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

La voie de signalisation Notch est une voie de signalisation conservée et primordiale aussi bien pour le développement des métazoaires que la maintenance de leurs tissus adultes. L'ubiquitine ligase Mind bomb1 (Mib1) est nécessaire à l'activation du récepteur Notch et contrôle l'ubiquitination et l'endocytose des ligands DLS (Delta/Serrate/Lag-2) de Notch. Afin d'identifier de nouveaux régulateurs de la signalisation des ligands DSL chez la drosophile, j'ai réalisé un crible modificateur sur un phénotype d'aile induit par des pertes partielles d'activité mib1. Une collection de lignées GS (Gene Search), permettant l'expression ectopique des gènes adjacents à leur point d'insertion, a permis d'identifier deux interacteurs génétiques de mib1 : α4GT1 et Hsc70-4. Hsc70-4 est un régulateur de la dynamique du manteau de clathrin dont la fonction dans la signalisation Notch et l'endocytose des ligands DSL n'a pas encore été analysée. α4GT1 est une α1,4- Nacétylgalactosaminyltranferase impliquée dans la voie de biosynthèse des glycosphingolipides (GSLs). L'obtention d'allèles mutants de cette enzyme a révélé qu'elle n'était pas essentielle à la signalisation Notch. En revanche, sa surexpression restaure le trafic des ligands DSL dans des contextes de pertes partielles d'activité ubiquitine ligase. Des analyses génétiques et biochimiques ont permis de montrer que cette fonction d'α4GT1 dans la signalisation Notch nécessite la synthèse d'un glycosphingolipide (GSL) particulier, N5, produit par α4GT1. L'identification d'un motif conservé d'interaction avec les GSLs dans le domaine Nterminal de Delta et Serrate suggère une interaction directe entre ces ligands et les GSLs.

Vascularization of tissue-engineered substitutes is imperative for successful implantation into sites of injury. Strategies to promote vascularization within tissue-engineered constructs have focused on incorporating endothelial or endothelial progenitor cells within the construct. However, since endothelial and endothelial progenitor cells are adult cell types and limited in number, acquiring quantities needed for regenerative medicine applications is not feasible. Pluriopotent stem cells have been explored as a cell source for tissue-engineered substitutes because of their inherent ability to differentiate into all somatic cell types, including endothelial cells (ECs). Current EC differentiation strategies require laborious and extensive culture periods, utilize large quantities of expensive growth factors and extracellular matrix, and generally yield heterogenous populations for which only a small percentage of the differentiated cells are ECs. In order to recapitulate in vivo embryonic stem cell (ESC) differentiation, 3D stem cell aggregates or embryoid bodies (EBs) have been employed in vitro. In the developing embryo, fluid shear stress, VEGF, and oxygen are instructive cues for endothelial differentiation and vasculogenesis. Thus, the objective of this work was to study the effects of fluid shear stress pre-conditioning of ESCs on EB endothelial differentiation and vasculogensis. The overall hypothesis is that exposing ESCs to fluid shear stress prior to EB differentiation will promote EB endothelial differentiation and vasculogenesis. Pre-conditioning ESCs with fluid shear stress modulated EB differentiation as well as endothelial cell-like cellular organization and EB morphogenesis. To further promote endothelial differentiation, ESCs pre-conditioned with shear were treated with VEGF. Exposing EBs formed from ESCs pre-conditioned with shear to low oxygen resulted in increased production of VEGF and formation of endothelial networks. The results of this work demonstrate the role that physical forces play in modulating stem cell fate and morphogenesis.

The objective of this work is to investigate the elastic modulus of gastrula-stage avian embryos and the effect of substrate stiffness on presumptive precardiac cell fate. Our overall hypothesis is that the mechanical microenvironment, specifically, tissue modulus and substrate stiffness, influences gastrulation and cardiac induction. Large-scale morphogenetic movements during early embryo development are driven by complex changes in biochemical and biophysical factors. Current models for amniote primitive streak morphogenesis and gastrulation take into account numerous genetic pathways but largely ignore the role of mechanical forces. Here, we used atomic force microscopy (AFM) to obtain for the first time precise biomechanical properties of the early avian embryo. Our data reveal that the primitive streak is significantly stiffer than neighboring regions of the epiblast, and that it is stiffer than the pre-primitive streak epiblast. To test our hypothesis that these changes in mechanical properties are due to a localized increase of actomyosin contractility, we inhibited actomyosin contractility via the Rho kinase (ROCK) pathway using the small-molecule inhibitor Y-27632. Our results using several different assays show the following: 1) primitive streak formation was blocked; 2) the time-dependent increase in primitive streak stiffness was abolished; and 3) convergence of epiblast cells to the midline was inhibited. Taken together, our data suggest that actomyosin contractility is necessary for primitive streak morphogenesis, and specifically, ROCK plays a critical role. To better understand the underlying mechanisms of this fundamental process, future models should account for the findings presented in this study. As presumptive cardiac cells traverse the course of differentiation into cardiac myocytes during cardiogenesis, the sequence, magnitude, and spatiotemporal map of biomechanical and biochemical signals has not been fully explored. There have been many studies detailing the induction of cardiogenesis on a variety of substrates and extracellular matrix (ECM) proteins, but none have completed a rigorous study of the effects of substrate stiffness on the induction of precardiac cells prior to the onset of cardiac gene expression (smooth muscle alpha actin [SMAA] at stage 5.) We investigate the effects of the mechanical environment on precardiac cell behaviors in an in vitro setting to elucidate the effect of substrate stiffness and inducing factors on precardiac tissue and the potential connection between them. The cells in the anterior portion of the primitive streak are fated to form the heart, and we show differing levels of SMAA expression on substrates of differing moduli, which suggests that substrate stiffness may play a role in cardiac differentiation. We cannot determine the physical mechanisms during morphogenesis without understanding the response of precardiac cells to changes in their mechanical environment.

<p>The proper modification and trafficking of plasma membrane proteins are essential for normal neuronal function, such as dendrite morphogenesis, spine formation and synaptic plasticity. The secretory organelles including endoplasmic reticulum and Golgi apparatus are critical for the trafficking of these molecules as shown in fibroblasts. Although these secretory organelles have been observed in neurons including dendritic branches, their spatial organization and function in protein trafficking, neuronal development and plasticity are not clear yet. Here, I used photobleaching and photoactivation approaches combined with electron microscopy to show that although rapidly diffusing within the continuous network of the somato-dendritic ER, membrane proteins such as nascent AMPA receptors are confined by ER spatial complexity. The spatial range of ER membrane protein mobility becomes progressively confined over neuronal development and is rapidly restricted by synaptic activity. Thus, constrained lateral mobility within the ER provides a novel mechanism for compartmentalized trafficking of nascent receptors throughout dendrites. I also identified an ER protein as a novel microtubule-associated protein regulating dendritic ER spatial complexity, neuronal dendrite elongation and spine formation. Together, these results describe the spatial organization of dendritic ER and its role in regulating membrane protein trafficking, neuronal morphogenesis and postsynaptic functions.</p> / Dissertation

<p>Fungal cells sense the amount and quality of external nutrients through multiple interconnected signaling networks, which allow them to adjust their metabolism, transcriptional profiles and developmental programs to adapt readily and appropriately to changing nutritional states. In organisms ranging from yeasts to humans, the Tor signaling pathway responds to nutrient-derived signals and orchestrates cell growth. While in the baker's yeast <italic>Saccharomyces cerevisiae</italic> Tor responds to nutrient-derived signals and orchestrates cell growth and proliferation, in <italic>Schizosaccharomyces pombe</italic> Tor signaling modulates sexual differentiation in response to nutritional cues. Thus, these differences provide a framework to consider the roles of Tor in other fungal organisms, in particular those that are pathogens of humans. </p><p>In this dissertation, I demonstrate that in the human fungal pathogen <italic>Candida albicans</italic>, Tor signaling also functions to promote growth. This study also uncovered a novel role for the Tor molecular pathway in promoting hyphal growth of <italic>C. albicans</italic> on semi-solid surfaces and in controlling cell-cell adherence. Gene expression analysis and genetic manipulations identified several transcriptional regulators (Bcr1, Efg1, Nrg1, and Tup1) that together with Tor compose a regulatory network governing adhesin gene expression and cellular adhesion. While the Tor kinases are broadly conserved, these studies further demonstrate the contrasting strategies employed by fungal organism in utilizing the Tor signaling cascade.</p><p>While extensive studies have focused on elucidating functions for the Tor signaling cascades among ascomycetes, little is known about the pathway in basal fungal lineages, in particular among zygomycetes and chytrids. Moreover, given that the Tor pathway is the target of several small molecule inhibitors including rapamycin, a versatile pharmacological drug used in medicine, there is considerable interest in Tor signaling pathways and their function. Capitalizing on emerging genome sequences now available for several basal fungal species, we show a remarkable pattern of conservation, duplication, and loss of the Tor signaling cascade among basal fungal lineages. Targeting the pathway with rapamycin results in growth arrest of several zygomycete species, indicating a conserved role for this pathway in regulating fungal growth. In addition, we show a potential therapeutic advantage of using rapamycin in a heterologous model of zygomycosis. Taken together, the Tor signaling cascade and its inhibitors provide robust platforms from which to develop novel antimicrobial therapies, which may include less immunosuppressive rapamycin analogs.</p> / Dissertation

<p>The means by which cells, tissues, and organisms undergo morphogenesis are variable and highly regulated, and the mechanisms that govern cellular changes in response to signaling cues are poorly understood. This study seeks to address the role of a newly characterized protein in zebrafish in translating signaling cues into physical changes within a cell.</p><p>The <italic>Cordon&ndash;bleu (Cobl)</italic> gene is widely conserved in vertebrates, with developmentally regulated axial and epithelial expression in mouse and chick embryos. <italic>In vitro</italic>, Cobl can bind monomeric actin and nucleate formation of unbranched actin filaments, while in cultured cells it can modulate the actin cytoskeleton. However, an essential role for Cobl <italic>in vivo</italic> has yet to be determined. We have identified the zebrafish <italic>cobl</italic> ortholog and have used zebrafish as a model to assess the requirements for Cobl in embryogenesis. We find that cobl shows enriched expression in ciliated epithelial tissues during zebrafish organogenesis. The utilization of antibodies developed against Cobl shows that the protein is concentrated along the apical domain of ciliated cells, in close proximity to the apical actin cap. </p><p>Reduction of <italic>cobl</italic> by antisense morpholinos reveals an essential role in embryonic morphogenesis and organ development. A requirement for Cobl was shown for the proper function of various and ciliated epithelial organs. Cobl appears to direct the elongation of motile cilia in organs such as Kupffer&rsquo;s vesicle and the pronephros. In Kupffer&rsquo;s vesicle, the reduction in Cobl coincides with a reduction in the amount of apical F-actin. Additionally, Cobl may play a role during gastrulation cell movements and convergence and extension morphogenesis during early embryonic development. Thus, Cobl may represent a molecular activity that couples developmental patterning signals with local intracellular cytoskeletal dynamics to support elongation of motile cilia and tissue morphogenesis.</p> / Dissertation

Aim of this thesis is to explain the role of property relations in the production of urban form. It is assumed that urban form is produced not only as a physical setting but also as a concrete and relatively fixed manifestation of property relations. In this respect, urban form should be considered in a relational conception of space. The study departs from the proposition that property relations are the main determinants of the formation of urban space, and private property constitutes the generator of the dynamics and contradictions of urban formation, through a continuous process of fragmentation. For this reason, in the control of urban formation, property rights are the basic element that city planners have to face. Therefore, this study presents a comprehensive framework that integrates the categories of urban morphology with a structural analysis of urban formation process. As the essential unit of capitalist city, &ldquo / production of the parcel as a commodity&rdquo / is elaborated as the core of urban formation process. In this framework, morphogenesis of Yenisehir&ndash / Ankara is analyzed in order to understand its historical transformation with reference to the context of property relations. Its morphological layers are depicted as a product of the contradictory relation between urban planning and property relations. It is seen that the morphogenesis of Yenisehir includes three distinct layers of formation, which are characterized by the gradual domination of commodity production in the formation process of urban space.

No description available.

Rinderpest virus is an enveloped Nonsegmented Negative Stranded RNA Virus (NNSV) belonging to the genus Morbillivirus in the Family Paramyxoviridae and the causative organism for “cattle plague”. The virion has a transport component and a replication component. The transport component consists of a lipid membrane with two external membrane-anchored glycoproteins, namely Hemagglutinin (H) and Fusion (F) proteins that are necessary for cell entry and release of newly formed virus particles. The replication component consists of viral genomic RNA encapsidated by the nucleoprotein (N) and a RNA polymerase complex (Large subunit L and phosphoprotein P). These two components are linked together by the matrix protein (M) that is believed to play a crucial role in the assembly and maturation of the virion particle by bringing the two major viral components together at the budding site in the host cell. To perform this function, M protein should be able to interact with the host cellular membrane, especially the plasma membrane in the case of Rinderpest virus, should be able to interact with itself to form multimers as well as with the nucleocapsid core. The function might include the interaction of M protein with the cytoplasmic tail of the other two envelope proteins namely F and H. To understand the role of matrix protein in Rinderpest virus life cycle, the following functions were characterized – 1) Matrix protein association with the host cell membrane. 2) Matrix protein association with nucleocapsid protein. Matrix protein association cellular membranes in rinderpest virus infected cells could be a result of its interaction with the cytoplasmic tails of the viral glycoproteins. Hence, this association was characterized in the absence of other viral proteins. In transiently transfected cells, M protein existed in two isoforms namely the soluble cytosolic form and membrane-bound form. The membrane-bound M protein associated stably with the membranes, most likely by a combination of electrostatic and hydrophobic interactions, which is inhibited at high salt or high pH, but not completely. Confocal microscopy analysis showed the presence of M protein in plasma membrane protrusions. When GFP was tagged with this protein, GFP was absent from nucleus and was present predominantly in the cytosol and the plasma membrane protrusions. However, M protein expression did not result in the release of membrane vesicles (Virus-like particles) into the culture supernatant implicating the requirement of other viral proteins in envelope acquisition. Matrix protein of RPV has been shown to co-sediment with nucleocapsid during mild preparation of RNP from virus-infected cells. This association was further investigated by virus solubilization. The matrix protein could be solubilised completely from virion only in the presence of detergent and high salt. This is in agreement with the previous observation from the laboratory that the purified matrix protein remained soluble in the presence of detergent and 1M NaCl. This suggested that M protein could oligomerise or associate with nucleocapsid. The purified M protein when visualized by Electron microscopy showed the presence of globular structures, which may be due to self association of M protein, which may be due to self-aggregation of M protein. The presence of GFPM in filamentous structures in transfected cells, as visualized by confocal microscopy could also be due to self-assembly of M protein. Interaction of matrix protein RPV nucleocapsid was confirmed using co- sedimentation and floatation gradient analysis. Results obtained from M-N binding assay using C-terminal deletions of nucleocapsid protein suggested that the matrix protein interacted with the conserved N-terminal core of nucleocapsid and non- conserved C-terminus 20% is dispensable. This is in agreement with the report that RPV M protein could be replaced with that of Peste-des-petits-ruminants virus(a closely related morbillivirus). The observation that the nucleocapsid protein interacts with both soluble and membrane-bound form suggests that the matrix protein can possibly interact itself to facilitate the assembly of replication component at the site of budding where the transport component is already assembled. Viral proteins of many RNA viruses interact with detergent-resistant host components that facilitate their transport inside the cell to the sits of assembly or replication. Rinderpest viral proteins acquire detergent resistance in infected cells. This acquisition is mediated by viral N protein. The relevance of this interaction in virus life cycle was studied using small molecule drugs that disrupt host cytoskeleton and lipid raft. The results obtained suggested that the host cytoskeleton, especially actin-filaments facilitate virus release from the plasma membrane. RPV matrix protein acquired detergent resistance in infected cells as well as in transfected cells. The pattern of detergent resistance suggested an association with the cytoskeleton or cytoskeleton associated proteins. However, results obtained from co-localisation studies in the presence of actin inhibitor and cold-ionic detergents are not consistent with the above observation. This property could be due to self-association of matrix protein.