Browsing by Subject "GTPase-Activating Proteins"
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Item Blood Vessel Development(2014-11-17) Koo, Yeon Seung; Carroll, Thomas J.; Cleaver, Ondine; Olson, Eric N.; Cobb, Melanie H.Cardiovascular system is the first developing functional organ in vertebrates, and one of the fundamental organ systems through adulthoods. Cardiovascular function depends on patent blood vessel formation by endothelial cells. In this dissertation, a careful analysis of different aspects of blood vessel development was conducted in order to expand our understandings of the biology of blood vessel. In brief, Chapter 2 provides an in depth description of vessel anatomy during formation of the first arteries and veins in developing murine embryos, and a stepwise acquisition of arteriovenous fate in these vessels. The dorsal aortae, the first intraembryonic vessels initiate arterial specification before complete circulation occurs, and gradually express a subset of arterial genes. The first veins then establish their fate later than the first arteries. It was also shown that the arteriovenous specification does not require hemodynamic flow, but the flow affects maintenance of select arterial genes. Chapter 3 focuses on the function of Rasip1 in vasculogenesis, angiogenesis and vessel maintenance in developing embryo and in adults. An observation of transient Rasip1 localization at apical membranes in endothelial cells during in vitro tube formation provided an insight where Rasip1 possibly functions. Mice lacking Rasip1 showed that Rasip1 is critical for lumen formation likely via recruiting Myosin to endothelial apical membranes to alter cell cytoskeleton during vasculogenesis. Rasip1 was also shown to be required for proper vessel remodeling and lumen formation during angiogenesis. I found, however, it is dispensable for adult established blood vessel lumen maintenance, making it a promising therapeutic target for anti-angiogenesis. Finally, in Chapter 4, I show my preliminary findings regarding the role of Arhgap29, a Rasip1 binding partner, during embryonic development. Genetic ablation of Arhgap29 in mice led to allantois-chorion fusion and cardiovascular defects during embryonic development. Arhgap29 expression patterns were correlated with the sites of defects, as Arhgap29 transcripts were seen in allantois and cardiovascular tissues. From these results, its potential role in regulation of cell adhesion, likely via RhoA, was suggested. Overall, this thesis describes basic aspects of vascular development and uncharacterized molecules in different aspects of cardiovascular function.Item Brain Molecules under the Influence: Intracellular Regulation of Behavioral Responses Induced by Ethanol(2013-01-17) Peru Y Colón de Portugal, Raniero Leonette; Rothenfluh, AdrianAlcohol abuse is a devastating condition affecting millions of individuals. Regulation of insulin receptor (InR) signaling is critical for ethanol-induced responses and consumatory ethanol behavior. However, the precise intracellular mechanisms regulating InR signaling, which in turn, affect ethanol-induced behaviors remain unknown. I describe an InR/Arf6/S6K pathway that controls acute ethanol responses in Drosophila. I show that Arf6 mutants are hypersensitive to ethanol's intoxicating effects, and that Arf6 is specifically required in the adult nervous system to regulate na•ve ethanol sensitivity. While Arf6 functionally integrates activated Rac1 to the InR signaling, neuronal S6K, an InR effector, is a key mediator of Arf6-dependent regulation of ethanol-induced behaviors. Ethanol vapor concentrations that produce moderate sedation increase S6K-P, while doses that confer total sedation completely abate S6K-P. Arf6 mutants are completely devoid of neuronal S6K-P at baseline, suggesting that lack of S6K-P pre-sensitizes Arf6 mutants to the intoxicating effects of ethanol, and thus sedate at low physiologic ethanol concentrations. Because Arf6 has been implicated in receptor-mediated endocytosis, and signal transduction pathways are largely regulated by receptor trafficking, I propose a model in which Arf6 regulates InR signaling via endocytosis to control behavioral ethanol responses. My doctoral work on the intracellular mechanisms that govern ethanol's intoxicating effects on behavior will be described. The present dissertation is divided in four main sections: 1) Introduction, 2) results and methods, which include figures and figure legends and 3) a discussion of the results. In the introduction, I will review the scientific literature on the regulatory mechanisms of ethanol-driven behaviors performed in humans and other vertebrate species, while the central focus of this thesis is on Drosophila research. In doing so, I will also highlight current issues and problems concerning the study of ethanol's direct and candidate targets, which affect behavioral responses to ethanol. In the result and methods section, I will describe my obtained experimental data and the methodology employed. In the discussion section, I will first illustrate on the initial part of the results dealing with neuronal Rac1, Arfip, and Arf6, which through a linear genetic pathway regulate acute ethanol sedation. Second, I will explain how Arf6 GTPase may integrate the Rho to InR signaling to control behavioral ethanol responses. Third, I will illuminate on recent data showing that Arf6 via neuronal S6K mediates behavioral sensitivity to ethanol. Moreover, I will propose a model in which Arf6 plays separable but intertwined roles in InR signaling and endocytosis, in order to regulate acute ethanol behaviors.