Browsing by Author "McNew, James"

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    Antiangiogenic Factor Receptor PlexinD1 is Required for Proper Formation of the Periocular Vasculature and Establishment of Corneal Avascularity
    (2015-08-03) Kwiatkowski, Sam C; Matthews, Kathleen; McNew, James; Stewart, Charles; Qutub, Amina
    The cornea is an avascular component of the visual system located in the anterior eye. Avascularity of the cornea is critical for proper vision since the cornea functions by transmitting light into the eye. Impaired vision from loss of avascularity may occur as a result of tissue damage which induces corneal neovascularization from the highly vascularized tissues of the anterior eye. Neovascularization in adult corneas is regulated by secreted pro- and anti-angiogenic factors. These factors function by maintaining corneal avascularity under healthy conditions while permitting neovascularization in damaged corneas. Several pro- and anti-angiogenic factors that function to maintain corneal avascularity during adult life have been identified, however little is known about how pro- and anti-angiogenic factors function to establish avascularity during corneal development. The purpose of this work was to study the role of pro- and anti-angiogenic factors during corneal development. First, the spatial and temporal expression patterns of numerous secreted pro- and anti-angiogenic factors were determined in the anterior eye during avian corneal development using semi-quantitative RT-PCR and RNA in situ hybridization. These techniques were also used to show that known receptors for secreted pro- and anti-angiogenic factors were simultaneously expressed in angioblasts and blood vessels located in the developing anterior eye. These experiments suggested that pro- and anti-angiogenic factor signaling mechanisms may contribute to the patterning of periocular vasculature and establishment of corneal avascularity. Next, I exemplified the role of pro- and anti-angiogenic factors during avian corneal development by using shRNA to knock down the expression of PlexinD1, an antiangiogenic factor receptor expressed in periocular angioblasts and blood vessels. Knockdown of PlexinD1 resulted in multiple patterning defects of the developing periocular vasculature including corneal neovascularization. These phenotypes implicated PlexinD1 as a critical component of the genetic mechanisms that establish corneal avascularity and were suggestive of the role that other pro- and anti-angiogenic factors may play during anterior eye development. These results demonstrate how pro- and antiangiogenic factors are used to simultaneously promote vascularization of the anterior eye and corneal avascularity during development. This information may lead to the creation of novel therapeutic treatments for vascular patterning abnormalities in the anterior eye and corneal neovascularization.
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    Discovery and Characterization of Lipid Disruption Activity in the Human Astrovirus Capsid
    (2022-12-02) Ykema, Matthew Ryan; McNew, James; Tao, Yizhi J
    Astroviruses (AstVs) are non-enveloped, positive single stranded RNA viruses that cause a wide range of inflammatory diseases in mammalian and avian hosts. The T=3 viral capsid is unique in its ability to infect host cells is a process driven by host proteases. Intercellular protease cleavages allow for viral egress from a cell, while extracellular cleavages allow for the virus to enter a new host cell to initiate infection. High resolution models of the capsid core indicate a large, exposed region enriched with protease cleavage sites. During maturation, the capsid goes through significant structural changes including the loss of many surface spikes. However, there is still a lack of structural information surrounding the immature VP90 capsid assembly, along with the matured VP70 and VP34/27/25 assemblies. I have expressed the VP90 protein and correlated the maturation of the viral protein to a previously uncharacterized function, disruption of lipid membranes in vitro. The original goal of this project was to model the stages of the capsid maturation cycle at high resolution. I was able to purify the VP90 state of the virus through expression and purification in a bacterial system, however full capsid assemblies were unable to be obtained. These purified VP90 proteins were used as a template for functional assays. It has been previously proposed that the AstV capsid can function as a toxic protein that causes an increased cellular membrane flux and disruption, but this function has not been observed in other conditions. I developed an assay to use artificially generated liposomes to test the lipid-disruption activity of AstV protein in vitro. I was the first to observe that the AstV VP90 protein has high levels of lipid-disruption activity only after maturation by trypsin treatment. This liposome assay was used to test many AstV proteins and lipid conditions. I have concluded that the lipid-disruption activity originates from the cleavage of trypsin sites from amino acids 299 to 313 in the AstV Outer Core domain, and the activity is dependent on the types of lipids present in the liposomes. Further understanding of the viral capsid structure and maturation process can contribute to characterizing the toxic properties of the AstV capsid, gastric therapeutics, and viral engineering applications.