Orientador: Pablo Agustin Vargas / Dissertação (mestrado) - Universidade Estadual de Campinas. Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-15T12:35:10Z (GMT). No. of bitstreams: 1 Alves_DanielBerrettaMoreira_M.pdf: 2353528 bytes, checksum: c4fc62eb44bbd4f300dbdb9d7dc74cd6 (MD5) Previous issue date: 2010 / Resumo: INTRODUÇÃO: As proteínas da família PLUNC ("palate, lung and nasalepithelium clone"), as quais são expressas no trato aéreo superior e na cavidade bucal humana, podem ser responsáveis por mediar a resposta imune inata e interagir com a superfície bacteriana. Entretanto, sabe-se muito pouco sobre a expressão destas proteínas nas glândulas salivares durante o período embrionário humano. OBJETIVO: O objetivo do presente estudo foi analisar a expressão imunoistoquímica de SPLUNC1, SPLUNC2 e LPLUNC1 em glândulas salivares maiores e menores, língua e palato de fetos humanos. MATERIAIS e MÉTODOS: A amostra do presente estudo foi composta por 26 fetos humanos com idade variando entre 12 e 25 semanas de vida intra-uterina. As glândulas parótidas (n=7), submandibulares (n=13) e sublinguais (n=5) e as glândulas salivares menores [lábio (n=18), palato (n=9) e língua (n=6)] foram classificadas de acordo com os estágios de morfodiferenciação. A marcação imunoistoquímica citoplasmática das glândulas salivares, epitélio respiratório palatino e epitélio lingual, foi avaliada como positiva ou negativa. RESULTADOS: Dentre os 26 fetos avaliados, 13 (50%) apresentaram positividade para SPLUNC1 nos plugues de mucina dos ductos estriados e no citoplasma dos ácinos mucosos. Os dois casos positivos para LPLUNC1 apresentaram expressão citoplasmática na porção apical das células do ducto estriado primitivo. Nenhum dos casos apresentou marcação positiva para os anticorpos SPLUNC2A e SPLUNC2B. Os estágios de morfodiferenciação avançados (estágio canalicular e broto terminal) prevaleceram entre os fetos que apresentaram positividade para SPLUNC1. Tecidos que estavam adjacentes as glândulas salivares avaliadas, como células do epitélio respiratório associado à região do palato (n=6) e curiosamente, do epitélio escamoso de superfície da língua associado às glândulas salivares menores da região lingual (n=3), também apresentaram marcação positiva para SPLUNC1. Os resultados do presente estudo sugerem que a expressão de SPLUNC2 em glândulas salivares inicia na vida pós-natal. A expressão da proteína LPLUNC1 nas células ductais durante a vida intra-uterina precisa ser melhor estudada, visto que apenas dois casos foram positivos para esta proteína. CONCLUSÕES: A proteína SPLUNC1 inicia sua expressão durante a vida intra-uterina, nas glândulas salivares em estágio de morfodiferenciação avançado (estágio canalicular e broto terminal). Estudos com métodos mais sensíveis devem ser realizados para avaliar e quantificar a expressão de SPLUNC1 e LPLUNC1 em glândulas salivares maiores e menores de fetos humanos, visando entender melhor a função destas proteínas na vida intra-uterina. / Abstract: OBJECTIVES: The expression of PLUNC proteins in human fetal tissue has not been previously studied. The aim of this study, therefore, was to determine expression in major and minor salivary glands, tongue and palate throughout fetal development and thus gain further insight into the function of these proteins. MATERIALS AND METHODS: 26 human fetuses (12 to 25 intra-uterine weeks) were collected retrospectively. Parotid (n=7), submandibular (n=13), sublingual (n=5) and minor salivary glands [lips (n=18)], palate (n=9) and tongue (n=6)] were classified according to their morphodifferentiation stage. Immunohistochemical analysis of three PLUNC proteins (SPLUNC1, SPLUNC2 and LPLUNC1) was performed and immunoreactivity was assessed as positive or negative. RESULTS: Mucin plugs in striated ducts and mucous cells of submandibular (n=4, 30.77%), sublingual (n=4, 80%) and minor salivary glands (n=9, 27.27%) were positive for SPLUNC1. The apical portion of primitive striated duct cells of submandibular and sublingual salivary glands was positive for LPLUNC1 (n=2). SPLUNC1 was detected in late morphodiferentiation stages (canalicular stage and terminal bud stage) of salivary glands. Tissues adjacent to the minor salivary glands, such as respiratory epithelial cells located in the palate (n=6) and squamous epithelial cells of the tongue surface (n=3), were also positive for SPLUNC1. All cases (n=26) were negative for SPLUNC2. CONCLUSIONS: Our results suggest that SPLUNC2, the major PLUNC protein in adult salivary glands, is either expressed very late in fetal development or only in adult tissues. Only 2 cases of LPLUNC1 protein in fetal duct cells were positive; further studies are needed to confirm expression. Salivary glands expressed SPLUNC 1 only in late morphodifferentiation stages (canalicular and terminal bud stages). Further studies using other sensitive methods, such as in situ hybridization, should be performed to assess and quantify the expression of SPLUNC 1 and LPLUNC1 in major and minor salivary glands, tongue and palate in order to better understand their function in intra-uterine life. / Mestrado / Estomatologia / Mestre em Estomatopatologia

As glândulas salivares são estruturas essenciais para a manutenção da homeostase da cavidade oral pela síntese e secreção do fluido salivar. A disfunção ou perda permanente das glândulas salivares causadas por radioterapia, doenças inflamatórias ou desordens congênitas elevam principalmente o risco de infecções da mucosa oral e de estruturas dentárias, além de potencialmente prejudicar funções fisiológicas como fala, mastigação e paladar, diretamente interferindo na qualidade de vida dos indivíduos afetados. Os tratamentos atualmente disponíveis são apenas paliativos, ressaltando a necessidade de se compreender melhor os processos embriogênicos a fim de desenvolver novas estratégias terapêuticas capazes de regenerar as glândulas salivares. O princípio da formação das glândulas salivares baseia-se na coordenação de diversos processos morfogenéticos, e este trabalho foca particularmente em investigar a formação do espaço luminal do sistema de ductos, uma vez que a adequada abertura dos lumens é um processo essencial para a secreção salivar. Relata-se que a remoção das células centrais dos cordões sólidos epiteliais por morte celular apoptótica é o principal mecanismo de abertura do espaço luminal dos futuros ductos glandulares em camundongos. Porém, pouco se sabe sobre o controle temporal da apoptose durante o desenvolvimento glandular e sobre seu comportamento em glândulas salivares humanas. Neste trabalho, o perfil de expressão de diversas proteínas envolvidas na cascata apoptótica em glândulas salivares fetais humanas foi analisado de acordo com cada estágio morfogenético por imunoistoquímica (Bax, Bak, Bad, Bid, Bcl-2, Bcl-x, Bcl-xL, caspase-3 clivada, caspases-6, -7 e -9, apaf-1, survivina e citocromo c). As análises semi-qualitativas resultaram em negatividade apenas para as proteínas Bcl-2, Bad, Bid e caspase-3 clivada em todas as fases de desenvolvimento. A expressão nuclear de Bax e Bak foi identificada em presumidos espaços luminais em estágios precoces, enquanto Bcl-xL foi o fator antiapoptótico da família Bcl-2 que exibiu expressão nuclear mais importante. Caspases-6, -7 e -9 foram positivas em todas as fases, e a ausência de caspase-3 clivada sugere caspase-7 como principal caspase efetora da apoptose em desenvolvimento de glândulas salivares humanas. Ambos os componentes do complexo apoptossomo foram positivos durante o desenvolvimento glandular, e o inibidor survivina demonstrou mais positividade nuclear em estágios mais avançados. Ao observar a expressão de reguladores apoptóticos durante o desenvolvimento glandular humano, foram realizados experimentos funcionais com culturas de tecido glandular de camundongos para avaliar o papel das caspases durante a formação desta estrutura. Inicialmente detectou-se a atividade apoptótica em glândulas salivares de camundongos albinos no centro dos cordões epiteliais primários a partir de estágios precoces de desenvolvimento através de TUNEL e caspase-3 clivada. A partir disso, foi realizada a inibição apoptótica funcional in vitro durante o mesmo período, que resultou em ductos significativamente mais amplos e em defeitos morfológicos importantes nas estruturas luminal e acinar. Este trabalho evidenciou portanto atividade apoptótica durante a formação de glândulas salivares humanas e de camundongo, expressando-se em fases mais precoces do que reportadas anteriormente. Além disso, a ausência de Bad e Bid indica que a via intrínseca está mais ativa que a extrínseca, e distintos perfis de expressão da maioria das moléculas sugere adicionais funções não-apoptóticas durante a morfogênese glandular. / Salivary glands are essential structures for the maintenance of homeostasis of the oral cavity by synthesizing and secreting saliva. Permanent dysfunction or loss of salivary glands caused by radiotherapies, inflammatory diseases or congenital disorders increase mainly the risk of infections of the oral mucosa and tooth surface, also impairing physiological functions as speech, mastication and taste, directly interfering in quality of life. Current treatments are only palliative-based, which highlights the need of having a better understanding of embryonic processes to develop therefore new therapeutic strategies able to regenerate salivary glands. The development of glandular secretory units and ductal system involves the coordination of several morphogenetic processes, and this study particularly focuses in investigating the formation of the lumenal space of the ductal system, as the proper lumen opening is an essential step for the salivary secretion. The clearance of the central cells of developing solid epithelial stalks by apoptotic cell death is the main mechanism of lumen space opening within presumptive ducts in mouse salivary glands. However little is known about its temporal regulation and its function in human salivary glands. Here we analysed the profile expression of several apoptosis-related proteins during human salivary gland development in correlation to each morphogenetic stage by immunohistochemistry (Bax, Bak, Bad, Bid, Bcl-2, Bclx, Bcl-xL, cleaved caspase-3, caspases-6, -7 e -9, apaf-1, survivin e citocromo c). Immunohistochemical results were analysed semi-qualitatively, and proteins Bcl-2, Bad, Bid and cleaved caspase-3 were considered completely negative at all stages of development. The nuclear expression of Bax and Bak were observed within the presumptive luminal spaces at early stages, while Bcl-xL was the antiapoptotic factor of Bcl-2 family that showed more prominent nuclear expression. Caspases-6, -7 and -9 were positive at all stages, and the absence of cleaved caspase-3 suggests caspase-7 as the main effector caspase during human salivary gland development. Both components of the apoptosome complex were also positive through all development, and the inhibitor of apoptosis survivin has shown more nuclear positivity at later stages. As the expression of apoptotic regulators was observed during human salivary gland development, functional experiments were then performed in mouse salivary gland cultures to determine the apoptotic activity of during the glandular formation. Initially, the apoptotic activity was detected in mouse salivary glands within the centre of primary epithelial stalks from early stages of development by TUNEL and cleaved caspase-3. Thus the in vitro apoptotic inhibition was performed at the same stages, which resulted in significant wider ducts and important morphological defects within luminal and acinar structures. This work has therefore evidenced the existence of apoptotic role in salivary gland lumen formation of both human and mouse models, having an earlier start point as reported before. Moreover, the absence of Bad and Bid indicates that the intrinsic pathway is more active than the extrinsic during human development, and the distinct subcellular expression of most molecules suggests additional non-apoptotic functions.

Made available in DSpace on 2017-07-10T17:47:58Z (GMT). No. of bitstreams: 1 Carolina_Aleteia_Mecabo.pdf: 1439256 bytes, checksum: c24dcfc3f28c7c0959b8b9303316bd0d (MD5) Previous issue date: 2014-02-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The study was conducted at Embrapa Beef Cattle, Campo Grande, Mato Grosso do Sul State, with the objective of evaluating the growth and nutritive value of grass in Piata agrosilvopastoral system with two densities of eucalyptus: 22 and14 meters between rows of eucalyptus. The experimental design was a randomized block design in a split plot design with four replications during winter. The plots treatments were the Integration Cattle Farming Forest at systems 1 and 2, which consisted of Brachiaria brizantha piatã planted after soybean harvest in the cropping system in consortium with eucalyptus (Eucalyptus urograndis) with 2m spacing between trees and between rows of 22 and 14 m, and the subplot treatments, points A, B, C, D and E, which were arranged between the rows of trees. The forage growth was evaluated, performing sampling 50 days after emergence, and every 15 days at each point (A, B, C, D and E) within each plot. During this period, also proceeded to analyze the morphogenesis of grass for 60 days with weekly reviews 5 tillers were marked within each section (A, B, C, D and E) within each portion. The variables analyzed were canopy height, canopy cover, plant density, leaf area index, leaf/stem ratio, dry matter production, Spad index, percentage of shade and morphogenesis (final leaf length, leaf appearance rate, rate of leaf elongation, stem elongation rate, lifespan of leaves and phyllochron number of live leaves per tiller). After the establishment of forage, which occurred at 110 days, the first court was held, where the entire sample was collected from each sub plot for the evaluation of the total production of forage and morphological components of nutritional assessment. There was a significant density of Eucalyptus for phyllochron variable. For the lifespan of leaves and plant density did not affect variable and this is not a variable influenced by shading. The dry matter content of the forage was the only variable that was higher in the near trees, where the highest percentage of shading points were found. The other morphogenetic and structural variables evaluated had a strong negative influence of shading, with lower values at the closest points to the trees. Treatment with 22 meters of distance between rows of trees, with a density of 227 eucalyptus trees/ha showed the best results of nutritional composition / O estudo foi desenvolvido na Embrapa Gado de Corte, em Campo Grande, no Estado do Mato Grosso do Sul, com o objetivo de avaliar o crescimento e o valor nutritivo do capim-piatã em sistema agrossilvipastoril com duas densidades de eucalipto: 22 e 14 metros entre fileiras de eucalipto. O delineamento experimental foi o de blocos casualizados em esquema de parcelas subdivididas, com quatro repetições, durante o inverno. Os tratamentos das parcelas foram os sistemas Integração Lavoura Pecuária Floresta 1 e 2, que consistiram no capim Brachiaria brizantha cv. Piatã, plantado após a colheita da soja, no sistema de cultivo em consórcio com o eucalipto (Eucalyptus urograndis), com espaçamento entre árvores de 2 m, e entre fileiras de 22 e 14 m, e os tratamentos das subparcelas, os pontos A, B, C, D e E, que eram marcados entre as fileiras das árvores. Avaliou-se o crescimento da forrageira, realizando-se amostragens 50 dias após a emergência, e a cada 15 dias, em cada ponto (A, B, C, D e E) dentro de cada parcela. Durante esse período, procedeu-se também a análise da morfogênese do capim, durante 60 dias, com avaliações semanais. Foram marcados 5 perfilhos dentro de cada ponto (A, B, C, D e E), dentro de cada parcela. As variáveis analisadas foram altura do dossel, cobertura do dossel, densidade de plantas, índice de área foliar, relação folha/colmo, produção de matéria seca, índice Spad, porcentagem de sombra e morfogênese (comprimento final de folha, taxa de aparecimento de folhas, taxa de alongamento de folhas, taxa de alongamento de colmo, duração de vida das folhas, filocrono e número de folhas vivas por perfilho). Após o estabelecimento da forragem, que se deu em 110 dias, foi realizado o primeiro corte, onde foi coletada toda a amostra de cada subparcela, para a avaliação da produção total, de componentes morfológicos da forragem e avaliação nutricional. Houve efeito significativo da densidade de eucalipto para a variável filocrono. Para a variável duração de vida das folhas e densidade de plantas não houve efeito significativo, não sendo esta uma variável influenciada pelo sombreamento. O teor de matéria seca da forragem foi a única variável que foi maior nos pontos mais próximos às arvores, ou seja, onde foram encontradas as maiores porcentagens de sombreamento. As demais variáveis morfogênicas e estruturais avaliadas tiveram forte influência negativa do sombreamento, sendo seus valores menores nos pontos mais próximos às árvores. No tratamento com 22 metros de distância entre fileiras de árvores, com densidade de eucalipto com 227 árvores/ha apresentou os melhores resultados de composição nutricional

Orientador: Beatriz Appezzato-da-Gloria / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T13:48:58Z (GMT). No. of bitstreams: 1 Fernando_JulianaAparecida_D.pdf: 4167657 bytes, checksum: 78dc365d5ec277447072a64005443c39 (MD5) Previous issue date: 2005 / Resumo: As células dos meristemóides são responsáveis pela expressão da via organogênica in vitro. Esses meristemóides podem ser formados no explante ou no calo originado do explante, caracterizando a organogênese direta e indireta, respectivamente. Tendo em vista que a organogênese in vitro é um pré-requisito ao desenvolvimento de estratégias de micropropagação e à transformação genética de plantas, o monitoramento histológico e ultra-estrutural das células envolvidas nesse processo de regeneração fornece subsídios para a otimização desses protocolos. Nesse contexto, a população FB ¿ 100 de Passiflora edulis Sims f. flavicarpa Deg. foi avaliada quanto ao processo de diferenciação dos meristemóides, à fonte de explante e à adição de água de coco ao meio de cultura. Para análises dos meristemóides, explantes foliares e hipocotiledonares foram inoculados em meio de cultura MS contendo 1,0 mg L-1 de BA e 5% de água de coco. Os estudos anatômicos dos explantes hipocotiledonares mostraram que os meristemóides eram formados a partir da camada epidérmica e das subepidérmicas. Os meristemóides originaram primórdios foliares e, esporadicamente, gemas. Em geral, os meristemóides continuavam o processo de divisão originando protuberâncias. Essas protuberâncias eram constituídas pela camada epidérmica e pelas subepidérmicas meristemáticas e células centrais parenquimáticas, sendo que somente as células periféricas eram capazes de originar gemas. Nos explantes foliares, o processo era similar ao descrito para os explantes hipocotiledonares. Porém, a camada epidérmica e as subepidérmicas das protuberâncias não eram definidas e o número de gemas formadas foi significativamente inferior ao obtido utilizando-se os explantes hipocotiledonares. Portanto, as análises estruturais e as análises estatísticas confirmaram a superioridade do explante hipocotiledonar em relação ao foliar. Os explantes hipocotiledonares desenvolveram um pequeno calo na superfície seccionada do explante. As camadas periféricas desse calo formaram meristemóides que originaram primórdios foliares, gemas esporádicas ou continuaram a se dividir formando protuberâncias. No presente trabalho foi caracterizada a ultra-estrutura das células das protuberâncias formadas diretamente nos explantes hipocotiledonares e aquelas originadas no calo. Os estudos mostraram que nas células meristemáticas das protuberâncias diretas o núcleo apresentou formato circular. Por sua vez, nas protuberâncias formadas no calo, o envoltório nuclear exibia grande quantidade de poros, profundas invaginações e fragmentação nuclear, caracterizando o processo amitótico. As análises ao microscópio eletrônico de varredura dos explantes hipocotiledonares inoculados em meio MS contendo 1,0 mg L-1 de BA suplementado ou não com 5% de água de coco mostraram que as gemas e as protuberâncias obtidas em ambas condições de cultivo apresentaram as mesmas características estruturais / Abstract: Meristemoids are responsible for the in vitro organogenesis expression. They may be formed from the explant (direct organogenesis) or from callus (indirect organogenesis). Once in vitro organogenesis is a prerequisite for developing micropropagation strategies and genetic transformation in plants, the ultrastructural analysis of the cells involved in such regeneration provides basic information that optimize protocols. In this context, FB - 100 population of Passiflora edulis Sims f. flavicarpa Deg. was evaluated as to the meristemoids differentiation, the source of explant and the coconut water supply to the culture medium. Meristemoids were analysed from leaf and hypocotyledonar explants cultured in MS medium supplemented with 1.0 mg L-1 BA and 5% coconut water. The histological analyses of the hypocotyledonar explants showed that meristemoids arose from epidermal and subepidermal layers. Meristemoids originated leaf primordia and, sometimes, buds. In general, meristemoids continued dividing, forming protuberances. Such protuberances consisted of meristematic epidermal and subepidermal cells, as well as central parenchymatic cells, although only peripheral layers of the protuberances originated buds. In leaf explants, this process was similar to the processes described for hypocotyledonar explants. However, epidermal and subepidermal layers of protuberances on leaf explants were not well-defined and the number of buds originated from leaf explants was significantly smaller than the number of buds from hypocotyledonar explants. Structural and statistical evaluations confirmed that the hypocotyledonar explants were better than leaf explants. Hypocotyledonar explants developed a callus in the cut region surface. Peripheral layers of the callus formed meristemoids that gave rise to leaf primordial and buds, or continued dividing to form protuberances. This work characterized the ultrastructure of protuberance cells originated directly on the hypocotyledonar explants, as well as those originated on callus. Meristematic cells of direct protuberances showed spherical nucleus. On the other hand, in indirectly-formed protuberances the nuclear envelope showed a large number of nuclear pore complexes, deep invaginations and nuclear fragmentation characterizing the amitotic process. Analyses under scanning electron microscope of the hypocotyledonar explants cultured on MS medium supplemented with 1.0 mg L-1 BA and either with or without 5% coconut water did not evidence structural differences between buds and protuberances. The quantitative evaluation demonstrated that coconut water was efficient to increase the number of buds / Doutorado / Biologia Vegetal / Doutor em Biologia Vegetal

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第17557号 / 工博第3716号 / 新制||工||1566(附属図書館) / 30323 / 京都大学大学院工学研究科マイクロエンジニアリング専攻 / (主査)教授 安達 泰治, 教授 楠見 明弘, 准教授 井上 康博, 教授 琵琶 志朗 / 学位規則第4条第1項該当

The bony fin rays of the zebrafish caudal fin are a convenient system for studying bone morphogenesis and patterning. Joints and bifurcations in fin rays follow predictable spatial patterns, though the mechanisms underlying these patterns are not well understood. We developed simulation models to explore ray pattern formation mechanisms in growing fins. In all models, the fin ray growth rates are based on quantitative experimental data. The different models simulate ray joint formation and bifurcation formation using different hypothetical mechanisms. In the most plausible model, ray joint and bifurcation formation result from the accumulation of two substances, arbitrarily named J and B. Model parameters were optimized to find the best fit between model output and quantitative experimental data on fin ray patterns. The model will be tested in the future by evaluating how well it can predict fin ray patterns in different fin shapes, mutant zebrafish fins, and other fish species.

(Constance) Stanzie Tooth’s thesis exhibition titled, Hand to Ground, explores ideas of origin; both the mythology of a personal origin as well as the origins of painting and representation. Interrogating the history of landscape painting, the exhibition skews notions of identity by reconsidering representations of the landscape. Inspired by Margaret Atwood’s novel Surfacing, and paired with reminiscences of her childhood in rural Ontario, Tooth’s paintings unfold oblique narratives of a communion with land. These questions of personal ecology are filtered through an intense material expression, reinforcing the idea of coming into being. The exhibition was on view at the Karsh Masson Gallery in Ottawa from August 1st to September 10th, 2015.

Cowpea, Vigna unguiculata (L.) Walp is an excellent source of protein, vitamins and minerals and a major food crop many parts of Africa. Optimal production levels are hampered by insect pests and diseases. Biotechnological techniques such as tissue culture and genetic engineering can aid in the development of varieties with resistance to insect pests and diseases. The objective of this study was to investigate conditions necessary for the development of a reproducible tissue culture system that can be applied to regenerate transformed cells from culture. The in vitro manipulation of cowpea using Murashige and Skoog (MS) medium, auxins and cytokinins resulted in the formation of callus and rhizogenesis. Calli that were formed were separated into six classes based on color and texture. Yellowish friable callus, yellowish compact, soft yellowish callus and green and white were composed of largely vacuolated cells and were non-regenerative. Friable green callus was the most prevalent callus type and could form of roots in some hormone combinations. Green spots were formed on hard compact green callus. The green spots became nodular, forming root primordia and ultimately giving rise to roots. None of the six calli types gave rise to the formation of shoots. Embryogenic callus was induced from cowpea explants cultured on MS medium supplemented with dicamba and picloram. Embryogenic suspension cultures were initiated from callus induced on MS supplemented with 3.0 mg/L dicamba or picloram and conditions for maintenance of embryogenic suspension cultures were evaluated. Somatic embryos were formed in suspension cultures. Attempts to convert and germinate the somatic embryos resulted in the formation of callus or formation of appendages on the somatic embryos or in the death of the embryos. The appendages formed roots on prolonged culture. Further research is needed to determine appropriate optimal conditions for embryo conversion and germination and ultimately plant recovery from culture.

Comprendre comment la coordination des cellules entre elles permet l’émergence d’une forme est une des questions les plus fascinantes en biologie du développement. Au cours de cette thèse, nous avons utilisé les premiers stades de développement des feuilles dentelées d'Arabidopsis thaliana comme modèle pour étudier la relation entre les évènements cellulaires et la morphogenèse. Pendant le développement des feuilles d'Arabidopsis thaliana, le contrôle fin de la prolifération et de l'expansion cellulaire permet la croissance différentielle au niveau de la marge foliaire, nécessaire à la formation des indentations. Dans ce modèle, la croissance différentielle est le résultat de l'interaction entre la signalisation de l’auxine et l’activité des facteurs de transcription CUP SHAPED COTYLEDONS impliqués dans le maintien de l'identité des domaines frontières. Pour affiner la compréhension des relations complexes entre les facteurs de transcriptions CUC, les réponses auxiniques et les événements cellulaires à l'origine des indentations foliaires, nous avons utilisé des expériences d’imagerie en temps réel sur des primordia foliaires de lignées exprimant des rapporteurs de développement et/ou de réponse auxinique. Nos résultats ont révélé un contrôle dynamique de la croissance différentielle à la marge des feuilles et l'implication critique de CUC3 dans la répression locale de la croissance cellulaire. / How a shape arises from the coordinated behavior of cells is one of the most fascinating questions in developmental biology. Here we used the early stages of development of serrated leaves in Arabidopsis thaliana as a model to study the tight relation between cellular behaviour and morphogenesis. During Arabidopsis thaliana leaf development the fine control of cell proliferation and cell expansion sustains differential growth at the margin required for the formation of leaf outgrowth named teeth. In this model, differential growth is the result of interplay between auxin signaling and CUC transcription factors that are involved in the maintenance of boundary domain identity. To clarify the interconnected relations between patterns of CUC TFs and auxin responses as well as the cellular events behind serrations we used time-lapse experiments on vegetative primordia of lines expressing developmental and/or auxin response reporters. Our results revealed a tight and dynamic control of differential growth at the leaf margin and the critical involvement of CUC3 in the local repression of cell growth in combination with low auxin responses.

Leaf size and shape are strongly influenced by the growth patterns of the epidermal tissue. Pavement cells are the prevalent cell type in the epidermis and during cell expansion they undergo a drastic shape change from a simple polyhedral cells to puzzled-shaped cell. The role of these cell protrusions, more commonly referred to as lobes, remains unknown but their formation has been proposed to help increase the structural integrity of the epidermal tissue. How the symmetry breaking event that initiates a lobe is controlled remains unknown, however pharmacological and genetic disruption of the microtubule system has been shown to interfere not only with lobe initiation but also with lobe expansion. Additionally, the role of microtubules in the pattering of microfibril deposition, the load-bearing structure of the cell wall, makes the microtubule system a good candidate to evaluate its dynamics as a function of shape change. Two main mechanical models for lobe initiation are evaluated here, one where microtubules serve as stable features suppressing local expansion and one where microtubules, similarly to the anisotropic expansion patterning in hypocotyl cells, pro-mote the local anisotropic expansion of the cell resulting in lobe formation. The main method to evaluate these models was through the use of long-term time-lapse image analysis using a plasma-membrane marker for accurate shape change quantification and a microtubule marker to quantify their location, persistence, and density as a function of cell shape change. Using the junctions where three cells come together,cells were sub-divided into segments and the shape of these segments were tracked using a new coordinate system that allowed the detection of new lobes as which can arise from ∼300 deflections. By mapping sub-cellular processes, such as microtubule persistence, to this coordinate system, correlations of microtubule organization and shape change was possible. Additionally, a subset of microtubules bundles that splay across the anticlinal and periclinal walls, perpendicular and parallel to the leaf surface respectively, were identified as marking the location and direction of lobe formation.Disrupting the cell boundary by partially digesting pectin, a main component in the middle lamella, revealed the cell-autonomous morphogenesis mechanism in pavementcells. Under pectinase treatment, cell invaginations were produced and similarly to lobes their initiation was microtubule and cellulose dependent. Lastly, stress prediction using finite-element models, based from live-cell images, co-localized regions of high cell wall stress with both microtubule persistence and shape shape locations in both lobing and invaginated segments. Together, a model of cellular shape change is presented where microtubules translate cell wall stresses to tissue morphogenesis.