O desenvolvimento ontogenético em peixes é considerado como uma das etapas mais importantes no fornecimento de informações para biologia do desenvolvimento, aquicultura e estudos taxonômicos. A Proteína Óssea Morfogenética 2 (BMP-2) é considerada uma molécula essencial como regulador no desenvolvimento embrionário e na formação óssea, sendo ainda pouco estudada em peixes. Neste trabalho, foram comparadas todas as fases do desenvolvimento de três espécies de peixes importantes da bacia do rio Pardo, curimbatá (Prochilodus hartii), piabanha (Brycon sp) e piau (Leporinus steindachneri). A superfície ovocitária, estádios e duração do desenvolvimento embrionário/larval foram classificados com a utilização de microscópio estereoscópio, havendo variações entre as espécies no desenvolvimento. A transformação dos alevinos também apresentaram variações, com 16 dias pós-eclosão em P. hartii, 5 dias em Brycon sp e 14 dias em L. steindachneri. Na imunohistoquímica, BMP-2 foi identificada na blastocele nas fases de blástula e gástrula; na formação da vesícula óptica, notocorda e somitos, na fase embrionária das espécies estudadas. Na fase larval e de juvenil, BMP-2 foi identificada na formação de brânquias, olhos, coração, estômago, intestino, fígado, nadadeiras, músculos e na ossificação em todas as espécies estudadas. Indivíduos adultos foram sacrificados, e depois de realizada a biometria, dissecados para mensuração dos órgãos internos e documentação fotográfica. Observou-se que o estômago de P. hartii tem forma de \"U\" com presença de uma estrutura semelhante a uma moela, enquanto em Brycon sp tem forma de \"J\" com presença de cecos pilóricos aderidos no piloro e em L. steindachneri, o mesmo possui forma de \"Y\" sem presença de cecos pilóricos. Esqueletos foram preparados através da técnica de maceração com insetos dermestídeos, sendo observadas diferenças, sendo interessante descrever que em P. hartii foram encontrados 4 ossos infraorbitais, 6 em Brycon sp e 4 em L. steindachneri. Conclui-se que os exemplares de P. hartii possuem hábito alimentar iliófago, enquanto Brycon sp e L. steindachneri são considerdados onívoros, baseado nos achados anatômicos e a expressão de BMP-2 está ligada com a morfogênese e organogênese na embriologia das espécies estudadas. / The ontogenetic development in fish is considered as one of the most important steps in providing information to developmental biology, aquaculture and taxonomic studies. The Bone Morphogenetic Protein 2 (BMP-2) is considered as a molecule essential for regulaton of embryonic development and bone formation, and has been poorly studied in fish. In this work, we have compared all the development phases of three important species of fish from the Pardo River basin, Curimbata (Prochilodus hartii) Piabanha (Brycon sp) and Piau (Leporinus steindachneri). The surface oocyte, stage and duration of embryonic/larval development were classified by using a stereoscopic microscope, and showed variations between the studied species. The transformation of the juveniles also showed variations, with 16 days post-hatching in P. hartii, 5 days in Brycon sp and 14 days in L. steindachneri. In immunohistochemistry, BMP-2 was identified in the blastocoel of the blastocyst and gastrula stages; forming the optic vesicle, notochord and somites on embryo of the ivestigated species. In larval and juvenil stages, BMP-2 has been identified in the formation of gills, eyes, heart, stomach, intestine, liver, fins, muscle and ossification in the species studies. Adults were sacrificed, dissected for biometrics measurements of the internal organs and photo documentation. The stomach of P. hartii showed a \"U\" shape with the presence of a structure similar to a gizzard, whereas in Brycon sp it was shaped like a \"J\" with the presence of pyloric caeca and in L. steindachneri it presented a \"Y\" form without the presence of pyloric caeca. Skeletons were prepared by retting technique with Dermestides beetles, interesting differences were observed between numbers of bones, where 4 infraorbital bones were found in P. hartii, 6 in Brycon sp and 4 in L. steindachneri. It is concluded that the specimens of P. hartii show ilyophagous eating habits while Brycon sp and L. steindachneri were considered omnivorous based on anatomical findings and that expression of BMP-2 is linked with morphogenesis and organogenesis of the studied species.

Avances en ingeniería de tejidos y en terapias celulares requerirán el desarrollo de un nuevo abanico de tecnologías. Estas incluirán sistemas de cultivo de líneas primarias en tres dimensiones (3D) con el fin de construir tejidos artificiales, sistemas de transducción eficientes y sistemas de regulación genética compatibles en humanos para ajustar intervenciones moleculares. Todo ello permitirá reprogramar las células animales con el objetivo de obtener fenotipos deseados y de optimizar la productividad celular.El objetivo de esta tesis es (i) estudiar y manipular las propiedades (electro) fisiológicas de los tejidos artificiales de miocardio mediante la regulación de BMP-2 para controlar y regenerar la funcionalidad cardiaca, (ii) estudiar, inducir y caracterizar los procesos de diferenciación y transdiferenciación de microtejidos adiposos y oseos mediante la sobre expresión de los genes BMP-2 y ΔFosB para tratar enfermedades relacionadas con el tejido oseo como la osteoporosis, y (iii) evaluar la productividad de los microtejidos artificiales para optimizar los procesos de producción con células animales. Por último, el objetivo es también automatizar la producción de microtejidos artificials para satisfacer las necesidades de la industria.Microtejidos libres de scaffold no solo presentan una alternativa a la ingeniería de tejidos para terapias celulares y regeneración de tejidos, sino también tienen un gran potencial en estudios celulares para aplicaciones en la industria bio-farmacéutica. Mientras en el área de investigación, los microtejidos artificiales presentan un modelo para el estudio de nuevos genes y proteínas implicadas en diferentes procesos celulares (diferenciación, proliferación, apoptosis, síntesis de proteínas, etc.), en el area de desarrollo, los microtejidos artificiales representan una herramienta para el análisis de dichos genes y proteínas por su acción terapéutica. Finalmente, una vez la proteína terapéutica ha sido identificada, los microtejidos artificiales podrían proveer un ambiente más beneficioso para la producción de proteínas terapéuticas. / Advances in tissue engineering and cell-based therapies will require the development of a range of specific technologies. These include systems for the cultivation of primary cells in three dimensions (3D) to form artificial tissues, efficient gene transduction technologies, and human-compatible gene regulation systems for adjustable molecular interventions all of which should enable the rational reprogramming of mammalian cells to achieve desired cell phenotypes, functionality, and to optimize cellular productivity. The aim of this thesis is to (i) study and modulate the (electro) physiological properties of artificial myocardial microtissues by BMP-2 regulation to control and to regenerate cardiac functionality, (ii) study, induce and characterize differentiation and transdifferentiation processes of adipose- and bone-microtissues by overexpression of BMP-2 and ΔFosB genes in order to treat bone-related diseases such as osteoporosis, and (iii) evaluate artificial microtissue productivity to optimize mammalian production processes. Finally, the purpose is also to automate artificial microtissue production in order to satisfy requirements for industrial scale applications.Scaffold-free artificial microtissues represent an alternative to existing tissue engineering strategies for cell-based therapies and tissue regeneration, as well as for cell-based biopharmaceutical applications. Within a research context, artificial microtissues could be used as a cell culture model to study/screen novel molecules/genes/proteins that are involved in different cellular processes (e.g. differentiation, proliferation, apoptosis, protein synthesis and secretion), while in a development context, artificial microtissues could be used as a tool to evaluate such target molecules/genes/proteins for their therapeutic action. Finally, once a therapeutic molecule/protein is identified, artificial microtissues may provide a more suitable or optimized environment for therapeutic protein production.

Hydroxyapatite (HA) based bioceramics have been developed to treat bone defects for the last 30 years. Doping HA with elements is a common approach to increase mechanical strength, biocompatibility and osteointegrity. Bone morphogenetic protein (BMP)-containing bioceramic composites enhance osteointegrity and induce bone formation. Strontium (Sr) is currently used to treat osteoporosis clinically as this element inhibits bone resorption and stimulates bone formation. In this study, HA was doped with silicon (Si), Sr, BMP-2 and evaluated in cortical bone defect healing. Ceramics were produced and tested mechanically after characterization. Sr release from ceramics was assessed. Ceramics were further evaluated in in vitro and in vivo conditions. X-ray diffraction analysis results of HA were in line with the literature and Sr-Si-HA ceramics showed similar intensities with HA. Ceramics had 36.9 to 41.6% porosity. Compression strength of Sr1000-Si-HA ceramics was 117.5 MPa which was more than that of the other groups. Consistent Sr release was observed in the Sr1000-Si-HA and the Sr250-Si-HA groups. Sr1000-Si-HA and Sr250-Si-HA ceramics showed higher cellular proliferation rates than the other groups in vitro. BMP addition increased alkaline phosphatase activities and DNA amounts. BMP-Sr-Si-HA group presented higher (0.304&plusmn / 0.02 g/cm2) bone mineral density values than the other groups 4 weeks after implantation however differences between groups were not significant in vivo. Sr-Si-HA and BMP-Sr-Si-HA composites stimulated new bone formation at cortical bone defects of tibia according to micro computerized-tomography and histological results. Findings of this study promote future research on Sr containing bioceramics in treatment of orthopedic problems.

Current treatment methods for non-union bone defects present problems. The objective of this study was to genetically engineer primary and immortalized cell types to express osteogenic molecules BMP2, RUNX2, OSX, or VEGF in a doxycycline dose-dependent manner for tissue regeneration. Coding cDNA sequences for all four factors were sub-cloned into the pRTS-1 expression plasmid and transfected into HUCPVCs, RBMCs, ROS cells. Electroporation was the most effective method of transfection for all cells but stably transfected cells could only be established for RBMCs and ROS cells. Cells achieved maximum expression within 72hours of induction and returned to basal levels after 18 days. Enhanced osteogenic bioactivity was only observed upon activation of BMP-2. The tight regulation of the pRTS-1 system allowed for a controlled gene expression. Future transplantation experiments using these engineered RBMC and ROS cells in vivo will evaluate the usefulness of the dox-inducible gene expression system in bone defects.

Current treatment methods for non-union bone defects present problems. The objective of this study was to genetically engineer primary and immortalized cell types to express osteogenic molecules BMP2, RUNX2, OSX, or VEGF in a doxycycline dose-dependent manner for tissue regeneration. Coding cDNA sequences for all four factors were sub-cloned into the pRTS-1 expression plasmid and transfected into HUCPVCs, RBMCs, ROS cells. Electroporation was the most effective method of transfection for all cells but stably transfected cells could only be established for RBMCs and ROS cells. Cells achieved maximum expression within 72hours of induction and returned to basal levels after 18 days. Enhanced osteogenic bioactivity was only observed upon activation of BMP-2. The tight regulation of the pRTS-1 system allowed for a controlled gene expression. Future transplantation experiments using these engineered RBMC and ROS cells in vivo will evaluate the usefulness of the dox-inducible gene expression system in bone defects.

A expressão das proteínas morfogenéticas ósseas (BMPs) está alterada em vários cânceres humanos. A BMP-2/4 e o BMPR-IA foram recentemente encontrados superexpressos em lesões malignas e pré-malignas de alto risco em epitélio oral. Este estudo analisou a expressão da BMP-2/4 e seu receptor BMPR-IA em 23 espécimes de Carcinoma Epidermóide Oral (CEO), utilizando a imuno-histoquímica. O grupo controle constou de 10 casos de Hiperplasia Fibro-epitelial da mucosa oral. O grupo experimental foi constituído por 16 casos de CEO não metastático e 7 casos de CEO metastático. Utilizou-se o parâmetro presença ou ausência de metástase nodal para avaliar o prognóstico da doença. Os resultados demonstraram imunorreatividade fraca para a BMP-2/4 e o BMPR-IA em todos os espécimes do grupo controle. No grupo experimental com metástase, a BMP-2/4 exibiu forte expressividade (71,4%), enquanto que o BMPR-IA mostrou fraca expressão (85,7%). No grupo experimental sem metástase, evidenciou-se forte expressão para a BMP-2/4 (62,5%) e para o BMPR-IA (100%). Encontrou-se significância estatística para a associação entre o prognóstico do CEO e a intensidade de marcação da BMP-2/4 (p=0,002). Para o BMPR-IA não houve significância estatística à sua associação com o prognóstico da doença (p>0,001), em função do tamanho da amostra. Portanto, os resultados sugerem que a fraca expressividade do BMPR-IA associada à forte expressão da BMP-2/4, no grupo experimental com metástase, tem relevância prognóstica, já que a perda de sensibilidade às BMPs, através da perda de expressão de seus receptores pode ser indicativo de desenvolvimento de metástase em CEO. _________________________________________________________________________________________ ABSTRACT: The expression of bone morphogenetic proteins (BMPs) is altered in a variety of human canceres. The BMP-2/4 and BMPR-IA were recently shown to be overexpressed in high-risk premalignant and malignant lesions of oral epithelium. The present study analysed the expression of BMP-2/4 and BMPR-IA in Oral Squamous Cell Carcinoma (OSCC) such as their implications in disease prognostic using munohistochemistry. Ten cases of Oral Fibroepithelial Hiperplasia were selected as a control group. The experimental group included 16 cases of OSCC without metastases and 7 cases of OSCC metastatic. The presence or absence of nodal metastases was used as parameter to evaluated the disease prognostic. The results demonstrated weak immunoreactivity for BMP-2/4 and BMPR-IA in every case of the control group. In the cases of OSCC with metastases an overexpression of BMP-2/4 (71,4%) was observed while the BMPR-IA showed weak expression (85,7%). In the cases of OSCC without metastases BMP-2/4 (62,5%) and BMPR-IA showed strong immunostaining standing out an overexpression of the receptor in all the specimens. Observed statistical significance for correlation between the oral cancer prognostic and the staining intensity of the BMP-2/4 (p=0,002). There wasn t statistical significance for association between the staining intensity of the BMPR-IA and the disease prognostic (p>0,001). In conclusion, this findings suggest that the overexpression of BMP-2/4 associated with the loss of expression of the BMPR-IA in OSCC metastatic has prognostic relevance, as the loss of sensitivity to BMPs can be an indicative of metastases development in OSCC.

No description available.

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Les amphibiens urodèles (e.g. les axolotls) possèdent la remarquable capacité de régénérer plusieurs parties de leur corps. Ils peuvent, entre autres, régénérer parfaitement un membre amputé par épimorphose, un processus biphasique comprenant une phase de préparation, spécifique à la régénération, et une phase de redéveloppement, commune à l’épimorphose et au développement embryonnaire. Durant la phase de préparation, les cellules du moignon se dédifférencient en cellules pseudo-embryonnaires, prolifèrent et migrent distalement au plan d’amputation pour former un blastème de régénération. Parmi les vertébrés, la dédifférenciation est unique aux urodèles. Afin de mieux comprendre le contrôle moléculaire de la régénération chez les urodèles, nous avons choisi d’étudier BMP-2, un facteur de croissance, en raison de son implication dans la régénération des phalanges distales chez les mammifères. Le facteur de transcription MSX-1 a également été sélectionné en raison de sa capacité à induire la dédifférenciation cellulaire in vitro et de son interaction potentielle avec la signalisation des BMPs. Les résultats présentés dans cette thèse démontrent que BMP-2 et MSX-1 sont exprimés lors des phases de préparation et de redéveloppement de l’épimorphose, et que leur profil d'expression spatio-temporel est très semblable, ce qui suggère une interaction de leurs signaux. En outre, chez les tétrapodes amniotes, l’expression de Shh est restreinte au mésenchyme postérieur des membres en développement et chevauche l’expression de BMP-2. Toutefois, l’expression de BMP-2 n’est pas restreinte à la région postérieure mais forme un gradient postéro-antérieur. Shh est le principal régulateur de la formation du patron de développement antéro-postérieur du ii membre. Étant donné les domaines d’expression chevauchants de BMP-2 et Shh et la restriction postérieure d’expression de Shh, on croit que Shh régule la formation du patron de développement de postérieur à antérieur par l’activation de l’expression de BMP-2. Fait intéressant, l’axolotl exprime également Shh dans la région postérieure, mais le développement des pattes se fait de la région antérieure à la région postérieure au lieu de postérieur à antérieur comme chez les autres tétrapodes, et ceci durant le développement et la régénération. Nous avons utilisé cette caractéristique de l’axolotl pour démontrer que la signalisation Shh ne structure pas l’autopode via BMP-2. En effet, l’expression de BMP-2 n'est pas régulée par l'inhibition de la signalisation Shh, et son expression est du côté opposé à celle de Shh durant le développement et la régénération des pattes de l’axolotl. Il a été observé durant le développement du membre chez la souris que MSX-1 est régulé par la signalisation Shh. Nos résultats ont démontrés que chez l’axolotl, MSX-1 ne semble pas régulé par l'inhibition de la signalisation Shh au cours de la régénération du membre. De plus, nous avons démontré que contrairement à l’expression de Shh, l’expression de BMP-2 est corrélée avec l’ordre de formation des phalanges, est impliquée dans la condensation cellulaire et dans l'apoptose précédant la chondrogenèse. L’ensemble de ces résultats suggère un rôle de BMP-2 dans l’initiation de l’ossification endochondrale. Enfin, nous avons démontré que la signalisation BMP est indispensable pour l’épimorphose du membre durant la phase de redéveloppement. / Urodele amphibians (e.g. the axolotls) have a remarkable ability to regenerate parts of their body. They will, among other things, fully regenerate an amputated limb by epimorphosis, a biphasic process comprising a preparation phase, specific to the regeneration, and a redevelopment phase, common to epimorphosis and embryonic development. During the preparation phase, the cells of the stump dedifferentiate into embryonic-like cells, proliferate and migrate distally from the level of amputation to form a regeneration blastema. Among vertebrates, the process of dedifferentiation is unique to urodeles. To better understand the molecular control of regeneration in urodeles, we chose to study BMP-2, a growth factor, because of its involvement in mammalian digit tip regeneration. The transcription factor MSX-1 has also been selected because of its ability to induce cellular dedifferentiation in vitro and its potential interaction with BMPs signaling. The results presented in this thesis show that BMP-2 and MSX-1 are expressed during phases of preparation and redevelopment of epimorphosis, and their spatio-temporal expression profiles are very similar at each stage of epimorphosis, suggesting an interaction of their signals during regeneration. In addition, in tetrapod amniotes, the expression of Shh is restricted to the posterior mesenchyme of developing limbs and overlaps with the expression of BMP-2. However, the expression of BMP-2 is not restricted to the posterior region but forms a posterior-anterior gradient. Shh is the main regulator of the anterior-posterior pattern formation of developing limbs. Given the overlapping expression domains of Shh and BMP-2, and the expression restriction of Shh in posterior, Shh is believed to iv regulate the pattern formation of developing limbs by the activation of BMP-2 expression. Interestingly, the axolotl also expresses Shh in the posterior region, but the limb develops from anterior to posterior rather than posterior to anterior as in other tetrapods, and this, during development and epimorphosis. We used this feature of the axolotl to demonstrate that Shh signaling does not regulate pattern formation through BMP-2. Indeed, the expression of BMP-2 is not regulated by the inhibition of hh signaling, and its expression is opposite to that of Shh during development and regeneration of the axolotl limb. It was observed, during limb development in mice that MSX-1 is regulated by Shh signaling. Our results suggest that in the axolotl, MSX-1 is not regulated by the inhibition of Shh signaling during limb regeneration. Furthermore, we demonstrated that unlike the expression of Shh, the expression of BMP-2 is correlated with the order of formation of the phalanges, is involved in cell condensation and apoptosis preceding chondrogenesis. Taken together, these results suggest a role for BMP-2 in the initiation of endochondral ossification. Finally, we demonstrated that BMP signaling is essential for the redevelopment phase of limb epimorphosis.

Le but de cette étude était d’évaluer si la bone morphogenetic protein (BMP)-2 pouvait aider à contrôler le phénotype de chondrocytes humains dans des conditions de culture à long terme nécessaires pour la transplantation de chondrocytes autologues (TCA). Nous avons aussi évalué le potentiel de la BMP-2 comme facteur de réparation du cartilage, en combinaison avec un biomatériau à base de collagène, pour étendre la technique aux lésions arthrosiques. Des chondrocytes humains ont été cultivés selon la procédure utilisée pour la TCA. Nous avons évalué la réponse des chondrocytes à la BMP-2 en culture monocouche ou dans les éponges de collagène par des analyses par PCR en temps réel, par Western blotting et Immunohistochimie.L’ajout de BMP-2 améliore le caractère chondrogénique des chondrocytes humains amplifiés en monocouche. L’effet stimulateur de la BMP-2 sur l’expression du collagène de type II a été observé au niveau des gènes mais aussi des protéines, ce qui est une propriété essentielle pour la reconstruction d’une matrice cartilagineuse. Nos résultats ont aussi montré que dans des chondrocytes tout d’abord amplifiés en monocouche puis cultivés en éponges de collagène en présence de BMP-2, la BMP-2 est capable de restaurer l’expression du gène COL2A1 et la synthèse de collagène de type II qui avaient été perdues pendant l’amplification. De manière importante, aucun signe de maturation hypertrophique ou d’induction ostéogénique n’a été détecté. Cette étude est la première à révéler le bénéfice de l’ajout de BMP-2 à des chondrocytes humains comme un agent thérapeutique pour la réparation du cartilage. / The aim of this study was to investigate if bone borphogenetic brotein (BMP)-2 could help to control human chondrocytes phenotype in long-term culture conditions necessary for autologous chondrocyte implantation (ACI). We also evaluated the potential of BMP-2 as a repair factor in combination with collagen-based biomaterials, to extend the technique to osteoarthritic lesions. Human chondrocytes were cultured independently, according to the procedure used for ACI. We evaluated the responsiveness of chondrocytes to BMP-2 when cultured in monolayer or within collagen sponges using Real-time PCR, Western blotting and Immunohistochemistry. Exogenous BMP-2 improved the chondrogenic character of human chondrocytes when amplified in monolayer. The stimulatory effect of BMP-2 on type II collagen expression was observed not only at the mRNA level but also at the protein level, and this is crucial for cartilage matrix reconstruction. Our data with human chondrocytes first amplified in monolayer then cultured in collagen sponges in the presence of BMP-2 have revealed that BMP-2 is able to restore COL2A1 gene expression and type II collagen synthesis that were lost during the amplification step. Importantly, no sign of hypertrophic maturation or osteogenic induction was detected beside the chondrogenic stimulatory effect of BMP-2. This study is the first to reveal the benefit of adding exogenous BMP-2 to human chondrocytes as a therapeutic agent for cartilage repair.