Orientador: Maria Alice da Cruz-Hofling / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T19:32:33Z (GMT). No. of bitstreams: 1 Fontana_Karina_D.pdf: 9531755 bytes, checksum: f7da29bd456d891709484931aa26063a (MD5) Previous issue date: 2008 / Resumo: O uso abusivo de esteróides anabólico androgênicos para melhorar a performance e aparência física tem sido associado a sérios efeitos adversos, alguns dos quais fatais, entretanto o seu uso controlado terapeuticamente pode trazer benefícios. O presente trabalho teve por objetivo avaliar no músculo sóleo (SOL), tibial anterior (TA) e gastrocnêmio (GAS), no músculo cardíaco e nos tendões, o efeito do uso oral do esteróide anabólico androgênico mesterolona (M, 17 beta-hydroxy-1 alpha methyl-5 alpha-androstan-3-one, C20H32O2) em camundongos sedentários e submetidos a um intenso programa aeróbico de corrida em esteira, usando um modelo murino transgênico (CETP+/-LDLr-/+). Grupos experimentais de camundongos machos (8 semanas no início dos experimentos) sedentários e exercitados (6 semanas de corrida involuntária em esteira, média de 16 m/min, 50 min/dia, 5 dias/semana) foram tratados com mesterolona dissolvida em goma arábica (veículo), ou com o veículo (ambos na concentração de 2 µg/g corpóreo: dose supra-fisiológica) nas últimas 3 semanas. Os animais eram semanalmente pesados e a pressão arterial aferida através de coxim caudal com pletismógrafo e os seus músculos foram pesados no momento do sacrifício. Foram delineados os tipos de fibras musculares com base na reação de m-ATPase, bem como a ação miotrófica da mesterolona, do exercício ou de ambos combinados nos diferentes tipos de fibras musculares. Ao microscópio de luz e eletrônico de transmissão foram avaliados no SOL, o número de miofibras, a área seccional transversa das miofibras e das mitocôndrias, a densidade mitocondrial/área de miofibra, o número de capilares/miofibra, a presença de células satélites ativadas, de "split fibers" (fibras fendidas) e a morfologia dos fusos musculares, bem como possíveis alterações da ultraestrutura em geral. A expressão da isoformas de óxido nítrico sintase neuronal (NOS I) e endotelial (NOS III) foi investigada através de imunohistoquímica e immunoblotting. Como parte, do sistema músculo esquelético, foi avaliada a resposta do tendão de Aquiles em termos de síntese de colágeno, concentração de hidroxiprolina, diâmetro e área (relativa) das fibras colágenas e aspecto ultraestrutural dos fibroblastos tendinosos (tenócitos). Finalmente, a investigação estendeu-se ao músculo cardíaco e métodos quantitativos, morfológicos e bioquímicos foram empregados para a avaliação da plasticidade cardíaca e do perfil lipídico desse modelo murino transgênico, o qual foi geneticamente modificado para estar mais próximo ao perfil lipídico humano. · Os resultados mostraram que os músculos soleo, tibial anterior e gastrocnêmio respondem diferentemente ao tratamento com mesterolona, ao exercício aeróbico ou a ambos associados. Igualmente, o mesmo se dá em relação à fibra tipo I ou aos vários tipos de fibras II, ou seja, dependendo do músculo em que estão inseridas, há variabilidade na sua resposta, isto é, elas podem ser recrutadas, não serem recrutadas, ou serem recrutadas diferencialmente. O mesmo pode ser dito em relação à sofrerem ou não hipertrofia e em que proporção ela ocorre. Igualmente, a expressão das óxido nítrico sintases, NOS I e NOS III, sofre modulação diferente dependendo do tratamento e músculo analisado. A associação do tratamento com mesterolona ao exercício físico intenso pode gerar efeitos sinérgicos ou antagônicos. · Os resultados mostraram que a mesterolona, o exercício físico e a combinação de ambos foram capazes de aumentar o conteúdo de hidroxiprolina, o diâmetro e área das fibrilas colágenas (contidas em uma área teste) do tendão e ao microscópio eletrônico os fibroblastos mostram-se hipertrofiados, com muitos prolongamentos e grande desenvolvimento da maquinaria sintética. · Os resultados mostraram o indesejável remodelamento e dano cardíaco, a indução de perfil pró-aterogênico nos animais tratados com mesterolona, e que o exercício neutraliza alguns desses efeitos. / Abstract: The abuse of anabolic-androgenic steroids (AAS) to improve physical performance is associated with serious adverse effects sometimes fatal, however benefits can be gain by its therapeutic controlled use. The present study aimed to evaluate the effects of the use of the anabolic androgenic steroid mesterolone (M, 17 beta-hydroxy-1 alpha methyl-5 alpha-androstan-3-one, C20H32O2) in the soleus (SOL), tibialis anterior (TA) and gastrocnemius (GAS), in the Achilles tendon and in the cardiac muscle remodeling and lipemic profile of transgenic mice (CETP+/-LDLr-/+), sedentary or submitted to a treadmill running aerobic exercise program. Experimental groups of male mice (aged 8 weeks at the beginning of the experiments) sedentary or exercised (6 weeks involuntary treadmill running, average of 16 m/min, 50 min/day, 5 days/week) were treated with mesterolone dissolved in gum arabic (vehicle), or with the vehicle (both at 2 µg/g body weight administered orally - supra physiological dose) in the last three weeks. The animals were weighed every week and the muscles were weighed after sacrifice of the animals. The arterial blood pressure was measured by tail-cuff plethismographys weekly. The delineation of the fibers types was done in the SOL, TA and GAS muscles through the m- ATPase reaction. The myotrophic action of mesterolone, exercise, or both combined, were also determined in relation to fiber types. The number of myofibers, the crosssectional area of the myofibers, and mitochondria, the density of mitochondria/area of myofiber, the number of capillaries/myofiber, the presence of activated satellite cells, split fibers and the morphology of the muscle spindles and of muscle tissue in general were evaluated by light and electron microscopy. The expression of the neuronal (NOS I) and endothelial (NOS III) nitric oxide synthase isoforms was investigated through immunohistochemistry and immmunoblotting. As part of the musculoskeletal system, the Achilles tendon response was evaluated in regard to the collagen synthesis, quantification of hydroxyproline, collagen fibers diameter and area (relative) and ultrastructure of the tendon fibroblasts (tenocytes). Finally, the investigation focused on the cardiac muscle remodeling and quantitative, morphological and biochemical approaches were employed for evaluating the cardiac plasticity and the lipid profile from this transgenic murine model whose lipid profile was transgenically modified to be more close to the human lipid profile. · The results showed that the soleus, tibialis anterior and gastrocnemius muscles respond differentially to the mesterolone treatment, to the aerobic exercise or both in association. Seemingly, fiber type I and the several types of II fibers showed variability. Depending on the muscle to which they belong to they can be recruited, not be recruited or be recruited differentially. Likewise, different degrees of hypertrophy, or absence of hypertrophy can be displayed by a given fiber type depending on the muscle. Similarly, the expression of the nitric oxide synthases, NOS I and NOS III, can be modulated differentially depending on the treatment and muscle analyzed. The association of mesterolone treatment and intense physical exercise can generate synergic or antagonistic effects. · The results showed that mesterolone, physical exercise or both combined were able to increase the hydroxyproline content, the diameter and area of tendon collagen fibrils (contained in an area probe), and to show evidences of increased synthesis of proteins by the fibroblasts, which besides showed several long and slender processes in agreement with characteristics of hypertrophy and enhancement of the synthetic machinery. · The results showed that mesterolone alone induced a pro-atherogenic profile and a pathogenic cardiac hypertrophy. The exercise counteracted these effects and modified favorably both the lipoprotein profile and the cardiac remodeling. / Doutorado / Doutor em Farmacologia

Para avaliar a eficácia do laser de arsenito de gálio em acelerar a cicatrização tendínea em cavalos PSI de corrida com lesão no tendão flexor digital superficial, foram realizados dois experimentos, primeiro (E1) com 14 casos clínicos onde as lesões foram decorrentes do esporte em um dos membros torácicos, o segundo (E2) com cinco casos experimentais, onde as lesões foram induzidas com colagenase em ambos os membros torácicos. No E1, após a detecção da lesão no tendão flexor digital superficial por exame ultra-sonográfico, todos os animais receberam antiinflamatório não esteróide associado à dimetilsulfóxido por via intravascular, crioterapia no local da lesão, e caminharam ao passo duas vezes ao dia por 15 minutos durante os 30 dias de acompanhamento. Os membros tratados pertenceram a onze animais que receberam uma sessão diária de laser de arsenito de gálio na dose de 20 J/cm2, realizadas durante 10 dias consecutivos, após término do antiinflamatório e da crioterapia. Os membros controle pertenceram a três animais, os quais não foram tratados com laser. No E2, após identificação das lesões, foram escolhidos aleatoriamente um membro torácico controle e outro a ser tratado por laser em cada animal. Da mesma forma, estes animais foram mantidos a passo e receberam o mesmo protocolo de laserterapia que os membros tratados do E1. Todos os membros foram avaliados através de exames ultra-sonográficos, utilizando-se como parâmetros o paralelismo das fibras tendíneas em corte longitudinal; a ecogenicidade, a área do tendão, a área da lesão e a porcentagem de ocupação da lesão em corte transversal. No E1, os membros controle não apresentaram diferença significativa (P>0,05) nos parâmetros avaliados entre os dias 0 e 30. Nos membros tratados, a área do tendão também não apresentou diferença significativa (P>0,05) entre os dias analisados, porém houve diminuição muito significativa nos escores de ecogenicidade e paralelismo (P<0,001), assim como diminuição no tamanho da lesão (P<0,05) e na porcentagem de ocupação da lesão (P<0,05). No E2 também não houve diferença significativa entre os dias em todos os critérios avaliados nos membros controles (P>0,05). Nos membros tratados a ecogenicidade, o paralelismo e o tamanho do tendão, não sofreram alterações significativas (P>0,05) entre os dias avaliados, porém o tamanho da lesão (P<0,05) e a porcentagem de ocupação da lesão (P<0,01) apresentaram diminuição significativa. O laser de arsenito de gálio na dose de 20 J/cm2 mostrou-se eficaz em acelerar a reparação da lesão tendínea nos membros tratados em relação aos membros controle, tanto no grupo de casos clínicos quanto no grupo experimental, quando comparados aos 30 dias do aparecimento da lesão. Esses resultados sugerem a participação positiva do laser de arsenito de gálio nos resultados e a validação do mesmo no tratamento da tendinite do flexor digital superficial de cavalos Puro Sangue Inglês de corrida. / This study conducted two trials to evaluate the efficacy of Gallium Arsenide Laser in the speed of the healing process of superficial digital flexor tendon (SDFT) lesions in thoroughbred horses. One trial group (T1) comprises 14 horses with lesions, which resulted from the sport, in the SDFT in one of the front limbs. The other trial group (T2) was formed by five horses that had lesions induced in both front limbs by collagenase injection. In the T1, after the detection of the lesion in the SDFT by ultrasonography all horses were treated by intravenous injection, once a day, over five days, with an association of two AINS: phenylbutazone and dimethylsulfoxide. Cryotherapy was also applied on the affected tendon (three times a day over five days) and the horses were kept in stalls and allowed controlled exercise (hand-walked twice daily for 15 minutes) during the 30 days of the study. The treated limbs belong to 11 horses that received laser sessions once a day for ten days at a dosage of 20J/cm2. These sessions started after the AINS association and cryotherapy. The control limbs belong to the other three horses that received the same treatment, except by the laser sessions. In the T2, after the detection of the lesion, a limb from each horse was randomly chosen to be the control limb and the other limb was treated by laser. These horses were also kept in stall under controlled exercise and the treated limb was under the same laser protocol that T1. All horses were evaluated by two ultrasonographic exams with a 30-day interval. The parameters evaluated were: the fiber alignment, the echogenicity, the tendon area, the lesion area and the proportion of the cross sectional area involved. In the T1, the control limbs showed no significant difference (p>0,05) in the echogenicity, the fiber alignment, the tendon area, the lesion area or the proportion of the cross sectional area involved, between the day 0 and 30. The treated limb, didn´t show any significant difference of the tendon area, but showed a significant difference on the echogenicity (P<0,001), the fiber alignment (P<0,001), the lesion area (P<0,05) and the proportion of the cross sectional area involved (P<0,05). In the T2, only one limb received the laser treatment, the other limb acted as a control. The control limbs showed no significant difference (P>0,05) in the echogenicity, the fiber alignment, the tendon area, the lesion area and the proportion of the cross sectional area involved between the day 0 and 30. The treated limbs showed no significant difference (P>0,05) in the echogenicity, the fiber alignment and the tendon area, but showed significant difference in the lesion area (P<0,05) and the proportion of the cross sectional area involved (P<0,01). The treatment dose of 20J/cm2 of gallium arsenide laser was efficient in speeding the healing process of SDFT lesions of the laser treated limbs in both groups (T1 and T2), when compared with the control limbs, at 30 days of the onset of the lesion. The group which lesions occurred due to the sport (T1) had a better response to the treatment than the group which lesions were induced by collagenasis (T2). The limbs treated by laser showed a positive response which validates the use of the Gallium Arsenide laser for the treatment of tendinitis in the Superficial Digital Flexor in race horses.

A estimativa das forças musculares durante o movimento humano tem inúmeras aplicações na área da Biomecânica. Nesse trabalho descrevo a aplicação de métodos de estimativa das forças musculares a dois diferentes problemas. O primeiro consiste em se quantificar as cargas a que os músculos e as articulações do membro inferior de adultos jovens estão sujeitos durante o andar em ambiente aquático e terrestre. Já o segundo consiste na investigação do efeito do aumento da complacência do tendão de Aquiles (TAq; tendão do triceps sural) na dinâmica de produção de força dos músculos gastrocnêmio e sóleo (G e S respectivamente; ambos músculos do triceps sural), visando compreender se o aumento da complacência, observado com o envelhecimento, poderia contribuir para as alterações na biomecânica da marcha de idosos. A hipótese no primeiro estudo era de que as forças desenvolvidas pelos músculos do membro inferior durante o andar em ambiente aquático não seriam menores em relação ao ambiente terrestre. Especificamente, esperávamos observar um aumento nas forças geradas nos flexores e extensores de quadril e joelho. No segundo estudo, nossas hipóteses eram de que o aumento na complacência do TAq faria com que as fibras musculares operassem mais distantes do seu comprimento ótimo, aumentando os níveis de ativação e o consumo metabólico dos músculos G e S. Com o objetivo de verificar as hipóteses levantadas nos dois estudos propostos utilizamos o software OpenSim 3.1 e dados experimentais dos movimentos estudados, para realizar simulações do andar humano e estimar as forças nos músculos do membro inferior durante essa tarefa. Em ambos os estudos os dados experimentais foram adquiridos através de sistemas de análise do movimento humano compostos por câmeras, para filmar os movimentos corporais dos voluntários, e plataformas de força, para medir as forças de reação do solo. Os resultados obtidos no primeiro estudo confirmaram nossas hipóteses, uma vez que indicam que durante determinadas fases do ciclo da marcha as forças geradas pelos músculos flexores e extensores de joelho e quadril, tais como os músculos isquiotibiais e o gluteo máximo, podem exceder as forças geradas em ambiente terrestre. Esses resultados corroboram a idéia de que o andar em ambiente aquático pode ser efetivo no ganho de força muscular. As hipóteses para o segundo estudo também foram confirmadas. Adicionalmente, nossos resultados previram que, para o triceps sural gerar a potência necessária para manter o indivíduo andando com velocidade confortável, os tendões dos músculos G e S devem se movimentar de maneira independente. A diferença no movimento dos feixes do TAq é tanto maior quanto maior a sua complacência. Além disso, verificamos que há um mínimo no consumo metabólico do gastrocnêmio em um nível específico de complacência do TAq, enquanto para o sóleo o consumo aumenta sistematicamente com o aumento da complacência. Esses resultados indicam que um aumento na complacência do TAq pode comprometer o desempenho dos músculos do triceps sural e aumentar o seu consumo energético, contribuindo para as alterações na biomecânica da marcha de idosos. As informações fornecidas nos dois estudos aqui apresentados podem auxiliar profissionais de área da saúde no planejamento de programas de treinamento e reabilitação para adultos e idosos, tanto no ambiente terrestre quanto no ambiente aquático. / Muscle force estimation during human motion has numerous applications in Biomechanics. In this work, we describe the application of methods of muscle force estimation to solve for two different problems. The first problem is to quantify lower limb muscle and joint loads that young adults are subjected to when walking in the aquatic and terrestrial environment. The second problem is to understand the effect of increased Achilles tendon compliance (AT; the triceps surae tendon) in the gastrocnemius and soleus contractile dynamics (G and S respectively; both triceps surae muscles), aiming at understanding if increased AT compliance, that occurs with aging, could play a role in the elderly gait alterations. Our hypothesis for this first study was that the forces developed by the lower limb muscles in water are not always lower than on land. Specifically, we hypothesized that the forces developed by the hip and knee flexors and extensors would be grater in water than on land. For the second study our hypothesis was that the compliant AT would cause the fibers to operate far from its optimal length resulting in higher levels of activation in both G and S, as well as higher specific metabolic consumption. In order to verify our hypotheses for both studies we used the software OpenSim 3.1 together with experimental data of volunteers walking in aquatic and terrestrial environments to simulate human walking and to estimate the forces developed by the lower limb muscles during this task. In both studies, experimental data were acquired through human movement analysis systems composed of cameras, to record the movements of the volunteers\' body, and force plates, to measure ground contact forces. We confirmed our hypotheses to the first study since our results showed that in certain periods of the gait cycle the forces developed in the knee and hip flexors and extensors, such as the hamstrings and the gluteus maximus, inside water exceeded the forces in corresponding periods of land walking. Those results corroborate the idea that walking inside water may be effective in muscle strengthening. We also confirmed our hypotheses to the second study. Additionally, we predicted that to generate the necessary power to walk with comfortable speed the triceps surae G and S tendon must move independently. This differential behavior becomes larger the greater the AT compliance. In addition, we also observed that G metabolic energy consumption was minimized for a specific level of AT compliance while S systematically increased. Those results suggest that increased AT compliance can compromise the triceps surae performance and increase metabolic consumption, contributing to the alterations on the elderly gait biomechanics. The results of our two studies may assist health professionals to better plan training and rehabilitation programs for adults and elderly in both, terrestrial and aquatic environment.

A maioria dos casos de pé torto congênito tratados pelo método de Ponseti requer a secção do tendão calcâneo para correção do equino residual. Evidências clínicas sugerem que há completa cicatrização entre os cotos tendíneos, mas este processo reparativo ainda não foi suficientemente estudado. Esta investigação teve como objetivo avaliar o processo de reparação que ocorre após a secção percutânea do tendão calcâneo para a correção do equino residual no pé torto congênito idiopático tratado pelo método de Ponseti. Por meio de estudo prospectivo, foram analisadas 37 tenotomias em 26 pacientes com pé torto congênito idiopático tratados pelo método de Ponseti, com seguimento mínimo de um ano após a secção. A tenotomia foi realizada percutaneamente com agulha biselada de grosso calibre, sob sedação e anestesia local. O exame ultrassonográfico foi feito logo após a secção tendínea para assegurar que ela tenha sido completa e mensurar o afastamento entre os cotos. A reparação foi estudada por meio da ultrassonografia realizada três semanas, seis meses e um ano após a tenotomia. A ultrassonografia, realizada imediatamente após o procedimento, mostrou que, em alguns casos, feixes tendíneos residuais persistiam entre os cotos, mas foram completamente seccionados, em seguida, sob controle ultrassonográfico. Houve afastamento médio de 5,65 mm ± 2,26 (2,3 a 11,0 mm) entre os cotos tendíneos logo após a secção. Em um caso ocorreu sangramento maior que o habitual, que foi controlado com pressão local e não provocou interferência no tratamento. Após três semanas, a ultrassonografia mostrou regeneração tendínea com preenchimento do espaçamento entre os cotos por tecido hipoecoico com ecotextura irregular e com restituição da continuidade entre os cotos demonstrada dinamicamente pela transmissão de movimentos do músculo tríceps sural para o calcanhar. Seis meses após a tenotomia, o exame ultrassonográfico evidenciou que o tecido de reparação apresentava ecotextura de aspecto fibrilar e, quando comparado ao tendão normal, havia leve ou moderada hipoecogenicidade e espessamento cicatricial. Um ano após a tenotomia, o exame ultrassonográfico mostrou estrutura fibrilar na região de reparação, com ecogenicidade semelhante ao tendão normal, mas ainda apresentando espessamento tendíneo cicatricial. Em termos gerais, ocorreu rápida cicatrização após a secção percutânea do tendão calcâneo, que restabeleceu a continuidade entre os cotos. Ao final do período de observação, o tecido de reparação tendínea apresentou aspecto ultrassonográfico semelhante ao lado normal, exceto por leve espessamento, o que sugere um mecanismo de reparação predominantemente intrínseco. / Most cases of congenital clubfoot treated by the Ponseti technique require percutaneous Achilles tenotomy in order to correct the residual equinus. Clinical evidences suggest that complete healing occurs between the cut tendon stumps, but there have not yet been any detailed studies investigating this reparative process. This study was performed to assess the Achilles tendon repair after percutaneous sectioning to correct the residual equinus of clubfoot treated by the Ponseti method. A prospective study analyzed 37 tenotomies in 26 patients with clubfoot treated by the Ponseti technique, with a minimum follow-up of one year after the section. The tenotomy was performed percutaneously with a large-bore needle bevel with patient sedation and local anesthesia. Ultrasonographic scanning was performed after section to ascertain that the tenotomy had been completed and to measure the stump separation. In the follow-up period, the reparative process was followed ultrasonographically at three weeks, six months and one year post-tenotomy. The ultrasonography performed immediately after the procedure showed that in some cases, residual strands between the tendon ends persisted, and these were completely sectioned under ultrasound control. A mean retraction of 5.65 mm ± 2.26 (range, 2.3 to 11.0 mm) between tendon stumps after section was observed. Unusual bleeding occurred in one case and was controlled by digital pressure, with no interference with the final treatment. After three weeks, ultrasonography showed tendon repair with the tendon gap filled with irregular hypoechoic tissue, and also with transmission of muscle motion to the heel. Six months after tenotomy, there was structural filling with a fibrillar aspect, mild or moderate hypoechogenicity, and tendon scar thickening when compared to a normal tendon. One year after tenotomy, ultrasound showed a fibrillar structure and the echogenicity at the repair site that was similar to a normal tendon, but with persistent mild tendon scarring thickness. It was observed that there was a fast reparative process after Achilles tendon percutaneous sectioning that reestablished continuity between stumps. The reparative tissue evolved to tendon tissue with a normal ultrasonographic appearance except for mild thickening, suggesting a predominantly intrinsic repair mechanism

Este trabalho investiga a influência de polimorfismos na região promotora do gene das metaloproteases 1, 3 e 8 na fisiopatogenia da insuficiência primária do tendão tibial posterior. A amostra de 150 pacientes selecionados é dividida em grupo-teste: 50 pacientes com diagnóstico clínico e anatomopatológico de tendinopatia do tendão tibial posterior e grupo-controle: 100 pacientes com tendão tibial posterior íntegro. O DNA dos voluntários é obtido a partir de células epiteliais da mucosa bucal mediante a extração com acetato de amônia. As técnicas de PCR e RFLP são utilizadas para análise dos genótipos. A análise estatística dos resultados é realizada pelo teste do qui-quadrado com nível de significância de 5%. Os resultados mostram que os polimorfismos -1607 da MMP-1 e -799 da MMP-8 estão relacionados com risco maior para tendinopatia primária do tendão tibial posterior, enquanto o polimorfismo -1612 da MMP-3 parece não influenciar essa tendinopatia / The aim of this study was to investigate the influence of polymorphisms in the promoter region of the gene of metalloproteinases 1, 3 and 8 in physiopatology of primary posterior tibial tendon insufficiency. The sample of 150 selected patients was divided into test group: 50 patients undergoing surgical procedures and pathological diagnosis of degenerative lesions of the posterior tibial tendon, and control group: 100 patients with posterior tibial tendon intact and no signs of degeneration. The DNA of the volunteers was obtained from oral mucosa epithelial cells, by extraction with ammonium acetate. PCR and RFLP were used for analysis of genotypes. Statistical analysis of results was performed by Chi-squared test with significance level of 5%. The results show that polymorphisms -1607 of MMP-1 and -799 of the MMP-8 are associated with increased risk for primary tendinopathy of the posterior tibial tendon, while the -1612 polymorphism of MMP-3 does not influence this tendinopathy

Os tendões são tecidos de transição das forças contráteis geradas pelos músculos e as transferindo para os ossos, podendo assim gerar movimento. A região em que o tecido tendíneo se insere ao ósseo é chamada de junção osteotendínea ou entese. Estas junções podem ser classificadas como fibrosas ou fibrocartilaginosas, as fibrosas são aquelas em que os tendões e/ou ligamentos se inserem no eixo (diáfise) dos ossos longos e as fibrocartilaginosas a inserção ocorre nas epífises dos ossos longos ou nos ossos curtos. Este estudo tem como propósito analisar as fibras colágenas e as células presentes na junção osteotendínea, utilizando microscopia de luz com as colorações de Hematoxilina-eosina, Azocarmim, Picro-sírius e Tricromo de Masson e os aspectos ultraestruturais empregando os métodos de microscopia eletrônica de varredura e de transmissão. Foram utilizados 30 ratos Wistar machos, sendo 15 adultos com idade de 4 meses (grupo A) e 15 idosos com idade de 18 meses (grupo B), sendo utilizados 5 ratos de cada grupo para microscopia de luz, 5 para microscopia eletrônica de varredura e 5 para microscopia eletrônica de transmissão. Os membros posteriores dos ratos foram retirados, dissecados e desmineralizados com EDTA a 7% para microscopia de luz e EDTA 0,15M pH 7,4 para microscopia eletrônica de transmissão durante 4 semanas, não foi necessário desmineralizar as amostras para microscopia eletrônica de varredura. Cortes de 6 m foram realizados e montados em lâminas histológicas para análise em microscópio de luz, a técnica de criofratura em nitrogênio líquido foi realizada para análises em microscópio eletrônico de varredura e cortes de 60 nm foram realizados, montados em telas de cobre de 200 mesh (EMS) para análises em microscópio eletrônico de transmissão. O processo de envelhecimento mostrou alterações das fibrilas colágenas, em que o tipo I predomina no grupo adulto e o tipo III no idoso, diminuição na quantidade de células de fibrocartilagem, processos citoplasmáticos destas células curtos e em número reduzido e uma diminuída capacidade de síntese devido a aparelhos de Golgi menores, poucas cisternas de retículo endoplasmático granular e escassas mitocôndrias. / Tendons are transition tissues of contractile forces generated by muscles and transferring to bone, generating this way move. The region where tendon attach to bone is called bone-tendon junction or enthesis. These junctions can be classified as fibrous or fibrocartilaginous, the fibrous are those in which tendons/ligaments are attached in the shaft (diaphysis) of the long bones and fibrocartilaginous attachment occur in the epiphyses of the long bones or short bones. This study aims to analyze collagen fibers and the cells present in the bone-tendon junction, using light microscope with Hematoxylineosin, Azocarmine, Picrosirius red and Masson\'s trichrome staining and ultrastructural aspects using scanning electron microscope, transmission electron microscope methods. Thirty male Wistar rats were using, 15 adults rats at 4 months-old (A group) and 15 old rats at 18 months-old (B group), were used 5 rats of each group to light microscopy, 5 rats to scanning electron microscopy and 5 rats to transmission electron microscopy. The hind limbs of the rats were removed, dissected and demineralized using 7% EDTA solution to light microscopy and 0,15M pH 7,4 EDTA solution to transmission electron microscopy for 4 weeks, was not necessary demineralize the samples to scanning electron microscopy. Cut in slices 6 m thickness were made and mounted on histological slides to analyze in light microscope, freeze-fracture technique with liquid nitrogen was performed to analysis in scanning electron microscope and cuts in slices 60 nm thickness were made, mounted on copper grids (200-mesh - EMS) to analysis in transmission electron microscope. The aging process showed changes of the collagen fibrils, in which type I predominates on adult groups and the type III on the old group, decrease in the amount of the fibrocartilage cells, few and short cytoplasmic processes of this cells and a decreased synthesis capacity due a small Golgi apparatus, a few cisternae of rough endoplasmatic reticulum and few mitochondria.

Abstract Type I collagen is the main connective tissue protein in vertebrates. The cross-linking and correct organisation of the molecules is crucial for the proper function of the tissue. Traditionally collagen cross-linking has been studied using chemical cross-link analyses. However, this does not distinguish between the collagen types or the location of the cross-link within the molecule. The focus in this work was to study the carboxyterminal telopeptide domain of type I collagen for the differently cross-linked forms. An immunochemical approach was used and a new immunoassay, SP4, was developed for the detection of immaturely cross-linked peptide forms. The differently cross-linked structures were purified and characterised from human bone by using SP4 together with the earlier developed ICTP assay for trivalently cross-linked C-terminal telopeptide form. It was found that the majority of the trivalent cross-links in the C-terminal telopeptide were presently unknown structures, other than pyridinoline. A non-cross-linked form of C-terminal telopeptide of α1-chain of type I collagen was also discovered in bone. The epitope of the ICTP assay was characterised and found to reside in the phenylalanine rich region of the ICTP peptide. MMP-9, but not cathepsin K, mediated breakdown of the collagenous matrix was found to produce a peptide detectable by the ICTP assay. Healthy human Achilles tendon comprises mainly of type I collagen. In ruptured Achilles tendons, an increased type III collagen content was found. Since the synthesis of type III collagen was not increased, it is postulated that the type III collagen must have accumulated over a long period of time indicative of a long-lasting microtraumatic process in the tendon before the total rupture occurred. The ICTP content was increased and the ratio of SP4 to ICTP decreased in calcified stenotic aortic valves suggesting a change in the molecular organisation and cross-linking towards the type found in human bone. The total collagen content was dramatically decreased in the calcified valves. Both in the Achilles tendons and in the aortic valves, the ICTP content was found to decrease with age with a concomitant increase in the variants of the C-terminal telopeptide structures detectable with the SP4 assay, pointing to a change in the molecular organisation of the collagenous matrix in these tissues.

Tendon disorders are painful, disabling, and a major healthcare problem, with millions of people affected by tendon injuries each year. Current treatment strategies are inadequate and knowledge of the underlying mechanobiological mechanisms is required to develop novel therapies. Although the tissue–level properties of tendon are well–documented there remains a lack of understanding of the deformation mechanisms of this complex tissue. Therefore, the aim of this thesis is to characterize the microstructural deformation of tendon through biological imaging, mechanical testing, and computational modeling. Emphasis is placed on the structure and function of elastic fibers in tendon, whose role is poorly understood. First, histology, immunohistochemistry, and multiphoton microscopy are used to characterize the organization of elastic fibers in healthy and damaged tendon providing detailed microstructural information on their morphology and location for the first time. Elastic fibers are found to have a sparse distribution in the extracellular matrix, but are highly concentrated in the endotenon sheath and pericellular matrix. Moreover, damaged specimens are found to have a severely disrupted elastic fiber network. Elastic fibers likely contribute to fascicular deformation mechanisms and the micromechanical environment of tenocytes, which are expected to be disrupted in damaged tendon. Second, mechanical testing and enzyme treatments are used to analyze the mechanical contribution of elastic fibers to tendon. Elastase is found to significantly affect the mechanical properties of the tissue and remove the elastin component of both tendon and a control collagen–elastin biomaterial. However, elastase is also found to degrade non–elastin structural molecules that may contribute to tendon mechanics. The mechanical changes associated with the elastase treatment suggest that elastic fibers do not contribute to the elastic recoil of tendon as previously hypothesized. Third, multiphoton microscopy in combination with a novel microtensile testing machine is used to observe the deformation of collagen fibrils and tenocytes in tissue exposed to load. Tissue displacement is consistent with a helical arrangement of fibrils and nuclei experience significant elongation under physiological conditions. These results suggest that a helical arrangement of fibrils is responsible for the nonlinear stress–strain response of tendon and that nuclei are prime candidates for sensing mechanical forces in tendon. Finally, computation modeling and structural imaging are used to generate a microstructural finite element model of tendon. A helical model with embedded pericellular matrix is able to reproduce the stress–strain response and cell–level deformation of the tissue. The pericellular matrix is found to amplify mechanical forces exposed to cells, which is required to initiate mechanobiological stimulation of tenocytes under physiological conditions. Therefore, the structure and composition of the PCM during health and disease is expected to significantly affect mechanobiological mechanisms of tendon. The work presented in this thesis has used new experimental methods to provide novel insight into the structure, function, and deformation mechanisms of tendon. The techniques and concepts developed are widely applicable to the study of collagenous tissues in health and disease. In particular, observations regarding the pericellular matrix may lead to the development of new tissue–engineered and pharmacological strategies for the treatment of tendon disorders.

The diploma thesis deals with design of plan – curved footbridge over Berounka river near the town of Čivice. Three different options were developed. First option includes a single – beam prestressed integral construction with haunches. The second option includes suspension footbridge construction, where the bridge deck is suspended at one side. In the third option the main span is built from concrete segments. Those are suspended on a pair of steel arches. The access ramps are cast – in – place, and they are supported by round steel piers. This variant was finally chosen as the best one and it was further processed. The construction was analyzed with Scia Engineer 2015 software, considering the construction process. The footbridge was loaded with dead loads and live loads including traffic and thermal actions. The internal forces were calculated and the construction was assess to the serviceability and ultimate limit states. In terms of serviceability, stress in concrete sections and deformations were checked. In terms of the ultimate limit state, the ultimate load capacities were calculated. The steel arches and piers were checked for possibility of loss of stability by buckling. The loss of stability was also checked by the software. Finally the dynamic behaviour of the construction was assessed, especially the harmonic excitation response. Then the construction plan and the drawings were made. The drawings include overview drawings and detailed drawings.

<div>As the soft tissue that transmits muscular forces to the bony skeleton, tendons play a key role in the human musculoskeletal system and must adapt over time to repeated mechanical loads to maintain functionality. Resistance exercise is one of the primary stimuli for increases in tendon size and strength in healthy, young individuals, but similar benefits are not observed in healthy, aged tendon. This failure in the elderly to adapt, along with the fact that tendons inevitably decline in morphology and function with age, puts older individuals at an increased risk of poor tendon health, subsequent injury, and a compromised quality of life. Alternative strategies to preserve and strengthen aged tendon has gone largely unexplored, highlighting a critical need to determine an effective stimulus for tendon adaptations in aging populations</div><div>The purpose of this study was to determine if age impacts the delivery of orally-consumed amino acids (AA) to the peritendinous Achilles space. If so, this investigation could serve as the foundation for future studies to evaluate the efficacy of supplemental amino acids for inducing positive adaptations in tendon during exercise. Furthermore, an enzyme-linked immunosorbent assay (ELISA) was performed to quantitively measure procollagen, a precursor of collagen, in the samples to evaluate the impact on supplemental amino acids on collagen synthesis. </div><div>To assess amino acid delivery, a microdialysis fiber was inserted into the peritendinous space anterior to the Achilles tendon in healthy young (n = 7, 21-30 years) and elderly (n = 6, 60-75 years) men and women after a twelve-hour fast. After baseline collection, subjects consumed a non-caloric, noncaffeinated AA beverage (16.65 g). Microdialysis samples were collected every fifteen minutes for four hours and analyzed using reverse-phase high-performance liquid chromatography. </div><div>Amino acid delivery to the peritendinous space was not compromised with age, and the administration of amino acids upregulated procollagen synthesis significantly more in healthy, elderly subjects than in those that are healthy and young. Though preliminary, these findings provide a strong foundation for future studies assessing the impact of amino acid supplementation as novel impetus for tendon adaptations in the elderly. </div><div><br></div>