Les membranes basales (MB) jouent un rôle majeur au cours des processus morphogénétiques. Elles et sont principalement composées de Collagène de type IV, de Perlecan et de Laminine. Les récepteurs d'adhésions/signalisations (Intégrines/Dystroglycans) localisés au pôle basal des cellules épithéliales, interagissent directement avec les MBs. De nombreuses études montrent l'importance de la composition des MBs dans le devenir cellulaire. Cependant, le rôle mécanique de la MB au cours du développement d'un organe multicouche n'est pas connu. Comme modèle de morphogenèse épithéliale, nous avons choisi d'étudier l'épithélium du follicule ovarien chez Drosophila melanogaster. La MB entoure chaque follicule ovarien qui est composé d'une monocouche de cellules épithéliales cuboïdes entourant un groupe interne formé de 16 cellules de la lignée germinale (15 cellules nourricières en postérieur et 1 ovocyte en antérieur). Au cours du développement folliculaire, les cellules épithéliales s'aplatissent suivant une vague antéro-postérieur. Cette transition cellulaire cuboïde – aplatie dépend du remodelage des jonctions d'adhérence et du cytosquelette. Mes travaux de thèse ont porté sur l'étude du rôle mécanique et moléculaire de la MB au cours de la morphogenèse épithéliale chez la Drosophile. J'ai ainsi pu montrer (i) que la rigidité de la MB augmente au fur et à mesure du développement du follicule, (ii) que l'aplatissement dépend de la structure de la MB et de la liaison a cette MB grâce aux intégrines (iii) que la MB s'assouplie lors de la transition cuboïde-squameux et que cette assouplissement dépend de ce processus. Ces résultats démontrent un dynamisme mécanique et moléculaire de la MB au cours de l'ovogenèse et de la morphogenèse, révélant le rôle central de la MB lors de ces processus. Parallèlement j'ai développé une approche par segmentation cellulaire afin d'extraire les valeurs métriques (hauteur, anisotropie, surface basale, volume) des cellules épithéliales et de mesurer les variations de ces paramètres au cours de la morphogenèse épithéliale (MARS-ALT) / Epithelial cell morphogenesis is an essential process for animal development. Epithelia are composed of polarized cells with a basal side interacting through Integrins, with a basement membrane (BM) and a lateral side containing cadherin-based junctional complexes. Integrins and Cadherins are, both, linked to actin filaments and are thus involved in cell shape regulation. While these links are well documented, it remains unclear how the components of the BM and the 3D organisation of this tissue influence epithelial cell morphogenesis. The model we are using to study this influence is the follicular epithelium in Drosophila melanogaster. It consists of a monolayer of 800 epithelial cells surrounding the egg chamber consisted of an internal cluster of 16 germline cells (15 nurse cells and one posteriorly-localized oocyte). An extracellular matrix (ECM), composed mainly of Collagen IV and Laminins, surrounds each follicle, directly secreted by follicular cells. During follicle development, the cuboidal epithelial cells become squamous around the nurse cells and columnar around the oocyte. The cuboidal-to-squamous transition depends on both Integrins (formed by the subunits aPS2/bPS) and Cadherin-based adherens junction remodelling. Here we designed an Atomic Force Microscopy (AFM) approach to investigate the elastic modulus of the ECM in a living ovarian follicle at different stages of development and particularly during epithelial cell morphogenesis. First, we found that the stiffness changes temporally during oogenesis with an increase of stiffness during Collagen IV deposition. Second, during cell morphogenesis, we observed a gradient of ECM stiffness. Third, by measuring the stiffness in mutants delaying or promoting cell flattening, we showed that the regional differences occurs in function of the cell ability to flatten. Fourth, to assess the involvement of Collagen IV or its structure for the ECM rigidity properties, we measured the stiffness of ECM produced by follicles mutant for Collagen IV or after collagenase treatment and concluded that collagen fibrils are the source of rigidity properties.Altogether, these results demonstrate the role of the regulation of the ECM stiffness for epithelial cell morphogenesis and highlight a new mechanical aspect in the comprehension of developmental processes

Ce travail de thèse concerne l'articulation trapézométacarpienne. La première partie du travail a concerné l'étude de la morphogénèse de l'arche palmaire par modélisation géométrique 3D. Cinq embryons ont été utilisés. Les résultats de cette étude montre le caractère asymétrique et l'opposition précoce du pouce chez l'embryon humain. La deuxième partie a consisté à partir d'un protocole IRM spécifique sur 5 sujets sains à déterminer la cinématique 3D de l'articulation trapézo-métacarpienne en utilisant les axes hélicoïdaux. La troisième partie, suivant le même protocole sur sujets pathologiques à partir d'image scanner a permis d'évaluer l'effet des différents types de traitement en cas de rhizarthrose. Les résultats trouvent leur application dans l'évaluation quantitative des pathologies de l'articulation trapézo-métacarpienne ainsi que dans le développement des prothèses. / This work concerned the first carpo-metacarpal joint. The first part of this work was the study of the morphogenesis of the palmar arch using three-dimensional geometrie modeling. Five embryos were used for this study. The results of this study support the hypothesis that opposition and asymmetry of the thumb appears early in embryological development. In the second part, with specifie MRI protocol on 5 normal subjects, the objective was to quantify the 3D motion of the trapezo-metacarpal joint using helical axes theory. In the last part, using the same protocol on pathological subjects (CT scan images), the objective was to evaluate the effect of different type of treatment of the first carpo­ metacarpal arthritis.The results of the work are of interest for the quantitative evaluation of pathological trapezo-metacarpal joint and in the development of prosthesis.

L'enveloppe cellulaire bactérienne joue plus qu'un rôle de barrière d'échange. Elle est au coeur des processus cellulaires essentiels comme la morphogenèse et la division. Cette structure abrite environ un quart des protéines codées par le génome. Le but de mon travail a été de mieux comprendre le rôle de deux protéines membranaires dans la construction et la dynamique de l'enveloppe chez Bacillus subtilis. GluP est une protéase intramembranaire rhomboïde. Ces protéases clivent des segments transmembranaires dans la membrane afin de moduler l'activité de diverses protéines. Elles participent à de nombreux processus cellulaires chez les eucaryotes. Cependant, les fonctions biologiques des rhomboïdes procaryotes sont pour l'heure presque totalement inconnues. Nos résultats suggèrent que GluP participe au contrôle qualité des protéines membranaires à la manière des pseudo-rhomboïdes associées au système ERAD eucaryote. Elle forme un complexe avec FtsH, une protéase majeure du contrôle qualité des protéines. Ce complexe est impliqué dans la dégradation d'un substrat de rhomboïde. Le rôle de GluP serait de permettre la dislocation du segment transmembranaire et faciliter la prise en charge du substrat par FtsH. Le second projet auquel j'ai participé a consisté à comprendre le rôle de la protéine CmmB dans la morphogenèse. Son absence conduit à une morphologie cellulaire élargie. CmmB semble faire partie de la machinerie de synthèse du peptidoglycane au cours de l'élongation de la paroi. Elle serait nécessaire au bon fonctionnement d'une ou de plusieurs penicillin-binding proteins (PBPs). En particulier, nous proposons que CmmB est un cofacteur de la transpeptidase PBP2a. / The bacterial cell envelope is an obligatory barrier. It is a fundamental component in essential cellular processes such as morphogenesis and cell division. It hosts about a quarter of the proteins encoded in the genome. My work was aimed at understanding the function of two membrane proteins in the building and the dynamics of the cell envelope in the model bacterium Bacillus subtilis.GluP is a rhomboid intramembrane protease. Usually, rhomboids cleave transmembrane segments within the membrane to modulate protein functions. In eukaryotes, they participate in many cellular processes and their dysfunction lead to several pathologies. However, prokaryotic rhomboid functions remain almost totally unknown. Our results suggest that GluP is involved in bacterial membrane protein quality control, in a process akin to pseudo-rhomboid dependent endoplasmic reticulum associated protein degradation in eukaryotes. GluP forms a complex with FtsH, a major protease in protein quality control. That complex is not involved in the cleavage of a membrane substrate but in its degradation. We propose that GluP is required for the dislocation of the transmembrane segment, thus facilitating full-length substrate degradation by FtsH in the cytoplasm. My thesis second objective was to understand the role of the CmmB protein in morphogenesis. The absence of CmmB leads to slightly enlarged cells. CmmB seems to belong to the peptidoglycan synthesis machinery for cell-wall elongation. Our data support the idea that it is required for the proper activity of one or several penicillin-binding proteins (PBPs). In particular, we propose that CmmB is a cofactor of the PBP2a transpeptidase.

O estudo da morfogênese e da dinâmica de acúmulo de forragem possibilita melhor entendimento dos processos fisiológicos da planta em resposta a práticas de manejo empregadas. O presente estudo foi conduzido em uma área pertencente ao Departamento de Zootecnia da ESALQ/USP, estabelecida com o capim-xaraés [Brachiaria brizantha (Hochst ex A. RICH) STAPF. cv. Xaraés], durante o verão agrostológico de 2006/2007. O objetivo do estudo foi comparar as respostas morfogênicas e estruturais do capim-xaraés submetido a estratégias de pastejo rotativo em janelas temporais pré-estabelecidas, perfazendo um verão agrostológico, para estabelecer possíveis contrastes nas respostas dessa espécie a intervalos de rebrotação variáveis. O experimento envolveu três tratamentos baseados em intervalo de pastejo: a cada 28 dias ou sempre que a interceptação de luz (IL) pelo dossel forrageiro atingisse 95% ou 100% durante a rebrotação. O delineamento experimental utilizado foi o completamente casualizado, com três repetições. Os dados foram coletados em três momentos durante o verão agrostológico, correspondentes aos meses de Out/Nov, Dez/Jan e Fev/Mar. Foram avaliadas as seguintes variáveis-reposta: densidade populacional de perfilhos (DPP) aéreos e basais, altura do dossel, massa de forragem, relação folha:colmo pré-pastejo (F:C), índice de área da folhagem (IAFo), taxa de alongamento de folhas (TAlF), taxa de senescência de folhas (TSeF), taxa de alongamento de colmos (TAlC), comprimento final da folha (CFF), comprimento do colmo (colmo + pseudo-colmo) (CC), número de folhas vivas por perfilho (NFV), filocrono (Fil), duração de vida da folha (DVF) e taxa de crescimento de folhas (TCF). O tratamento 100% IL resultou nos maiores valores de massa de forragem pré- (5200 kg ha-1 MS) e pós-pastejo (1330 kg ha-1 MS), assim como no maior acúmulo médio de forragem (3870 kg ha-1 MS). O tratamento de 28 dias resultou em níveis variáveis de IL pré-pastejo, embora, na média, tenha sido semelhante ao tratamento 95% IL. O tratamento 95% IL e o 28 dias resultaram nos menores valores de IAFo pré-pastejo (3,62 e 3,75, respectivamente), mas os maiores IAFos pós-pastejo. A relação F:C não variou entre os tratamentos, assim como a DPP aéreos e total. O tratamento 100% IL gerou a menor DPP basal (1189 perfilhos m-2) e o tratamento 95% IL a maior (1260 perfilhos m-2). Não houve diferença entre os tratamentos para a TApF, Fil, DVF e NFV nas épocas avaliadas. O tratamento 100% IL resultou nos maiores valores de CFF e TCF e 95% IL nos menores. Conclui-se que o uso da IL como indicador do momento de interrupção da rebrotação e início do pastejo resulta em variações no comprimento do período de descanso em pastos sob lotação intermitente. Além disso, fica evidente que o componente colmo é o principal responsável pelas altas taxas de acúmulo de forragem de gramíneas tropicais em situações de períodos prolongados de descanso. Estudos que estabeleçam padrões de idades fisiológicas podem explicar melhor os contrastes existentes entre tratamentos cujos intervalos de pastejo se baseiam em níveis de interceptação luminosa pelo dossel forrageiro durante a rebrotação. / The study of morphogenesis and the dynamics of forage accumulation allows for a better understanding of plant physiological processes in response to management practices. This study was conducted in Piracicaba, SP, on a pasture established with Xaraés palisadegrass [Brachiaria brizantha (Hochst ex A. Rich) Stapf. cv. Xaraés], during the summer growing season of 2006/2007. The objective was to compare the structural and morphogenic responses of this forage grass subjected to intermittent defoliation strategies during the summer growing season, and try to establish possible contrasts in the responses of this species to varying regrowth intervals. Treatments were three grazing frequencies applied as rest periods: grazing every 28 days or when the light interception (LI) by sward reached 95% or 100% during the regrowth. The experimental design was completely randomized, with three replications. Data were collected and then divided into three portions of the growing season, corresponding to the months of Oct/Nov, Dec/Jan and Feb/Mar. The following responses were measured: tiller (basal and aerial) population density (TPD), canopy height, pre- and post-graze forage mass, pre-graze leaf:stem ratio (L:S), leaf area index (LAI), leaf elongation rate (LER), stem elongation rate (SLR), final leaf length (FLL), stem length (stem+pseudo-stem) (SL), number of live leaves per tiller (NLL), phyllochron (Phy), leaf life span (LLS), and leaf growth rate (LGR). The 100% LI treatment resulted in higher pre-graze forage mass (5200 kg DM ha-1) as well as and post-graze forage mass (1330 kg DM ha-1), as well as higher mean herbage accumulation (3870 kg DM ha-1). The 28-d treatment resulted in varying levels of pre-graze LI, although, on average, it was similar to the 95% LI treatment. The 95% LI and the 28-d resulted in lower pre-graze LAI (3.62 and 3.75, respectively), but the highest post-graze LAI. The L:S ratio did not vary across treatments, and neither did the TPD (total and aerial). The 100% LI treatment resulted in lowest basal DPP (1189 tillers m-2) and the 95% LI in the highest DPP (1260 tillers m-2). There was no difference among treatments for LAR, Phy, LLS, and NLL in any of the periods within the growing season. The 100% LI treatment resulted in higher FLL and LGR and the 95% LI in the lowest values for these responses. It is concluded that the use of LI as an indicator of the time for termination of regrowth and the beginning of grazing results in variations in the length of rest period in pastures under intermittent stocking. In addition, it is clear that the stem is the main component responsible for high rates of accumulation of tropical forage grass in situations of prolonged periods of rest. Studies to establish standards of \"physiological age\" may explain the contrast between the grazing treatments whose intervals are based on levels of light interception by the canopy during pasture regrowth.

Candida albicans est un pathogène fongique opportuniste de l’Homme, qui peut causer des infections superficielles mais aussi systémiques chez les patients immunodéprimés. Sa virulence est associée à sa capacité de changer d’une forme bourgeonnante à une forme hyphale. La petite GTPase de type Rho, Cdc42, est critique pour la croissance filamenteuse et, sous forme activée, sa localisation est restreinte à l’extrémité des hyphes. J’ai utilisé un système photoactivable, constitué des domaines d’Arabidopsis thaliana Cry2PHR-CibN, pour contrôler le recrutement de Cdc42 constitutivement actif à la membrane plasmique. J'ai déterminé comment le photo-recrutement de Cdc42 constitutivement actif perturbe la croissance filamenteuse et où, quand et comment une nouvelle croissance filamenteuse est ré-initiée. Mes résultats démontrent que, lors du photo-recrutement de Cdc42 constitutivement actif, l'extension du filament cesse puis un nouveau site de croissance s’établit dans la cellule. La localisation de ce nouveau site de croissance est corrélée à la longueur du filament. J'ai étudié les mécanismes moléculaires qui sous-tendent le désassemblage du site de croissance initial et l'emplacement spécifique du nouveau site de croissance filamenteuse. Dans les hyphes en croissance, un «cluster» de vésicules, appelé Spitzenkörper, est localisé à l'extrémité du filament. Lors du photo-recrutement de Cdc42 constitutivement actif, un nouveau «cluster» de vésicules, de composition similaire à celui du Spitzenkörper initial, apparaît dans la cellule mère. J'ai suivi la dynamique du Spitzenkörper et la localisation de Cdc42 sous forme activée, des sites d'endocytose, des vésicules de sécrétion et des câbles d’actine suite à la perturbation du site de croissance initial dans le filament. Dans l’ensemble, mes résultats indiquent qu'il existe une compétition pour la croissance entre le Spitzenkörper et le «cluster» de vésicules qui se forme immédiatement après le photo-recrutement de Cdc42 constitutivement actif et qu'un axe de polarité dynamique peut être établi en l'absence de croissance directionnelle. / Candida albicans is a fungal human pathogen that can cause life-threatening infections in immunocompromised patients, in part, due to its ability to switch between an oval budding form and a filamentous hyphal form. The small-Rho GTPase Cdc42 is crucial for filamentous growth and, in its active form, localizes as a tight cluster at the tips of growing hyphae. I have used a light-activated membrane recruitment system comprised of the Arabidopsis thaliana Cry2PHR-CibN domains to control the recruitment of constitutively active Cdc42 to the plasma membrane. I have determined how photorecruitment of constitutively active Cdc42 perturbs filamentous growth and where, when and how new filamentous growth is subsequently initiated. My results demonstrate that, upon photorecruitment of constitutively active Cdc42, filament extension is abrogated and a new growth site can be established in the cell. Location of a new filamentous growth site correlates with the length of the initial filament. I have investigated the molecular mechanisms that underlie the disassembly of an initial growth site and the specific location of the new filamentous growth site. In growing hyphae a cluster of vesicles, referred to as a Spitzenkörper, is localized at the tip of the filament. Upon photorecruitment of constitutively active Cdc42, a new cluster of vesicles, with a composition similar to that of the initial Spitzenkörper, appears in the mother cell. I have followed the dynamics of the Spitzenkörper, active Cdc42, sites of endocytosis, secretory vesicles and actin cables subsequent to disruption of the initial growth site in the filament. Taken together, my results suggest that there is competition for growth between the Spitzenkörper and the cluster of vesicles that forms immediately after the photorecruitment of constitutively active Cdc42 and that a dynamic polarity axis can be established in the absence of directional growth.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22687号 / 医博第4631号 / 新制||医||1045(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 木村 剛, 教授 浅野 雅秀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Morphogenesis is the fundamental and remarkable biological process that produces elaborate and diverse tissues and organs from simple groups of cells, which can happen on timescales as short as minutes or as long as days. One of the biggest challenges in understanding morphogenesis is the gap between our knowledge of the molecular-scale activities of genes and proteins, and the large-scale behaviors of cells and tissues. To fill this gap, a complete understanding of both biochemical and mechanical factors involved in morphogenesis is needed. Morphogenesis is naturally a mechanical process in which tissues are physically sculpted by mechanical stress, strain, and movements of cells that are induced by these genetic and molecular programs. However, many of the mechanical factors involved in morphogenesis remain poorly understood partially due to the strong coupling of mechanical factors and biological factors, the active responses of living tissues to the environment, and the lack of experimental methods to study the mechanics of tissues in vivo. Epithelial tissues play crucial roles in shaping early embryos and are widely spread in mature animals to serve as boundaries and barriers. They are robust tissues that not only support the structure of embryos and organs, but also actively change shape and structure, displaying a fluid behavior during morphogenesis. Contractile tension and cell-cell adhesion are thought to be the main mechanical factors involved in epithelial tissue morphogenesis, but how the balance between these two determines epithelial tissue mechanics remains unclear. To build a fundamental understanding of the mechanical mechanisms underlying epithelial tissue morphogenesis, this dissertation studies the germband epithelial tissue in the early Drosophila melanogaster embryo and addresses two important open questions in the field of mechanics in morphogenesis: (1) what mechanical factors are involved in the morphogenesis of epithelial tissues; (2) how does a cell control these factors to tune tissue mechanical behaviors. In this dissertation, we developed a systematic, quantitative, in vivo experimental approach to explore mechanics of epithelial tissue morphogenesis in the Drosophila embryo by integrating molecular genetics approaches, live confocal fluorescence imaging, and quantitative image analysis. Combining our experimental studies in the Drosophila embryo with our collaborators’ theoretical modeling approaches, we showed that the shapes and alignment of cells within tissues can help us understand and predict epithelial tissue mechanical behaviors, such as tissue fluidity, during morphogenesis and how defects in these processes can result in abnormalities in embryo shape. We also observed that the Drosophila germband tissue transitions from more solid-like to more fluid-like behavior to help accommodate dramatic tissue flows during convergent extension, which indicates that the mechanical properties of developing tissues might be tuned during morphogenetic events. To elucidate molecular mechanisms underlying how tissue mechanical properties may be regulated during morphogenesis, this dissertation explores the role of cell-cell adhesion in controlling epithelial tissue mechanics. By systematically modulating cell-cell adhesion levels in the Drosophila germband tissue and combining live imaging and quantitative image analysis, we studied the effects of cell-cell adhesion levels on cellular and tissue behaviors. We found biphasic dependencies of cell rearrangements, cell shape, and tissue fluidity on cell-cell adhesion levels, which are surprisingly linked to each other by cell patterns in the tissue. In particular, tissues comprising cells with either lower or higher cell-cell adhesion levels tend to rearrange faster and show cell patterns indicating more fluid-like tissue behaviors. Further studies suggested that cell-cell adhesion works with cytoskeletal molecules to achieve these effects. The experimental approaches developed for exploring mechanics in 2-D in the Drosophila germband epithelial tissue are expanded upon in order to investigate germband tissue mechanics in 3-D. These approaches are also used to study mechanics in the inner ear round window membrane of the guinea pig for clinical application. This dissertation advances our understanding of mechanics of epithelial tissue morphogenesis in vivo and provides a practical, quantitative, and appealing platform for exploring mechanics in living tissues during morphogenesis. This helps fill the gap in our knowledge of molecular-scale activities and tissue-level behaviors, provides insight into building tissues with precise shapes and structures in the lab, and sheds light on human diseases associated with improper regulation of tissue mechanics such as birth defects, aberrant wound healing, and cancer metastasis.

Les cellules épithéliales changent en forme et en nombre au cours de divers processus morphogénétiques pendant le développement. La dynamique du réseau d’acto-myosine en interaction directe avec les jonctions adhérentes (JA) est à la base de ces mouvements cellulaires. Cependant, les mécanismes qui régulent cette dynamique cellulaire et moléculaire dans l’espace et le temps sont peu étudiés. Durant les stades précoces de l’ovogenèse chez la drosophile, le follicule ovarien est une sphère composée d'un cyste germinal recouvert d'un épithélium folliculaire monocouche d'origine somatique. Aux pôles de cette structure, un groupe de cellules, les Cellules Polaires (CP), sont produites en excès (3-6 cellules) au début de l'ovogenèse, et ensuite subissent une mort cellulaire programmée apoptotique entre les stades 2 et 4 de l’ovogenèse. De cette façon, à partir du stade 5 tous les pôles contiendront 2CP. Les CP sont l’unique source de sécrétion du ligand de la voie de signalisation JAK/STAT, Unpaired. Notre équipe a démontré que l’activation autonome et non-autonome cellulaire de la voie JAK/STAT est nécessaire pour l'apoptose développementale des CP. Grâce à l’utilisation de l’imagerie confocale en temps réel ainsi que sur des tissus fixés, j’ai établi une séquence d’évènements stéréotypés qui a lieu pendant l’élimination des CP surnuméraires. Trois phases ont été identifiées dans cette séquence: 1) une phase lente de remodelage cellulaire dépendante de la voie de signalisation JAK/STAT au cours de laquelle chaque CP à être éliminée est totalement enveloppée par les CP voisines (plus de 7h) ; 2) une phase d’activation de la cascade canonique de l’apoptose, commençant lorsque la PC est entièrement enveloppée, suivie d’un détachement puis d’une extrusion latérale des corps apoptotiques (1h) ; et 3) une phase de phagocytose des corps apoptotiques par les Cellules Folliculaires (CF) voisines (plus de 5h). Ensuite, en utilisant une approche gènes candidats, j’ai effectué des perturbations génétiques de la Myosine, de la Cadhérine et de différents régulateurs de l’Actine dans les CF et/ou dans les CP, ainsi que des analyses de la dynamique de certaines de ces molécules. Ces expériences m’ont permis de déterminer que la fonction de ces molécules est nécessaire dans les CF pour le processus d’élimination des CP surnuméraires. Finalement un lien entre la signalisation JAK/STAT et la dynamique de la Myosine a été mis en évidence. / Epithelial cells change in shape and number over the various morphogenetic processes occurring during development. The dynamics of the acto-myosin network in direct interaction with adherens junctions is the basis of these cell movements. However, the mechanisms regulating these cellular and molecular dynamics in space and time have not been much studied. During the early stages of oogenesis in Drosophila, the ovarian follicle is a sphere composed of a germline cyst surrounded by a mono-layered follicular epithelium of somatic origin. At the poles of this structure, a group of cells, the Polar Cells (PCs), which are produced in excess (3-6 cells) during early oogoenesis, undergo apoptotic programmed cell death between stages 2-4 of oogenesis, thus that as of stage 5 all poles contain exactly 2 PCs. PCs are the only source of the secreted ligand of the JAK/STAT signaling pathway, Unpaired. Our group has demonstrated that cell autonomous and cell non-autonomous activation of the JAK/STAT pathway is necessary for this developmental apoptosis. Through the use of confocal imaging in real time and on fixed tissues, I established a stereotyped sequence of events that occurs during the elimination of supernumerary PCs. Three phases were identified in this sequence: 1) a slow phase of cellular remodeling dependent on JAK/STAT signaling in which the PC to be eliminated is completely enveloped by its PC neighbors (more than 7 hours); 2) activation of the canonical apoptosis cascade, occurring when the PC is fully enveloped, followed by cell detachment and lateral extrusion of apoptotic corpses (1h); and 3) phagocytosis of apoptotic corpses by the surrounding Follicular Cells (FCs) (over 5 hours). Then, using a candidate gene approach, I conducted genetic perturbation of Myosin, Cadherin and actin regulators in the FCs and/or PCs, and the analysis of the dynamics of some of these molecules. These experiences allowed me to determine that the function of these molecules is required in FCs for the process of elimination of supernumerary PCs. Finally, evidence obtained suggests a link between JAK/STAT signaling and Myosin dynamics.

Exocyst is protein complex evolutionary conserved in yeasts, animals and plants, which plays a role in control of cell morphogenesis and polarity. It is a tethering complex whose function is to attach secretory vesicles to specifi c foci on plasma membrane. Complex exocyst is formed by eight subunits. Subunit EXO70 is encoded by 23 paralogue genes in genome of Arabidopsis thaliana. Mutation in paralogue subunit EX070H4 causes defect in trichome maturation. Mutant trichomes have thin, not reinforced cell wall, making them soft and elastic. Transcription of EXO70H4 gene is induced by UV radiation, therefore observations of plants cultivated on UV-B radiation were done. Analysis of mutants cultivated on UV-B radiation revealed hyperaccumulation of vesicules in cytoplasm, which were visible by light microscope. Hyperaccumulation was not observed in control plants cultivated on UV-B radiation, but thickening of cell wall was induced. Th is reaction to UV in trichomes hasn't been described yet. Analysis of cellular localization made with YFP tagged constructs revealed that EXO70H4 localizes into mobile corpuscules associating with Golgi apparatus. It was found with yeast two hybrid system that EXO70H4 interacts with TRS120, subunit of tethering complex TRAPPII which is active in Golgi apparatus....

Orientador: Glaucia Amorim Faria / Resumo: A passicultura vem sofrendo com patógenos, os quais limitam muito a produtividade e a viabilidade do cultivo de maracujá. Dentre os principais patógenos estão a antracnose (Colletotrichum gloesporioides Penz.), a mancha bacteriana (Xanthomonas campestris pv. passiflorae) e a morte-prematura (Fusarium oxysporum f. passiflorae), esta é a principal doença e gargalo da cultura, limitando o tempo da passicultura de perene para anual. Alternativas que visem a melhoria deste cenário devem ser estudadas, deste modo os objetivos deste trabalho foram: a) avaliar a germinação, contaminação endógena e exógena em sementes de maracujá-azedo comercial (Passiflora edulis f. flavicarpa; b) avaliar o estabelecimento in vitro de Passiflora caerulea, Passiflora foetida e Passiflora tenuifila, através do Teste exato de Fisher, Teste do Qui-Quadrado e Índice Kappa para as variáveis de natureza qualitativas e Teste de Tukey e análise de variância para as quantitativas; c) encontrar o tamanho ótimo de parcelas para experimentos em casa de vegetação com Passiflora setacea e Passiflora alata utilizando o método da máxima curvatura modificado. Conclui-se que na germinação o substrato orgânico pode substituir o meio MS, que a giberelina teve um efeito positivo na germinação e que o melhor tratamento físico de semente foi a escarificação. No estabelecimento, as espécies Passiflora caerulea e Passiflora tenuifila apresentaram melhores resultados que a Passiflora foetida, indicando que se adaptaram melhor ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Passiculture has been suffering with a several pathogens problems, which limit the productivity and viability of the passion-fruit culture. Between the mainly pathogens are the antracnosis (Colletotrichum gloesporioides Penz.), the bacterial spot (Xanthomonas campestris pv. passiflorae) and the premature-death (Fusarium oxysporum f. passiflorae), this one, the mainly bottleneck disease of the crop, limiting time of passiculture from perennial to annual. Alternatives aiming at improving of this scenario must been study, that way the objectives of this study was: evaluate the germination, endogen and hexogen contamination of commercial passion-fruit seed (Passiflora edulis f. flavicarpa) in factorial design of 10x3 (10 substrates and 3 physical treatments); evaluate the in vitro establishment of Passiflora caerulea, Passiflora foetida and Passiflora tenuifila, through Fisher’s Exact Test, Chi-Square Test and Cohen’s Kappa for the qualitative variables and Tukey’s Test and variance analysis for the quantitatives; find the optimum plot sizes for the experiments on green house with Passiflora setacea and Passiflora alata. Concluded that for the germination the organic substrate can substitute the MS medium, that the gibberellin had a positive effect in germination and that the best physical treatments for seed was scarification. In establishment, the species Passiflora caerulea and Passiflora tenuifila shown better results than Passiflora foetida pointing that those one adapts its... (Complete abstract click electronic access below) / Mestre