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dc.contributor.authorRoche, Rebecca Ien_US
dc.date.accessioned2014-03-14T20:37:07Z
dc.date.available2014-03-14T20:37:07Z
dc.date.issued2002-05-02en_US
dc.identifier.otheretd-05162002-100901en_US
dc.identifier.urihttp://hdl.handle.net/10919/32843
dc.description.abstractAngiogenesis is the formation of new blood vessels from existing vasculature. Vascular Endothelial Growth Factor (VEGF), a known angiogenic protein, stimulates endothelial cell proliferation and migration via interactions with its receptors, KDR and Flt-1. A secreted form of Flt-1 (sFlt-1), derived from alternatively-spliced RNA, can inhibit actions of VEGF in vivo. It has been suggested that alterations in sFlt-1 expression could significantly change the angiogenic VEGF activity. This project focuses on characterizing intronic elements that regulate Flt-1 mRNA splicing. A "wild-type" construct (pFIN13), containing the first 13 exons, intron 13 and exons 14-30 of mouse Flt-1, was shown to produce both forms of Flt-1 mRNA after transfection into HEK293 cells. To gauge the strength of the native splicing signals in intron 13 of Flt-1, a series of point mutations were made in the polypyrimidine tract using pFIN13. After transient transfection, the levels of Flt-1 and sFlt-1 protein and mRNA were compared using quantitative PCR, RNA hybridization analysis, and protein immunoblotting. Results from quantitative PCR showed that purine substitutions were associated with 120 to 350 fold decreases in Flt-1 mRNA (normalized against neor), consistent with less efficient splicing. These large decreases in Flt-1 mRNA were accompanied by increases in sFlt-1 mRNA. Modest (20 to 100%) increases in Flt-1 mRNA, reflecting improved splicing, resulted from increasing the uridine complement in the polypyrimidine tract. These results suggest that the wild-type polypyrimidine tract is of intermediate strength and may be a regulatory locus for modulating Flt-1: sFlt-1 ratios.en_US
dc.publisherVirginia Techen_US
dc.relation.haspartRocheThesis.pdfen_US
dc.rightsI hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Virginia Tech or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.en_US
dc.subjectAngiogenesisen_US
dc.subjectVascular Endothelial Growth Factoren_US
dc.subjectRNA Splicingen_US
dc.subjectFlt-1en_US
dc.subjectPolypyrimidine Tracten_US
dc.subjectsFlt-1en_US
dc.titleRole of the Intron 13 Polypyrimidine Tract in Soluble Flt-1 Expressionen_US
dc.typeThesisen_US
dc.contributor.departmentVeterinary Medical Sciencesen_US
thesis.degree.nameMaster of Scienceen_US
thesis.degree.levelmastersen_US
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen_US
dc.contributor.committeechairHuckle, William R.en_US
dc.contributor.committeememberRobertson, John L.en_US
dc.contributor.committeememberGillaspy, Glenda E.en_US
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-05162002-100901/en_US
dc.date.sdate2002-05-16en_US
dc.date.rdate2005-10-13
dc.date.adate2002-05-22en_US


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