O presente trabalho teve por objetivo avaliar a atuação de biorreguladores (ANA, BAP, TDZ e 2iP) no desenvolvimento morfogenético de microplantas de pupunheiras. Para tanto, a cada 28 dias aferiram-se o número de propágulos, o desenvolvimento radicular, o comprimento e a taxa de crescimento da parte aérea, número de folhas, oxidação, número de centros meristemáticos na região basal dos explantes e nas bainhas e/ou nos primórdios foliares dos propágulos, número de complexos celulares pró-embriogênicos e as rotas e padrões morfogênicos. Todos os experimentos foram conduzidos em delineamento inteiramente casualizado. Os dados qualitativos foram apresentados como porcentagens e médias gerais e os dados quantitativos foram submetidos à análise de regressão polinomial ou exponencial. As avaliações morfofisiológicas evidenciaram que o TDZ isoladamente favoreceu o desenvolvimento de gemas adventícias, embora curtas e por vezes, com acúleos. Embriões somáticos somente não se desenvolveram em tratamentos contendo a presença isolada de BAP, TDZ e 2iP, nos demais tratamentos observou-se um comportamento exponencial, cujo número foi reduzido e variou apenas em função do período de cultivo. Maior número de raízes foi observado no tratamento contendo ANA em associação ao BAP, embora o tratamento controle tenha induzido maior porcentagem de explantes enraizados e com maior comprimento, sendo o único a evidenciar ramificação. A presença isolada do TDZ promoveu maior comprimento da parte aérea e sua associação ao ANA promoveu a maior taxa de crescimento. O melhor desenvolvimento da parte aérea de microplantas e explantes de pupunheiras ocorreu em meio de cultura MS isento de biorreguladores ou em meio constituído pela associação entre ANA/TDZ aos 140 dias de cultivo, sendo que o acréscimo isolado das citocininas BAP, TDZ e 2iP no meio de cultura MS promoveu tardiamente a taxa máxima de crescimento. A adição isolada do 2iP no meio de cultura promoveu intensa oxidação nas primeiras folhas desenvolvidas das microplantas em explantes de pupunheiras e a aplicação isolada do TDZ ou associada à ANA promoveu anomalias como dobramento foliar e desenvolvimento de acúleos. O monitoramento das rotas e padrões morfogênicos evidenciou que embriões somáticos com origem multicelular originaramse diretamente na região central da base dos explantes nos tratamentos isentos de biorreguladores, na presença isolada de ANA e de TDZ ou na associação de ambos. Pró-embriões com origem unicelular foram induzidos também na presença isolada de ANA ou TDZ e nas associações ANA/BAP ou ANA/TDZ, sendo que pró-embriões com origem multicelular foram induzidos na presença isolada do TDZ a partir da atividade meristemática apical caulinar e a partir da (bi) polarização de elevado número de centros meristemáticos presentes na extremidade distal da base destes explantes. Gemas adventícias com origem multicelular diretamente da extremidade proximal da base dos explantes foram induzidas em maior freqüência no tratamento isento de biorreguladores e no tratamento contendo ANA/TDZ. Entre os primórdios foliares dos explantes, estas estruturas foram freqüentemente induzidas em tratamentos contendo a presença isolada de ANA. / This study aimed to evaluate the role of bioregulators (NAA, BAP, TDZ and 2iP) on the morphogenetic development of pejibaye microplants. For this, every 28 days, the number of propagules, the root development, the length and the growth rate of shoots, the number of leaves, the oxidation, the number of meristematic centers in the explants basal region and in the leaf sheaths and/or in the propagules leaf primordia, the number of pro-embryogenic cell complex and the morphogenic routes and patterns were measured. All experiments were conducted in a randomized design. Qualitative data were presented as percentages and general averages, and quantitative data were subjected to polynomial regression or exponential analysis. The morphophysiological evaluations showed that the TDZ alone favored the development of adventitious buds, although short, and sometimes with thorns. Only in the treatments with the isolated presence of BAP, TDZ and 2iP, somatic embryos were not developed. For the other treatments there was an exponential behavior, in which the number of somatic embryos was reduced and varied according only to the cultivation period. Although a greater number of roots was observed in the treatment, in which NAA was associated with BAP, the control treatment has induced the highest rate of rooted explants and with greater root length, and has been the single one that produced branches. The isolated presence of TDZ promoted the greater shoot length, and its association with ANA promoted the highest growth rate. The best shoot development of pejibaye microplants and explants occurred in the MS bioregulator-free culture medium of or in the medium with NAA/TDZ association, at140 days of cultivation. Moreover, the isolated addition of the cytokinins BAP, TDZ and 2iP in the medium, later promoted the highest growth rate. In pejibaye explants the addition of 2iP alone promoted intense oxidation of the first developed leaves and the application of TDZ alone or associated with ANA promoted anomalies such as folding leaf and thorn development. The monitoring of the morphogenetic routes and patterns showed that in the treatments that were bioregulator-free, or with the isolated presence of NAA and TDZ, or the association of both, somatic embryos, from multicellular origin, raised up directly in the central region of the explants base. Pro-embryos from unicellular origin were also induced in the isolated presence of NAA or in NAA/BAP and NAA/TDZ associations. Moreover, in the isolated presence of TDZ, pro-embryos from multicellular origin were induced from shoot apical meristem activity and from the (bi) polarization of a large number of meristematic centers in the distal extremity of the explants base. Adventitious buds with multicellular origin from the proximal extremity of the explants base were induced more frequently in the bioregulator-free treatment and in the treatment with NAA/TDZ. Between the leaf explants primordia, adventitious buds were often induced by the treatments with the isolated presence of ANA.

As condições divergentes do ambiente in vitro, como elevadas concentrações de sacarose, de reguladores de crescimento, de substâncias tóxicas, quando comparadas com o ambiente ex vitro, refletem-se em um desequilíbrio da relação entre compostos antioxidantes versus compostos oxidantes, resultando em elevada formação de espécies reativas de oxigênio, culminando no estabelecimento de um estresse oxidativo in vitro. No entanto, mesmo sendo capazes de conduzirem a morte celular, as espécies reativas de oxigênio atuam como importantes sinalizadoras do crescimento e desenvolvimento vegetal, por alterarem o padrão de expressão gênica, o metabolismo e a competência celular, sendo nesse aspecto considerado benéfico um nível moderado de estresse oxidativo para desencadear uma determinada rota morfogênica. Diante disso, o presente trabalho objetivou induzir o estresse oxidativo, suplementando o meio de cultura com reguladores de crescimento do grupo das citocininas e auxinas, com a finalidade de compreender a atuação deste evento nas respostas morfogênicas in vitro do pinhão manso (Jatropha curcas L.). Para tanto, utilizou-se sementes de pinhão manso de três procedências (CNPAE 101: Rio Verde, GO; CNPAE 115: Xambrê, PR e CNPAE 224: São Francisco do Glória, MG), as quais foram germinadas in vitro para a obtenção das plântulas e utilização do terço mediano do hipocótilo como explante nos experimentos de indução de estresse oxidativo in vitro. A análise morfofisiológica permitiu selecionar os tratamentos que induziram respostas organogênicas, que juntamente com o grupo controle, tiveram a quantificação da peroxidação lipídica, as atividades das enzimas antioxidantes catalase, superóxido dismutase e ascorbato peroxidase, determinadas, assim como o estabelecimento do perfil proteico de cada tratamento e a realização de análises histológicas e histoquímicas. Os resultados evidenciaram que as respostas morfogênicas foram dependentes do tipo e combinação de regulador de crescimento presente no meio de cultura; e ficou evidente pela quantificação da peroxidação lipídica e das atividades das enzimas antioxidantes, que a ausência de regulador de crescimento no meio de cultura foi o principal indutor do estresse oxidativo, permitindo constatar que quanto mais suave o estresse oxidativo, mais promissoras eram as respostas organogênicas constatadas nos explantes hipocotiledonares. Desta forma, o maior nível de estresse oxidativo constatado no grupo controle, relacionou-se negativamente com a resposta morfogênica obtida neste grupo, quando comparado aos demais tratamentos que apresentaram um nível de estresse oxidativo mais ameno e, consequentemente, uma resposta morfogênica mais promissora. O perfil proteico evidenciou o padrão existente de algumas bandas, independentemente da procedência considerada, confirmando a proximidade genética dos diferentes acessos de pinhão manso no Brasil. As análises histológicas demonstraram a ocorrência de organogênese indireta, com o desenvolvimento de calos organogênicos, ao passo que nas análises histoquímicas somente a presença de lipídios não foi detectada, enquanto proteínas totais, compostos fenólicos e amido foram constatados em pelo menos um dos três materiais analisados, para as três procedências de pinhão manso. Os resultados obtidos possibilitaram estabelecer uma relação entre o estresse oxidativo in vitro, a resposta morfogênica e o efeito dos reguladores de crescimento presentes no meio de cultura, além do potencial sucesso de produção de pinhão manso pela cultura de tecidos. / The environmental divergent conditions in vitro culture , such as high concentrations of saccharose , growth regulators, toxic substances, and others, when compared with the ex vitro conditions, reflected in an imbalance of antioxidants versus oxidant compounds relationship, resulting in an elevated formation of reactive oxygen species, which culminates in the establishment of an oxidative in vitro stress. However, even being able to conduce cell death, reactive oxygen species act as an important signaling of plant growth and development by altering the pattern of gene expression, cellular metabolism and cellular competence, being considerate beneficial in this context in which a moderate level of oxidative stress is required to trigger a morphogenetic route. Therefore, this study aimed to induce oxidative stress, supplementing the culture medium with growth regulators group of cytokines and auxin, with the purpose of understand the role of these event in vitro morphogenetic responses of physic nut (Jatropha curcas L.). Hence, were used physic nut´s seeds of three provenances (CNPAE 101: Rio Verde, GO; CNPAE 115: Xambrê, PR and CNPAE 224: São Francisco do Glória, MG ), which were in vitro germinated to obtain the seedlings and the middle third of hypocotyls that was used as explant in experiments to induction in vitro oxidative stress. The morphophysiological analysis allowed to select the treatments that induced the organogenic responses, which along with the control group, had the quantification of lipid peroxidation, activities of antioxidant enzymes catalase, superoxide dismutase and ascorbate peroxidase, determined as well as the establishment of the protein profile of each treatment and the realization of histological and histochemical analyses. The results showed that the morphogenic responses were dependent on the type and combination of growth regulators present in the culture medium; and with the lipid peroxidation quantification as well as antioxidant enzyme activities, it became evident that the oxidative stress was mainly caused by the absence of growth regulators in the culture medium, allowing noted that the softer oxidative stress, were the most promising responses organogenic found in hypocotyls. Thus, the higher level of oxidative stress observed in the control group correlated negatively with the morphogenic response obtained in this group when compared to the other treatments that showed a level of milder oxidative stress and, consequently, a more promising morphogenic response. The protein profile showed some band pattern, regardless of the provenances considered, confirming the low genetic distance between different accessions of Jatropha in Brazil. Histological analysis demonstrated the occurrence of indirect organogenesis, with the development of organogenic calli, whereas in histochemical analyzes only the presence of lipids was not detected while total proteins, phenolic compounds and starch were found in at least one of the three materials analyzed for the three provenances of physic nut. Taking together all the obtained data, it was possible to establish a relationship between oxidative stress, the morphogenic response in vitro and the effect of growth regulators in the culture medium as well as prove the potential success of the production of jatropha tissue culture.

Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-06-29T15:50:34Z No. of bitstreams: 1 derikribeirodepaiva.pdf: 8307169 bytes, checksum: 3b1448fabaff436fa6ccdfcbe5e4e90d (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-07-03T13:52:19Z (GMT) No. of bitstreams: 1 derikribeirodepaiva.pdf: 8307169 bytes, checksum: 3b1448fabaff436fa6ccdfcbe5e4e90d (MD5) / Made available in DSpace on 2018-07-03T13:52:19Z (GMT). No. of bitstreams: 1 derikribeirodepaiva.pdf: 8307169 bytes, checksum: 3b1448fabaff436fa6ccdfcbe5e4e90d (MD5) Previous issue date: 2018-03-28 / O crescente interesse na representação dos aspectos do relevo tem como justificativa a ampla gama de possibilidades de aplicação dos produtos oriundos dessa técnica cartográfica no que se refere ao planejamento. Diante disso, a demanda por documentos cartográficos que subsidiem os processos de intervenção no relevo tem aumentado. Essa crescente atenção facultada à cartografia geomorfológica nas pesquisas ambientais tem culminado num subsidio não só a Geomorfologia especificamente, como também às diversas ciências que envolvem as mais distintas esferas do planejamento. Diante dessa realidade tornam-se de substancial importância os esforços em prol da pesquisa e desenvolvimento de trabalhos voltados para a produção de mapas e cartas geomorfológicas em escala de alto nível de detalhamento, visando que estas possam vir a subsidiar o processo decisório, em âmbito local, acerca da disposição espacial e tipos de intervenção passíveis ou não de serem desenvolvidas no ambiente em questão. Assim, a presente dissertação tem como objetivo geral a elaboração da carta geomorfológica da bacia hidrográfica do rio Novo, intentando-se que esta possa subsidiar futuras pesquisas que visem a compreensão dos processos e fatores responsáveis pela conformação do relevo local. Como objetivos específicos intenta-se que a mesma possa subsidiar o processo decisório voltado para o planejamento na Zona da Mata Mineira, podendo também vir a fornecer dados básicos de entrada para outras pesquisas. A metodologia adotada para o alcance dos objetivos supracitados concerne à proposta elaborada no âmbito do IBGE, cujo princípio básico trata-se da ordenação dos fatos geomorfológicos conforme uma classificação que leva em consideração aspectos temporais e espaciais, e que viabiliza a distinção dos modelados como unidade básica do relevo, bem como os grupamentos que mantém relações hierárquicas com essas unidades básicas. Dessa forma, foram identificados os tipos de modelados, sendo estes posteriormente diferenciados e caracterizados de acordo com fatores intrínsecos à sua natureza estrutural, litológica, pedológica, climática e morfodinâmica. Como forma de expandir a compreensão acerca dos processos e dinâmicas condicionantes da conformação das paisagens da região foi levada a efeito uma interpretação de cunho morfoestrutural da área, tendo esta possibilitado a identificação de cinco distintos compartimentos morfoestruturais regionais que, em parte, abrangem a área cerne da presente pesquisa. / The increasing interest in the representation of the aspects of the relief has as justification the wide range of possibilities of application of the products coming from this cartographic technique when it comes to planning. In view of this, the demand for cartographic documents that subsidize relief intervention processes has increased. This growing attention given to geomorphological cartography in environmental research has culminated in a subsidy not only to Geomorphology specifically, but also to the various sciences that involve the most distinct spheres of planning. Faced with this reality, efforts to research and develop geomorphic maps and geomorphic charts on a high level of detail are becoming of great importance, aiming at supporting the decision-making process at the local level, about the spatial arrangement and types of intervention that may or may not be developed in the environment in question. Thus, the present dissertation has as general objective the elaboration of the geomorphological chart of the river basin of the rio Novo, trying to be able to subsidize future researches that aim at the understanding of the processes and factors responsible for the conformation of the local relief. As specific objectives, it is intended that it can support the decision-making process geared to planning in the Zona da Mata Mineira, and may also provide basic input data for other surveys. The methodology adopted for the achievement of the aforementioned objectives concerns the proposal elaborated within the scope of the IBGE, whose basic principle is the ordering of geomorphological facts according to a classification that takes into account temporal and spatial aspects, and which makes possible the distinction of those modeled as a unit as well as the groupings that maintain hierarchical relationships with these basic units. In this way, the types of modeling were identified, being later differentiated and characterized according to factors intrinsic to its structural, lithological, pedological, climatic and morphodynamic nature. As a way to expand the understanding of the processes and dynamics conditioning the landscape's conformation, a morphostructural interpretation of the area was carried out, making possible the identification of five distinct regional morphostructural compartments that, in part, cover the core area of this research.

Le bourgeon de membre est, dès son émergence dans le flanc de l'embryon, divisé en deux domaines distincts, l'un antérieur, l'autre postérieur. Nous analysons le rôle des gènes Msx dans cette polarisation. Chez la souris, les gènes Msx constituent une petite famille multigénique comprenant Msx1 et Msx2: ces gènes codent pour des facteurs de transcriptions à homéodomaine. Les souris mutantes pour Msx1 et Msx2 présentent également une surcroissance de la région antérieure du bourgeon de membre, associée à la perte d'un domaine apoptotique, qui conduit à des polydactylies antérieures. La projet vise à élucider les mécanismes par lesquels les Msx agissent sur la morphogenèse de cette région du membre. Au cours de cette investigation, nous avons fait d'intéressantes observations qui indiquent que les Msx interagissent avec Gli3, et nous avons obtenu d'intéressants résultats expérimentaux par co-immunoprécipitation des Msx et de Gli3 (co-IP) dans des cellules en culture transfectées, et par test de ligation de proximité (PLA) sur le bourgeon de membre in situ. La stratégie repose aussi sur l'analyse différentielle des transcriptome d'embryons doubles mutants pour Msx1 et Msx2 et d'embryons normaux, de façon à identifier les cibles qu'ils peuvent avoir en commun avec Gli3, ces dernières étant déjà décrites. Ces modèles devraient permettre de corréler le niveau d'apoptose avec la surcroissance du bourgeon et la polydactylie qui en résulte, apportant une importante contribution aux mécanismes de la morphogenèse qui sont encore très mal compris. / The vertebrate limb is a paradigmatic model for morphogenesis. The anteroposterior (AP) growth and patterning of the limb bud rely on an intricate regulatory genetic network involving many genetic players such Shh and Gli3. Shh is produced in the posterior region of the limb mesoderm and acts as a morphogen along the AP axis. It regulates both digit number and identity of different AP positions. As the main function of Shh is to prevent proteolysis of Gli3FL into Gli3R, Gli3R concentrates anteriorly which is also where apoptosis takes place. In the mouse, paradoxically, despite a quasi-symmetrical expression profile, Msx1/2 null mutants show systematic anterior defects with an overgrowth in the anterior limb domain, resulting in anterior polydactyly. Both Gli3 and Msx mutant limb defects are concentrated anteriorly in a similar fashion suggesting an interaction between these genes and a possible role of this interaction in AP patterning in the limb bud as well as dysregulation of apoptosis. Indeed, we demonstrate interactions between Msx and Gli3 genes and even proteins. Mutations of Msx and Gli3 result in anterior polydactylous phenotypes and a similar loss of anterior apoptosis. This morphological analysis is associated with a search for common Gli3 and Msx transcriptional targets and partners in an attempt to link gene activity with changes in cell physiology that underlie morphogenesis. We have performed a differential transcriptome (RNA-Seq) on whole limb buds of Msx1-/-Msx2 narrowing down on a number of potential common targets of Gli3 and Msx. We demonstrate that Msx and Gli3 work together in regulating the AP axis of limb development, independent of Shh.

Developing tissues are out-of-equilibrium systems that grow and reshape to form organs in adult animals. They are typically composed of a large number of cells. The constitutive cells of a tissue perform different roles in tissue development and contribute to the overall tissue shape changes. In this thesis, we construct a hydrodynamic theory of developing epithelial tissues. We use it to investigate the developing wing of the fruit fly Drosophila melanogaster. This theory relates the coarse-grained cell scale properties to the large-scale tissue flows. We explicitly account for the active cellular processes in the tissue that drive tissue flows. In our description of the tissue, we also include the memory effects that are necessary to account for the experimental observations. These memory effects have a significant influence on the tissue rheology. Using this hydrodynamic theory we analyze shear flow in a developing fruit fly wing tissue. We find that the active cellular processes contribute to overall tissue flows and that memory effects are present in the wing tissue. We investigate consequences of these findings on the rheology of tissue shear flow. We find that the memory effects give rise to an inertial response that leads to oscillations in the tissue but it does not stem from the wing mass. Finally, we describe how the tissue rheology is affected by different boundary conditions. We then investigate the area changes during the pupal wing development and we construct a mechanosensitive model for the cell extrusion rate in the pupal wing. Analysis of cell extrusions in the context of this model also allows us to extract information about the cell division properties. Boundary connections between the wing tissue and surrounding cuticle are crucial for the proper development of the pupal wing. A dumpy mutant wing is strongly misshaped during the pupal wing morphogenesis. We use a simple model for the wing to show that the dumpy mutant wing can be described as a wild type wing with compromised boundary conditions. Finally, we analyze cell properties and tissue flows in a developing wing disc epithelium. Motivated by the observation of radially oriented active T1 transitions in the wing disc epithelium, we use the hydrodynamic theory to investigate the influence of such T1 transitions on stresses in the tissue. We show that sufficiently strong radially oriented active T1 transitions can contribute to the control of the tissue size. Results obtained in this thesis extend our understanding of the fly wing tissue rheology and the role of internal and external forces in the proper shaping of the wing epithelium. The hydrodynamic theory we use to describe the fly wing development provides a set of phenomenological parameters that characterize the tissue mechanics and can be experimentally measured. Therefore, we expect that future research will include and extend the hydrodynamic theory presented in this thesis.

Parmi les processus fondamentaux qui ont lieu pendant le développement d'un organisme, la morphogenèse est un des plus complexes. De nombreuses études expérimentales ont contribué à mieux comprendre les mécanismes morphogénétiques dans les organismes vivants. Cependant peu de modèles mathématiques ont été proposés afin d'étudier la morphogenèse dans les tissus vivants. Dans ce contexte, la thèse se propose de développer de nouveaux modèles mathématiques pour étudier les changements de forme dans les tissus mous tubulaires. L'approche adoptée est macroscopique où le tissu biologique est considéré comme un milieu continu déformable. L'hypothèse principale sur laquelle se basent les modèles proposés est la suivante: pendant les processus de croissance et remodelage, des contraintes résiduelles peuvent s'accumuler dans le tissu et, une fois une valeur critique dépassée, le mener à un changement morphologique sous la forme d'une instabilité élastique. Pour cela, les modèles développés intègrent les théories modernes de croissance et remodelage, dans le cadre de la thermomécanique des systèmes ouverts. Ensuite, l'analyse de stabilité linéaire permet de calculer les seuils et modes de l'instabilité élastique en utilisant la méthode des déformations incrémentales superposées aux déformations finies. L'ensemble de ces techniques (théorie morpho-élastique) est utilisé dans cette thèse afin de modéliser deux différents processus morphogénétiques ayant lieu dans les tissus mous tubulaires : la formation d'une variété des formes dans le système gastro-intestinal et le flambage hélicoïdal dans les organes tubulaires avec précontraintes. / Among the fundamental processes involved in the development of an organism, morphogenesis is one of the most complex. During the past centuries, an amount of experimental studies have improved our actual knowledge of the mechanisms which drive many morphogenetic processes in living organisms. Only recently, experiments have been complemented with mathematical modeling as a tool for proving novel insights on morphogenesis in soft tissues. In this context, this thesis aims at developing new mathematical models for the formation of patterns and forms in soft tubular organs. A macroscopic approach is adopted, where the tissue is considered as a continuum body undergoing growth and remodeling. The main idea behind the proposed models is that during growth and remodeling, residual stresses can arise and once they exceed a critical value, an elastic instability can occur in the tissue and lead to a morphological change. Therefore, the morphoelastic models are developed integrating the modern theories of growth and remodeling within the framework of the thermo-mechanics of open systems. The occurrence of the elastic instability is investigated using the method of incremental deformations superposed on finite deformations. The critical thresholds for the onset of the instability are determined together with the modes of the associated instability pattern. The morphoelastic theory is applied to the modeling of different morphogenetic processes occurring in soft tubular organs and gives useful insights in two interesting problems: the formation of the wide range of patterns in the gastro-intestinal system and the occurrence of torsional instabilities in pre-stressed tubular organs.

ABSTRACT: Formation of organ shape is an intriguing yet largely unanswered question in developmental biology. Shapes arise as a result of tightly controlled spatial variation in the rates and directions of tissue expansion over the course of development; therefore, quantifying these growth patterns could provide information about the underlying mechanisms of morphogenesis. Here we present a novel technique and computational tools for quantifying growth and shape changes in developing leaves, with a few unique capabilities. This includes the ability to compute growth from three-dimensional (3D) coordinates, which makes this the first method suitable for studying leaf growth in species or mutants with non-flat leaves, as well as small leaves at early stages of development, and allows us to simultaneously capture 3D shape changes. In the following, we apply these methods to study growth and shape changes in the first rosette leaf of Arabidopsis thaliana. Results reveal clear spatiotemporal patterns in growth rates and directionality, and tissue deformation maps illustrate an intricate balance involved in maintaining a relatively flat leaf surface in wild type leaves. Semi-automated tools presented make a high throughput of data possible with this method, and algorithms for generating mean maps of growth will make it possible to perform standardized comparative analyses of growth patterns between wild type and mutants and/or between species. The methods presented in this thesis will therefore be useful for studying leaf growth and shape, to further investigate the mechanisms of morphogenesis.   RÉSUMÉ: Comment un organe acquiert sa forme particulière au cours du développement est une question intéressante mais largement non résolue. La forme d’un organe résulte de la façon dont les taux et directions de croissance de ses tissues varient dans l’espace et dans le temps. Quantifier les motifs de croissance est donc nécessaire pout élucider les mécanismes sous-jacents de la morphogenèse. Nous présentons ici une nouvelle méthodologie pour quantifier la croissance et les changements de forme dans les feuilles en développement. Cette méthodologie s’appuie sur le développement de nouvelles techniques expérimentales et de programmes informatiques, et présente des avantages uniques : la croissance de la surface des feuilles et le changement de forme peuvent être analysés en trois dimensions (3D), pour une longue période et de large déformations. De plus l’analyse de multiples échantillons permet de générer une cartographie moyenne des motifs de croissance à la surface des feuilles au cours de leur développement, ainsi qu’une description quantitative de la déformation des tissus sous l’effet de leur croissance. Dans cette thèse, nous présentons les résultats de croissance et de changements de forme de la première feuille de rosette d'Arabidopsis thaliana au cours de son développement. Les cartes moyennes de croissance révèlent des motifs spatio-temporels évidents tant pour les taux que pour les directions de croissance. De plus, la description de la déformation des tissus démontre l'équilibre complexe impliqué dans le maintien d'une surface relativement plane dans les feuilles. La méthode proposée et les logiciels associés permettra d’effectuer des analyses comparative de la croissance entre feuilles de type sauvage et feuilles de mutants aux formes altérées, afin d’élucider les mécanismes de la morphogenèse foliaire.

In order to effectively treat disorders whose pathology is marked by neovascularization, a better understanding of the pathways that mediate the processes involved in angiogenesis is needed. To this end we have identified two important pathways that regulate endothelial cell capillary morphogenesis, a key process in angiogenesis. We have identified the small GTPase RhoB as being induced by vascular endothelial growth factor (VEGF) in human umbilical vein endothelial cells (HUVECs). Depletion of RhoB inhibited endothelial cell VEGF - mediated migration, sprouting, and cord formation. Cells depleted of RhoB showed a marked increase in RhoA activation in response to VEGF. Defects in cord formation in RhoB - depleted cells could be partially restored through treatment with the Rho inhibitor C3 transferase or ROCK I/II inhibitors, indicating increased RhoA activity and enhanced downstream signaling from RhoA contribute to the phenotype of decreased cord formation observed in cells depleted of RhoB. Interestingly, we did not observe a significant change in RhoC activity in RhoB - depleted cells suggesting differential regulation of RhoA and RhoC by RhoB in HUVECs. We have also identified microRNA - 30b (miR - 30b) as being negatively regulated by VEGF and as being a negative regulator of HUVEC capillary morphogenesis. Overexpression of miR - 30b significantly reduced HUVEC cord formation in vitro, while inhibition of miR - 30b enhanced cord formation. Neither overexpression nor inhibition of miR - 30b affected migration or viability of endothelial cells. Interestingly, miR - 30b regulated the expression of TGFβ2 but not TGFβ1, with overexpression of miR - 30b inducing expression of TGFβ2 mRNA and protein, and inducing phosphorylaton of Smad2 , suggesting TGFβ2 produced in response to miR - 30b overexpression functions in an iii autocrine manner to stimulate HUVECs . MiR - 30b effects on TGFβ2 expression were found to be regulated to an extent by ATF2, as miR - 30b overexpressing cells exhibited increased levels of phosphorylated ATF2 , with depletion of ATF2 via siRNA resulting in inhibition of miR - 30b - induced TGFβ2 expression. Treatment of HUVECs with TGFβ2 inhibited cord formation, while TGFβ1 had no effect, indicating a major difference in how endothelial cells respond to these two related growth factors. Inhibition of TGFβ2 with a neutralizing antibody restored cord formation in miR - 30b overexpressing cells to levels similar to control cells, thus identifying TGFβ2 expression as contributing to the inhibitory effects of miR - 30b overexpression on capillary morphogenesis. Thus, we have identified two signaling pathways regulated by VEGF in HUVECs that further our understanding of the process of angiogenesis and may provide novel targets for therapeutic intervention into diseases involving angiogenesis.

Comment les signalisations dynamiques et les mouvements morphogénétiques régionalisent et permettent la formation de tissus complexes durant l'embryogenèse est très peu compris. J’ai caractérise au cours de ma thèse, les évènements signalisants qui sont mis en place au cours de la myogenèse précoce chez l'embryon de poulet. J'ai montre que les progénitures musculaires présents dans les somites requièrent l'activation dynamique des voies de signalisation Wnt et Notch. L’activation transitoire de la signalisation Notch est requise pour adopter un destin myogénique. Le ligand de Notch Dll1 est exprime de manière mosaïque dans les cellules migrantes des crêtes neurales qui passent près du somite. Gain et perte de fonction de Dll1 dans les crêtes neurales modifient la signalisation Notch dans les somites, résultant en un délai ou une prématuré myogenèse. Nos résultats indiquent que les crêtes neural régulent la formation précoce du muscle par un mécanisme unique mené par la migration des cellules des crêtes neurales exprimant Dll1 qui déclenche l'activation transitoire de la signalisation Notch dans certains progénitures musculaires sélectionnes. Cette dynamique signalisation garantie une différentiation progressive du pool de progénitures musculaires. / How dynamic signalling and extensive tissue rearrangements interplay to generate complex patterns and shapes during embryogenesis is poorly understood. During my PhD, I have characterized the signalling events taking place during early morphogenesis of chick skeletal muscles. I observed that muscle progenitors present in somites require dynamic activation of Wnt and Notch signalling. I showed that a transient activation of NOTCH signalling is required to undergo terminal differentiation. The NOTCH ligand Delta1 is expressed in a mosaic pattern in neural crest cells that migrate past the somites. Gain and loss of Delta1 function in neural crest modifies NOTCH signalling in somites, which results in delayed or premature myogenesis. These results suggest that the neural crest regulates early muscle formation by a unique mechanism that relies on the migration of Delta1-expressing neural crest cells to trigger the transient activation of NOTCH signalling in selected muscle progenitors. This dynamic signalling guarantees a balanced and progressive differentiation of the muscle progenitor pool.

La structure polygonale des épithéliums mono-stratifiés exerce une certaine fascination sur les biologistes depuis les observations originales par Robert Hooke en 1665. Cependant, il est difficile d‘expliquer comment la stabilité de la morphogenèse est atteinte, i.e. comment les structures polygonales maintiennent la régularité au sein d'un individu, entre les individus et au sein des phylums. Dans ces travaux, nous introduisons une nouvelle mesure quantitative de la stabilité de la morphogenèse entre individus appelée l'homéostasie topologique. Nous démontrons que les épithéliums non-prolifératifs, formés par un processus d'accrétion, sont plus stables que les épithéliums prolifératifs. Dans le contexte de prolifération, l'homéostasie topologique dépend du rapport apoptose/mitose comme en témoigne le modèle Drosophila où l'homéostasie épithéliale diminue drastiquement quand l'apoptose est inhibée dans les disques imaginaux. Ainsi, l'apoptose agit comme un régulateur positif dans la canalisation de la stabilité de la morphogenèse. En outre, des simulations numériques reproduisant la morphogenèse épithéliale, basées sur la physique des milieux divisés, décrivent comment les mécanismes d'accrétion dans les épithéliums non prolifératifs et l'apoptose dans les épithéliums prolifératifs sont des moyens efficaces pour parvenir à la stabilité morphogénétique. / The polygonal structure of mono-stratified epithelia exerts a unique fascination among biologists since the original observations of Robert Hooke in 1665. However, it is always unclear how the stability of morphogenesis is achieved, i.e., how these polygonal structures maintain regularity among individual, between individuals and among all phyla, and among individuals for each tissue within each species. Here, we introduce a new and quantitative measure of the level of morphologic stability between individuals, referred to as topological homeostasis. We demonstrated that non-proliferative epithelia, formed by an accretion process, are significantly more regularly stabilized than proliferative ones. In proliferative context, topological homeostasis directly depends on the apoptosis/mitosis ratio, as evidenced in the Drosophila imaginal disc model, where topological homeostasis drastically drops down when apoptosis is inhibited. Apoptosis therefore acts as an unexpected positive regulator in the canalization of morphogenetic stability. In addition, numerical simulations of epithelial morphogenesis, based on the physics of devided media, described how accretion mechanisms in non-proliferative epithelia, and, apoptosis in proliferative ones, are efficient means to achieve morphogenetic stability.