Dermatomiosite é doença idiopática inflamatória crônica que afeta musculatura estriada, pele e outros órgãos. Este trabalho tem o objetivo de caracterizar os pacientes com a doença encontrados no HC-FMUSP no período de janeiro de 1992 a dezembro de 2002, avaliando a classificação, as manifestações cutâneas e sistêmicas, achados laboratoriais, terapêuticos e evolutivos em relação à literatura. Este estudo foi baseado nos dados obtidos dos prontuários de 109 pacientes com critérios diagnósticos definidos por Bohan e Peter e modificados por Drake. Os pacientes foram divididos em cinco grupos: 23 dermatomiosites juvenis, 59 dermatomiosites primárias idiopáticas, 6 dermatomiosites amiopáticas, 7 dermatomiosites associada a neoplasias e 14 dermatomiosites associadas a outras doenças do tecido conectivo. Sessenta pacientes foram caracterizados como diagnóstico definido, 33 como possíveis, 4 como prováveis e 12 como amiopáticos. A maior representação foi do sexo feminino (85/109) e a idade média do diagnóstico da doença foi de 36 anos. Manifestações cutâneas foram observadas em todos os pacientes; em relação às alterações sistêmicas a manifestação muscular mais freqüente foi a perda de força proximal (88%); manifestação pulmonar mais comum foi a pneumopatia intersticial (16,5%) e a manifestação digestiva mais observada foi a gastrite (20,2%). Documentaram-se neoplasias durante o seguimento da doença em 6,42% dos casos, sendo mais freqüente nos pacientes acima de 60 anos (71,4%).A enzima muscular alterada na maioria dos casos foi a desidrogenase lática (78,6%). Realizou-se biópsia cutânea em 68 pacientes com alterações compatíveis ao exame anatomopatológico em 78% dos casos. Dentre os casos, 53 pacientes se submeteram à biópsia muscular e 96% deles apresentaram miosite ao exame anatomopatológico. Das 58 eletroneuromiografias efetuadas, mostrou-se padrão miopático compatível com a enfermidade em 81% dos casos. A terapia mais utilizada foram os esteróides e a mortalidade foi de 14,7%, sendo as causas de óbito mais freqüentes a neoplasia maligna, septisemia e infecção pulmonar. / Dermatomyositis is a chronic idiopathic inflammatory disorder which affects the striated skeletal muscles, the skin, and other organs. This present study aims at characterizing the patients who have been affected by this disease and were under treatment at HCFMUSP from January, 1992 to December, 2002 Its classification, the cutaneous and systemical manifestations, as well as laboratorial, therapeutical and evolutive findings were checked out in accordance with the literature. The base for this study was the data obtained from the analysis of 109 patients´records who met not only Bohan\'s and Peter\'s criteria for dermatomyositis diagnosis but also the ones modified by Drake. The patients were divided into five groups: 23 juvenile dermatomyositis, 59 idiopatic primary dermatomyositis, 6 amyopatic dermatomyositis, 7 dermatomyositis associated to malignancy and 14 dermatomyositis associated to connective tissue disorder Sixty of these patients were classified as definite diagnostic, 33 as possible, 4 as probable and 12 as amyopathic. Most patients were female (85/109) and the average age for the diagnosis of the illness was 36. Skin manifestations were observed in all patients, while regarding the systemical alterations, the most frequent was the loss of proximal strength (88%). As the lung manifestation, the most common was an interstitial pneumopathy (16,5%) and as the digestive manifestation, the one which was more commonly observed was gastritis (20,2%). Neoplasies were documented after the following up of the disease in 6,42% of the cases, which was more frequent in patients over 60 years of age (71,4%). The muscle enzyme modified in the majority of the cases was the lactic dehydrogenize (78,6%). Skin biopsy procedure was carried out in 68 patients who showed compatible changes to anatomo-pathological exam in 78% of the cases; 53 patients went under muscular biopsy and 96 % of them proved to have myositis after anatomo-pathological exams. From the fifty-eight electroneuromiographies that were carried out, 81% of the cases showed to have compatible myopathic patterns. Steroidal drugs were the most common therapy used. The mortality rate was 14,7% and the most usual causes of death were malignant neoplasm, sepsis and pulmonary infection.

A ordem Cetacea compreende golfinhos e baleias, mamíferos totalmente adaptados ao ambiente aquático. A língua de duas espécies de cetáceos, Pontoporia blainvillei e Sotalia fluviatilis, foram utilizadas para descrição da morfologia das papilas linguais e da superfície de interface epitélio-tecido conjuntivo da lâmina própria, empregando as técnicas de microscopia de luz e eletrônica de varredura. As línguas foram fixadas e divididas em três porções: rostral, médio e caudal e processadas conforme os protocolos das técnicas utilizadas. A língua da Pontoporia blainvillei e Sotalia fluviatilis estava revestida por um epitélio estratificado queratinizado, sendo possível observar as camadas basal, espinhosa, granulosa e córnea. Pode-se notar que na língua, o tecido conjuntivo denso apresentava vasos sanguíneos e profundamente relacionado com o tecido muscular. A superfície de interface epitélio-tecido conjuntivo da lâmina própria apresentou papilas conjuntivas de várias formas. Elas correspondem às papilas filiformes e valadas, mostrando feixes de fibras colágenas em disposição original. Na microscopia de luz polarizada revelou-se fibras colágenas do tipo I e III. No terço médio e caudal de ambas as espécies foram encontradas ácinos de glândulas salivares. As amostras tratadas com solução de hidróxido de sódio, tiveram a camada epitelial removida, e as papilas conjuntivas foram observadas em aspecto tridimensional. Foi possível observar que a superfície dorsal de ambas as espécies é totalmente recoberta por papilas conjuntivas de aspecto filiforme. Observa-se também a presença dos forames dos ductos das glândulas salivares nas porções média e caudal da língua, sendo eles mais numerosos nos filhotes. Morfometricamente, as papilas conjuntivas apresentaram maior comprimento na espécie Sotalia fluviatilis do que na Pontoporia blainville. Foi observado somente na espécie Pontoporia blainvillei estruturas conjuntivas arredondadas na região média e caudal. Embora as áreas dessas estruturas tenham sido maiores na região caudal que na média, estatisticamente estes resultados não foram significativos (P>0,05). / The Cetacea order comprises dolphins and whales, mammals totally adapted to the aquatic ambient. The tongue of two cetaceans species, Pontoporia blainvillei and Sotalia fluviatilis, was studied for description of the morphology of the lingual papillae and of interface epithelium the connective tissue surface, using the light and scanning electron microscopic techniques. The tongues were fixed and divided in three portions: rostral, medium and caudal and treated according to the protocols of techniques used. The tongues of the Pontoporia blainvillei and Sotalia fluviatilis were covered by keratinized stratified epithelium, being that is possible to verify the basal, spinosa, granular and cornetiel ayers. In the tongue the dense connective tissue of lamina propria showed blood vessels and muscular layer was deeply related. The interface epithelium-connective tissue surface of the lamina propria presented connective tissue papillae which are various in shape. They correspond to the filiform and vallate papillae showing the collagen fibers bunches in original disposition. In the polarized light microscopy revealed collagen fibers type I and III. In the medium and caudal third of both species various groupins of salivary gland acini. The samples treated with sodium hydroxide solution, the epithelial layer was removed and the connective tissue papillae were observed in three-dimensional SEM images. It was possible to observe that the dorsal surface of the both species is totally covered by connective tissue papillae of filiform aspect. We observed also the presence of the ducts of the salivary glands in the media and caudal portions of the tongue, being them more numerous in pups. Morphometric analysis showed that the connective tissue papillae presented larger length in the Sotalia fluviatilis than in the Pontoporia blainville samples. The round connectives structures in the media and caudal portions were observed only the Pontoporia blainvillei species. Although, the areas of those structures have been major in the caudal region than media, and these results were not signifficant (P>0,05).

Le système musculo-squelettique permet la mobilité. Le développement des muscles, du tissu conjonctif (TC) et des os est coordonné de manière très précise. Osr1 encode pour un facteur de transcription qui est exprimé au niveau du TC musculaire au cours du développement. Le but de cette thèse est d'élucider la fonction d’Osr1 dans la régulation cellulaire non-autonome de la formation des muscles au niveau des membres dans le modèle murin. Le traçage génétique a révélé que les cellules Osr1+ sont à l’origine de plusieurs TC, y compris musculaire, cutané et pulmonaire, mais aussi à l’origine du muscle lisse et des adipocytes bruns. L’analyse phénotypique des embryons de souris Osr1GCE/GCE à E13.5 a révélé des défauts dans l’organisation des muscles. L’analyse transcriptomique montre deux caractéristiques moléculaires causées par le manque d'activité d’Osr1. Tout d'abord, Osr1 réprime l'expression de gènes associés au développement du cartilage et du tendon, ce qui suggère qu’Osr1 confère une identité «tissu conjonctif musculaire». Ensuite, Osr1 régule positivement l'expression des composants de la matrice extracellulaire (MEC). De plus, l’expression de nombreuses molécules de signalisation est diminuée dans les cellules déficientes pour Osr1. Ces résultats montrent l’importance des cellules Osr1+ du TC dans la formation des muscles des membres. Ces résultats montrent également qu’Osr1 régule la transcription des composants de la MEC au niveau du tissu conjonctif musculaire. Enfin, ils suggèrent qu’Osr1 exerce sa fonction par l'intermédiaire de facteurs sécrétés pour assurer le bon développement musculaire. / The musculoskeletal system allows body motion. Despite the distinct mesodermal origins of its components, the development of muscle, connective tissue (CT) and bone is highly coordinated. Osr1 encodes a zinc-finger transcription factor expressed in muscle CT in limbs. The aim of the PhD was to elucidate Osr1 function in the non-cell autonomous regulation of mouse limb muscle formation. Genetic lineage tracing revealed that Osr1+ cells are progenitors for several CTs, including muscle, dermal and lung CTs, but also for smooth muscle and brown adipocytes. Comprehensive phenotypic analysis of skeletal muscles in E13.5 Osr1GCE/GCE mouse embryos revealed impaired muscle formation. Transcriptomic analysis highlighted two major molecular characteristics caused by the lack of Osr1 activity. First, Osr1 actively repressed the expression of genes associated with cartilage and tendon development, suggesting that Osr1 confers a muscle connective tissue identity. Second, Osr1 positively regulated the expression of components of the extracellular matrix (ECM). In addition to the decrease of ECM components, numerous signaling molecules were significantly down-regulated in Osr1-deficient cells of mutant embryos. This highlights the function of Osr1+ resident connective tissue cells in limb muscle formation. It also establishes that Osr1 regulates the transcription of ECM components in limb muscle CT. Lastly, it suggests that Osr1 exerts its function via chemokines and secreted factors to ensure proper muscle development.

This thesis presents work on investigations into the mechanical properties of connective tissue. A model system of hydrogels was used to investigate how volume change through water flow is coupled to relaxation. This was done using digital image correlation (DIC) and a custom built setup. It was found, in hydrogels, that water loss is directly coupled to an increase in tension and water intake is directly coupled to tension relaxation. The experimental setup was tested by investigating the mechanical properties of the well known material polydimethylsiloxane (PDMS) and the novel materials of carbon nanotube (CNT) elastomers, cholesteric liquid crystal elastomers (CLCEs), and 3D polydomain liquid crystal elastomers (3DLCEs). The setup accurately demonstrated the incompressibility of PDMS, even at short time scales, and demonstrated how DIC can map the inhomogeneity of material by locating clusters of CNTs in CNT elastomers by how they deform. Novel results for 3DLCEs were also found, where it was discovered that there is a softening of the bulk modulus at small time scales resulting in a volume increase following deformation, the bulk modulus then recovers and there is over all no volume change. This is in stark contrast to the typical case, where it is the shear modulus that becomes comparable to the bulk modulus, resulting in increased volume. A theoretical investigation was carried out into critical damping in viscoelastic oscillators, where the aim was to apply to the findings to connective tissue. The fractional Maxwell model and zener model where both solved for, where it was found that damping decreases as the material becomes more solid and the peak of critical damping becomes broader. Finally, investigations into how strain relates to the viscoelastic properties of connective tissue were carried out on horse tendon and rat fascia. How relaxation changes was determined through the relaxation constant, where a large constant means it takes the sample longer to relax and it is more solid like. It was found, that in general, the relaxation constant increases quickly with an imposed strain and then either stabilises or increases more slowly. This growth of relaxation constant also occurs during the initial stages of tissue injury, where irreversible deformation occurs.

A seleção de linhagens de frangos de corte com altas taxas de crescimento e aumento de rendimento muscular vem contribuindo para o surgimento de perdas significativas na indústria avícola em decorrência de modificações anatômicas. Dentre estas alterações, destaca-se uma miopatia que se caracteriza pelo aparecimento de graus de estrias esbranquiçadas no músculo Pectoralis major, as quais seguem a direção da fibra muscular, referida como white striping. A etiologia da white striping ainda é desconhecida, mas avaliações histológicas demonstraram que esta alteração é usualmente associada à degeneração muscular e alterações miopáticas. Problemas de qualidade da carne podem ser resultantes de mudanças estruturais, morfométricas e bioquímicas do tecido muscular, com consequente alteração nas fibras musculares. Para tanto este trabalho, conduzido através de dois experimentos, teve como objetivos caracterizar histopatologicamente a miopatia white striping em diferentes graus de severidade (NORM, MOD, SEV) no músculo Pectoralis major de frangos de corte e as características morfométricas das fibras musculares, bem como avaliar a incidência desta condição em frangos de corte de linhagem Cobb abatidos aos 42 dias de idade. No primeiro experimento, a avaliação histopatológica demonstrou uma miopatia degenerativa nos graus de severidade classificados como MOD e SEV, entretanto as amostras de grau MOD apresentaram necrose ou necrose multifocal moderada, enquanto as de grau SEV apresentaram necrose difusa e acentuada, sugerindo avanço do processo degenerativo. Não foi observada proliferação de tecido conjuntivo em nenhum dos graus de white striping analisados, caracterizando este caso como de ocorrência aguda. No segundo experimento, foram verificados menor área de tecido muscular, menor diâmentro e maior densidade de fibras nos músculos Pectoralis major de frangos acometidos com white striping (MOD e SEV) quando comparado aos peitos sem esta condição (NORM). Conforme os resultados obtidos nesta pequisa, pode-se concluir que as fibras com os graus MOD e SEV de white striping demonstraram o início de um processo de regeneração muscular, de característica hiperplásica. / Genetic selection of broiler chickens for growth rate and increase of muscle yield has contributed to the appearance of significant losses in the poultry industry caused by anatomical changes. Among these alterations stands out a myophathy characterized by the occurrence of white striations on Pectoralis major muscle, seen parallel to the direction of muscle fibers, referred as white striping. The etiology of white striping is still unknown but histological changes demonstrated that this alteration is usually associated with muscle degeneration and myopatic changes. Meat quality problems may result from structural, morphological and biochemical changes of muscle tissue with consequent alterations in muscle fibers. To this study, two experiments were conducted, and aimed to histopathologically characterize the myopathy white striping at varying degrees of severity (NORM, MOD, SEV) on broilers Pectoralis major muscle and the morphometric muscle fibers characteristics as well as evaluate the incidence of this condition on Cobb 500 broiler breast fillets slaughtered at 42 days of age. The histopathological evaluation on experiment 1 showed a degenerative myopathy in both MOD and SEV degrees, however the samples classified as MOD showed microscopically necrosis or necrosis multifocal moderate, while the classified as SEV showed necrosis diffuse and strong suggesting an advancement in the degenerative process. The proliferation of connective tissue was not observed for either of the degrees of white striping investigated, characterizing this profile as an acute occurrence. In experiment 2, small area and diameter and higher densities of muscle fiber were observed in broilers Pectoralis major muscle with white striping (MOD and SEV) compared to NORM. According to the results, we can conclude that muscle fibers with white striping showed the beginning of a muscle regeneration process with hyperplasic characteristics.

La perception de la viande bovine par le consommateur est un enjeu crucial pour l’industrie de la viande. La tendreté reste l’une des qualités organoleptiques essentielle aux yeux du consommateur, avec la jutosité et la flaveur. L’objectif de ma thèse a été d’étudier les propriétés structurales et biochimiques du tissu conjonctif intramusculaire responsables de la dureté de base et de faire le lien avec la tendreté et plus largement avec les qualités organoleptiques de la viande. Le développement et la mise au point d’une méthode d’analyse d’images a permis d’étudier objectivement les caractéristiques des composantes périmysiale et endomysiale du tissu conjonctif intramusculaire et de montrer que la surface occupée par ces deux structures influençait la tendreté de la viande. Préalablement à l’étude biochimique et moléculaire du tissu conjonctif intramusculaire le dosage des protéoglycanes totaux a été développé et mis en place au laboratoire. Les résultats de cette deuxième partie ont permis de mettre en avant la complémentarité entre protéoglycanes, collagène total et liaisons de réticulation au sein du muscle et leur rôle sur la jutosité. Par ailleurs, nous avons pu mettre en évidence le rôle positif des protéoglycanes de haut poids moléculaire sur la jutosité, alors que les protéoglycanes de faible poids moléculaire, telle que la décorine,joueraient plutôt un rôle sur la dureté de la viande. L’ensemble des résultats obtenus confirme nos hypothèses, à savoir que la composante structurale ainsi que les molécules minoritairement présentes dans le tissu conjonctif peuvent influencer les qualités organoleptiques de la viande bovine. / The perception of beef by consumers is a crucial issue for the meat industry. Tenderness remains one of the sensory qualities essential to the consumer, with the juiciness and flavour. The aim of my thesis was to study the structural and biochemical properties of intramuscular connective tissue responsible for the background toughness and study relationships with tenderness and more widely with the sensory qualities of beef. The development of a method of image analysis was used to study objectively the characteristics of the perimysial and endomysial component of the intramuscular connective tissue. The results showed that the area occupied by these two structures influenced the beef tenderness. An assay for total proteoglycan measurement has been developed previously to the study of biochemical and molecular characteristics intramuscular connective tissue. The results obtained in this second part have highlighted the complementarities between proteoglycans, total collagen and cross-links within the muscle and their role on the juiciness. Furthermore, we were able to highlight the positive role of high molecular weight proteoglycans on juiciness, while low molecular weight proteoglycans, such as decorin, rather played a role in beef toughness. The overall results confirm our hypothesis that the structural components of the connective tissue and the small quantity of extracellular matrix molecules could influence the sensory qualities of beef.

Content: Leather is a fairly durable and flexible material created by tanning animal rawhides and can be found in many household and personal products. However, ensuring that the product endures attack from the environmental elements that contribute to its wear and tear is the key concern of the general consumer. Animal rawhides are soft collagenous connective tissues. The most important function of collagen is a mechanical one - to withstand loads acting on the leather material. The purpose of this paper is to show how findings from recent studies on the mechanics of collagen in connective tissues lend to the goal of structural biologists to establish a complete understanding of the functional significance of collagen in connective tissues. In particular, 28 different types of collagen have been identified - about 90% being type 1 collagen - in the human body. Most types of collagen participate in higher-order assemblies such as networks, filaments, microfibrils, fibrils, fibres/fascicles. These assemblies collectively form a hierarchical architecture in the tissue from the molecular level to the macroscopic level. A complete understanding the functional significance of collagen in connective tissues could direct the development of new technology, e.g. leather design and production. In this paper, I shall discuss findings related to the higher-order assemblies. The conventional understanding of the collagenous fibre-like structures - embedded in a hydrated ground substance - in connective tissue finds an analogy to engineering fibres reinforcing composite materials such as carbon fibre reinforced polymer composites. The macroscopic stress- strain response of the connective tissue to external loads acting on it is consistent with fibre composite behaviour. A structure-mechanical framework, underpinning the hierarchical architecture of the connective tissue, is proposed to explain this mechanical response of the tissue. By integrating models specific to the different levels of the tissue to enable better understanding of the macroscopic nature of the tissue, the framework serves as a representation of reality for guiding further research, especially for the purpose of exploring hypotheses and revealing properties for which only sparse (or no observational data) is available. This paper ends with a discussion on the prospect and challenges for future studies on collagen in connective tissues. Take-Away: A fresh look at the degree of collagen fibril alignment in tissue Rethinking the mechanics of cross-linking between fibrils Interfibrillar mechanics is governed by plastic stress transfer Influence of fibril diameter on interfibrillar stress transfer

MicroRNAs (miRNAs) represent a class of small noncoding RNAs that regulate gene expression. Recent studies have shown that miRNAs are mis-expressed in various human cancers and that some miRNAs have the potential to act as tumor suppressors or oncogenes. MiR-10b is one miRNA that has been shown to be deregulated in breast cancer. However, current findings regarding miR-10b’s role in breast cancer are controversial. MiR-10b was originally reported to be downregulated in breast cancer compared to normal breast tissue. Subsequently, miR-10b was argued to be upregulated in metastatic breast cancer cell lines, acting as a potent pro-metastatic agent via regulation of HOXD10. This report was soon challenged by another group who reported that miR-10b expression in a large patient cohort correlated inversely and significantly with tumor size, grade, and vascular invasion, but did not correlate with development of distant metastases or survival. These latter data suggest that miR-10b may impede specific functions associated with breast cancer progression. In this thesis, I present my analysis of miR-10b function in breast carcinoma cells, which revealed that it suppresses their migration and invasion. To define a mechanism that accounts for this suppressive function, I identified T-lymphoma invasion and metastasis 1 (TIAM1), a guanine nucleotide exchange factor for Rac1, as a miR-10b target and demonstrated that miR-10b inhibits TIAM1-dependent Rac1 activation, migration, and invasion. In addition, I identified the VEGF receptor fms-related tyrosine kinase 1 (FLT-1) as a second target of miR-10b and discovered a novel function for FLT-1 in promoting breast carcinoma cell migration and invasion. My results show, for the first time, that Rac activation can be regulated by a specific miRNA and provide a novel mechanism for the regulation of TIAM1 and FLT-1 in breast cancer. These data support the conclusion from clinical data that miR-10b expression correlates inversely with breast cancer progression, and suggest that miR-10b functions to impede breast carcinoma progression by regulating key target genes involved in cell motility.

<p>Glomerulosclerosis (GS) is the irreversible scarring of glomerular tissue which underlies the development of chronic kidney disease (CKD). Increased intraglomerular capillary pressure (P_gc) is a major contributor to the development of GS and can occur in both hypertensive and diabetic patients. With elevated P_gc, <em>in vitro</em> and <em>in vivo</em> evidence suggest that mesangial cells (MC) experience cyclic stretch and secrete pro-fibrotic factors such as connective tissue growth factor (CTGF) which contributes to GS. The signaling pathways that are activated in response to elevated P_gc and lead to extracellular matrix (ECM) production in MCs are the main focus of this thesis.</p> <p>Previous data demonstrated activation of the Rho GTPase, Rac1, with cyclic stretch in MCs. Furthermore, the most characterized effector of Rac1, p21-activated kinase (PAK), has been observed to have a role in endothelial cells (ECs) exposed to mechanical stress. We thus proposed that the Rac1-PAK signaling pathway is involved in mechanical stress signaling in MCs.</p> <p>Our data demonstrate that Rac1-PAK signaling was activated in response to cyclic stretch and required for stretch-induced CTGF production in MCs. RhoA activation was also regulated by Rac1-PAK signaling, and RhoA/ROCK were observed to mediate CTGF upregulation with stretch. Further investigation on the role of Rac1-PAK signaling and how it regulates CTGF in MCs exposed to stretch, will provide insight into potential therapeutic targets to delay the progression of hypertension-mediated CKD.</p> / Master of Science in Medical Sciences (MSMS)

Im Mittelpunkt dieser kumulativ angefertigten Arbeit stehen fünf verschiedenartige biomechanische Untersuchungen faseriger Bindegewebe, welche in einer Gesamtschau zusammengeführt werden. Die einzelnen Beiträge setzen sich zusammen aus Untersuchungen zum Einfluss zellulärer Bestandteile auf die mechanischen Eigenschaften faseriger Bindegewebe und die Beeinflussung dieser Ergebnisse durch Messfehler, speziell am Beispiel des Materialschlupfs. Über diese beiden Beiträge wird eine Verbindung hergestellt zur rechnergestützten Simulation der Wirkung eines Beckenkompressionsgurts auf die Bänder des Beckenrings und dem Transmissionsverhalten faseriger Bindegewebe bei Zugbelastung. Im fünften Beitrag wird am Beispiel des Zusammenwirkens von Achillessehne, Fersenfettpolster und Plantarfaszie in vitro die Komplexität der Betrachtung faseriger Bindegewebe aufgezeigt. Die Zusammenführung der einzelnen Untersuchungen wird begleitet von der Frage, ob die bestehenden biomechanischen Untersuchungsansätze ausreichend sind, um ein umfassendes Verständnis zur funktionellen Bedeutung faseriger Bindegewebe aufbauen zu können.