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Contrôle spatio-temporel de la croissance filamenteuse chez Candida albicans / Temporal and spatial control of fungal filamentous growth in Candida albicansSilva, Patricia Maria de Oliveira e 22 May 2018 (has links)
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.
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Quantitative analysis of 3D tissue deformation reveals key cellular mechanism associated with initial heart looping / 初期心ループ形成時における3次元組織動態の定量解析と細胞機構の解明Kawahira, Naofumi 27 July 2020 (has links)
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
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Mechanics of Epithelial Tissue MorphogenesisWang, Xun January 2021 (has links)
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.
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Lien entre signalisation JAK/STAT, remodelage cellulaire et extrusion d’un groupe de cellules épithéliales dans l’ovaire de drosophile / Link Between JAK/STAT signaling, cell remodeling and extrusion from the follicular epithelium in the Drosophila ovaryTorres Espinosa, Alba Yurani 16 December 2016 (has links)
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.
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Úloha vybraných podjednotek komplexu exocyst ve vývoji epidermis Arabidopsis. / Subunits of exocyst complex in the development of Arabidopsis epidermisVojtíková, Zdeňka January 2013 (has links)
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....
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Propagação, estabelecimento in vitro e tamanho de parcelas experimentais de espécies de maracujazeiro /Pigari, Lucas Bernardo. January 2018 (has links)
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
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Morphogenesis and Protein Composition of Valve Silica Deposition Vesicles from DiatomsHeintze, Christoph 05 April 2022 (has links)
The silica-based cell walls of diatoms are outstanding examples of nature’s capability to synthesize complex porous structures with genetically controlled patterns from the nanometer scale to the range of hundreds of micrometers. Formation of the cell wall building blocks (valves and girdle bands) occurs in membrane-bound compartments, termed silica deposition vesicles (SDVs), which are unique organelles in silica-forming protists. Isolation of the SDVs has not yet been achieved, which has severely hampered the efforts to understand the mechanisms of biological silica morphogenesis. The present thesis aimed to address this shortcoming. The foundation was the development of an improved cell cycle synchronization and a fluorescence labeling method for the model diatom Thalassiosira pseudonana that enabled rapid identification of valve SDVs in a cell lysate. Correlative fluorescence and electron microscopy allowed visualizing the development of valve silica with unprecedented spatio-temporal resolution. Elemental analysis and demineralization of immature valves provided the first direct chemical evidence that silica morphogenesis is an interplay of inorganic and organic molecules inside the valve SDVs. Cryo TEM imaging of valve SDVs indicated the formation of organic patterns that precede silica depostion. From these observations, an organic biomolecule dependent, liquid-liquid phase separation based model for pore formation in the diatom T. pseudonana was proposed. The second part of this thesis was focused on the enrichment of valve SDVs from T. pseudonana and the subsequent proteomics based identification of more than 40 potential valve SDV proteins. Among these, three diatom-specific proteins contained conserved protein protein interaction domains (ankyrin-repeats) and were surprisingly predicted to be located in the cytoplasm. The fluorescent tagging of the three proteins (termed dANK1-3) confirmed their association with the valve SDVs. When the respective dank genes were knocked out by CRISPR/Cas9, the valves displayed permanent anomalies in the quantity and the pattern of ~22 nm sized pores. Double knockout mutants lacking both dank1 and dank3 were almost completely devoid of pores. The analysis of valve morphogenesis in the single and double knockout mutants revealed phenotypic changes that were consistent with the liquid-liquid phase separation based model for pore pattern formation in diatom biosilica. The work of this thesis has provided for the first time direct access to valve SDVs, which has opened entirely new possibilities for studying the composition, properties, and working mechanism of an organelle that forms a complex shaped mineral.
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Capillary Morphogenesis Gene Protein 2 (CMG2) Mediates Matrix Protein Uptake and is Required for Endothelial Cell Chemotaxis in Response to Multiple Vascular Growth FactorsTsang, Tsz Ming Jeremy 09 April 2020 (has links)
Pathological angiogenesis, or new blood vessel formation, is involved in many pathologies, including cancer and serious eye diseases. While traditional anti-angiogenic therapies target vascular endothelial growth factor receptors to reduce or inhibit new vessel formation, this approach has several downsides, including unpleasant side effects and low efficacy over time. Therefore, identifying new targets to treat pathological angiogenesis is still needed. CMG2, one of the two identified anthrax toxin receptors, has been proposed as an alternative target to treat pathological angiogenesis. CMG2’s role as a cell surface receptor that mediates anthrax toxin internalization is very well documented. One physiological function for CMG2, not related to anthrax intoxication, is suggested by the observation that loss-of-function mutations in CMG2 cause hyaline fibromatosis syndrome (HFS), a genetic disease that results in accumulations of extra-cellular matrix (ECM) protein in different parts of the body. While the complete molecular mechanism for CMG2’s role in regulating angiogenesis has not been determined, this dissertation addresses multiple ways CMG2 regulates pathological angiogenesis. We have discovered that CMG2 plays a role in mediating ECM homeostasis via endocytosis of ECM proteins and protein fragments as a way to generate angiogenic signals from the cell. We have also demonstrated that a fragment from Col IV, S16, is endocytosed into the cells by interacting with CMG2, and S16 treatment to endothelial cells leads to a significant reduction in cell migration. Also, an endothelial cell migration assay with CMG2 knockout cells results in abolished directional migration, indicating that CMG2 is required for endothelial cell chemotaxis. Notably, we have identified that bFGF, VEGF, and PDGF are involved in CMG2 mediated chemotaxis but not insulin and sphingosine-1-phosphate (S1P). While recent literature reports show that CMG2 works closely with RhoA GTPase, which is commonly known to regulate cell migration, we have also observed that inhibition of RhoA also reduced cell chemotaxis towards VEGF but not S1P. These results could be leveraged to develop new classes of therapeutic molecules to treat pathological angiogenesis induced by multiple various growth factors via targeting CMG2.
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Nrg1p and Rfg1p in Candida albicans yeast-to-hyphae transitionLacroix, Céline. January 2008 (has links)
No description available.
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Varicose/ Senz'Aria, A MAGUK Required for Junctional Assembly During Epithelial Morphogenesis in DrosophilaMoyer, Katherine Ellen 10 1900 (has links)
Scaffolding proteins belonging to the Membrane Associated GUanylate Kinase (MAGUK) superfamily function as adaptors linking cytoplasmic and cell surface proteins to the cytoskeleton to regulate cell-cell adhesion, cell-cell communication and signal
transduction. We have identified a novel Drosophila MAGUK member, Varicose (Vari), the homologue of vertebrate scaffolding protein PALS2. Similar to its vertebrate counterpart, Varicose localizes to pleated Septate Junctions (pSJs) of all embryonic,
ectodermally derived epithelia and peripheral glia. In vari mutants, essential SJ proteins NeurexinIV and FasciclinIII are mislocalized basally and the cells develop a leaky paracellular seal. Localization of SJ protein Discs Large is not affected, indicating Vari is not involved in cell polarization. In addition, vari mutants display irregular tracheal tube diameters and have reduced lumenal protein accumulation suggesting involvement in tracheal morphogenesis. We found that Vari is distributed in the cytoplasm of optic lobe neuroepithelium and is required for proper ommatidial patterning. As well, Vari is expressed in a subset of neuroblasts and differentiated neurons of the nervous system. We also present a novel MAGUK function in wing hair alignment during adult
morphogenesis. We conclude that Varicose is involved in scaffold assembly at the SJ and has a role in patterning adult epithelia and in nervous system development. / Thesis / Doctor of Philosophy (PhD)
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