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[eng] Myotonic dystrophy type 1 (DM1), one of the more complex diseases already known, is the most common and multisystemic rare neuromuscular disease among adults, without cure and limited pharmacological treatments. In the light of the ´Leave No-one Behind´ commitment behind Sustainable Development Goals, the nutritional management of DM1 may provide a valuable strategy to improve the very limited quality of life of people living and suffering this minority disease. Our previous background research suggested that caffeine could elicit some positive effects on DM1. Therefore, the present research aimed to evaluate the potential of methylxanthines, to scientifically substantiate properly formulated food products based on methylxanthines, as a novel nutritional management approach in Myotonic Dystrophy type 1. Accordingly, the research disclosed herein comprises: technology surveillance on nutritional studies in DM1; natural sources of methylxanthines; food content and consumption of methylxanthines from natural sources; the regulatory framework for food products, particularly, food supplements and food for special medical purposes; as well as the European requirements for launching said food products into the market. In order to do so, the project required the complete food product research and development, from the definition of composition through in vitro and in vivo studies on DM1 models, to dose definition and development of galenic form, while carefully considering intellectual property constraints, such as patent protection status and freedom to operate studies. Then, an observational clinical study evaluating the background nutritional habits in a DM1 adults’ cohort precede the interventional clinical trial (study code NCT04634682) to measure the effect of the investigational food product on quality of life, fatigue and hypersomnia in DM1 adult patients. The data shown herein suggest that the controlled and dosed administration of the investigational food product could provide statistically significant improvements in diminishing excessive daytime sleepiness and increasing the 6-minute walking distance, at least in adult DM1 male. Excessive daytime sleepiness has been scored as one of the main aspects limiting the quality of life in DM1. Although the present pilot study encompassed a limited dataset, the obtained results encourage further clinical trials in larger cohorts, ideally to be performed beyond the era of COVID-19 pandemic., [cat] La Distròfia Miotònica de tipus 1 (DM1), una de les malalties més complexes que es coneixen, és una malaltia rara neuromuscular i multisistèmica, la més freqüent en adults, sense cura i amb tractaments farmacològics limitats. En el marc de ‘No deixar ningú enrere’ associat als Objectius de Desenvolupament Sostenible, el maneig nutricional de la DM1 pot proporcionar una estratègia valuosa per millorar la tant limitada qualitat de vida de les persones que conviuen i pateixen aquesta malaltia minoritària. La nostra recerca prèvia suggeria que la cafeïna podia induir alguns efectes positius en DM1. Per tant, la recerca actual tenia com a objectiu avaluar el potencial de la família de metilxantines per fonamentar científicament productes alimentaris basats en metilxantines, adientment formulats, com a una nova aproximació al maneig nutricional de la DM1. En aquest sentit, la recerca que s’ha dut a terme inclou: vigilància tecnològica d’estudis nutricionals en DM1; fonts naturals de metilxantines; contingut i consum de metilxantines presents en fonts naturals alimentàries; el marc regulatori dels productes alimentaris, particularment, complements alimentosos i aliments per a usos mèdics especials; així com els requeriments europeus necessaris per poder comercialitzar aquests productes. Per tal d’aconseguir els objectius, el projecte requeria de la recerca i desenvolupament complerts del producte en investigació, des de la definició de la seva composició a partir de resultats d’estudis en models in vitro i in vivodeDM1, a la definició de dosi i desenvolupament de la forma galènica, i a la vegada considerant especialment possibles limitacions en matèria de propietat intel·lectual i industrial, com ara l’estat de protecció en patents i estudis de llibertat operacional. A continuació, l’estudi clínic observacional per avaluar els hàbits nutricionals de base en una cohort d’adults afectats per DM1 va precedir l’estudi clínic intervencionista (codi estudi a clinicaltrials.govNCT04634682) per mesurar l’efecte del producte en investigació en la qualitat de vida, fatiga i somnolència excessiva en malalts adults amb DM1. Els resultats assolits descrits en aquesta memòria suggereixen que l’administració controlada i dosificada del producte en investigació pot proporcionar millores estadísticament significatives en la disminució de la somnolència diürna excessiva i un increment en la distància recorreguda durant sis minuts, almenys en homes adults afectats per DM1. La somnolència diürna excessiva s’ha considerat un dels principals factors que limiten la qualitat de vida en DM1. Malgrat que aquest estudi pilot es va realitzar en un grup limitat de participants, els resultats obtinguts encoratgen dur a terme nous estudis clínics en cohorts més grans, idealment per dur a terme un cop superada la Pandèmia de COVID-19.

[eng] The composition and architecture of the extracellular matrix (ECM), and their dynamic alterations, play an essential regulatory role in numerous cellular processes. Furthermore, structural, and biochemical properties of the ECM are central in regulating cell response via mechanical, chemical, and topological cues detected by receptors on the cell membrane, which induce cytoskeleton and/or cell nucleus rearrangements affecting gene expression. Indeed, distinct ECM architectures in the native stroma depend on tissue type, function, and composition. For instance, ECM anisotropy and stiffness are associated with altered ECM degradation and remodeling in cancer. In turn, abnormal ECM architecture favors tumor progression and invasion. Moreover, numerous diseases are associated with mutations in genes encoding ECM components, leading to deficient mechanical properties and altered ECM structure. Thus, there is an increasing interest to exploit and consolidate this knowledge to improve patients’ diagnosis, treatment, and care. In this work, we examined the use of cutting-edge open-source software to characterize the properties of ECM fibers and fibril bundles in patients’ samples and in vitro model tissues. We focused on Collagen I-III in lung cancer and on Collagen VI-related muscular dystrophies (COL6-RMD) because these pathologies present a clear link between altered ECM architecture and patients’ outcomes, which requires further investigation and may be exploited at the clinical level. For this purpose, we initially generated phantom images of fibrils networks (created artificially in silico or obtained from bioengineered models in vitro mimicking isotropic/anisotropic ECM) to test and validate the analysis of ECM fibrils with different bioengineering tools. Then, we focused on ECM images obtained from patient cell cultures or tissue biopsies and evaluated ECM descriptors as novel potential biomarkers. We collaborated with the Institut de Recerca Sant Joan De Déu (IRSJD) to analyze ECM models obtained from fibroblasts of patients affected by COL6-RMD. This is a rare subset of neuromuscular diseases related to deficiency in collagen type VI (COL6) expression, which affects young patients causing a broad range of severe disabilities, which worsen with time and shorten their lifespan. Currently, analysis of in vitro cultures of patients’ fibroblasts is employed as the first screening to select cases of COL6-RMD, and image-based classification has been suggested to improve this assay. Employing similar protocols, fibroblasts of different sources and malignant origin have been used to obtain decellularized cell-derived matrices (CDM), which retain topography and biochemical features, preserving their native microenvironment. Therefore, we produced and analyzed patients’ CDM to provide new in vitro personalized ECM models and improve early recognition of COL6-RMD subtypes with novel fiber-based ECM descriptors. In parallel, in collaboration with the CIBERES (Centro de Investigación Biomédica en Red de Enfermedades Respiratorias), we characterized the fibrillar properties of Non-Small Cell Lung Cancer (NSCLC) patients’ tissues. NSCLC is among the tumors with the highest death rate and recurrence; therefore, novel biomarkers may provide helpful complementary data with clinical impact. Fiber descriptors extracted by CT-FIRE have been demonstrated to capture relevant prognostic changes in the collagen structure of several tumors. In NSCLC, collagen architecture influences immune system activation, and increased fibrosis is already reported to correlate with patients’ prognosis. Therefore, we studied alterations of collagen fiber networks in the two most common NSCLC subtypes, Adenocarcinoma and Squamous Cell Carcinoma, to provide novel collagen structure-based biomarkers. Ultimately this work, along with the tools presented, paves the way for addressing the need for novel ECM-based descriptors, to stratify patients and evaluate their response to experimental treatments., [spa] La composición y arquitectura de la matriz extracelular (MEC), y sus alteraciones dinámicas, juegan un papel regulador importante en numerosos procesos patológicos. Por ejemplo, la anisotropía y la rigidez de la MEC están asociadas con la remodelación alterada de la MEC en el cáncer. A su vez, esta arquitectura favorece la progresión e invasión tumoral. Además, numerosas enfermedades están asociadas con mutaciones en los genes que codifican los componentes de la MEC, lo que conduce a propiedades mecánicas deficientes y una estructura alterada de la MEC. Por lo tanto, existe un interés creciente por explotar y consolidar este conocimiento para mejorar la atención de los pacientes. En este trabajo, nos centramos en las distrofias musculares relacionadas con el colágeno VI (COL6-RMD) y el cáncer de pulmón de células no pequeñas (NSCLC), para investigar biomarcadores relacionados con la MEC in vitro y en los tejidos de los pacientes que se implementarán en el entorno clínico. Empleamos la segmentación automática de imágenes para cuantificar las características fibrilares e investigamos la asociación con la información clinicopatológica de los pacientes. Por lo tanto, producimos y analizamos matrices derivadas de células de pacientes para proporcionar nuevos modelos de ECM personalizados in vitro y nuevos descriptores de ECM basados en fibra de los subtipos COL6-RMD. Se ha demostrado que los descriptores de fibra basados en imágenes capturan cambios pronósticos relevantes en la estructura del colágeno de varios tumores. En el NSCLC, la arquitectura del colágeno influye en la activación del sistema inmunitario y ya se reconoce que el aumento de la fibrosis se correlaciona con el pronóstico de los pacientes. Por lo tanto, estudiamos las alteraciones de las redes de fibras de colágeno en los dos subtipos de NSCLC más comunes, el adenocarcinoma y el carcinoma de células escamosas, y demostramos que los descriptores de la estructura del colágeno son posibles biomarcadores basados en imágenes con aplicaciones de diagnóstico y pronóstico. En última instancia, este análisis, junto con las herramientas presentadas, es prometedor para abordar la necesidad de nuevos descriptores, para estratificar a los pacientes y evaluar su respuesta a los tratamientos experimentales.

The collagen VI-related disorders (COL6-RDs) are a group of severe congenital neuromuscular disorders with no available treatment. New insight into its molecular characterisation together with the advance in the microscopy field offers the opportunity of making early diagnosis a quotidian process. In this study, three state-of-the-art microscopy techniques such as light sheet fluorescence microscopy (LSFM), second-harmonic generation (SHG) microscopy and fluorescence lifetime imaging microscopy (FLIM) are used to study a mouse model of the disorder. With these techniques parameters that define the pathological phenotype such as the cross-sectional area (CSA) of the muscle fibres, the distance between the myosin bands within the sarcomeres, and the differential lifetimes of the muscles autofluorescence can be compared to the wild type.

Collagen VI-related dystrophies (COL6-RDs) are a group of rare congenital neuromuscular dystrophies that represent a continuum of overlapping clinical phenotypes that go from the milder Bethlem myopathy (BM) to the severe Ullrich congenital muscular dystrophy, for which there is no effective treatment. Mutations in one of the three Collagen VI genes alter the incorporation of this protein into the extracellular matrix (ECM), affecting the assembly and the structural integrity of the whole fibrillar network. Clinical hallmarks of COL6-RDs are secondary to the ECM disruption and include muscle weakness, proximal joint contractures, and distal hyperlaxity. Although some traits have been identified in patients’ ECMs, a correlation between the ECM features and the clinical phenotype has not been established, mainly due to the lack of predictive and reliable models of the pathology. Herein, we engineered a new personalized pre-clinical model of COL6-RDs using cell-derived matrices (CDMs) technology to better recapitulate the complexity of the native scenario. We found that CDMs from COL6-RD patients presented alterations in ECM structure and composition, showing a significantly decreased Collagen VI secretion, especially in the more severe phenotypes, and a decrease in Fibrillin-1 inclusion. Next, we examined the Collagen VI-mediated deposition of Fibronectin in the ECM, finding a higher alignment, length, width, and straightness than in patients with COL6-RDs. Overall, these results indicate that CDMs models are promising tools to explore the alterations that arise in the composition and fibrillar architecture due to mutations in Collagen VI genes, especially in early stages of matrix organization. Ultimately, CDMs derived from COL6-RD patients may become relevant pre-clinical models, which may help identifying novel biomarkers to be employed in the clinics and to investigate novel therapeutic targets and treatments. Copyright © 2022 Almici, Chiappini, López-Márquez, Badosa, Blázquez, Caballero, Montero, Natera-de Benito, Nascimento, Roldán, Lagunas, Jiménez-Mallebrera and Samitier.

Muscular dystrophies are a group of disorders that cause progressive muscle weakness. There is an increasing interest for the development of biomarkers for these disorders and specifically for Duchene Muscular Dystrophy. Limited research however, has been performed on the biomarkers' development for the most rare muscular dystrophies, like the Facioscapulohumeral Muscular Dystrophy, Limb-Girdle Muscular Dystrophy and Myotonic Dystrophy type 2. Here, we aimed to identify novel serum-based miRNA biomarkers for these rare muscular dystrophies, through high-throughput next-generation RNA sequencing. We identified many miRNAs that associate with muscular dystrophy patients compared to controls. Based on a series of selection criteria, the two best candidate miRNAs for each of these disorders were chosen and validated in a larger number of patients. Our results showed that miR-223-3p and miR-206 are promising serum-based biomarkers for Facioscapulohumeral Muscular Dystrophy type 1, miR-143-3p and miR-486-3p for Limb-Girdle Muscular Dystrophy type 2A whereas miR-363-3p and miR-25-3p associate with Myotonic Dystrophy type 2. Some of the identified miRNAs were significantly elevated in the serum of the patients compared to controls, whereas some others were lower. In conclusion, we provide new evidence that certain circulating miRNAs may be used as biomarkers for three types of rare muscular dystrophies. This Project (POST-DOC/0916/0235) was co-financed by the European Regional Development Fund and the Republic of Cyprus through the Research and Innovation Foundation. A.C.K, A.O., M.T. and G.M.S. were funded by the European Commission Research Executive Agency Grant BIORISE [number 669026], under the Spreading Excellence, Widening Participation, Science with and for Society Framework. H.L. receives support from the Canadian Institutes of Health Research (Foundation Grant FDN-167281), the Canadian Institutes of Health Research and Muscular Dystrophy Canada (Network Catalyst Grant for NMD4C), the Canada Foundation for Innovation (CFI-JELF 38412), and the Canada Research Chairs program (Canada Research Chair in Neuromuscular Genomics and Health, 950-232279)

Muscular dystrophies are a group of rare and severe inherited disorders mainly affecting the muscle tissue. Duchene Muscular Dystrophy, Myotonic Dystrophy types 1 and 2, Limb Girdle Muscular Dystrophy and Facioscapulohumeral Muscular Dystrophy are some of the members of this family of disorders. In addition to the current diagnostic tools, there is an increasing interest for the development of novel non-invasive biomarkers for the diagnosis and monitoring of these diseases. miRNAs are small RNA molecules characterized by high stability in blood thus making them ideal biomarker candidates for various diseases. In this study, we present the first genome-wide next-generation small RNA sequencing in serum samples of five different types of muscular dystrophy patients and healthy individuals. We identified many small RNAs including miRNAs, lncRNAs, tRNAs, snoRNAs and snRNAs, that differentially discriminate the muscular dystrophy patients from the healthy individuals. Further analysis of the identified miRNAs showed that some miRNAs can distinguish the muscular dystrophy patients from controls and other miRNAs are specific to the type of muscular dystrophy. Bioinformatics analysis of the target genes for the most significant miRNAs and the biological role of these genes revealed different pathways that the dysregulated miRNAs are involved in each type of muscular dystrophy investigated. In conclusion, this study shows unique signatures of small RNAs circulating in five types of muscular dystrophy patients and provides a useful resource for future studies for the development of miRNA biomarkers in muscular dystrophies and for their involvement in the pathogenesis of the disorders. This Project (the european regional development fund and the republic of Cyprus through the research and innovation foundation POST-DOC/0916/0235) was co-financed by the European Regional Development Fund and the Republic of Cyprus through the Research and Innovation Foundation. A.C.K, A.O., M.T. and G.M.S. were funded by the European Commission Research Executive Agency Grant BIORISE [number the spreading excellence, widening participation, science with and for society framework 669026], under the Spreading Excellence, Widening Participation, Science with and for Society Framework. HL receives support from the Canadian Institutes of Health Research (Foundation Grant the canadian institutes of health research FDN-167281), the Canadian Institutes of Health Research and Muscular Dystrophy Canada (Network Catalyst Grant for NMD4C), the Canada Foundation for Innovation (the Canada foundation for innovation CFI-JELF 38412), and the Canada Research Chairs program (Canada Research Chair in Neuromuscular Genomics and Health, the Canada research chairs program 950-232279)

[eng] The skeletal muscle is the largest tissue of the human body. Its main function is to generate contractile forces, essential for locomotion, thermogenesis, and metabolism. Fundamental research on skeletal muscle in health and disease, and preclinical research for new therapies, are currently based on 2D in vitro cell cultures and in vivo animal models. However, these strategies have important shortcomings. For instance, conventional cell culture models cannot emulate the complex 3D architecture of native skeletal muscle, and the species-specific differences in animal models limit their relevance to humans. In contrast, engineered skeletal muscle tissues are emerging as in vitro 3D cell culture models that complement existing 2D strategies. These engineered tissues can offer an improved microenvironment resembling native muscle tissue, comprised of bundles of aligned, multinucleated fibers. Therefore, the main objective of this thesis was to develop 3D skeletal muscle tissues for in vitro studies of muscle metabolism and disease modeling. Skeletal muscle precursor cells were encapsulated in microfabricated hydrogel scaffolds, introducing the appropriate topographical and microenvironmental cues to guide muscle fiber formation. First, photocrosslinkable gelatin methacryloyl (GelMA)-based composite hydrogels were synthesized and evaluated as cell-laden bioinks for 3D bioprinting of murine skeletal muscle tissue. The fabrication conditions were optimized to ensure the biocompatibility of the process and promote in vitro myogenesis. Our results demonstrated that the composite hydrogels have a higher resistance to degradation than GelMA hydrogels. Thus, the bioprinted scaffolds maintained their 3D structure over a prolonged culture period. Furthermore, the shear stress during extrusion bioprinting combined with the appropriate scaffold geometry resulted in highly aligned myoblasts that correctly differentiated into multinucleated myotubes. Considering these results, GelMA-carboxymethylcellulose methacrylate (CMCMA) hydrogels were then used to generate skeletal muscle microtissues in long-lasting cell cultures. Photomold patterning of cell-laden GelMA-CMCMA filaments led to the formation of highly aligned 3D myotubes expressing sarcomeric proteins. Moreover, the presented protocols were highly biocompatible and reproducible. Murine skeletal muscle microtissues were fabricated in a microfluidic platform integrated with an electrical stimulation system and biosensors for monitoring muscle metabolism in situ. Here, we measured the contraction-induced release of muscle-secreted cytokines upon electrical or biological stimulation. The obtained results confirmed the endocrine function of the bioengineered tissues, obtaining in vivo-like responses upon exercise or endotoxin-induced inflammation. Then, the photomold patterning protocol was optimized for human cells to develop the first in vitro 3D model of myotonic dystrophy type 1 (DM1) human skeletal muscle. DM1 is the most prevalent hereditary myopathy in adults, and there is no effective treatment to date. We proved that 3D micropatterning enhances DM1 myotube formation compared to 2D cultures. Furthermore, we detected the reduced thickness of 3D DM1 myotubes compared to healthy controls, which was proposed as a new in vitro structural phenotype. Thus, as a proof-of-concept, we demonstrated that treatment with an antisense oligonucleotide, antagomiR-23b, could rescue both molecular and structural phenotypes in these bioengineered DM1 muscle tissues. Finally, animal-derived components were eliminated to develop in vitro functional tissues in xeno-free cell culture as a next step towards improving bioengineered human skeletal muscle tissues. Cell-laden nanocomposite hydrogels consisting of human platelet lysate and functionalized cellulose nanocrystals (HUgel) were fabricated in hydrogel casting platforms that implemented uniaxial tension during matrix remodeling. We modulated the content of cellulose nanocrystals to tune the mechanical and biological properties of HUgel and favor the formation of long, highly aligned myotube bundles. Additionally, we performed in situ force measurements of electrical stimulation-induced contractions. Altogether, the results presented in this thesis provide promising approaches to advanced cell culture models of skeletal muscle tissue that could be valuable tools for fundamental studies, disease modeling, and future personalized medicine., [spa] El músculo esquelético tiene funciones esenciales para la salud que pueden verse afectadas por enfermedades neuromusculares o metabólicas. Actualmente, la investigación fundamental y preclínica se basa en cultivos celulares en 2D y modelos animales. Sin embargo, estos ensayos tienen relevancia limitada para la salud humana. En cambio, modelos in vitro de tejidos 3D que mimeticen la arquitectura y funcionalidad del músculo esquelético, podrían complementar las estrategias 2D tradicionales. Por lo tanto, el objetivo principal de esta tesis fue desarrollar tejidos de músculo esquelético en 3D para estudios sobre el metabolismo muscular y modelos de enfermedades in vitro. Los tejidos fueron desarrollados mediante diferentes técnicas de microfabricación de hidrogeles, en los que se encapsularon células precursoras del músculo esquelético introduciendo las señales topográficas adecuadas para guiar la formación de fibras musculares. Las propiedades de estos biomateriales fueron optimizadas para garantizar su biocompatibilidad y promover la miogénesis. Estos biomateriales mantienen su estructura durante periodos de cultivo prolongados, permitiendo la formación y diferenciación de miotubos 3D altamente alineados. La función endócrina de los tejidos fue evaluada utilizando un dispositivo músculo-en-un-chip, con el que fue posible medir la liberación de citoquinas secretadas tras estimulación eléctrica o biológica. Posteriormente, se desarrolló el primer modelo 3D de músculo esquelético humano para la distrofia miotónica tipo 1. Como prueba de concepto, demostramos que el tratamiento con un oligonucleótido antisentido, antagomiR-23b, podría rescatar fenotipos moleculares y estructurales en los tejidos fabricados a partir de células de pacientes. Finalmente, se desarrollaron tejidos funcionales en cultivos celulares xeno-free, con el objetivo de incrementar la relevancia de modelos humanos en los que fue posible medir las fuerzas generada por tejidos contráctiles. En conjunto, los resultados de esta tesis proporcionan enfoques prometedores para modelos avanzados de músculo esquelético que podrían ser herramientas valiosas para estudios fundamentales, modelos de enfermedades y medicina personalizada.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Esta pesquisa situa-se no campo dos estudos sobre a inclusão de crianças com deficiência e doença crônica na educação infantil. Nesse sentido, almeja identificar e compreender desafios e experiências relacionados à presença de crianças com distrofia muscular congênita nessa etapa na educação básica. Assume, para tanto, como referencial teórico-metodológico, a psicologia histórico-cultural, com destaque para as contribuições de Vigotski centradas, sobretudo, na gênese social do desenvolvimento humano, no papel dos contextos educacionais e das interações estabelecidas em seu interior como fonte privilegiada para a promoção de aprendizagem e desenvolvimento de todas as crianças. A pesquisa contempla uma revisão da literatura nacional sobre a temática distrofia muscular, empreendida tanto na esfera da saúde, buscando colocar em evidência suas principais características e sintomas, bem como no campo da educação, revelando um cenário de escassa produção sobre esse assunto. Em seu cerne, conta com uma imersão em campo, mais precisamente em uma escola pública situada na cidade de Guarulhos, centrada em observação participante junto a um grupo do Estágio II, onde está matriculada uma criança de 5 anos com distrofia muscular congênita e deficiência física. A análise dos dados selecionados aponta para a ausência de investigações sobre crianças com distrofia muscular na educação infantil, pontua, como desafios, a partir da imersão em campo, a ausência de recursos de acessibilidade que permitam que a criança usufrua plenamente de todos os ambientes escolares, colocando em evidência o contexto de precariedade em que as crianças público-alvo da educação especial são recebidas na educação infantil. No campo das experiências, ao analisar as relações, interações e mediações ali estabelecidas, nas quais assumimos uma dimensão partícipe, destacam-se o papel do professor como agente organizador daquele meio e central para a ampliação efetiva das possibilidades de participação dessas crianças, aspectos que coadunam com a potencialização e emergência de sua aprendizagem e desenvolvimento. A investigação aponta, também, para a necessidade de políticas públicas que oportunizem condições concretas de acessibilidade, bem como possibilitem a ampliação da formação inicial e continuada de educadores em um perspectiva de educação inclusiva. ABSTRACT This research is in the studies field about the children inclusion with disabilities and chronic illness in early childhood education. Thus, it aims to identify and comprehend challenges and experiences related to children with congenital muscular dystrophy in basic schooling education. It assumes, therefore, as a theoretical-methodological framework, the cultural-historical psychology, with emphasis on Vigotski’s contributions centered, above all, by the social genesis of human development, in the role of educational contexts and interactions established within them as the privileged to source for the promotion of learning and all children's development. This research includes a national literature review about muscular dystrophy theme, undertaken both in the health sphere, to highlight its main characteristics and symptoms, just as in the education area, revealing a scarce production scenario about this subject. Besides, it has immersion in the field, more precisely in a public school located in the Guarulhos city, focused on participant observation with a II Stage group, where a 5-year-old child with congenital muscular dystrophy and physical disability is enrolled. Selected data analysis points to a lack of investigation about children with muscular dystrophy in early childhood education, showing as challenges, from field immersion, deprivation accessibility resources for a child to fully enjoy all school environments, highlighting the precarious context in which children target, audience from special education, are received in early childhood education. In the experiences field, analyzing relationships, interactions, and established mediations, in which we assume a participative dimension, we stand out the role educator as an organizing agent in this environment and central effective expansion of this child's participation possibilities, aspects that fit together with the enhancement and urgency from their learning and development. The investigation also points to the need for public policies that provide concrete conditions for accessibility, as well as enable the expansion of initial and continuing education for educators in an inclusive education perspective. 001

Orientador: Humberto Santo Neto Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: A Distrofia Muscular de Duchenne (DMD) e o camundongo mdx, modelo experimental da doença, caracterizam-se pela ausência de distrofina e pela necrose das fibras musculares. Alguns músculos são protegidos da mionecrose e admite-se que nestes a expressão das proteínas reguladoras de cálcio está aumentada. Os músculos intrínsecos da laringe (ILMs) apresentam características anatômicas e fisiológicas semelhantes aos músculos extra-oculares (EOMs), que são protegidos da mionecrose em pacientes portadores de DMD e camundongos mdx. Assim levantamos a hipótese de que os ILMs são protegidos da necrose de suas fibras e apresentam expressão diferenciada de proteínas reguladoras do cálcio. Foram avaliados os ILMs e músculos apendiculares de camundongos C57Bl/10 (controles) e mdx, adultos e idosos. Foi analisado a porcentagem de núcleos centrais, como um sinal de fibras lesionadas e em reparação e foi utilizado o azul de Evans, como marcador de lesão miofibrilar. Após a caracterização desses músculos, foi estudado por imunohistoquímica e imunobloting, o nível da expressão de proteínas reguladoras do cálcio, calsequestrina e Ca2+-ATPase do retículo sarcoplasmático (SERCA1). Observou-se que, exceto o músculo cricotireóideo (CT), nenhum ILMs dos camundongos mdx adultos ou idosos apresentaram sinais de miopatia, entretanto núcleos centrais foram visíveis no músculo tibial anterior do mesmo animal. Houve aumento significativo da porcentagem de núcleos centrais no músculo CT comparado com outros ILMs, o qual piorou com envelhecimento. A expressão de SERCA1 e calsequestrina está aumentada nos ILMs distróficos em relação aos controles, distintamente do que ocorre nos músculos apendiculares. Assim, conclui-se que os ILMs dos camundongos mdx são protegidos da mionecrose e mostram níveis elevados de SERCA1 e calsequestrina, sugerindo que a manutenção da homeostase do cálcio pode estar envolvida na proteção desses músculos Abstract: Duchenne muscular dystrophy (DMD) and mdx mice, a model for DMD, is characterized by the lack of dystrophin expression and muscle fiber necrosis. Some muscle are enigmatically protected and admitted that an elevated expression of calcium-binding proteins. The intrinsic laryngeal muscles (ILMs) share many anatomical and physiological properties with extra-ocular muscles, which are unaffected in both Duchenne muscular dystrophy and mdx mice. We hypothesized that ILMs are spared from myonecrosis in the mdx and investigated whether this possible protection is related to an increased expression of calcium-binding proteins, SERCA1 and calsequestrin, which may be protective against the elevated calcium levels seen in dystrophic fibers. ILMs and limb muscles of adult and aged control C57Bl/10 and mdx mice were used. The percentage of central nucleated fibers, as a sign of muscle fibers that had experienced injury and regeneration, and myofibers labeling with Evans blue dye, as a marker of myofiber damage, were studied. After this characterization, the expression of Sarco-endoplasmic-reticulum Ca2+-ATPase (SERCA1) and calsequestrin was examined using immunofluorescence and immunoblotting. Except for the cricothyroid muscle (CT), none of the ILMs from adult and old mdx mice showed signs of myofiber damage. Central nucleation was readily visible in tibialis anterior of the same mdx mice. A significant increase in the percentage of central nucleated fibers was observed in adult CT compared to the other ILMs, which was worsened by age. Dystrophic ILMs presented a significant increase in the proteins studied, in comparison to controls. These proteins were reduced in the non-spared mdx muscles. Thus we show that the ILMs are spared from the lack of dystrophin and the increase of SERCA1 and calsequestrin may permit a better maintenance of calcium homeostasis with the consequent absence of myonecrosis Mestrado Anatomia Mestre em Biologia Celular e Estrutural

Dystroglycanopathies are diseases characterized by progressive muscular degeneration and impairment of patient's quality of life. They are associated with altered glycosylation of the dystrophin-glycoprotein (DGC) complex components, such as α-dystroglycan (α-DG), fundamental in the structural and functional stability of the muscle fiber. The diagnosis of dystroglycanopathies is currently based on the observation of clinical manifestations, muscle biopsies and enzymatic measures, and the available monoclonal antibodies are not specific for the dystrophic hypoglycosylated muscle condition. Thus, modified α-DG mucins have been considered potential targets for the development of new diagnostic strategies toward these diseases. In this context, this work describes the synthesis of the hypoglycosylated α-DG mimetic glycopeptide NHAc-Gly-Pro-Thr-Val-Thr[αMan]-Ile-Arg-Gly-BSA (1) as a potential tool for the development of novel antibodies applicable to dystroglycanopathies diagnosis. Glycopeptide 1 was used for the development of polyclonal antibodies and recombinant monoclonal antibodies by Phage Display technology. Accordingly, polyclonal antibodies were reactive to glycopeptide 1, which enables the application of anti-glycopeptide 1 antibodies in immune reactive assays targeting hypoglycosylated α-DG. Regarding monoclonal antibodies, for the first time variable heavy (VH) and variable light (VL) immunoglobulin domains were selected by Phage Display, identified by NGS and described by in silico analysis. The best-characterized VH and VL domains were cloned, expressed in E. coli Shuffle T7 cells, and used to construct a single chain fragment variable that recognized the Glycopeptide 1 (GpαDG1 scFv). Molecular modelling of glycopeptide 1 and GpαDG1 scFv suggested that their interaction occurs through hydrogen bonds and hydrophobic contacts involving amino acids from scFv (I51, Y33, S229, Y235, and P233) and R8 and α-mannose from Glycopeptide 1.

Orientador: Maria Julia Marques Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: O camundongo mdx é o animal mais utilizado como modelo da distrofia muscular de Duchenne (DMD), diferindo dos humanos doentes por apresentar ciclos de regeneração muscular e reduzida fibrose. Este trabalho tem como objetivos: 1. desenvolver protocolo experimental para promover fibrose muscular através de exercício de corrida em esteira e 2. verificar se a suramina inibe a fibrose induzida experimentalmente. A suramina tem efeito anti-fibrótico, sendo um potencial agente farmacológico para tratamento da DMD visando sucesso de terapias celulares. Foram utilizados camundongos mdx (n=42) e C57BL/10 (n=11) com seis meses de idade. Os camundongos mdx foram divididos em quatro grupos experimentais: grupo sedentário e tratado com salina (n=11), grupo sedentário e tratado com suramina (n=11), grupo exercitado e tratado com salina (n=10) e grupo exercitado e tratado com suramina (n=10). Os animais foram submetidos à corrida em esteira diariamente e o tratamento com suramina (60mg/kg) foi realizado em dias alternados, via intra-peritoneal. Após sete semanas, os animais foram sacrificados e o músculo tibial anterior, bíceps braquial, diafragma e coração coletados e congelados para análise histológica e protéica por western blot. O plasma sanguíneo foi submetido à análise de creatina-quinase. A força de tração dos membros anteriores foi medida no início e no final do protocolo experimental utilizando-se Grip Strength Meter e o músculo diafragma submetido ao estudo in vitro para verificar a força de contração. Verificamos que o protocolo de corrida em esteira foi adequado para induzir a fibrose e inibir a regeneração nos músculos da pata dos camundongos mdx. O aumento da área de fibrose foi acompanhado pelo aumento dos níveis de TGF-?1, aumento de creatina-quinase e diminuição da força de tração. O tratamento com suramina diminuiu a fibrose nos músculos exercitados e acelerou o processo de regeneração. Adicionalmente, observamos que a suramina reduziu o número de fibras marcadas com azul de Evans, diminuiu os níveis da CK e impediu a perda da força de tração bem como a força de contração do músculo diafragma. Concluímos que o protocolo de corrida em esteira foi eficaz na indução de fibrose nos músculos tibial anterior e bíceps braquial. O efeito anti-fibrótico da suramina torna-a droga potencialmente útil para a terapia farmacológica da DMD Abstract: The mdx mouse is commonly used as a model to study Duchenne muscular dystrophy (DMD) however shows cycles of muscle regeneration and reduced fibrosis. The purposes of this study were (1) to induce muscle fibrosis through eccentric running exercise in mdx mice and (2) to verify the effects of suramin on muscle fibrosis. Six-month-old mdx (n=42) and control (C57BL/10, n=11) mice were used. Mdx mice were divided in four groups: sedentary and saline-treated (n=11); sedentary and suramintreated (n=11); exercised and saline-treated (n=10) and exercised and suramin-treated (n=10). The mdx mice belonging to the exercised groups were placed on treadmill to run daily, for seven weeks. Suramin was injected at a dose of 60mg/kg i.p. on alternate days. At the end of the experimental protocol, the mice were sacrificed and the tibialis anterior, biceps brachii, diaphragm and heart muscles were dissected, frozen in liquid nitrogen and used to histological and western blot analysis. Blood was obtained to determine creatine-kinase (CK) levels. The forelimb force was measured by an adapted Grip Strength Meter. Force of contraction of diaphragm in vitro was verified. Our results showed that the experimental protocol was adequate to induce muscle fibrosis in mdx mice. The occurrence of fibrosis was accompanied by elevated levels of TGF- ?1 and serum CK and decreased forelimb force. Suramin reduced muscle fibrosis, decreased the number of muscle fibers stained with Evans blue, reduced serum CK levels and prevented the loss of muscle force in exercised mdx and diaphragm strips in vitro. We concluded that the downhill running protocol was effective for inducing fibrosis in tibialis anterior and biceps brachii muscles of mdx mice. Suramin seems to be a potential useful therapeutic alternative for DMD treatment Doutorado Anatomia Doutor em Biologia Celular e Estrutural

Orientador: Elaine Minatel Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: A peroxidação lípidica causada pelo aumento de espécies reativas de oxigênio (EROs) no período que antecede o início da degeneração muscular no camundongo mdx, sugere que o estresse oxidativo pode ser um dos mecanismos primários da degeneração muscular distrófica, ao invés de ser um efeito secundário deste processo. No presente trabalho verificamos se o tratamento com ácido ascórbico antes que se iniciem os ciclos de degeneração/regeneração diminui a degeneração muscular em camundongos mdx. Camundongos mdx com 14 dias de vida pós-natal receberam por gavagem doses diárias de 0,1mg/kg de Ácido Ascórbico diluído em água por 7 (grupo mdx T7) e 14 dias (grupo mdx T14) e/ou dose de 200mg/kg por 14 dias (grupo mdx T14AA). Animais mdx controle receberam solução salina. Após este período, os músculos tibial anterior (TA), esternomastóide (STN) e diafragma (DIA) foram retirados. Na análise histológica, o ácido ascórbico diminuiu a degeneração muscular nos músculos TA do grupo mdx T7 e no DIA do grupo mdx T14AA (P0,05, Student's t Test). Aumento significativo no conteúdo de TNF-", pela técnica de Western Blotting, foi observado nos músculos dos camundongos mdx tratados com ácido ascórbico. Os resultados indicam que o tratamento precoce de camundongos mdx com o ácido ascórbico diminuiu significativamente a mionecrose nos músculos tibial anterior e diafragma. O presente trabalho também sugere que o aumento do TNF-" não está necessariamente correlacionado ao aumento da mionecrose. Abstract: Increased production of reactive oxygen species (ROS) can cause lipid peroxidation in the period preceding muscle necrosis in the mdx mice. This suggests that ROS may be involved as a primary, rather than secondary, cause of degeneration. In the present study, we verified whether ascorbic acid treatment before the cycles of muscle degeneration-regeneration decreases muscular degeneration in mdx mice. Mdx mice at 14 days of age received 0.1mg/kg of acid ascorbic daily in the water for 7 (group mdx T7) or 14 days (group mdx T14) and/or 200mg/kg for 14 days (group mdx T14AA). Control mdx mice received saline. After this period the tibialis anterior (TA), sternomastoid (STN) and diaphragm (DIA) muscles were removed. Hislogical analysis showed that ascorbic acid significantly decreased muscle degeneration in the TA of mdx T7 group and in the DIA of mdx T14AA group (P0,05, Student's t Test). Immunoblots showed a significant increase in the content of TNF-" in the muscles of the treated mdx mice. The present results indicate that ascorbic acid treatment before the cycles of muscle fiber degeneration-regeneration significantly decreases myonecrosis in TA and DIA dystrophic muscles. The present findings also sugest that increase TNF-" not necessarily correlates with increased myonecrosis. Mestrado Anatomia Mestre em Biologia Celular e Estrutural