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.

Stem cell differentiation is regulated by the complex interplay of multiple parameters, including adhesive intercellular interactions, cytoskeletal and extracellular matrix remodeling, and gradients of agonists and antagonists that individually and collectively vary as a function of spatial locale and temporal stages of development. Directed differentiation approaches have traditionally focused on the delivery of soluble morphogens and/or the manipulation of culture substrates in two-dimensional, monolayer cultures, with the objective of achieving large yields of homogeneously differentiated cells. However, a more complete understanding of stem cell niche complexity motivates tissue engineering approaches to inform the development of physiologically relevant, biomimetic models of stem cell differentiation. The capacity of pluripotent stem cells to simultaneously differentiate toward multiple tissue-specific cell lineages has prompted the development of new strategies to guide complex, three-dimensional morphogenesis of functional tissue structures. The objective of this project was to characterize the spatiotemporal dynamics of stem cell biophysical characteristics and morphogenesis, to inform the development of ESC culture technologies to present defined and tunable cues within the three-dimensional spheroid microenvironment. The hypothesis was that the biophysical and biochemical cues present within the 3D microenvironment are altered in conjunction with morphogenesis as a function of stem cell differentiation stage. Understanding biochemical and physical tissue morphogenesis, including the relationships between remodeling of cytoskeletal elements and intercellular adhesions, associated developmentally relevant signaling pathways, and the physical properties of the EB structure together elucidate fundamental cellular interactions governing embryonic morphogenesis and cell specification. Together, this project has established a foundation for controlling, characterizing, and systematically perturbing aspects of stem cell microenvironments in order to guide the development of complex, functional tissue structures for regenerative therapies.

Stem cell-based therapies are an important developing technology for treating cardiovascular ischemic disease, including subsequent co-morbidities such as ulcerative wounds. Mesenchymal stem cells (MSCs) have a proven ability to augment wound healing and neovascularization processes and have been more recently investigated for their endothelial-like behavior. This doctoral work aims to understand mechanisms underlying matrix-driven MSC tubulogenesis within PEGylated fibrin gels, specifically (1) why this behavior occurs and (2) if this behavior has clinical utility. Briefly, a three-dimensional morphological quantification pipeline was first developed for analyzing the maturity of vascular networks (Chapter 2). This method was applied in later studies that examined the full spectrum of MSC behavior in PEGylated fibrin gels, linking biomaterial properties with network development (Chapter 3). Mechanisms underlying the cell-matrix relationship were more fully clarified through gain-of-function cell studies. These studies indicated that PEGylated fibrin promotes endothelial-like MSC behavior through a combination of hypoxic stress and bioactive fibrin cues (Chapter 4). Notably, this endothelial-like MSC behavior closely mirrored vasculogenic mimicry, a process whereby tumors establish non-endothelialized vasculature in response to hypoxic stress. The functionality of these tumor vessels suggests that mature endothelial differentiation of MSCs may not be necessary to achieve therapeutically beneficial tissue perfusion. This hypothesis opens up new mechanisms for exploitation in vascular tissue engineering strategies. / text