The thesis deals with the role of a family in particular life phases of a person suffering from the rare disease, spinal muscular atrophy. There are three main parts. The first one defines the term family, namely its system, features and functions, and explores the particularities of a family living with a handicapped member. The second part focuses on the spinal muscular atrophy, primarily on the development objectives of individual life phases of the people suffering from the disease. Thirdly, there is a section aiming at describing the role of the people taking care of the people challenged by spinal muscular atrophy from the early childhood to the middle adulthood. Furthermore, the diploma thesis points out the effort of these people to become independent and live their lives to the fullest with the help of care assistants, partners as well as start their families. For a better comprehension of the situation, there have been made semi-structured interviews with twelve families with a member of various age suffering from spinal muscular atrophy, inquiring not only the challenged persons, but also their parents and siblings.

published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

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

A síndrome da dor patelofemoral (SDPF) é o diagnóstico mais comum em populações fisicamente ativas. A SDPF está relacionada com o mau alinhamento dos membros inferiores durante tarefas de descarga de peso, causando maior estresse e dor na articulação patelofemoral. Esse mau alinhamento está relacionado com um aumento da inclinação ipsilateral do tronco, adução do quadril, abdução do joelho e maior grau de rotação interna da tíbia durante atividades dinâmicas, como agachamento unipodal, corrida, salto e subir e descer escadas. Fatores anatômicos e biomecânicos estão relacionados a alterações ao redor da articulação femoropatelar, como menor força de extensão do joelho, atraso na ativação do vasto medial em relação ao vasto lateral e atrofia do músculo quadríceps. Recentemente, alterações do quadril (fatores proximais), tornozelo e pé (fatores distais) têm sido propostas como fatores contribuintes da SDPF. No entanto, as evidências sobre ativação e alteração da morfologia muscular dos membros inferiores, principalmente nos fatores proximais e distais, são escassas. Esta tese teve como objetivo verificar as alterações neuromusculares dos membros inferiores e determinar se algum parâmetro neuromuscular explicava a cinemática durante tarefas unipodais. Após a apresentação dos motivos para realização deste estudo (Capítulo I), no Capítulo II objetivamos verificar as alterações neuromusculares (ativação muscular e morfologia muscular) relacionadas aos fatores proximais e distais na SDPF por meio de uma revisão sistemática. As buscas foram realizadas nas bases de dados Medline (via PubMed), Scielo, Scopus, PEDro, Cochrane Central, Embase e ScienceDirect databases até abril de 2018 para estudos avaliando ativação muscular ou parâmetros de morfologia muscular das articulações do tronco, quadril e tornozelo/pé. Dois revisores independentes avaliaram cada trabalho para inclusão e qualidade. Dezenove estudos foram identificados (SDPF, n = 319; GC, n = 329). Três estudos investigaram os músculos ao redor das articulações do tronco e tornozelo/pé. Quinze estudos investigaram os músculos ao redor da articulação do quadril. As evidências foram inconclusivas sobre a ativação do transverso do abdome/oblíquo interno (TrA/OI) na SDPF durante atividades de alta velocidade. Os níveis de ativação, duração e atraso na ativação de Glúteo Médio (GMed), glúteo máximo (GMax), biceps femoral (BF) and semitendinoso (ST) foram inconclusivos nos estudos incluídos. Não foram observadas diferenças na ativação de gastrocnêmio lateral (GL), gastrocnêmio medial (GM), sóleo (SOL), tibial anterior (TA) e fibular longo (FIB). Apenas um estudo incluído avaliou parâmetros da morfologia muscular, sem alterações na espessura muscular e na intensidade do eco GMed e GMax. Com base na falta de evidências sobre alterações na ativação muscular em torno das articulações do quadril, tornozelo e pé durante tarefas dinâmicas, e no fato de que um único estudo avaliou os resultados da morfologia muscular (GMed e GMax) na SDPF, propusemos um artigo original (Capítulo III) que teve como objetivo comparar os parâmetros neuromusculares dos membros inferiores e a cinemática no plano frontal durante tarefas unipodais de descarga de peso em mulheres com SDPF e determinar se algum resultado neuromuscular explicava o índice dinâmico de valgo (IVD) durante as tarefas. As buscas foram realizadas nas bases de dados Medline (via PubMed), Scielo, Scopus, PEDro, Cochrane Central, Embase e ScienceDirect databases até abril de 2018 para estudos avaliando ativação muscular ou parâmetros de morfologia muscular das articulações do tronco, quadril e tornozelo/pé. Dois revisores independentes avaliaram cada trabalho para inclusão e qualidade. Dezenove estudos foram identificados (SDPF, n = 319; GC, n = 329). Três estudos investigaram os músculos ao redor das articulações do tronco e tornozelo/pé. Quinze estudos investigaram os músculos ao redor da articulação do quadril. As evidências foram inconclusivas sobre a ativação do transverso do abdome/oblíquo interno (TrA/OI) na SDPF durante atividades de alta velocidade. Os níveis de ativação, duração e atraso na ativação de Glúteo Médio (GMed), glúteo máximo (GMax), biceps femoral (BF) and semitendinoso (ST) foram inconclusivos nos estudos incluídos. Não foram observadas diferenças na ativação de gastrocnêmio lateral (GL), gastrocnêmio medial (GM), sóleo (SOL), tibial anterior (TA) e fibular longo (FIB). Apenas um estudo incluído avaliou parâmetros da morfologia muscular, sem alterações na espessura muscular e na intensidade do eco GMed e GMax. Com base na falta de evidências sobre alterações na ativação muscular em torno das articulações do quadril, tornozelo e pé durante tarefas dinâmicas, e no fato de que um único estudo avaliou os resultados da morfologia muscular (GMed e GMax) na SDPF, propusemos um artigo original (Capítulo III) que teve como objetivo comparar os parâmetros neuromusculares dos membros inferiores e a cinemática no plano frontal durante tarefas unipodais de descarga de peso em mulheres com SDPF e determinar se algum resultado neuromuscular explicava o índice dinâmico de valgo (IVD) durante as tarefas. Quinze mulheres com SDPF e quinze mulheres saudáveis pareadas por 5 idade (grupo controle - GC) foram comparadas com os seguintes testes: (1) questionário funcional; (2) espessura muscular ao redor do quadril (GMed e tensor da fáscia lata - TFL), joelho (VL e VM) e tornozelo/pé (TA e FIB); (3) IVD e ativação muscular durante agachamento e salto vertical unipodais; (4) torque isométrico máximo para abdução do quadril, extensão do joelho e eversão/ inversão do pé; e (5) ativação muscular durante testes isométricos e funcionais. Uma regressão linear múltipla (modelo Stepwise) foi usada para verificar se alguma variável neuromuscular explicava o IVD durante as tarefas unipodais. O tamanho de efeito (ES) foi usado para determiner a magnitude da diferença entre os grupos. Comparado ao GC, o grupo SDPF apresentou: (1) menor espessura do GMed (-10.02%; ES = -0.82) e maior espessura do TFL (+18.44%; ES = +0.92) e do FIB (+14.23%; ES = +0.87); (2) menor ativação do TA durante o agachamento unipodal (-59,38%; ES = -1.29); (3) menor ativação do GMed durante o salto vertical unipodal (-28.70%; ES = -1.35) e (4) maior ativação do GMed durante o teste isométrico de abdução de quadril (+34.40%; ES = +0.77). IVD durante o agachamento unipodal foi explicado pela ativação do VL durante a tarefa somente no GC, enquanto a espessura do TA no GC e o torque de eversores do pé no SDPF explicou o IVD durante o salto vertical unipodal. Com base em nossos resultados, as mulheres com SDPF apresentaram alterações neuromusculares significativas nas articulações do quadril e tornozelo/pé. No entanto, apenas fatores distais explicaram o IVD no grupo SDPF. / Patellofemoral pain syndrome (PFPS) is the most common diagnoses in physically active populations. PFPS is related with lower limbs poor alignment during weight-bearing tasks, causing higher patellofemoral joint stress and pain. This poor alignment is related with an increase of ipsilateral trunk lean, hip adduction, knee abduction and greater tibial internal rotation during dynamic activities such as single-leg squat, running, jumping, and stepping tasks. Anatomical and biomechanical factors are related with unwanted changes around the patellofemoral joint, such as lower knee extension strength, delayed onset of vastus medialis activation relative to vastus lateralis and quadriceps muscle atrophy (knee joint muscles intrinsic changes). Recently, hip (proximal), ankle and foot (distal) changes have been proposed as PFPS contributing factors. However, the evidences about lower limb muscle activation and morphology changes, mainly in proximal and distal factors, are scarce. This thesis aimed to create clinical subgroups based in lower limb neuromuscular changes and determine if some neuromuscular outcome explained kinematics during single-leg tasks. After displaying the reasons to perform this study (Chapter I), in Chapter II we aimed to verify neuromuscular changes (muscle activation and muscle morphology) related to proximal and distal factors in PFPS through a systematic review. Medline (via PubMed), Scielo, Scopus, PEDro, Cochrane Central, Embase and ScienceDirect databases were searched until April 2018 only for retrospective studies evaluating muscle activation or muscle morphology parameters of trunk, hip and ankle/foot joints. Two independent reviewers assessed each paper for inclusion and quality. Twenty retrospective studies were identified (PFPS, n=319; CG, n=329). Three studies investigated muscles around trunk and ankle/foot joints. Fifteen studies investigated muscles around the hip joint. Evidences were inconclusive about transversus abdominis/internal oblique (TrA/IO) activation in PFPS during high-speed activities. Gluteus medius (GMed), gluteus maximus (GMax), bíceps femoris (BF) and semitendinous (ST) activation level, activation duration and activation onset were inconclusive in the included studies. No differences were observed in gastrocnemius lateralis (GL), gastrocnemius medialis (GM), soleus (SOL), tibialis anterior (TA) and fibularis (FIB) muscle activation. Only one included study evaluated muscle morphology parameters, without changes in the GMed and GMax muscle thickness and echo intensity. Based in the lack of evidences about muscle activation changes in PFPS patients’ muscles around hip, ankle and foot joints during dynamic tasks, and in the fact that a single study evaluated muscle morphology outcomes (GMed), we proposed an original article (Chapter III) that aimed to compare lower limb neuromuscular parameters and frontal plane kinematics during single-leg tasks in women with PFPS, and determine if some neuromuscular outcome explained dynamic valgus index (DVI) during tasks. Fifteen PFPS women and fifteen healthy age-matched women (control group - CG) were compared with the following tests: (1) functional questionnaire; (2) hip (GMed and tensor fasciae latae - TFL), knee (VL and VM) and ankle/foot (TA and FIB) muscle thickness; (3) DVI and muscle activation during single-leg squat and vertical jump; (4) maximal isometric torque for hip abduction, knee extension and foot eversion/inversion; and (5) muscle activation during isometric and functional tests. A multiple- 7 stepwise regression analysis was used to test if neuromuscular outcomes explained DVI during single-leg tasks. Effect sizes (ES) were used to determine the magnitude of between-groups differences. Compared to the CG, PFPS showed: (1) smaller GMed (-10.02%; ES = -0.82) and greater TFL (+18.44%; ES = +0.92) and FIB muscle thickness (+14.23%; ES = +0.87); (2) lower TA muscle activation during single-leg squat (-59,38%; ES = -1.29); (3) lower GMed muscle activation during single-leg jump (-28.70%; ES = -1.35) and (4) greater GMed muscle activation during hip abduction isometric test (+34.40%; ES = +0.77). DVI during single-leg squat was explained by VL activation during this task only in CG, whereas lower TA muscle thickness in the CG and higher foot eversion torque in PFPS explained DVI during single-leg vertical jump. Based in our results, females with PFPS showed significant neuromuscular changes at the hip and ankle/foot joints. However, only distal factors explained DVI in the PFPS group.

Selective neuronal loss in response to loss or dysfunction of a ubiquitously expressed protein is a hallmark of neurodegenerative disease. Proximal spinal muscular atrophy (SMA) is caused by homozygous loss of the ubiquitously expressed survival motor neuron 1 (SMN1) gene, resulting in progressive neuromuscular weakness that eventually leads to flaccid paralysis and death from respiratory failure by two years of age in the most severely affected patients. Despite widespread motor neuron loss, certain motor pools are clinically spared. Type I SMA patients exhibit intercostal recession in conjunction with diaphragmatic sparing that produces a characteristic “bell-shaped chest.” Additionally, patients retain extraocular and external sphincter function, even in late disease stages. In order to fully define this differential vulnerability, I performed an extensive characterization of neuromuscular autopsies from Type I SMA patients and age-matched control patients. I found highly divergent degrees of motor unit degeneration, even within individual cranial nerves or a select anatomical region such as the neck. Remarkably, the diaphragm in a Type I SMA patient kept alive on life support for 17 years was still relatively preserved, despite virtually complete fibro-fatty infiltration in other muscles. Extraocular functions were also normal in this patient. These findings suggest that the molecular determinants of SMA-resistance provide indefinite protection against low SMN protein. Thus, identification and modulation of these genes and pathways represents a promising potential therapeutic strategy. Remarkably, this exquisite pattern of selectivity was preserved in the SMNΔ7 mouse, a widely used SMA mouse model. This suggests that the molecular determinants of differential vulnerability are conserved between mouse and human. Given the high degree of diversity between motor pools, I performed a comparative transcriptional microarray between multiple SMA-vulnerable and –resistant motor pools in healthy mice. This analysis revealed a small number of candidate therapeutic genes that segregate closely with vulnerability. I present a series of preliminary studies evaluating these targets in the SMNΔ7 mouse. Ongoing and future studies combine pharmacological, viral, and genetic approaches to modulate these candidate targets in the SMNΔ7 mouse and assess for improvements in neuromuscular pathology. Given the remarkable preservation of select motor pools in SMA patients, changing expression levels of the candidate targets I have identified may provide substantial clinical benefit.

Neuronal circuit perturbations are emerging as important determinants in the pathogenesis of neurodegenerative diseases, including Alzheimer’s disease, Huntington’s disease, and spinal muscular atrophy (SMA). SMA is a motor neuron disease caused by deficiency in the ubiquitously expressed survival motor neuron (SMN) protein. The hallmarks of SMA include loss of motor neurons, muscle atrophy, and abnormal postural reflexes. Although cell-autonomous mechanisms of motor neuron death have received much attention, recent studies in animal models of SMA revealed that motor circuit deficits resulting from early impairment of synaptic function and sensory-motor connectivity precede motor neuron death. It remains to be established whether motor circuit dysfunction is a consequence of SMN-deficiency in the motor neuron or SMN-dependent alterations in the activity of premotor neurons. Here I sought to address these outstanding issues through the development and characterization of a simplified in vitro model of the motor circuit based on the use of embryonic stem cell-derived motor neurons and interneurons. I found that SMN deficiency caused death of motor neurons in co-culture with other neurons as well as in isolation, demonstrating the cell autonomous origin of this defect. SMN requirement for motor neuron function was investigated using intracellular patch clamp recordings to measure both passive and active membrane properties. Remarkably, SMN deficiency induced hyperexcitability of motor neurons only when they are cultured in the presence of interneurons but not in isolation, providing initial evidence that SMN deficiency induces motor neuron hyperexcitability in a non-cell autonomous manner and that dysfunction and death of motor neurons are uncoupled. To determine the role of SMN-dependent interneuron dysfunction on motor neuron hyperexcitability, the effect of selective SMN depletion in either motor neurons or interneurons was investigated. Importantly, I found that SMN-deficient motor neurons cultured in the presence of wild type interneurons are not hyperexcitable, while the presence of SMN-deficient interneurons is necessary and sufficient to elicit hyperexcitability of wild type motor neurons. Therefore, in the context of SMN deficiency, increased excitability of motor neurons is a homeostatic response to interneuron dysfunction. Although the exact mechanism is currently unknown, reduced glutamatergic drive appears to play a role since glutamatergic receptor blockers phenocopied SMN deficiency in inducing motor neuron hyperexcitability but not neuronal death. Moreover, SMN deficiency caused reduction of excitatory VGluT2 synapses on motor neurons. In addition to changes in intrinsic membrane properties, SMN deficiency caused severe reduction in the spontaneous activity and firing pattern of motor neurons. However, in contrast to death and hyperexcitability, SMN-dependent deficits in both motor neurons and interneurons appear to underlie this complex phenotype. The findings presented in this study validate the use of in vitro models to study SMA disease mechanisms and shed new light on the cellular basis of motor circuit dysfunction induced by SMN deficiency that can have predictive value in vivo.

Neste trabalho, avaliamos os efeitos da suplementação crônica de leucina e do treinamento de força sobre a miopatia diabética. 40 ratos machos da linhagem Wistar Hannover foram distribuídos em 5 grupos: controle, não diabético (C), diabético não tratado (D), diabético treinado (DT), diabético suplementado com leucina e treinado (DLT). O início das intervenções ocorreu na 4ª semana de vida dos animais, e perdurou por 8 semanas. Foram avaliados: massa corporal, consumo de ração e água, concentrações sanguíneas de glicose, insulina e perfil lipídico; capacidade funcional muscular voluntária através de testes de força de preensão e de ambulação; conteúdo intracelular de proteínas relacionadas à via anabólica mTOR e p70S6K, totais e fosforiladas, no músculo extensor longo dos dedos. Os ratos diabéticos não tratados (grupo D) apresentaram hiperglicemia e hipoinsulinemia moderada, menor massa corporal, maior consumo de ração e água, menor peso absoluto dos músculos extensor longo dos dedos e gastrocnêmio, menor força de preensão, menor capacidade de ambulação e menor atividade das proteínas mTOR e p70S6K comparado ao grupo C, o que caracteriza o quadro de miopatia diabética. O peso relativo do músculo gastrocnêmico (peso absoluto/100g de peso do animal) foi maior nos grupos DT e DLT comparado com o grupo D, e maior no grupo DLT comparado com o grupo DL (p < 0,05). Não houve diferença estatística entre os grupos DL e D sobre os pesos relativos dos músculos, ou seja, a suplementação crônica de leucina não afetou este parâmetro nos ratos diabéticos. Interessantemente, houve diferença estatística entre os grupos DL e D sobre a força muscular (p < 0,05), sem haver diferença entre grupos DL e C quanto à glicemia; ou seja, a dieta suplementada com leucina foi capaz de controlar a glicemia e atenuar a perda de força muscular. O treinamento de força também controlou a glicemia, recuperou a força muscular e melhorou a capacidade de ambulação, bem como a regulação da via mTOR-p70S6K. A fosforilação da via mTOR-p70S6K foi maior nos grupos DT e DLT comparado com o grupo D (p < 0,05), e sem diferença entre estes grupos treinados e o grupo C, sugerindo que o treinamento de força combinado com a suplementação de leucina recuperou a atividade da via do mTOR-p70S6K nos animais diabéticos, que pode refletir em maior síntese proteica muscular. O colesterol total do grupo D foi maior comparado com o do grupo C; e nos grupos diabéticos treinados (DT e DLT), este parâmetro foi menor do que no grupo D (p < 0,05). Adicionalmente, o HDL-c aumentou nos grupos treinados (DT e DLT) quando comparado com o grupo D, mas não alterou no grupo que recebeu apenas a suplementação de leucina (grupo DL). Portanto, neste estudo, a suplementação crônica de leucina por si só normalizou a glicemia e melhorou a força muscular dos animais diabéticos. Além disso, o treinamento de força foi responsável pelo maior aumento de força e da massa muscular, bem como pela normalização da glicemia, pela elevação da concentração de HDL-c e pela redução do colesterol total dos animais diabéticos e ambas foram capaz de recuperar a via mTOR-p70S6K. / In this study, we evaluated the effects of chronic supplementation with leucine and resistance training on diabetic myopathy. 40 Wistar Hannover rats were divided into 5 groups: control, non-diabetic (C), untreated diabetic (D), trained diabetic (DT), diabetic supplemented with leucine and trained (DLT). The beginning of the interventions occurred in the 4th week of life of the animals, and lasted for 8 weeks. Were evaluated: body weight, food and water intake, blood concentrations of glucose, insulin and lipid profile; voluntary muscle functional capacity through grip strength and ambulation test; intracellular content of proteins related to the anabolic mTOR and p70S6K pathway, total and phosphorylated in the extensor digitorum longus muscle. Diabetics untreated mice (group D) had hyperglycemia and moderate hypoinsulinemia, lower body mass, food and water intake, reduced absolute weights of the muscles of the long extensor digitorum, and gastrocnemius, the lower grip strength, lower ambulation capacity and lower activity of mTOR and p70S6K protein compared the C group, featuring diabetic myopathy. The relative weight of the gastrocnemius muscle (absolute weight / 100g of body weight) was greater in DT and DLT groups compared with group D, and higher in the DLT group compared to the DL group (P < 0.05). No statistical difference between the DL and D groups on the relative weights of the muscles, that is, chronic supplementation of leucine did not affect this parameter in diabetic rats. Interestingly, there was statistical difference between the DL and D groups on muscle strength (P < 0.05), with no difference between groups DL and C on the blood glucose; that is, the diet supplemented with leucine was able to control glycemia and avoid loss of muscle strength of diabetic animals. Resistance training also controlled glycemia, recovered muscle strength and improved the capacity of ambulation of diabetic animals and the regulation of the mTOR-p70S6K pathway. The phosphorylation of mTOR-p70S6K pathway was higher only in the DT and DLT groups compared with the D group (P < 0.05), and no difference between the DT and C groups, suggesting that the training recovered muscle mass in diabetic animals. Total cholesterol was greater in Group D compared to the group C; and trained diabetic groups (DLT and DT), this parameter was lower than that of the D group (P < 0.05). In addition, HDL-C increased in trained groups (DT and DLT) as compared to group D, but had no effect the group that received only leucine supplementation (DL group). Therefore, in this study, chronic supplementation of leucine alone normalized glucose and improved muscle strength of diabetic animals. In addition, resistance training was responsible for the largest increase in strength and muscle mass, as well as the normalization of glucose, elevated concentrations of HDL-C and reduction in total cholesterol of animals diabetics and both were able to recover mTOR- p70S6K pathway.

Spinal muscular atrophy is an autosomal recessive genetic disease that results from the loss and/or degeneration of alpha motor neurons in the lower part of the spinal cord. With ~ 1 in 6000 live births per year being affected, this disease is the second leading cause of infant death and is caused by the loss or decrease of the Survival of Motor Neuron protein (SMN). While a lot is known about the role that SMN plays in the cytoplasmic assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs), it remains a crucial question in the field to gain a better understanding of what specific/distinct function(s) SMN might have in motor neurons. We have identified novel interactions between SMN and two RNA-binding proteins (RBPs) known to be components of axonal RNA granules. More specifically, we demonstrated that SMN interacts with HuD and SERBP1 in a direct fashion in foci-like structures along neurites of motor neuron-derived cells. We have also demonstrated that the SMN/HuD interaction is required for the localization of HuD into RNA granules in neurites of motor neuron-derived cells. Furthermore, I have shown that SERBP1 is down-regulated in the absence of normal levels of SMN and, most importantly, that over-expression of SERBP1 can rescue SMA-like neuronal defects using a cell culture model of the disease. These findings may help shed light on the non-canonical molecular pathway(s) involving SMN and RBPs in motor neurons and underscores the possible therapeutic benefits of targeting these RBPs in the treatment of SMA.

Muscle wasting is a significant complication of many diseases including diabetes mellitus, renal and liver failure, HIV/AIDS, and cancer. Sustained loss of skeletal muscle can severely impair a patient's quality of life and often results in poor tolerance and responsiveness to disease treatments. The increased protein breakdown observed during muscle atrophy has been attributed to accelerated activity of the ubiquitin-proteasome pathway, but the precise mechanisms by which this activation stimulates muscle protein loss are poorly understood. Previous work showed that the deubiquitinating enzyme USP19 is upregulated in rat skeletal muscle in various forms of muscle wasting, including streptozotocin induced diabetes, cancer, and dexamethasone treatment. 1 To further explore the role of USP19 in muscle wasting, siRNA-mediated depletion of the enzyme was carried out in L6 myotubes. Knockdown of USP19 resulted in more rapid differentiation of myoblasts into myotubes, with a greater extent of myoblast fusion. It also produced tubes that were visibly larger than those formed by myoblasts transfected with a control siRNA. At the molecular level, silencing of USP19 increased the amount of myosin heavy chain (MHC) and tropomyosin proteins. It also increased levels of MHC transcript, suggesting that USP19 acts at the level of gene transcription or mRNA stability rather than protein degradation. USP19 may mediate its effects on muscle-specific gene expression through the myogenic transcription factor myogenin, since depletion of USP19 increased protein and mRNA levels myogenin but did not affect protein levels of the related transcription factor Myf5. Moreover, the increased tropomyosin and MHC observed upon USP19 knockdown could be abolished when myogenin was simultaneously depleted using siRNA. Collectively, these results suggest that USP19 functions to inhibit the synthesis of key muscle proteins and may therefore be a promising target for the treatment of muscle atrophy.

Knee function was evaluated by knee score, activity level, clinical findings and performance tests, muscle size by computerized tomography (CT), morphology by light (LM) and electron microscopy (EM), muscle function by electromyography (EMG) and isokinetic performance in 29 patients with chronic anterior cruciate ligament (ACL) tear. Preoperatively CT disclosed a significant mean atrophy of the quadriceps and nonsignificant changes of the other muscle areas of the injured leg. Morphology of m vastus lateralis of the injured leg was normal in more than half of the biopsies preoperatively, the rest showed signs of nonoptimal activation. Significant decreases in all isokinetic parameters were noticed together with significantly decreased EMG of the quadriceps muscle of the injured leg. Âfter surgical reconstruction the knees were immobilized in a cast for 6 weeks at either 30° or 70° of knee flexion. After cast removal CT showed significant decreases of all areas which also remained after training. The 30° group showed larger fibres (intracellular oedema) and more frequent morphological abnormalities than the 70° group. Fourteen weeks postoperatively the patients were allocated to either a combination of isometric and progressive resistance training or isokinetic training for 6 weeks. CT showed slightly larger areas at 20 weeks postoperatively than at 6 weeks. Morphological abnormalities were still prominent at 20 weeks postoperatively. Maximum isokinetic knee extensor mechanical output and endurance were markedly decreased at 14 weeks postoperatively but both improved progressively during the one year rehabilitation, mostly during the intensive 6 week training period but irrespective of training programme used. Fatiguability/endurance level improved over the preoperative level. Muscular work/integrated EMG was stable while EMG/t increased indicating neuromuscular relearning. The clinical result at 28 months foliowup was excellent or good in 93% of the patients and clinical stability improved in 66%. Independent upon primary knee immobilization angle or training programmes no differences could be demonstrated with respect to stability, range of motion, function or isokinetic mechanical output. Isokinetic performance was still significantly lower in the injured compared to the noninjured leg and not significantly different from the preoperative values. Morphology, only 6 cases, showed abnormalities similar to preoperative findings. In conclusion, the reason for the decreased maximum and total knee extensor performance in these patients with ACL tears is suggested to be nonoptimal activation of normal functioning muscle fibres depending on changes in knee joint receptor afferent inflow. No differences concerning the markedly improved postoperative clinical result could be seen between the different treatment modalities used. A nonoptimal muscular activation might explain the still decreased isokinetic performance present at followup. / <p>S. 1-40: sammanfattning, s. 43-137: 5 uppsatser</p> / digitalisering@umu.se