Submitted by Alison Vanceto (alison-vanceto@hotmail.com) on 2017-02-07T10:35:47Z No. of bitstreams: 1 TeseCMP.pdf: 2337703 bytes, checksum: 27d55740b52010374ef6d09b5fb74efa (MD5) / Approved for entry into archive by Camila Passos (camilapassos@ufscar.br) on 2017-02-08T12:04:25Z (GMT) No. of bitstreams: 1 TeseCMP.pdf: 2337703 bytes, checksum: 27d55740b52010374ef6d09b5fb74efa (MD5) / Approved for entry into archive by Camila Passos (camilapassos@ufscar.br) on 2017-02-08T12:08:43Z (GMT) No. of bitstreams: 1 TeseCMP.pdf: 2337703 bytes, checksum: 27d55740b52010374ef6d09b5fb74efa (MD5) / Made available in DSpace on 2017-02-08T12:10:37Z (GMT). No. of bitstreams: 1 TeseCMP.pdf: 2337703 bytes, checksum: 27d55740b52010374ef6d09b5fb74efa (MD5) Previous issue date: 2016-02-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Peripheral nerve injury disrupts the normal functions of neurons and leads to rapid and progressive alterations in structural skeletal muscle, such as muscle atrophy and fibrosis, causing functional deficits. Electrical stimulation (ES) has been recommended to treat denervated muscles. The best parameters of ES to minimize muscle atrophy due to denervation is controversial. Furthermore, it is not clear if ES can, in fact, affect reinnervation of denervated muscles. Thus, this thesis has two main objectives: 1) to verify if ES, applied to denervated muscles by surface electrodes, can affect neuromuscular recovery after nerve crush injury in rats; and 2) to assess the impact of ES on fibrosis establishment in denervated muscles. Two manuscripts were produced and used the same experimental groups. Thirtyfive Wistar rats were divided into 5 groups: (1) Normal (N); (2) 7-day denervation (D7d); (3) 7-day denervation and ES (DES7d); (4) 15-day denervation (D15d); and (5) 15-day denervation and ES (DES15d). Tibialis anterior (TA) muscle denervation was induced by crushing the sciatic nerve. The ES protocol to stimulate TA muscles consisted of: 200 contractions per day divided into 4 consecutive series of 50 contractions, with 10-minute rests between each set. The following parameters were used: exponential monophasic pulse; width time: 2x chronaxie; amplitude: motor level; time On: 3s and Off: 6s. The sciatic functional index was calculated. Muscle excitability was assessed considering the rheobasis, chonaxie and accommodation. Morphometric analyses, such as the muscle fiber cross-sectional area and percentage of connective tissue proliferation were used to characterize muscle morphology. Molecular markers related to reinnervation (neural cell adhesion molecule, NCAM), neuromuscular junction organization and maintenance (MuSK, Dok-7 and nicotinic Acetylcholine Receptors (nAChR) subunits), muscle mass control (atrogin-1, MuRF1, myoD and myostatin), fibrosis (TGF-β and myostatin), extracellular matrix remodeling (metaloproteinases, MMPs) and inflammation (TWEAK/Fn14) were investigated by molecular biology techniques such as western-blot, qPCR or zymography. The main results showed ES impaired natural recovery of denervated muscles accentuating disability, muscle atrophy and fibrosis, as well as reducing muscle excitability. These morphofunctional and electrophysiological changes were related to different modulations of all molecular markers investigated in a timely manner. Overall, this thesis provides evidence that ES can delay the reinnervation process by modulating factors related to NMJ stability and organization, as well as induced disability and muscle atrophy, and decreased muscle excitability. In addition, ES applied to denervated muscles induced muscle fibrosis by modulating inflammatory pathways and also extracellular matrix production and remodeling. Warnings should be given to rehabilitation teams when recommending ES to treat denervated muscles. / A lesão nervosa periférica interrompe as funções normais dos neurônios e leva a alterações rápidas e progressivas no músculo esquelético, tais como a atrofia muscular e a fibrose, causando perdas funcionais. Para o tratamento dos músculos desnervados a estimulação elétrica (EE) tem sido utilizada. A escolha dos melhores parâmetros de EE para minimizar a atrofia muscular devido a desnervação, é controversa. Além disso, não está claro se a EE pode afetar, de fato, a reinervação de músculos desnervados. Assim, esta tese tem dois objetivos principais: 1) verificar se a EE, aplicada aos músculos desnervados com eletrodos de superfície, pode afetar a recuperação neuromuscular após axonotmese do nervo ciático em ratos; e 2) avaliar o impacto da EE no estabelecimento da fibrose do músculo desnervado. Dois manuscritos foram produzidos e contavam com os mesmos grupos experimentais. Trinta e cinco ratos Wistar foram divididos em 5 grupos: (1) Normal (N); (2) Desnervado 7 dias (D7d); (3) Desnervado e ES 7 (DES7d); (4) Desnervado 15 dias (D15d); e (5) Desnervado e ES 15 dias (DES15d). Foi realizada a axonotmese no nervo isquiático. O protocolo de EE do músculo tibial anterior consistiu em: 200 contrações por dia, divididas em 4 séries consecutivas de 50 contrações, com 10 minutos de descanso entre cada série. Foram utilizados os seguintes parâmetros: pulso monofásico exponencial; tempo: 2 vezes cronaxia; amplitude: nível motor; TON: 3s e TOFF: 6s. O índice funcional do ciático foi calculado. A excitabilidade muscular foi avaliada considerando a reobase, cronaxia e acomodação. Análises morfométricas, tais como área de secção transversa da fibra muscular e a porcentagem de proliferação do tecido conjuntivo foram utilizados para caracterizar a morfologia. Marcadores moleculares relacionados à reinervação como a N-CAM (molécula de adesão celular neural), organização e manutenção da junção neuromuscular (JNM) (MuSK, Dok-7 e receptores de acetilcolina), controle de massa muscular (atrogin-1, MuRF1, myoD e miostatina), fibrose (TGF-β e miostatina), remodelação da matriz extracelular (metaloproteinases) e, inflamação (TWEAK / Fn14) foram investigados por técnicas de biologia molecular como western-blot, qPCR ou zimografia. Os principais resultados mostraram que a EE provocou perda da recuperação natural dos músculos desnervados acentuando a perda funcional, a atrofia muscular e a fibrose, assim como, reduzindo a excitabilidade muscular. Estas alterações morfofuncionais e eletrofisiológicas foram relacionadas à diferentes modulações de todos os marcadores moleculares, no decorrer do tempo estudado. De modo geral, a presente tese forneceu provas de que a EE pode atrasar o processo de reinervação por fatores relacionados com a estabilidade e a organização da JNM, bem como a induzir à incapacidade e à atrofia muscular, com diminuição da excitabilidade muscular. Além disso, a EE aplicada aos músculos desnervados induziu fibrose através da modulação da via inflamatória e também pela produção e remodelamento da matriz extracelular. Cuidados devem ser tomadas pelas equipes de reabilitação ao utilizar a EE no tratamento de músculos desnervados.

Pressure-overload-induced cardiac hypertrophy represents one cause of the development of heart failure. The aim of this study is to characterize the influence of the TIR-domain-containing adapter-inducing interferon- (TRIF) during afterload-induced myocardial remodeling. After transaortic constriction (TAC), cardiac pressure overload leads to an early increase in MyD88- (Myeloid differentiation primary response gene 88) and TRIF-dependent cytokines. The maximum cytokine expression appeared within the first week and decreased to its control level within five weeks. While cardiomyocyte hypertrophy was comparable, the myocardial accumulation of the inflammatory cells was lower in TRIFmice. At d7, TRIF deficiency reduced transcription factors and TRIF-dependent cytokines. Through the modulation of the TGF-signaling pathway and anti-fibrotic microRNAs, TRIF was involved in the development of interstitial fibrosis. The absence of TRIF was associated with a decreased expression of proapoptotic proteins. In echocardiography and working heart analyses, TRIF deficiency slowed left-ventricular wall thickening, myocardial hypertrophy, and reduces the ejection fraction. In summary, TRIF is an important adapter protein for the release of inflammatory cytokines and the accumulation of inflammatory cells in the early stage of maladaptive cardiac remodeling. TRIF is involved in the development of cardiac fibrosis by modulating inflammatory and fibrotic signal transduction pathways.