Roundabout (Robo) receptors were first identified in neurons as guidance molecules, however growing evidence suggests that they also play a role in other cells. The aim of this thesis was to characterise the expression and function of a novel endothelial specific member of this family, Robo4. This study revealed that Robo4 is expressed primarily in vessels but also differentially expressed in tumour vessels. Interestingly Robo4 was primarily located within cytoplasmic vesicles coated with clathrin, suggesting that the presence of Robo4 on the cell surface is being tightly regulated. Overexpression of Robo4 induced filopodia and pseudopodia formation and actin re-organisation into stress fibres. It co-localised with actin and tubulin suggesting an important interaction between Robo4 and the cytoskeleton. Robo4's function in endothelial cells was directly investigated using two approaches, overexpression using adenovirus and knockdown using small interfering RNA. Functional cell-based assays revealed that disrupting Robo4's level of expression negatively affects endothelial cell functions that are required during angiogenesis, such as proliferation, migration and tubulogenesis. Overexpression of a truncated version of Robo4, which lacks the C-terminus, provided clues regarding Robo4's function. The intracellular domain is critical for Robo4's localisation and its association with the cytoskeleton. It is also required for pseudopodia formation. Other findings include possible cleavage of Robo4 and Robo4 homodimerisation and heterodimerisation with Robo1. Taken together, the findings presented in this study strongly suggest a role for Robo4 in endothelial cell guidance. Cell guidance during angiogenesis is poorly understood therefore the identification of a new molecule potentially involved in this mechanism will hopefully help elucidate the process.

The retina is one of the most metabolically active tissue in the body, rendering it sensitive to vascular dysfunction. Consequently, diseases that disrupt normal retinal blood supply, including retinal vein occlusions (RVO) and diabetic retinopathy, are the leading causes of blindness in working-age adults. Despite available therapies, an estimated 50% of patients do not respond to treatment. We employed a mouse model of retinal vein occlusion (RVO), achieved by tail-vein injection of rose bengal, followed by laser photocoagulation of retinal veins. In vivo analyses – optical coherence tomography (OCT), fluorescein angiography, and electroretinograms (ERGs) - were conducted with the Micron IV system (Phoenix Research Labs). RVO induces acute retinal edema, which peaks during the first 24 hours following injury. Over a 7 day time course the edema resolves, revealing a permanent retinal thinning due to death of retinal neurons. We identified caspase-9, a protease traditionally associated with apoptosis, as an essential mediator of edema. Increased levels of activated caspase-9 were detected in vascular endothelial cells 1 hour following RVO. We tested RVO in mice with inducible endothelial-cell-specific deletion of caspase-9 (iC9 ECKO). Compared to littermate controls, iC9 ECKO mice develop less edema, and sustain less retinal degeneration after RVO injury. ERG analysis showed preservation of retinal function in iC9 ECKO mice. To study whether inhibiting caspase-9 would provide protection against RVO we utilized a highly specific caspase-9 inhibitor, which we can deliver to the retina using simple eyedrops. Treatment of wildtype mice with the caspase-9 inhibitor immediately after induction of RVO provided morphologic, biochemical and functional retinal protection. Inhibition of caspase-9 reduces edema, protects retinal morphology, and helps prevent vision loss following RVO injury. Our studies indicate that endothelial caspase-9 plays an essential role in regulating edema pathogenesis. Moreover, our novel cell permeant caspase-9 inhibitor abrogates the edema and may be a potential therapy for individuals suffering from vascular eye disease.

No description available.

Includes bibliographical references.

Bibliography: leaves 157-177.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Blood vessel injury was found to release intracellular pools of protein D-aspartyl/L-isoaspartyl carboxyl methyltransferase (PIMT) into the extracellular milieu, where it became trapped. Trapped PIMT was able to utilize radiolabeled S-adenosyl-L-methionine (AdoMet) introduced into the circulation to methylate blood vessel proteins containing altered aspartyl residues specifically at the site of injury. In vitro studies more fully characterized this endogenous PIMT activity in thoracic aorta and inferior vena cava. At least 50% of the PIMT activity released during injury, was resistant to non-ionic detergent extraction, suggesting that the enzyme activity can become trapped within or behind the extracellular matrix (ECM). Analysis of inferior vena cava, found that 90% of the altered aspartyl residues in blood vessels are inaccessible to methylation by intracellular PIMT under physiological conditions. Subfractionation of inferior vena cava on the basis of solubility found that at least 40% of the altered aspartyl containing proteins in blood vessels are insoluble in non-ionic detergent containing buffers and are highly resistant to extraction by protein denaturants. Analysis of peptides revealed that the majority of the altered aspartyl groups in blood vessels are located extracellularly. Digestion of these extracellular matrix proteins with cyanogen bromide (CNBr), followed by methylation with (PIMT), found that about 60% of the altered aspartyl residues in the ECM are solubilized by this treatment. The presence of hydroxyproline in amino acid hydrosolates of this fraction and acidic pH gel electrophoresis of methylated peptides, allowed the identification of collagen as the major PIMT substrate in the CNBr-soluble material. CNBr peptides derived from both type I and type III collagen were found to methylated. It is estimated that one centimeter of blood vessel contains on the order of 5 x 10����� altered aspartyl residues involving 1% to 5% of the total extracellular protein. / Graduation date: 1997

Thesis (Ph.D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Health and Human Performance. Title from title-page of electronic thesis. Includes bibliographical references.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 73-90). Also available in print.

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 15, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Dr. Gabor Forgacs. Vita. Includes bibliographical references.