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.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Este trabalho tem como objetivo 1) avaliar o efeito da BMP-15 e do FSH, sozinhos ou em diferentes associaÃÃes sobre o crescimento, sobrevivÃncia e formaÃÃo de antro em folÃculos secundÃrios bovinos e 2) avaliar os nÃveis de expressÃo do RNAm para os receptores de BMP-15 (BMPR-IB e BMPR-II) em folÃculos crescidos in vitro. Com relaÃÃo ao cultivo, folÃculos secundÃrios foram microdissecados e cultivados por doze dias em &#945;-MEM+ sozinho, ou suplementado com BMP-15 e/ou FSH, sozinhos ou em diferentes associaÃÃes. ApÃs o cultivo, foram avaliados o diÃmetro folicular, a viabilidade e a taxa de formaÃÃo de antro. A anÃlise de expressÃo gÃnica foi realizada por PCR em tempo real, utilizando folÃculos cultivados nos diferentes tratamentos, para comparaÃÃes dos nÃveis de RNAm. ApÃs o perÃodo de cultivo verificou-se que a utilizaÃÃo da BMP+FSH (0-12) estimulou um aumento significativo no diÃmetro folicular em relaÃÃo aos demais tratamentos (p<0,05), bem como estimulou a formaÃÃo da cavidade antral de forma mais efetiva que o controle (MEM) e a BMP-15 sozinha (p<0,05). No entanto, em relaÃÃo à sobrevivÃncia, apÃs12 dias o tratamento BMP+FSH (0-12) reduziu significativamente a viabilidade folicular em relaÃÃo aos demais tratamentos (p<0,05). Os resultados mostraram que os nÃveis de RNAm para o receptor de BMP-15 do tipo I (BMPR-IB) foram significativamente maiores nos folÃculos cultivados com MEM quando comparado aos demais tratamentos, enquanto que os nÃveis de RNAm para o receptor do tipo II (BMPR-II) nÃo diferiram entre os tratamentos. Os maiores nÃveis de RNAm para o receptor de FSH foram detectados em folÃculos cultivados com BMP-15 sozinha. Em conclusÃo, este estudo demonstrou que a combinaÃÃo de BMP-15 + FSH durante todo o perÃodo de cultivo (D0-D12) acelera o crescimento de folÃculos prÃ-antrais bovinos cultivados por 12 dias, mas este efeito reduz a viabilidade folicular, afetando a integridade ultra- estrutural dos folÃculos. AlÃm disso, a adiÃÃo de BMP isoladamente aumentou os nÃveis de expressÃo de RNAm para FSHR apÃs 12 dias de cultivo,mostrando que BMP-15 e FSH podem contribuir significativamente para o desenvolvimento in vitro de folÃculos prÃ-antrais cultivados por 12 dias nesta espÃcie. / This study aims to 1) evaluate the effect of BMP-15 and FSH, alone or in different combinations on the in vitro development, survival and antrum formation of bovine secondary follicles and 2) quantify the levels of mRNA expression for BMP-15 receptors (BMPR-IB and BMPR-II), FSH receptor and Kit Ligand in follicles grown in vivo and in vitro. With regard to culture, secondary follicles were microdissected and cultured for twelve days in &#945;- MEM+ alone, or supplemented with BMP-15 and/or FSH, alone or in various associations. After culture, follicular diameter were evaluated, viability and rate of antrum formation. For the analyzes gene expression, by real time PCR, were used follicles cultured in the different treatments for comparisons of the levels of mRNA. After the culture period it was found that the association of BMP+FSH (0-12) stimulated a significant increase in follicular diameter in relation to other treatments (p<0.05), as well as stimulated the formation of antral cavity more effectively the control (MEM) and BMP-15 alone (p<0.05). However, for survival, after 12 days treatment BMP+FSH (0-12) significantly reduced follicular viability when compared to other treatments (p<0.05). The results showed that the levels of mRNA for BMP receptor type I (BMPR-IB) were significantly higher in follicles cultured with MEM when compared to other treatments, while levels of mRNA for the receptor type II (BMPR-II) did not differ between treatments. The highest levels of mRNA for the FSH receptor were detected in follicles cultured with BMP-15 alone. In conclusion, this study demonstrated that combination of BMP-15+FSH (D0-D12) accelerates the growth of bovine preantral follicles cultured for 12 days, but this effect reduces the follicular viability, affecting the ultrastructural integrity of the follicles. Furthermore, the addition of BMP alone increased expression levels of RNAm for FSHR after 12 days of culture , showing that BMP-15 and FSH may contribute significantly to the in vitro development of preantral follicles cultured for 12 days in this specie.

Objetivo: O propósito deste estudo é avaliar a regeneração óssea nos defeitos alveolares congênitos com proteína morfogenética óssea (rhBMP-2) em membrana de colágeno reabsorvível. Material e Métodos: Vinte e quatro pacientes com fissura labiopalatina completa unilateral, em momento ideal, foram distribuídos ao acaso entre os grupos controle (porção medular de ilíaco) e teste (rhBMP-2 em membrana de colágeno absorvível). Foram avaliados em 6 meses e 1 ano de pós-operatório por meio de radiografias periapicais e tomografias computadorizadas pela escala de Chelsea. Resultados: Encontraram-se índices de sucesso quanto à altura do septo ósseo semelhantes entre os grupos: em 6 meses, na análise radiográfica, 81,82% no controle e 91,67% no teste; em 1ano, 83,33% no controle e 100% no teste; na análise tomográfica, observaram-se 70% no controle e 91,67% no teste em 6 meses, 75% no controle e 100% no teste em 1 ano. Na análise volumétrica, os valores encontrados foram 45,34% de preenchimento ósseo da fissura no grupo controle aos 6 meses e 40,63% no teste, 53,33% no controle e 53,1% no teste em 1 ano. Não houve diferença estatisticamente significante. Conclusão: A rhBMP-2 em membrana reabsorvível de colágeno produziu resultados semelhantes ao enxerto autógeno de medula de crista ilíaca ao reparar defeitos alveolares de fissuras labiopalatinas completas. / Objective: The purpose of this study is to assess the bony regeneration of alveolar clefts using bone morphogenetic protein (rhBMP-2) in a collagen sponge carrier. Material and Methods: Twenty four patients with unilateral complete cleft lip and palate, in the very best moment, were randomly assigned to groups control (iliac crest cancellous bone) and test (rhBMP-2 in an absorbable collagen sponge). They were evaluated 6 months and 1 year postoperative with periapical radiographs and computed tomographs using Chelsea scale. Results: For control group, radiographic examination revealed success index for bone height 81,82% 6 months and 83,33% 1 year postoperative; for test group, findings were 91,67% first control and 100% 1 year. Computed tomograph revealed 70% 6 months and 75% 1 year for control group, 91,67% first control and 100% 1 year for bone height test group. Volume ratios were 45,34% bone filling 6 months and 53,33% 1 year for control group and 40,63% 6 months and 53,1% 1 year for test. There was no statistical significance. Conclusion: rhBMP-2 in a collagen sponge carrier induced bony regeneration close to that from iliac crest cancellous bone to repair complete alveolar clefts.

Les cellules multiciliées (MCC) possèdent à leur surface apicale des centaines de cils mobiles générant un flux directionnel liquidien nécessaire par exemple pour le nettoyage des voies respiratoires. La fabrication de ces cils (multiciliogénèse) requiert une séquence d’évènements cellulaires dont un arrêt du cycle cellulaire, une réorganisation du réseau apical d’actine, une multiplication massive des centrioles suivie de leur migration au pôle apicale et de leur maturation en corps basal, à partir desquels les cils s’allongent.Mon laboratoire d’accueil a mis en évidence le rôle conservé de la famille de microARN miR-34/449 dans le contrôle de la multiciliogénèse en inhibant la voie de signalisation Notch ainsi qu’en induisant un arrêt du cycle. Au cours de ma thèse, j’ai mis en évidence un nouveau niveau de régulation de ces microARN par lequel ils contrôlent la réorganisation apicale du cytosquelette d’actine, en modulant l’expression et l’activité de certaines petites GTPases. Par ailleurs, j’ai identifié et caractérisé des séquences variantes des miR-34/449 canoniques, appelées isomiR. Tandis que ces isomiR partagent des fonctions semblables à celles de leurs homologues canoniques, ils apportent également une complémentarité d’action en modulant des transcrits cibles spécifiques. Enfin, le dernier axe de mon travail a permis d’identifier le rôle de la voie de signalisation BMP dans la multiciliogénèse ainsi que d’élucider certains des mécanismes moléculaires par lesquels elle contrôle ce phénomène. L’ensemble de nos découvertes offre une opportunité inédite pour développer des stratégies thérapeutiques dans le traitement de maladies associées à des désordres ciliaires / Vertebrate multiciliated cells (MCC) project hundreds of motile cilia at their apical surface which coordinately beat to generate a directional fluid flow necessary for many biological functions including airway cleansing. Biogenesis of multiple cilia (multiciliogenesis) follows different key cellular steps corresponding to a cell cycle arrest, a massive multiplication of centrioles which then migrate to the apical surface to form basal bodies, from which cilia elongate. In 2011, my host laboratory evidenced that the miR-34/449 family of microRNAs control vertebrate multiciliogenesis by inducing the cell cycle arrest and by repressing the Notch pathway. My thesis work has revealed a new role of miR-34/449 by demonstrating that they modulate expression and activity of small GTPases to drive the apical reorganization of the actin network, a prerequisite for basal body anchoring. Besides, I have identified and characterized variant sequences of canonical miR-34/449 family, named isomiRs. Whereas these isomiRs share common biological functions with canonical miR-34/449 miRNAs, they may also contribute to a complementary effect by targeting specific transcripts. Finally, the last part of my work has contributed to the identification of the conserved role of the BMP pathway in the control of multiciliogenesis. I have evidenced some molecular mechanisms by which the BMP signal controls this phenomenon. Importantly, I demonstrated that BMP inhibition promotes regeneration of tracheal MCC in vivo in an asthmatic mouse model. Overall, our findings offer an unprecedented opportunity to develop novel therapeutic strategies to treat diseases associated with ciliary disorders

The passage of the Federal Water Pollution Control Act of 1972 required states to develop forestry BMPs to help reduce potential nonpoint source pollution from forest operations. Properly applied forestry best management practices (BMPs) have since been proven to protect water quality from forest operations. This research project reviewed BMP effectiveness studies in the U.S., assessed current state developed and implemented of forestry BMPs, and developed a simple method to estimate potential erosion from forest operations for the Piedmont physiographic region based on previous studies. Eighty-one BMP effectiveness studies were reviewed. The review of past effectiveness studies indicates that water quality protection is increased when BMPs are implemented correctly. These effectiveness studies provide states with valuable information on how their BMP guidelines are achieving the goals defined by the Federal Water Pollution Control Act. Every U.S. state has forestry BMP guidelines. These guidelines may be non-regulatory, quasi-regulatory, or regulatory depending on the state. Twenty states reported implementing non-regulatory BMP guidelines, 19 quasi-regulatory BMP guidelines, and 11 regulatory BMP guidelines. State forestry agencies were reported as being the lead agency responsible for BMP monitoring in 35 states. The national forestry BMP implementation rate was 91% (32 states). However, states did report deficiencies for specific BMP guideline categories. Supplementary to the reviewed BMP effectiveness studies, forest erosion studies in the southeastern U.S. that quantified erosion rates from forest operations were also reviewed. Erosion rates obtained from the literature were reviewed by operation categories (timber harvesting, forest roads, skid trails, log landings, stream crossings, and streamside management zones) and physiographic region (Mountains, Piedmont, Gulf Coastal Plain, and Atlantic Coastal Plain). There were numerous research gaps regarding erosion rates from forest operations for all the regions except the Piedmont region. The Piedmont region was selected for developing a method to estimate potential erosion from forest operations. This erosion estimation method is a quick and potentially useful tool for estimating potential erosion; however, it is based on limited data from the Piedmont region only. The basic method approach might be considered for the other physiographic regions, but further research is needed to fill current knowledge gaps. / Ph. D.

Master of Landscape Architecture / Department of Landscape Architecture/Regional and Community Planning / Mary Catherine E. Kingery-Page / Currently, about 15-20 percent of the worldwide tourist industry, or 70-90 billion dollars can be accounted for annually by mountain tourism (Price et al. 1997). The base of mountain tourism lies within the profound natural features of the surrounding mountain landscape. Therefore, maintaining clean cool air, dramatic variations in topography, and scenic beauty of both the physical and cultural landscapes are imperative. The following project addresses the issue of designing a sustainable mixed use community within the broader context of a top rated mountain resort. Within the last decade as public awareness of green practices has increased, so has the public’s knowledge of terms such as LEED (Leadership in Energy and Environmental Design), sustainability, and mixed-use development. Starting in 1995 a sustainable visioning plan emerged for the future development of the Greater Wasatch in Utah. The project, titled Envision Utah Quality Growth Plan, has been widely recognized as one of the country’s most successful efforts to involve the public in regional visioning. The proposed addition to Park City Mountain Resort in the Wasatch Front has been fueled by a desire to create an ecologically, socially, and economically sustainable mixed-use development. The Envision Utah Quality Growth Plan and a precedent study in Whistler, combined with literature by Peter Calthorpe, Sherry Dorward, and Ian McHarg, provided the foundation for a sustainability assessment framework. The framework is applied to test the sustainable viability of existing resorts, as a guide for the design of resorts, and as a tool for comparative analysis between mountain resorts. The result is a conceptual master plan for Park City, Utah that employs the use of stormwater and architectural best management practices, recycled materials, mixed use design, alternative forms of energy, and an efficient public transportation system.

Les BMPs (Boue Morphogenetic Proteins) sont des morphogènes membres de la superfamille du TGF-[bêta] qui interagissent avec des récepteurs de surface cellulaire à activité sérine/thréonine kinase. L'interaction des BMPs avec ces récepteurs entraîne l'activation d'une cascade de signalisation cellulaire impliquant les facteurs de types R-Smads (Smad1, 5 et 8). La voie de signalisation des BMPs joue des rôles cruciaux dans des processus biologiques tels que l'embryogenèse et l'organogenèse des tissus ainsi que des processus cellulaires tels que la prolifération, la migration et la différenciation cellulaire. De plus, ces derniers semblent aussi impliqués dans les processus de mort cellulaire et dans la tumorigenèse. Malgré un intérêt grandissant pour la signalisation des BMPs, il existe très peu d'études sur les rôles spécifiques joués individuellement par les différents Smads dans la morphogenèse de l'intestin in vivo . Ceci est principalement dû au fait que la délétion classique des différents effecteurs de la voie des BMPs entraîne la mort in utero à cause de multiples défauts dans l'embryogenèse. Le système Cre/loxP, sous le contrôle d'un promoteur tissu spécifique, a été utilisé dans notre laboratoire, dans le but de générer une lignée murine possédant une délétion conditionnelle de l'effecteur Smad5 strictement au niveau de l'épithélium intestinal. Une analyse comparative à l'aide d'un modèle de délétion conditionnelle du récepteur BmpR1a au niveau de l'épithélium intestinal a été effectuée afin de décortiquer spécifiquement le rôle de l'effecteur Smad5 dans le développement de cet organe. Afin de valider les résultats obtenus in vivo nous avons généré un modèle cellulaire nous permettant de mimer l'effet de la délétion de l'effecteur Smad5 à l'aide de la technologie du shRNA.Les résultats obtenus dans les deux modèles suggèrent que Smad5 serait un facteur clé impliqué dans la régulation de la migration cellulaire des entérocytes. En effet, l'invalidation de la voie des BMPs et plus particulièrement de l'effecteur Smad5 dans les souris entraîne une augmentation de la vitesse de migration des cellules le long de l'axe crypte villosité. Ces résultats sont corroborés dans un modèle cellulaire dans lequel l'expression de Smad5 a été inhibée par interférence d'ARN. Dans ce modèle, la migration se fait de façon beaucoup plus compacte en comparaison aux cellules contrôles. L'augmentation de la vitesse de migration cellulaire pourrait être due à un phénomène de relocalisation des protéines de jonctions adhérentes ainsi qu'à une modulation de l'actine filamenteuse. Ce phénomène pourrait faire intervenir les petites protéines G Rho/Rac ainsi que les kinases Src. En plus de l'actine filamenteuse, différentes protéines impliquées dans la formation des complexes de jonctions adhérentes semblent relocalisées dans nos deux modèles (E-cadhérine/[bêta]-caténine). En conclusion, les différents résultats recueillis au cours de mes travaux de maîtrise dans le laboratoire du Dr. Nathalie Perreault nous suggèrent que l'effecteur Smad5 de la voie des BMPs serait un facteur impliqué dans la stabilité des complexes de jonctions adhérentes, régulant ainsi la migration des cellules le long de l'axe crypte villosité.

Cranial placodes are transient thickenings of the vertebrate embryonic head ectoderm that will give rise to sensory (olfactory, lens, and otic) and non-sensory (hypophyseal) components of the peripheral nervous system (PNS). In most vertebrate embryos, these four sensory placodes undergo invagination. Epithelial invagination is a morphological process in which flat cell sheets transform into three-dimensional structures, like an epithelial pit/cup. The process of invagination is crucial during development as it plays an important role for the formation of the lens, inner ear, nasal cavity, and adenohypophysis. Using the chick as the model system the following questions were addressed. What signals are involved in placode invagination? Is there any common regulatory molecular mechanism for all sensory placode invagination, or is it controlled by unique molecular codes for each individual placode? Are placode invagination and acquisition of placode-specific identities two independent developmental processes or coupled together? To address this we used in vivo assays like electroporation and whole embryo culture. Our in vivo results provide evidence that RhoA and F-actin rearrangements, apical constriction, cell elongation and epithelial invagination are regulated by a common BMP (Bone morphogenetic protein) dependent molecular mechanism. In addition, our results show that epithelial invagination and acquisition of placode-specific identities are two independent developmental processes. BMP signals have been shown to be essential for lens development and patterning of the retina. However, the spatial and temporal requirement of BMP activity during early events of lens development has remained elusive. Moreover, when and how retinal cells are specified, and whether the lens plays any role for the early development of the retina is not completely known. To address these questions, we have used gain- and loss-of-function analyses in chick explant and intact embryo assays. Here, we show that during lens development BMP activity is both required and sufficient to induce the lens specific marker, L-Maf. After the L-Maf upregulation the cells are no longer dependent on BMP signaling for the next step of fiber cell differentiation, which is characterized by up-regulation of δ-crystallin expression. Regarding the specification of retinal cells our results provide evidence that at blastula stages, BMP signals inhibit the acquisition of eye-field character. Furthermore, from optic vesicle stages, BMP signals emanating from the lens are essential for maintaining eye-field identity, inhibiting telencephalic character and inducing neural retina cells.

No description available.

The brain is our most complex organ, with an estimated 1011 neurons. With the spinal cord, it forms the central nervous system which controls our movements and our senses, holds our memories and creates our thoughts. Because of this, neurodegenerative disorders can be extremely distressing and a thorough understanding of how the nervous system develops is essential if progress is to be made in finding ways to treat them. Critically, this includes understanding how the nervous system forms, i.e., the nature of the signals that promote neural identity (neural induction) and determine correct positional information (patterning). The zebrafish (Danio rerio) has become established as a model for embryological studies due to ease of experimental manipulation. Taking advantage of this, the aims of this PhD were to contribute to unravelling the molecular mechanisms of neural induction and patterning, using a variety of embryological and molecular methods. In the first project, functional analyses of the eve1 gene identified a key factor for posterior neural development. Eve1 was found to be a critical posteriorising factor, with an additional role in posterior neural induction. An outstanding question in neural induction is the relative contribution to this process of two key developmentally important signalling pathways, Bmp and Fgf. In the second project, differential analyses of maternal versus zygotic Bmp and Fgf signalling revealed crucial maternal roles for these two pathways in neural development as neural and epidermal capacitators. The results further suggested that Fgf signalling may be the critical neural inducer. Finally, as a third project, a zebrafish ectodermal explant assay was developed using the organiser-deficient ichabod mutant. The aim was to develop a system to analyse how key molecules directly affect ectoderm and neural development, free of mesoderm and endoderm influences, as signalling from these layers can directly or indirectly influence neural development.