS/NM. Os resultados mostraram que os NM cultivados na ausência das CS mostraram-se mais susceptíveis à morte. Os NM cocultivados com as CS ELA mostraram maior número de perfis neurodegenerativos em comparação com os NM co-cultivados com as CS controle. Após o tratamento com o meio condicionado das CS ELA, os NM mostraram redução no tamanho dos prolongamentos e aumento do número de células em neurodegeneração em comparação com o grupo controle. Quantidades reduzidas dos fatores neurotróficos foram encontradas no meio condicionado das culturas de CS ELA. Alterações na expressão gênica das CS e no nervo periférico evidenciaram disfunções na unidade CS/NM que podem estar contribuindo para o processo neurodegenerativo visto na ELA. Conclui-se que a falência nos mecanismos de neuroproteção pelas CS ELA é um importante mecanismo implicado na morte neuronal, com grande potencial terapêutico / Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the selective loss of upper and lower motor neurons (MN). Recently, central glia (astrocytes, microglias and olygodendrocytes) were toxic to the MN, but the molecular aspects have not fully described. In relation to the peripheral glia, electrophysiological changes in the sciatic nerve of ALS animal model in the presymptomatic stage have been reported by our group and early denervation findings in both animal models and patients suggests the participation of Schwann cells (SC) in the retrograde neuronal death of ALS , theory known as dying back. In this context, the SC proved to be able to induce axonal retraction and denervation of the neuromuscular junctions, early events in the disease, possibly occurring in the pre-symptomatic phase. The aim of this thesis was to investigate the influence of SC of pre-symptomatic experimental model and from patient with recent evolution of ALS sporadic form, in the survival and axonal length of MN in vitro and understand the molecular nature of the phenomenon. Highly purified SC cultures were obtained from the sciatic nerve of the animal model and from ALS patient\'s peripheral nerve. MN from the newborn mouse spinal cord were co-cultured with SC and the neurodegeneration was assessed by the presence of the marker Fluoro-Jade C (FJC). MN were also treated with conditioned medium from cultures of SC of the animal model or ALS patients. MN had their neuronal length measured and neuronal degeneration was identified by the presence of the FJC. Several neurotrophic factors were measured in conditioned medium of mice and ALS patient\'s SC cultures by ELISA. The chain reaction quantitative polymerase (qPCR) was performed to detect changes in the SC and peripheral nerve that could be related with dysfunction in the functional unit SC/MN. The MN co-cultured with ALS SC showed a greater number of neurodegenerative profiles compared with MN cocultured with control SC. After treatment with ALS SC conditioned medium, MN showed a reduction in the neuronal length and increased number of cells in neurodegeneration compared with the control group. Lower levels of neurotrophic factors were found in the conditioned medium of ALS SC cultures. Changes in the gene expression of SC and peripheral nerve showed dysfunctions in SC/MN unit, which may be contributing to the neurodegenerative process seen in ALS. In conclusion, the failure of neuroprotection by ALS SC is an important mechanism implicated in the MN cell death, with great therapeutic potential

Esta pesquisa se desenvolveu a partir da parceria entre o Projeto APOIAR do Laboratório de Saúde Mental e Psicologia Clinica Social do Instituto de Psicologia da USP e a ABRELA (Associação Brasileira de Esclerose Lateral Amiotrófica). O estudo refere-se à investigação e compreensão de aspectos afetivos, emocionais e de qualidade de vida dos profissionais da área de saúde, enfocando sua relação com cuidadores e pacientes portadores de Esclerose Lateral Amiotrófica (ELA), doença degenerativa dos neurônios motores, sem prognóstico de cura, embora com a consciência mantida até a morte. Foi empregado o método clínico de pesquisa em onze profissionais, médicos, assistentes sociais, fisioterapeutas respiratórios e motor, nutricionista e terapeuta ocupacional, no setor da Neuromuscular da UNIFESP/Escola Paulista de Medicina em parceria com a ABRELA. Foram empregados como instrumentos: Entrevista Psicológica Semi Estruturada, Método de Rorschach pelo Sistema de Avaliação Aníbal Silveira (sendo alguns índices selecionados) e o Questionário de Vida de McGill. Os dados foram tratados considerando o grupo de forma geral e não de modo individualizado. Principais resultados obtidos apontam para a maturidade do grupo de profissionais, a sensibilidade da maioria à dor e ao sofrimento dos pacientes, com respostas que denotam afetividade, frustração diante da falta de cura e sucesso no tratamento. Demonstram qualidade de vida preservada, e os dados sugerem que trabalham na área que escolheram apesar das dificuldades que sentem. Sugere-se que sejam implementados programas que possam dar aos profissionais de saúde o suporte necessário para que se mantenham como o grupo estudado nessa pesquisa, em condições de dar conta do contato com pacientes com enfermidades graves e crônicas como a ELA. / The actual research was possible due to the partnership between the Project APOIAR (SUPPORT) of the Laboratory of Mental Health and Clinical Psychology of the Institute of Psychology of USP University of Sao Paulo, and ABRELA ( Brazilian Association of Amyotrophic Lateral Sclerosis ( ALS ). This study intends to investigate and better understand the Quality of Life and emotional aspects of health professionals in their relationship with ALS patients and their carers. ALS is a degenerative illness which affects motor neurons causing severe physical impediments, even tough its patients continue lucid up to their death. This illness does not have a prognosis of cure. We have applied the Clinical Method of survey and research on eleven professionals physicians, social workers, motor and respiratory physiotherapists, nutritionist and occupational therapist from de Department of neuromuscular of UNIFESP/ Federal Paulista Medical School and partnership with ABRELA. The study Instruments used were the Semi- Structured psychological Interview, the Rorschach Method ( the Evaluation System by Anibal Silveira) and the Questionnaire of Quality of Life. The collected data considered the group as a whole and not individuals. The main results point towards the maturity of the group of health professionals and their sensitivity related to the pain and suffering of patients, with answers showing affection and frustration due to impossibility of cure and success of treatment. The results show preserved Quality of Life and suggest that those health professionals work within their chosen specialty despite their difficulties.The study suggests the need of implementing programs which give health professionals the necessary support, as shown in this study, so that they can deal with their patients with serious and chronic illnesses such ALS.

A Esclerose Lateral Amiotrófica (ELA) é uma doença crônica, progressiva e neurodegenerativa causada pela morte dos neurônios motores. O diagnóstico destes pacientes pode levar até 12 meses para acontecer, sendo que estes vão à óbito entre 3-5 anos do início dos sintomas. Há, porém, grande variabilidade de quadro clínico, com alguns pacientes falecendo com menos de 1 ano do início dos primeiros sinais, e outros que sobrevivem por décadas. A identificação da ELA8, causada por uma mutação missense no gene VAPB (c.C166T, p.P56S), tem contribuído significativamente com o conhecimento dos mecanismos moleculares por trás da ELA. A literatura recente tem evidenciado que a diminuição dos níveis de VAPB está presente em modelos celulares e murinos da doença, e também em amostras de pacientes, sugerindo que esta proteína teria papel central na doença e uma contribuição significativa para a morte dos neurônios motores. O presente trabalho buscou três objetivos principais: (1) o diagnóstico molecular através de um painel de sequenciamento de nova geração que inclui os genes SOD1, FUS, TARDBP, SETX, SPG11, FIG4 e VAPB; (2) a avaliação dos níveis de RNAm de VAPA, VAPB e EPHA4 em pacientes de ELA8, controles familiares e outros pacientes de ELA, com o intuito de investigar possíveis papéis destes genes na doença; e por fim, (3) o desenvolvimento de um ensaio quantitativo para as proteínas VAPA, VAPB e VAPC baseado em cromatografia líquida acoplada à espectrometria de massas em tandem (LC-MS/MS), para a posterior avaliação de VAPB como possível biomarcador em ELA, e de suas isoformas VAPA e VAPC como modificadores da doença. Para a análise genômica, foram avaliados 67 pacientes, sendo que 31 (ou 46%) apresentaram a mutação c.C166T em VAPB; 4 pacientes (6%) em SOD1, sendo que um destes apresentou uma mutação também em FIG4; 1 paciente (1.5%) foi identificado uma mutação patogênica em FUS; outro, duas mutações deletérias em trans em SPG11. Os níveis de RNAm de VAPB, VAPA e EPHA4 não são estatisticamente distintos entre pacientes e controles; porém, os níveis de EPHA4 estavam significativamente elevados em dois pacientes de início bulbar da doença. Para o desenvolvimento do método quantitativo por LC-MS/MS, foram escolhidos 8 peptídeos inequívocos para análise, estabelecidos dos parâmetros de corrida, e desenvolvidos dois padrões internos (linhagens SILAC e VAPB recombinante) para a quantificação. Esta ferramenta desenvolvida poderá auxiliar não apenas os estudos moleculares que envolvem os mecanismos por trás ELA8, responsável por uma elevada taxa dos casos familiais brasileiros, mas também poderá determinar o potencial de VAPB como biomarcador para Esclerose Lateral Amiotrófica / Amyotrophic Lateral Sclerosis is a chronic, progressive neurodegenerative disorder caused by the death of motor neurons. Diagnosis can take up to 12 months, with no molecular marker to expedite this process. In this scenario, patients die within 3 to 5 years of symptom onset, although a large clinical variability is seen, with severe patients dying less than one year after onset, and others surviving for decades. The identification of ALS8, caused by a missense mutation in the VAPB gene (c.C166T; p.P56S), has contributed significantly to the knowledge of molecular mechanisms behind ALS. Recent literature has evidenced that the decrease of VAPB levels is present in cellular and murine models, and also in patient samples, suggesting a central role in motor neuron death in ALS. The present work sought three main objectives: (1) a molecular diagnosis through a NGS sequencing panel including the SOD1, FUS, TARDBP, SETX, SPG11, FIG4 and VAPB genes; (2) analyze the expression levels of VAPA, VAPB and EPHA4 in patients, family controls and other forms of ALS, in order to investigate their possible roles in ALS8; and (3) the development of a targeted quantitative mass spectrometry based assay, gold standard in protein quantification due to its precision and sensitivity, for the VAPA, VAPB and VAPC proteins, seeking the analysis of VAPB as a potential biomarker in ALS and of its isoform\'s potential roles as modifiers in the disease. The genomic analyses revealed that out of 67 patients, 31 presented the ALS8 mutation in VAPB, 4 patients (6%) presented a mutation in SOD1, with one patient carrying a second mutation in FIG4; 1 (1.5%) patient was identified with a pathogenic mutation in FUS; and another presented two pathogenic mutations in trans in the SPG11 gene. Thus, we were able to diagnose over half of the patients included in this study with a panel of only 7 genes. VAPB, VAPA and EPHA4 mRNA levels are not statistically different between patients and controls; however, EPHA4 was shown to be highly elevated in two bulbar-onset non-ALS8 patients. For the development of the LC-MS/MS targeted assay, 8 surrogate peptides were chosen for analysis, run parameters were established, and two internal standards for quantification were developed (SILAC cell lines and recombinant VAPB). This tool will prove to be useful not only towards elucidating the molecular mechanisms behind ALS8, one of the most prevalent forms of familial ALS in Brazil, but also to determine VAPB\'s potential as a biomarker for ALS

Introdução: A Esclerose Lateral Amiotrófica (ELA) é uma doença degenerativa e progressiva que afeta neurônios motores da medula espinhal, tronco cerebral e/ ou córtex motor. Sua manifestação clínica é bastante variada, sua etiologia desconhecida e a progressão, fatal. Não existe ainda um tratamento curativo para a ELA, porém alguns medicamentos e a realização de fisioterapia podem auxiliar, fornecendo ao paciente uma melhor qualidade de vida. Objetivos: Testar a hipótese de que técnicas quantitativas de Ressonância Magnética (RM) são eficazes para detectar a lesão neuronal no encéfalo de pacientes com ELA no estágio inicial da doença e, verificar se existe correlação entre a lesão encefálica e a perda funcional do paciente. Dessa forma, pretende-se estabelecer um protocolo capaz de contribuir para o diagnóstico precoce da ELA. Metodologia: Quinze pacientes com diagnóstico de ELA definida ou provável (12 homens e três mulheres), com idade entre 37 e 79 anos e seus respectivos controles foram submetidos a um protocolo de avaliação por RM, que incluiu um estudo estrutural volumétrico e quantitativo do dano neuronal e mielínico por razão de sequências ponderadas em T1 e FLAIR, da Transferência de Magnetização (MT), Relaxometria, Anisotropia Fracionada (FA) e Difusão (DTI), além da Espectroscopia de prótons. Alguns dos sujeitos foram submetidos também à uma avaliação física de força muscular, de funcionalidade através da Amyotrophic Lateral Sclerosis Functional Rating Scale Revised (ALSFRS-R), de qualidade de vida através da Amyotrophic Lateral Sclerosis Assessment Questionnaire (ALSAQ-40) e de quantificação da fadiga pela Fadigue Severity Scale (FSS). Os dados da RM dos dois grupos foram comparados através da análise de variância multi e univariada e submetidos à correção de múltiplas comparações de Bonferroni. Nas variáveis com diferença significante entre os grupos, foi estudada a validade preditiva da medida, calculando-se a área sob a curva ROC e estabelecidos os valores de sensibilidade, especificidade e acurácia. Os dados das escalas foram comparados pelo teste de Mann-Whitney e correlacionados entre si e com as estruturas através da correlação de Spearman. Resultados: As várias técnicas da RM, com exceção da MT, identificaram pelo menos uma estrutura com diferença significante entre os dois grupos, totalizando 11 estruturas mais a razão dos metabólitos NAA/Cre. Os valores de sensibilidade, especificidade e acurácia foram satisfatórios variando entre 0,60 e 1,00 , com destaque para o Volume do Giro Superior Frontal Direito e Giro Superior Frontal Esquerdo que apresentaram valores 1,00 , 0,93 e 0,97 , respectivamente. As escalas ALSFRS-R e ALSAQ-40 apresentaram diferença significativa entre os dois grupos, mas a FSS não apresentou. As escalas apresentaram correlação significativa entre si em quase todos os escores totais e domínios. Já a correlação das escalas com as estruturas foi significativa apenas para o Volume. Conclusões: Técnicas como DTI, FA, Relaxometria e Volume se mostraram mais eficazes no diagnóstico precoce de pacientes com ELA do que as outras. A redução de volume de substância cinzenta se correlacionou positivamente com a ALSFRS-R. Por fim, propomos um protocolo para avaliação de pacientes com ELA, que inclua imagem volumétrica de alta resolução para cálculo da Volumetria e DTI. / Introduction: Amyotrophic Lateral Sclerosis (ALS) is a progressive and degenerative disease that affects motor neurons in the spinal cord, brain stem and/ or motor cortex. Their clinical presentation is varied, its unknown etiology and fatal progression. There isnt still a curative treatment for ALS, but some medications and physical therapy can help by providing the patient a better quality of life. Objectives: To test the hypothesis that quantitative techniques of magnetic resonance imaging (MRI) are effective to detect neuronal damage in the brain of patients with ALS at the inicial stage of the disease and see if there is a correlation between brain injury and functional loss of the patient. Thus, we intend to establish a protocol can to contribute to early diagnosis of ALS. Methods: Fifteen patients with definite or probable ALS (12 men and three women) aged between 37 and 79 and their respective controls underwent an MRI evaluation protocol, including a volumetric and quantitative structural study of damage neuronal and myelin by reason of T1-weighted sequences and FLAIR , Magnetization Transfer (MT), Relaxometry, Fractional Anisotropy (FA), Diffusion (DTI) and Proton magnetic resonance spectroscopy. Some of the subjects also underwent a physical assessment of muscle strength, functionality by Amyotrophic Lateral Sclerosis Functional Rating Scale Revised (ALSFRS-R), quality of life through the Amyotrophic Lateral Sclerosis Assessment Questionnaire (ALSAQ-40) and quantification of fatigue by Fadigue Severity Scale (FSS). The MRI data of the two groups were compared using analysis of variance multivariate and univariate and submitted to correction for multiple comparisons of Bonferroni. In the variables with significant differences between groups, we studied the predictive validity of the measure, by calculating the area under the ROC curve and set the sensitivity, specificity and accuracy. The data of the scales were compared using Mann-Whitney test and correlated with each other and with the structures by Spearman correlation. Results: The various MRI techniques, with the exception of MT, identified at least one structure with a significant difference between the two groups, a total of 11 structures over the reason for the metabolites NAA/Cre. The sensitivity, specificity and accuracy were satisfactory ranging from 0.60 to 1.00 , with emphasis on Volume of Gyros Superior Frontal Right and Gyros Superior Frontal Left that averaged 1.00 , 0.93 and 0,97 , respectively. The ALSFRS-R and ALSAQ-40 scales showed significant differences between the two groups, but the FSS did not. The scales were significantly correlated with each other in almost all domains and total scores. The correlation with the structures of the scales was significant only for the Volume. Conclusions: Techniques such as DTI, FA, Relaxometry and Volume are more effective in early diagnosis of ALS patients than others. The decrease in gray matter volume was positively correlated with the ALSFRS-R. Finally, we propose a protocol for evaluation patients with ALS, including high-resolution volumetric image to calculate the Volume and DTI.