Em quadros mais avançados da insuficiência cardíaca (IC), além do comprometimento do miocárdio, observa-se uma importante perda de massa muscular esquelética, a qual contribui para o mau prognóstico e orbimortalidade dos pacientes. As espécies reativas de oxigênio (ROS) parecem estar diretamente envolvidas no desenvolvimento e progressão da atrofia muscular em doenças crônico-degenerativas. De fato, já é sabido que a IC está associada ao estresse oxidativo na musculatura esquelética, o qual parece contribuir para o catabolismo proteico culminando em atrofia muscular na síndrome, apesar desta relação de causa e efeito ainda ser pouco investigada. No entanto, as fontes envolvidas na produção exacerbada de ROS na musculatura esquelética em ratos com infarto do miocárdio ainda não foram caracterizadas. Como a NADPH oxidase é um complexo enzimático especializado em produzir ROS, e sabendo que esta família de enzimas pró-oxidantes é ativada for agentes pró-inflamatórios e alguns agonistas de receptores acoplados à proteína G que estão aumentados na IC, na primeira parte do projeto de pesquisa testou-se a hipótese de que as NADPH oxidases estariam hiperativadas nos músculos plantar e sóleo de ratos submetidos ao infarto do miocárdio, contribuindo para um quadro de estresse oxidativo e consequente hiperativação do sistema proteolítico ubiquitina proteassoma (SUP), culminando assim na atrofia deste tecido. Para testar esta hipótese, ratos Wistar foram submetidos à cirurgia de infarto do miocárdio ou Sham e, quatro semanas pós-cirurgias, foram submetidos a oito semanas de tratamento com uma substância inibidora da NADPH oxidase (apocinina) ou placebo. Foram quantificados nos músculos plantar e sóleo: níveis de mRNA de componentes da família Nox da NADPH oxidase presentes no tecido muscular, bem como a atividade desse complexo enzimático; marcadores de estresse oxidativo; atividade de enzimas antioxidantes e concentração total de glutationa; níveis de mRNA de componentes do SUP e atividade do proteassoma; e o trofismo muscular (Subprojeto 1). A segunda parte deste projeto testou a hipótese de que o treinamento físico aeróbico (TFA) previniria a hiperativação das NADPH oxidases na musculatura esquelética de ratos submetidos ao infarto do miocárdio, contribuindo para uma diminuição do quadro de estresse oxidativo e menor ativação do SUP, prevenindo assim a atrofia deste tecido. Para testar esta hipótese, ratos Sham e infartados foram submetidos a oito semanas de TFA ou permaneceram sedentários (Subprojeto 2). As variáveis estudadas foram as mesmas relacionadas ao subprojeto 1. Os resultados do subprojeto 1 demonstraram que o infarto do miocárdio em ratos promove atrofia do músculo plantar desencadeada em parte pela ativação da NADPH oxidase, promovendo uma maior produção de ROS e consequente hiperativação do SUP. Além disso, o infarto do miocárdio promove atrofia do músculo sóleo, a qual também está associada a um aumento dos níveis de ROS e da atividade do proteassoma, porém independente da ativação da NADPH oxidase. Em relação ao subprojeto 2, nossos dados também demonstram que o TFA previne parcialmente a atrofia do músculo plantar de ratos infartados, prevenindo a hiperativação da NADPH oxidase e a hiperativação do SUP induzida pelo infarto do miocárdio. Essa intervenção não farmacológica também previne a atrofia do músculo sóleo dos ratos infartados associada à redução das ROS e à redução da hiperativação do SUP induzida pelo infarto do miocárdio, porém de forma independente da atividade do complexo enzimático NADPH oxidase. Dessa forma, concluímos que a NADPH oxidase está envolvida de maneira músculo-específica com a produção de ROS levando a ativação do SUP e à atrofia muscular associada ao infarto crônico do miocárdio. O TFA é capaz de prevenir a atrofia muscular esquelética induzida pelo infarto do miocárdio associada a redução das ROS em ambos os músculos estudados. No musculo plantar, esta resposta esta relacionada a uma redução na hiperativação do complexo enzimático NADPH oxidase, ressaltando a contribuição desse complexo para a geração de ROS em músculos glicolíticos de ratos infartados. O TFA consiste em importante ferramenta não farmacológica agindo na homeostase redox e proteica no musculo esquelético de ratos infartados / Although heart failure (HF) is a syndrome of cardiac origin, it promotes significant skeletal muscle atrophy in more advanced stages, which contributes to poor prognosis and increased mortality rate. Reactive oxygen species (ROS) seem to be directly involved in the development and progression of muscle atrophy in chronic degenerative diseases. In fact, HF is associated with skeletal muscle oxidative stress, which seems to contribute to protein catabolism and muscle atrophy. However, it is important to highlight that the sources involved in the exacerbated skeletal muscle ROS production in HF have not been characterized yet. NADPH oxidase enzyme complex is an important source of ROS production activated by proinflammatory cytokines and some G-protein-coupled receptors, which are increased in HF. Therefore, in the first part of the thesis, we tested whether NADPH oxidases would be overactivated in plantaris and soleus muscles of rats submitted to myocardial infarction, thus contributing to oxidative stress and consequent ubiquitin proteasome proteolytic system (UPS) hyperactivation, ultimately leading to muscle atrophy. To test this hypothesis, Wistar rats underwent myocardial infarction or Sham surgery and, four weeks post-surgery, rats underwent eight weeks of treatment with an NADPH oxidase inhibitor (apocynin) or placebo. It was quantified in plantaris and soleus muscles: mRNA levels of skeletal muscle-NOX family, as well as NADPH oxidase activity; oxidative stress markers; antioxidant enzymes activity and total glutathione concentration; mRNA levels of UPS components and proteasome activity; and muscle trophicity (Subproject 1). In the second part of the thesis we tested whether aerobic exercise training (AET) would prevent NADPH oxidases overactivity in plantaris and soleus muscles of rats submitted to myocardial infarction, thus decreasing oxidative stress and UPS hyperactivation, preventing skeletal muscle atrophy. To test this hypothesis, Sham and infarcted rats underwent eight weeks of AET or remained sedentary (Subproject 2). The variables studied were the same as in Subproject 1. The results of the subproject 1 demonstrated that myocardial infarction in rats induces plantaris muscle atrophy triggered in part by overactivation of NADPH oxidase promoting increased ROS production paralleled by UPS hyperactivation. Moreover, myocardial infarction induced soleus muscle atrophy, which was also associated with increased ROS levels and proteasome overactivity, but independently of NADPH oxidase activation. Regarding the subproject 2, our data showed that AET partially prevented plantaris muscle atrophy in infarcted rats, preventing myocardial infarction-induced NADPH oxidase hyperactivation and UPS hyperactivation. AET also prevented soleus muscle atrophy in infarcted rats, which was associated with a ROS levels reduction and prevention of myocardial infarction-induced UPS hyperactivation, but independently of NADPH oxidase activity. Collectively, our data give support for the involvement of NADPH oxidase as a source of ROS production leading to UPS activation and skeletal muscle atrophy associated with chronic myocardial infarction, however this occurs in a muscle specific pattern. AET prevents myocardial infarction-induced skeletal muscle atrophy and exacerbated ROS levels in both plantaris and soleus muscles. In plantaris muscle, this response is related to a prevention of NADPH oxidase hyperactivation, highlighting the contribution of this enzyme complex to ROS production in glycolytic muscles of infarcted rats. Finally, AET is an important nonpharmacologic tool acting in skeletal muscle redox and protein homeostasis of infarcted rats

Hipotrofía nos músculos que estabilizam a coluna tem sido identificada nos pacientes com lombalgia. Entretanto, não se sabe se a perda muscular é causa ou conseqüência desta disfunção, nem se ela é influenciada pelo tipo de alteração discal que o indivíduo apresenta. Este estudo testou a hipótese de que a hipotrofía dos pacientes com lombalgia seja dependente do tipo de alteração discal. Para avaliar a condição muscular em diferentes tipos de alteração discal, optou-se por um estudo retrospectivo e por uma seleção aleatória de 78 exames de ressonância magnética de indivíduos com lombalgia. Em cada exame foram realizadas medidas quantitativas da porcentagem de gordura na área de secção transversa dos mm. multífidus e psoas, nos três últimos níveis da coluna lombar. A alteração discal foi encontrada em 95% dos exames, sendo o abaulamento o achado de imagem mais freqüente, seguido pela protrusão discal. A porcentagem de gordura variou conforme o tipo de alteração discal. Nos níveis com abaulamento há em ambos os músculos estudados 6% a mais de tecido gorduroso do que nos níveis onde há protrusão e esta diferença foi estatisticamente significante. Músculos nos níveis onde há protrusão sem fissura no anel fibroso apresentaram maior substituição gordurosa do que aqueles onde há protrusão com fissura. A porcentagem de gordura foi influenciada por características anatômicas como músculo estudado e nível da coluna, e por características como idade e sexo dos sujeitos. Estes resultados indicam que a hipotrofía muscular em pacientes com lombalgia não é um processo uniforme e generalizado, mas sim correlacionado a diferentes variáveis, entre elas o tipo de alteração discal que o paciente apresenta. / Low back pain patients present atrophy on muscles responsible for spine stabilization. However, it is not clear if muscle waste is related to the cause or if it is a consequence of this disfunction. Nor it is clear if muscle athophy is affected by the type of disc pathology. This study tested the hypothesis that muscle waste in low back pain patients influenced by the type of disc derangement. Magnetic resonance scans of 78 low back pain patients were randomly analysed. Cross sectional area percentage of fat tissue in multifidi and psoas major muscles was measured on the lower levels of the lumbar spine. Disc pathology was found in 95% of the exams and disc bulge was the most frequent abnormality, followed by disc protrusion. Fat percentage varied according to disc pathology and this difference was statistically significant. Muscles on levels with disc bulge presented 6% more fat deposits than muscles on levels with disc protrusion. Muscles on levels with discs without anular tear present more fat infiltration than muscles on levels with anular tear. Fat percentage was also influenced by anatomic aspects such as evaluated muscle and spine level, and sample characteristics as age and sex. The results indicated that muscle atrophy in low back pain patients is not a uniform and generalized feature. It is correlated to different variables, such as type of disc pathology

L’objectif général de cette thèse est d’étudier la production quantitative (i.e., nombre de mots) et qualitative (i.e. regroupements et alternances) du verbe d’action dans le vieillissement normal et dans les pathologies du mouvement à travers la tâche de fluence d’action, comparativement aux fluences classiques (i.e., sémantique et littérale). Nous avons montré que la production lors d’une tâche de fluence d’action est altérée chez des adultes âgés (60 ans et plus) comparativement à des adultes jeunes (30 ans et moins) et qu’elle est en lien avec les fonctions exécutives (Expérience 1) et la mémoire de travail (Expérience 3) chez l’adulte âgé. L’effet de l’âge paraît être modifié par le temps de production, les adultes âgés produisant moins de verbes d’action que les adultes jeunes pour un temps de trois minutes (Expérience 1) mais pas pour des temps d’une (Expérience 2) et de deux minutes (Expérience 3). De plus, les adultes âgés prenaient davantage en considération la consigne temporelle lors des tâches de fluence verbale que les adultes jeunes (Expérience 2). Les données d’une tâche d’association verbale (Expérience 4) indiquaient que les associations entre les noms et les verbes sont modifiées par l’âge. Nous avons comparés les performances de patients atteints de pathologiesdu mouvement à celles de participants contrôles lors de la tâche de fluence d’action. Nous avons montré que la production quantitative lors cette tâche est altérée chez des patients atteints de maladie de Parkinson (Expérience 5) et d’atrophie multisystématisée (Expérience 6). Dans le tremblement essentiel (Expérience 7), nous avons montré une atteinte de la fluence d’action alors que le traitement des verbes, lors d’une tâche de décision lexicale, n’était pas altéré. De plus, la chirurgie (stimulation cérébrale profonde) modifiait qualitativement la production dans la tâche de fluence d’action. Nos données montrent un impact du vieillissement normal et des pathologies du mouvement sur la production du verbe d’action, enraison de la nature fortement exécutive de ce processus. / The aim of this thesis is to investigate the production of action verbs through the action fluency task, in comparison to classical fluency tasks (i.e., semantic and letter). In first instance, production has been studied, both quantitatively and qualitatively, in aging. We have shown that action fluency is impacted by healthy aging and is related to executive functions (Experiment 1) and working memory (Experiment 3) in older adults. This effect of age appearsto be affected by production time, with older adults producing fewer action verbs than younger adults in three minutes (Experiment 1) but not in one (Experiment 2) and two minutes (Experiment 3). In addition, older adults took more into account the time instruction during verbal fluency tasks (Experiment 2). The verbal association task’s data (Experiment 4) indicated that associations between nouns and verbs are modified by age. In second instance,the production of action verbs has been studied within movement disorders. We have shown that the production in the action fluency task and the adaptation to time instruction are impacted by Parkinson's disease (Experiment 5). We have also shown that action fluency is altered within an atypical parkinsonism, multiple system atrophy (Experiment 6). In essential tremor (Experiment 7), we have shown an impairment of action fluency while verbs treatment, in a lexical decision task, was not altered. In addition, surgery (deep brain stimulation) qualitatively modified the production of verbs in action fluency. Our data highlight the impact of healthy aging and movement disorders on the production of action verb, due to the highly executive nature of this process.

A estimulação elétrica neuromuscular (EENM) é um importante recurso utilizado na medicina esportiva para acelerar processos de recuperação. O objetivo deste estudo foi analisar os efeitos da EENM durante a imobilização do músculo gastrocnêmio, em posições de alongamento (LP) e encurtamento (SP). Para tanto, 60 ratas fêmeas jovens WISTAR foram distribuídas em seis grupos e acompanhadas durante 7 dias: controle (C), eletroestimuladas (EE), imobilizadas em encurtamento (ISP), imobilizadas em alongamento (ILP), imobilizadas em encurtamento e eletroestimuladas (ISP+EE) e imobilizadas em alongamento e eletroestimuladas (ILP+EE). Para a imobilização, o membro posterior direito foi envolvido por uma malha tubular e ataduras de algodão juntamente à atadura gessada. A EENM foi utilizada com uma freqüência de 50 Hz, 10 minutos por dia, totalizando 20 contrações em cada sessão. Após 7 dias os animais foram submetidos à eutanásia e os músculos gastrocnêmios foram retirados para a realização do ensaio mecânico de tração em uma máquina universal de ensaios (EMIC®). A partir dos gráficos carga versus alongamento foram calculadas as seguintes propriedades mecânicas: alongamento no limite de proporcionalidade (ALP), carga no limite de proporcionalidade (CLP), alongamento no limite máximo (ALM), carga no limite máximo (CLM) e rigidez. As imobilizações SP e LP promoveram reduções significativas (p<0,05) nas propriedades de ALP, CLP, ALM e CLM sendo mais acentuada, principalmente, no grupo ISP. Quando utilizada a EENM, houve acréscimo significativo (p<0,05) destas propriedades somente no grupo ISP. Já, em relação à rigidez, foi observada redução significativa (p<0,05) somente do grupo C para o grupo ISP. Quando utilizada a EENM, a rigidez do grupo ILP+EE foi significativamente (p<0,05) maior e mais próxima do grupo C que a do grupo ISP+EE. Neste modelo experimental, a imobilização dos músculos em alongamento atrasou a queda das propriedades e a estimulação elétrica, contribuiu para a manutenção das propriedades mecânicas durante o período de imobilização, principalmente no grupo ILP+EE. / The neuromuscular electric stimulation (NMES) is an important tool used in sport medicine to accelerate the recovery process. The objective of this study was to analyze the effects of NMES during the immobilization of the gastrocnemius muscle, in lengthened (LP) and shortened positions (SP). Sixty young female rats WISTAR were distributed into six groups and followed for 7 days: control (C), electric stimulation (ES), immobilized in shortened (ISP), immobilized in lengthened (ILP), immobilized in shortened and electric stimulation (ISP+ES) and immobilized in lengthened and electric stimulation (ILP+ES). For the immobilization, the right hind limb was involved by a tubular mesh and cotton rolls and plaster. NMES was used in a frequency of 50 Hz, 10 minutes a day, totaling 20 contractions in each session. After 7 days the animals were killed and their gastrocnemius muscles of the right side were submitted to a mechanical test in traction in an universal test machine (EMIC®). From the curves load versus elongation the following mechanical properties were obtained: elongation in the yield limit (EPL), load in the yield limit (LPL), elongation in the ultimate load (EUL), ultimate load (UL) and stiffness. The immobilizations SP and LP promoted significant reductions (p<0,05) in the properties of EPL, LPL, EUL and UL being more accentuated mainly in group ISP. When used NMES, there was significant increment (p<0,05) of such properties only in group ISP. As for stiffness, significant reduction was observed (p<0,05) only of the group C for group ISP. When the NMES was used, the stiffness of group ILP+EE was significantly (p<0,05) higher and closer to group C than for group ISP+EE. We conclude that in this experimental model the immobilization of the muscles in the lengthened position delayed the reduction of the properties and the electric stimulation contributed to the maintenance of the mechanical properties during the immobilization period, mainly for group ILP+ES.

Spinal Muscular Atrophy (SMA) is a neurodegenerative, inherited disease caused by an insufficient amount of functional Survival of Motor Neurone protein (SMN), though the exact mechanism underlying this is not fully understood. The primary function of SMN is assembling a ring of Sm proteins around small nuclear RNA (snRNA) in an early, cytoplasmic stage of small nuclear ribonucleoprotein (snRNP) biogenesis, a process essential in eukaryotes. SMN, together with several mRNA binding proteins, has been linked to neural transport of mRNA towards areas of growth in Motor neurons for local translation of transcripts. Previous research in our group has found that this may involve Coatomer protein-containing vesicles transported by Dynein and requiring the Sm family protein, SmB, for maintenance. Little is known, however, about what other proteins are also present and required for correct transport and localisation of these vesicles. To further investigate this, we have produced plasmids expressing each Sm protein tagged to fluorescent proteins to help track their behaviour, in some cases for the first time, and developed a detergent-free fractionation protocol to enrich for SMN containing vesicles, providing tools that can be used to further probe behaviour and interactions in the future. Using these approaches, SmN, a neural specific Sm protein, was identified to also be present in SMN-containing vesicles similarly to SmB. Analysis of the interactomes of different Sm proteins identified a novel interactor of SMN, Neurochondrin (NCDN), that appears to be required for the correct localisation of SMN in neural cells. NCDN was found to not associate with snRNPs, indicating an snRNP-independent interaction with SMN. NCDN and SMN both independently associated and co-enriched with Rab5, indicating a potential endocytic and cell polarity role for the interaction. This interaction has the potential to be key in SMA pathology and may have therapeutic potential.

Objetivos: Comparar as alterações de espessura das camadas da retina macular adquiridas com tomografia de coerência óptica (TCO) e as amplitudes das ondas de eletrorretinograma multifocal (ERGmf) convencional e com estímulo lentificado entre olhos com hemianopsia temporal sequelar por compressão quiasmática e controles saudáveis, além de avaliar as correlações entre TCO, ERGmf e campo visual (CV) central. Métodos: Quarenta e três olhos (30 pacientes) com hemianopsia temporal sequelar por tumores hipofisários previamente submetidos a descompressão quiasmática e 37 olhos saudáveis (19 controles) foram submetidos a TCO-DS da mácula, ERGmf e CV tipo 10-2. Após segmentação, foram medidas as espessuras maculares da camada de fibras nervosas da retina (CFNR), camada de células ganglionares (CCG), camada plexiforme interna (CPI), camada nuclear interna (CNI), camada plexiforme externa (CPE), camada nuclear externa (CNE) e camada de fotorreceptores (CFR). Amplitudes e potenciais oscilatórios (PO) multifocais foram adquiridos com o ERGmf convencional e com estímulo lentificado, respectivamente, analisados em valores médios por quadrantes e hemicampos. Resultados: Espessuras da CFNR, CCG e CPI foram significantemente menores em todos os quadrantes, enquanto CNI, CPE e CFR tiveram medidas significantemente maiores nos quadrantes nasais nos pacientes comparados com os controles. Correlações significantes entre a TCO e os valores de CV 10-2 foram positivas para CFNR, CCG e CPI, e negativas para CNI, CPE e CFR. As amplitudes médias dos POs e de N1 do ERGmf se mostraram significantemente reduzidas na hemirretina nasal dos pacientes. Correlações significantes foram encontradas entre as amplitudes de PO e ERGmf para com medidas de TCO da hemirretina nasal interna e externa, respectivamente. Conclusões: Pacientes com hemianopsia temporal sequelar por compressão quiasmática previamente tratada apresentam adelgaçamento significante de CFNR, CCG e CPI e espessamento de CNI, CPE e CFR, associados com amplitudes reduzidas de PO e N1, sugerindo que o dano axonal à retina interna desencadeia lesão secundária das camadas externas da retina nesta condição / Purpose: To compare optical coherence tomography (OCT)-measured macular retinal layers in eyes with permanent temporal hemianopia from chiasmal compression and controls, compare regular and slow-flash multifocal electroretinography (mfERG) in patients and controls, and assess the correlation between OCT, mfERG and central visual field (VF) data. Methods: Forty-three eyes (30 patients) with permanent temporal hemianopia from pituitary tumors previously submitted to chiasm decompression and 37 healthy eyes (19 controls) were submitted to macular SD-OCT, mfERG and 10-2 VF testing. After segmentation, macular retinal nerve fiber layer (RNFL), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL) and photoreceptor layer plus (PRL+) thickness was measured. Amplitudes and oscillatory potentials (OP) were measured on regular and slow-flash mfERG, respectively, and expressed as mean values per quadrant and hemifield. Results: RNFL, GCL and IPL thickness measurements were significantly reduced in all quadrants while INL, OPL and PRL were significantly increased in the nasal quadrants in patients compared to controls. Significant correlations between OCT and 10-2 SAP measurements were positive for RNFL, GCL and IPL and negative for INL, OPL and PRL. OPs and mfERG N1 amplitudes were significantly reduced in the nasal hemiretina of patients. Significant correlations were found between OP and mfERG amplitudes for inner and outer nasal hemiretina OCT measurements, respectively. Conclusions: Patients with permanent temporal hemianopia from previously treated chiasmal compression displayed significant RNFL, GCL, IPL thinning and INL, OPL and PRL thickening associated with reduced OP and mfERG N1 amplitudes, suggesting axonal injury to the inner retina leads to secondary damage to the outer retina in this condition

OBJETIVO: avaliar a capacidade do eletrorretinograma de padrão reverso de campo total (PERG) e multifocal (PERGmf) de detectar alterações funcionais da retina em pacientes com afecções desmielinizantes e compressivas da via óptica anterior; estudar a correlação entre as amplitudes do PERG e PERGmf com as espessuras das camadas internas da retina obtidas pela tomografia de coerência óptica (TCO) e com a perda de campo visual avaliada pela perimetria computadorizada padrão (PCP). MÉTODOS: cento e cinquenta e dois olhos de 28 pacientes com esclerose múltipla (EM), 38 com espectro da neuromielite óptica (NMO) com ou sem neurite óptica (NO) prévia e 30 olhos de 26 controles normais foram submetidos ao PERG, à TCO para avaliação da camada de fibras nervosas da retina (CFNR) peripapilar e camadas internas da retina na mácula e à PCP. Os olhos estudados foram divididos em 4 grupos: grupo 1. EM com NO; grupo 2. EM sem NO; grupo 3. NMO; grupo 4. Mielite transversa longitudinal extensa. Os achados foram comparados utilizando-se as equações de estimativas generalizadas. As correlações entre os achados do PERG, da TCO e da PCP foram avaliadas pela correlação de Pearson ou Spearman e pela análise de regressão linear. Para avaliação dos pacientes com doenças compressivas da via óptica anterior, foram estudados 25 olhos de pacientes com defeito campimétrico temporal e atrofia em banda (AB) do nervo óptico decorrente de compressões quiasmáticas já tratadas e 25 olhos de 25 controles normais pelo PERGmf, a TCO e a PCP. As comparações entre olhos com AB e controles foram feitas usando análise de variância (ANOVA). As correlações entre achados do PERGmf, da TCO e da PCP foram avaliadas pela correlação de Pearson ou Spearman e pela análise de regressão linear. RESULTADOS: comparado com controles, as amplitudes do PERG foram significativamente reduzidas em olhos de pacientes com NMO e EM com NO, mas não mostraram diferenças significativas em olhos de pacientes com EM sem NO e mielite transversa longitudinal extensa. Correlações significantes foram encontradas entre os valores de amplitude de N95, a espessura da camada de células ganglionares da retina e a espessura da CFNR peripapilar. As reduções de amplitudes do PERG também foram significativamente associadas à perda de sensibilidade do campo visual. Na avaliação de pacientes com AB do nervo óptico, as amplitudes do PERGmf foram significativamente reduzidas nos quadrantes temporais quando comparados com controles. Não houve diferenças de amplitude nos setores nasais. Correlações significativas foram encontradas entre as amplitudes do PERGmf, a espessura macular total e a CFNR peripapilar no setor temporal e a perda de sensibilidade do campo visual. CONCLUSÕES: O PERG foi capaz de detectar perda funcional em pacientes com EM e NMO com NO prévia e suas amplitudes foram significativamente correlacionadas com a espessura das camadas internas da retina e camada de fibras nervosas peripapilar. As amplitudes do PERGmf foram capazes de demonstrar perda funcional em pacientes com AB do nervo óptico e apresentaram correlações significantes com a perda de sensibilidade do campo visual e as medidas de espessura macular total e da CFNR peripapilar / PURPOSE: To evaluate the ability of full-field and multifocal (mf) pattern electroretinogram (PERG) parameters to detect functional changes of the retina in patients with demyelinating and compressive diseases of the anterior visual pathway; to study the relationship between the PERG (full field and multifocal) amplitudes, optical coherence tomography (OCT)-measured inner retinal layers measurements and visual field sensitivity loss on standard automated perimetry (SAP). METHODS: one hundred fifty-two eyes of 28 patients with multiple sclerosis (MS), 38 with neuromyelitis optica (NMO) spectrum with or without previous episodes of optic neuritis (ON) and 30 eyes of 26 normal controls underwent PERG, OCT macular and peripapillary retinal nerve fiber layer measurements and SAP. The eyes of the patients were divided into 4 groups: group 1. EM with ON; group 2. EM without ON; group 3. NMO; group 4. Longitudinally extensive transverse myelitis (LETM). The findings were compared using the generalized estimating equations (GEE). The correlations between the findings of the PERG, OCT and SAP were assessed by Pearson correlation coefficients or Spearman\'s rank correlation coefficients and linear regression analysis. For evaluation of patients with compressive diseases of the anterior visual pathway, 25 eyes of patients with temporal visual field defect and band atrophy (BA) of the optic nerve due to chiasmal compression and 25 eyes of 25 normal controls were studied by mfPERG, OCT and SAP. Comparisons between BA and control eyes were made using variance analysis (ANOVA). The correlations between the findings mfPERG, OCT and SAP were evaluated by Pearson correlation coefficients or Spearman\'s rank correlation coefficients and linear regression analysis. RESULTS: compared to controls, PERG amplitudes were significantly reduced in eyes of patients with MS with ON and NMO, but not in eyes with MS without ON and longitudinal extensive transverse myelitis. Significant correlations were found between N95 amplitudes values, OCT-measured macular ganglion cell layer thickness, total retinal thickness, and temporal peripapillary RNFL thickness.The reduced mfPERG amplitudes were also significantly associated with loss of visual field sensitivity. In the evaluation of patients with BA of the optic nerve, mfPERG amplitudes were significantly reduced in the temporal quadrants when compared with controls. There were no differences in the amplitudes values in the nasal sectors. Significant correlations were found between mfPERG amplitudes, OCT-measured total macular thickness and peripapillary RNFL in the temporal sector and the loss of sensitivity of the visual field. CONCLUSIONS: full-field PERG measurements were able to detect functional loss in patients with MS and NMO with previous episodes of ON and PERG amplitudes were significantly correlated with inner retinal layers thicknesses and peripapillary RNFL. Multifocal PERG amplitudes were able to demonstrate functional loss in patients with BA of the optic nerve and showed significant correlations with visual field sensitivity loss and OCT-measured total macular and peripapillary RNFL thickness

A hipóxia-isquemia (HI) encefálica neonatal pode gerar sequelas neurológicas permanentes nos indivíduos que sobrevivem a este evento precoce. Dentre estas sequelas, o diagnóstico de Transtorno de déficit de atenção e hiperatividade (TDAH) já foi relacionado em pesquisa clínica. Sabendo que não há consenso de um modelo adequado para o estudo do TDAH em pesquisa experimental, novas abordagens que contribuam para o desenvolvimento desse modelo são necessárias. Assim, o objetivo do presente estudo foi investigar se a HI neonatal contribui para o desenvolvimento das características comportamentais relacionadas ao TDAH na fase adulta em ratos e correlacionar os resultados comportamentais com o volume da lesão encefálica. Para isso, ratos Wistar machos foram divididos em dois grupos: hipóxia-isquemia (HI, n=12) e controle (CT, n=10). O procedimento de HI consistiu na combinação da oclusão da artéria carótida comum direita no 7º dia pós-natal com exposição a uma atmosfera hipóxica (8% O2 e 92% N2, durante 90 minutos). Durante a fase adulta, ao atingir dois meses de idade, os animais foram testados no teste attentional set-shifting (ASS) para avaliar flexibilidade comportamental atencional e no teste de tolerância ao atraso da recompensa, para avaliação da escolha impulsiva. Os resultados mostraram que os animais submetidos à HI apresentaram prejuízo na função executiva, avaliado no ASS, evidenciado por uma inflexibilidade comportamental quando a regra para a execução da tarefa era mudada (p ≤ ,05 para o número de tentativas para passar dos estágios de Reversão 2 e Reversão 3, assim como o número de erros nesses estágios, além do estágio de mudança extradimensional – Teste t não-pareado). No teste de tolerância ao atraso da recompensa, não foi observada uma maior impulsividade dos animais HI, tendo os dois grupos um comportamento similar neste teste. Além disso, as avaliações do volume encefálico pelo Método de Cavalieri demonstraram uma atrofia no grupo HI no hemisfério total, córtex cerebral, substância branca, hipocampo e estriado, principalmente no lado ipsilateral à lesão (p ≤ ,05, Teste t não-pareado). Considerando esses resultados, podemos inferir que a HI neonatal é um fator ambiental que pode contribuir para o desenvolvimento das características comportamentais observadas no TDAH, e que estas são associadas a uma atrofia encefálica geral. / Neonatal hypoxic-ischemic encephalopathy (HI) can cause permanent neurological sequelae in survivors of this early event. Among these sequelae, the diagnosis of attention deficit hyperactivity disorder (ADHD) has already been linked in clinical research. There is no consensus about an ideal ADHD model in experimental research, being necessary new approaches that contribute to the development of this model. Thus, the aim of this study was to investigate whether HI contributes to the development of characteristics related to ADHD in adult rats and correlate the behavioral results with brain damage volume. Male Wistar rats were divided into two groups: hypoxia-ischemia (HI, n=12) and control (CT, n=10). The HI procedure consist of a permanent occlusion of the right common carotid artery followed by a period of hypoxia (90 min; 8% O2 and 92% N2), at seventh postnatal day (PND). Two months later, animals were evaluated in attentional set-shifting test (ASS) for assessment of attentional flexibility and in the tolerance to delay of reward, for evaluation of impulsivity choice. Our results demonstrated that animals submitted to HI manifest impairments in executive function, evidenced by a behavioral inflexibility when the rule for the execution of the ASS task was changed (p ≤ ,05 for number of trials to reach the criterion in Reversion 2 and 3 stages, as well as in number of erros in these stages, in addition to the Extradimensional shift stage – Unpaired t test). In the tolerance to delay of reward, no greater impulsivity of HI animals was observed, with both groups demonstrating similar behavior in this task. Moreover, the assessments of brain volume by Cavalieri method demonstrated atrophy in HI group in total hemisphere, cerebral cortex, white matter, hippocampus and striatum, especially on the side ipsilateral to the lesion (p ≤ ,05 – Unpaired t test). Considering these results, we can infer that neonatal HI is an environmental factor that could contribute to the development of behavioral characteristics observed in ADHD which are associated to general brain atrophy.

Le Syndrome de Wolfram (SW; OMIM #222300, prévalence 1-9 / 1000 000) est une maladie neurodégénérative, qui se présente avec un début juvénile, intégrant le diabète insipide, diabète sucré, l’atrophie optique (AO), et la surdité. AO est généralement son premier symptôme neurologique, commençant à l’âge de 11 ans et se terminant par la cécité 8 ans plus tard. Malheureusement, un modèle murin du SW approprié aux symptômes ophtalmologiques n'a pas encore été trouvé, donc la recherche de la thérapie pour sauver la vision en est à ces débuts. Dans cette thèse j’ai étudié l’atteinte visuelle de deux modèles de souris mutantes pour le SW et succès d’une approche de thérapie génique (TG) avec le gène humain WFS1.Premièrement, les souris Wfs1exon8del sont été examinées à 3 et 6 mois pour l’acuité visuelle (AV) et la sensibilité aux contrastes (SC) via changements dans le reflexe optomoteur (ROM), la fonction rétinienne neurale par électrorétinogramme (ERG), ainsi que la physiologie de l’œil par la fondoscopie et tomographie par cohérence optique (TCO). De plus, la proportion des cellules ganglionnaire de la rétine (CGRs) et la perte axonale dans le nerf optique (NO) à 7 mois ont été examinés avec marquage anti-Brn3a et microscopie électronique, respectivement. Il y avait une perte progressive de l’AV et la SC chez les souris KO à partir du 1 mois. Elle était accompagnée d'une pâleur du disque optique (DO), d'amincissement de la rétine ainsi que des lésions axonales. Par contre, il n’avait pas de perte des CGRs ni stress du réticulum endoplasmique dans la rétine. Brièvement, les souris KO présentent un phénotype ophtalmique du SW significatif et peuvent servir comme modèle.Deuxièmement, à la recherche d'un autre modèle du SW, les fonctions visuelles de la lignée Wfs1E864K de la souris ont été étudiées. Déjà à 1 mois, les souris Wfs1E864K/E864K avait une perte drastique de la fonction des CGRs, mais en gardant le nombre de cellules à un niveau normal. Ceci a été accompagné par un amincissement de la rétine et d’un sévère dommage du NO, comme montré par le TCO et la fondoscopie, respectivement. En conséquence, les souris Wfs1E864K/E864K, avec leur fort phénotype ophtalmique, pourraient servir comme modèle du SW classique.Enfin, pour enquêter sur les futures options de traitement contre le SW, les souris de la lignée Wfs1exon8del à 1 mois ont subi une TG intravitréenne avec AAV-2/2-CMV-WFS1. Les examens à 3 et 6 mois ont montré une amélioration de l’AV, ainsi que le sauvetage de la pâleur du DO et réduction des lésions axonales chez les souris KO. En outre, aucun effet indésirable lié à des injections TG n’ont été noté. Suivant cette idée, les souris Wfs1E864K/E864K ont également été soumis à la TG intravitréenne, délivrée à P14, mais sans succès.En conclusion, la lignée Wfs1exon8del de la souris est un modèle fiable du SW, y compris les aspects visuels. Je propose le modèle Wfs1E864K/E864K comme une alternative, en particulier pour enquêter sur la fonction de Wfs1 dans l'œil. Enfin, la GT intravitréenne avec WFS1 a un potentiel pour sauver partiellement le phénotype ophtalmique, ouvrant la voie vers le traitement pour les patients du SW / Wolfram Syndrome (WS; OMIM #222300, prevalence 1-9 / 1 000 000) has a juvenile onset and incorporates diabetes insipidus, diabetes mellitus, optic atrophy (OA), and deafness; leading to death in middle age. OA is its first neurological symptom, starting in adolescence and ending with blindness within 8 years. Unfortunately, a suitable WS mouse model comprising ophthalmologic symptoms has not yet been found, therefore the search for its treatment is delayed. In this thesis, I studied visual impairment in two WS mouse models along with a success of a gene therapy (GT) approach with the human WFS1 gene.Firstly, 3 and 6 months old Wfs1exon8del mice were examined for the visual acuity (VA) and contrast sensitivity via changes in the opto-motor reflex (OMR), the neural retinal function via electroretinogram (ERG), as well as the eye physiology via fundoscopy and optic coher-ence tomography (OCT). Also, the proportion of retinal ganglion cells (RGC) and the axonal loss at the age of 7 months were determined with anti-Brn3a immuno-labeling of retinal sections and electron microscopy of optic nerve (ON) sections, respectively. There was a progressive loss of VA and contrast sensitivity in Wfs1exon8del-/- mice, starting already at 1 month of age. It was accompanied by optic disc pallor, retinal thinning as well as axonal damage. However, there was no RGC loss and the endoplasmic reticulum (ER) stress in the retina was at a normal level. It suggested a presence of another cause for the reported degeneration in KO mice; in opposition to what was proposed in the literature. I brief, KO mice exhibit significant WS ophthalmic phenotype.Secondly, in search for another model, visual functions of Wfs1E864K mouse line were investigated. This line was originally a model of Wolfram-like Syndrome, characterized by dominant mutations in WFS1 leading to congenital progressive hearing impairment, diabetes mellitus and OA. Only homozygous mutants, however, showed expected visual impairment. Already at 1 month of age, Wfs1E864K/E864K mice had drastic loss of RGC function, albeit keeping the cell number at a normal level. This was accompanied by retinal thinning and a severe ON damage, as shown with OCT and fundoscopy, respectively. In contrast, the RGC function in Wfs1E864K/+ mice dropped slightly only at the age of 7 and 12 months, showing that the pathology of the E864K mutation-driven disease in mice is different than in humans. Therefore, Wfs1E864K/E864K mice, with their strong ophthalmic phenotype, could potentially serve as a model of the classical WS.Finally, to investigate future treatment options, 1 month old Wfs1exon8del+/+ (WT) and Wfs1exon8del-/- (KO) mice underwent a uni- and bi-lateral intravitreal gene therapy (GT) with AAV-2/2-CMV-WFS1. Exams at 3 and 6 months of age showed improved VA, as well as optic pallor and axonal damage rescue in KO mice. Also, no adverse effects related to either GT or sham injections were noted. Following this idea, the Wfs1E864K/E864K mice were also subjected to intravitreal GT, delivered at P14, but without success.In conclusion, Wfs1exon8del mouse line is a reliable model of WS, including the visual aspects. I propose the Wfs1E864K/E864K model as an alternative, especially to investigate Wfs1 function in the eye. Finally, the intravitreal AAV-driven GT with WFS1 has a potential to partially rescue the ophthalmic phenotype, paving the wave towards the treatment for WS patients.

Introdução. A insuficiência cardíaca (IC) é caracterizada por exacerbação da atividade nervosa simpática muscular (ANSM), baixa tolerância ao esforço e dispneia. Além disso, é característico nessa população o desequilíbrio entre o anabolismo e catabolismo, favorecendo dessa maneira uma acentuada perda da massa magra muscular, o que agrava ainda mais a qualidade de vida nos pacientes com IC. Dentre as alterações anabólicas observadas na IC avançada destaca-se a diminuição dos hormônios GH, IGF-1 e testosterona. A testosterona, um importante hormônio para as características masculinizantes e na manutenção da massa muscular, apresenta acentuada redução com o avançar da doença. Esta perda da massa magra, leva ao processo de caquexia muscular e consequente atrofia, com diminuição da força e da capacidade funcional do paciente com IC. A reposição de testosterona nesses pacientes tem sido estudada e se mostra uma importante terapêutica para melhorar a capacidade funcional e força muscular. Porém, não se conhece claramente o papel deste tratamento medicamentoso sobre o processo anabólico muscular, bem como na melhora da composição corporal. O exercício físico como tratamento não medicamentoso tem sido amplamente recomendado na IC por reduzir a ANSM, melhorar o fluxo sanguíneo periférico, aumentar a força muscular e melhorar a qualidade de vida. Entretanto, a combinação das estratégias do exercício físico associado à terapia de reposição de testosterona, não é conhecido em pacientes com IC. Métodos. 24 pacientes com IC foram randomizados em 3 grupos: Treinamento (TR, n=9), Testosterona (T, n=8) e Treino+Testosterona (TRT, n=7). A ANSM foi avaliada pela técnica de Microneurografia. O fluxo sanguíneo do antebraço foi avaliado pela pletismografia de oclusão venosa. A composição corporal foi avaliada pela densitometria (DEXA). A biópsia do músculo vasto-lateral foi feita para avaliarmos a área de secção transversa da fibra e a tipagem de fibras musculares. A qualidade de vida foi avaliada pelo questionário de Minnesota. O treinamento físico aeróbio em bicicleta foi realizado 3 vezes por semana, com 40 minutos de exercício por sessão, pelo período de 4 meses. A reposição de testosterona foi realizada pela administração intramuscular de undecilato de testosterona pelo período de 4 meses. Resultados. Após 4 meses de intervenção, observamos restauração dos níveis de testosterona em todos os grupos. A ANSM reduziu nos grupos TR e TRT. Não houve aumento do fluxo sanguíneo entre os grupos. O consumo de oxigênio aumentou em todos os grupos, porém apenas o grupo TRT aumentou a potência máxima ao exercício. A massa magra apresentou aumento significativo apenas no grupo TRT. Não observamos mudança no conteúdo mineral ósseo entre os grupos. Apenas o grupo TRT aumentou de maneira significativa a área de secção transversa das fibras tipo I (oxidativas). A qualidade de vida melhorou apenas nos grupos TR e TRT. Conclusões. O exercício físico associado à terapia de reposição de testosterona se mostrou mais eficaz em reduzir a ANSM, aumentar a capacidade funcional, a força muscular, a massa magra com um importante aumento das fibras do tipo I. Nossos resultados enfatizam a importância do exercício físico em pacientes com IC e traz uma nova perspectiva com a associação da testosterona para pacientes com hipogonadismo / Introduction. Heart failure (HF) is characterized by exacerbation of muscle sympathetic nerve activity (MSNA), exercise intolerance and dyspnea. Furthermore, is characteristic in this population the imbalance between anabolism and catabolism which lead to loss of skeletal muscle mass worsening quality of life in HF patients. Prior studies have demonstrated decrease in anabolic hormones such as GH, IGF-1 and testosterone. Testosterone, an important hormone for masculinization feature and maintenance of muscle mass, shows sharp decline in advanced HF. Loss muscle mass leads to cachexia and atrophy which decrease strength and functional capacity in HF patients. Testosterone replacement in these patients has been studied and shows an important therapeutic to enhance functional capacity and muscle strength. However it is not known the role of this medical treatment on muscle anabolic process as well as on body composition. Physical exercise as a non-medication treatment has been widely recommended to reduce MSNA, enhance peripheral blood flow, increase muscle strength and improve quality of life. However, the combination of the strategies of physical exercise associated with testosterone replacement therapy is not known in HF patients. Methods. 24 HF patients were randomized in 3 groups: Training (TR, n=9), Testosterone (T, n=8) and Training+Testosterone (TRT, n=7). MSNA was recorded by microneurography technic. Forearm blood flow was evaluated by venous occlusion plethysmography. Body composition was measured by densitometry (DEXA). Muscle biopsy was done in vastus lateralis to evaluate the cross-sectional area and type of fibers. Quality of life was assessed by Minnesota living with heart failure questionnaire. Aerobic exercise training on a bicycle was performed 3 times per week, with 40 minutes of exercise per session, for a period of 4 months. Testosterone replacement was performed by intramuscular administration of testosterone undecylate for a period of 4 months. Results. After 4 months testosterone levels were restored in all groups. MSNA decreased in TR and TRT groups. There was no increase in blood flow between groups. Oxygen consumption increased in all groups, but only the TRT group showed increase in maximum power to exercise. Lean body mass increased significantly only in the TRT group. We did not observe changes in bone mineral content between groups. Only TRT group significantly increased the cross-sectional area of type I fibers (oxidative). The quality of life improved only in TR and TRT groups. Conclusions. Exercise training associated with testosterone replacement therapy was more effective in reducing MSNA, increase functional capacity, muscle strength, lean mass with a significant increase in type I fibers. Our results emphasize the importance of physical exercise in patients with HF and bring a new perspective to association testosterone for patients with hypogonadism