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An Examination of the Human Fibrinogen-Like Protein2: Sequence Variations and Genetic Expression by Human Endothelial Cells

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An Examination of the Human Fibrinogen-Like Protein2: Sequence Variations and Genetic Expression by Human Endothelial Cells Georgia State University ScholarWorks @ Georgia State University Biology Theses Department of Biology 1-12-2006 An Examination of the Human Fibrinogen-like Protein2: Sequence Variations and Genetic Expression by Human Endothelial Cells Meredith E. Jenkins Follow this and additional works at: https://scholarworks.gsu.edu/biology_theses Part of the Biology Commons Recommended Citation Jenkins, Meredith E., "An Examination of the Human Fibrinogen-like Protein2: Sequence Variations and Genetic Expression by Human Endothelial Cells." Thesis, Georgia State University, 2006. https://scholarworks.gsu.edu/biology_theses/1 This Thesis is brought to you for free and open access by the Department of Biology at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Biology Theses by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. AN EXAMINATION OF THE HUMAN FIBRINOGEN-LIKE PROTEIN 2: SEQUENCE VARIATIONS AND GENETIC EXPRESSION BY HUMAN ENDOTHELIAL CELLS by MEREDITH JENKINS Under the Direction of Roberta Attanasio ABSTRACT A role for Fgl2 (fibroleukin) in thrombophilic-associated human fetal loss has been indicated by previous studies. Clotting of vascular vessels of the placenta and fetus interferes with adequate blood circulation. The human Fgl2 gene was sequenced with identification of six SNPs, suggesting an association with a population of women suffering from recurrent fetal losses. A small sample size however, prevented precise statistical analyses of this association. In vitro, human endothelial cells (HUVECs) were not found to constitutively express Fgl2, but were shown to up-regulate its expression when challenged with IFN-γ and TNF-α. Interestingly, TNF-α was only shown to induce expression of Fgl2 in HUVECs from male donors and not female donors. A larger case-control study is needed to examine the relationship of Fgl2 with recurrent fetal loss. The role of estrogen in the Th1-induced expression of Fgl2 by HUVECs should also be examined. INDEX WORDS: Fibroleukin, Fgl2, thrombosis, recurrent fetal loss, endothelial cells, HUVEC, Th1 cytokines, IFN-γ, TNF-α AN EXAMINATION OF THE HUMAN FIBRINOGEN-LIKE PROTEIN 2: SEQUENCE VARIATIONS AND GENETIC EXPRESSION BY HUMAN ENDOTHELIAL CELLS by Meredith Jenkins A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science In the College of Arts and Sciences Georgia State University 2005 Copyright by Meredith Jenkins 2005 AN EXAMINATION OF THE HUMAN FIBRINOGEN-LIKE PROTEIN 2: SEQUENCE VARIATIONS AND GENETIC EXPRESSION BY HUMAN ENDOTHELIAL CELLS by MEREDITH ESTHER JENKINS Major Professor: Roberta Attanasio Committee: P.C. Tai W.C. Hooper Electronic Version Approved: Office of Graduate Studies College of Arts and Sciences Georgia State University December 2005 iv Dedication This thesis is dedicated to my loving family: Robert, Erin, and William Pyle and to my incredibly supportive parents, William and Elberta Jenkins. I could not have accomplished all that I have in life without you. v Acknowledgements I would like to thank Dr. Craig Hooper and the Centers for Disease Control and Prevention Division of Hereditary Blood Disorders for the use of laboratory facilities and general support for this research activity. I would also like to thank Dr. Christine DeStaercke for her continuing encouragement and faith in my abilities, without which I would not have been “properly” motivated to complete this project before the birth of William. vi TABLE OF CONTENTS ACKNOWLEDGEMENTS.............................................................................................................v LIST OF TABLES.........................................................................................................................vii LIST OF FIGURES.................................................................................................................viii-ix LIST OF ABBREVIATIONS..........................................................................................................x INTRODUCTION........................................................................................................................1-4 MATERIALS AND METHODS.................................................................................................4-9 DATA ANALYSIS........................................................................................................................10 QUALITY CONTROL.............................................................................................................10-12 RESULTS.................................................................................................................................12-40 DISCUSSION ..........................................................................................................................40-43 REFERENCES.........................................................................................................................44-46 vii LIST OF TABLES 1. Table 1: PCR and Sequencing Primers...............................................................................6 2. Table 2: PCR Protocols.......................................................................................................6 3. Table 3: ExoSAP-IT Clean-up Reaction.............................................................................7 4. Table 4: Sequencing PCR Protocol.....................................................................................7 5. Table 5: Ethanol Precipitation Clean-up Reaction..............................................................7 6. Table 6: RNA Purification Protocol...................................................................................9 7. Table 7: Reverse Transcriptase Reaction............................................................................9 8. Table 8: Real-Time PCR Protocol......................................................................................9 9. Table 9: Variant Allele Frequencies................................................................................ 14 10. Table 10: Sequencing Results: Raw Data and Allelic Frequencies.............................15-26 viii LIST OF FIGURES 1. Figure 1: FGL2 Single Nucleotide Polymorphism Map..................................................13 2. Figure 2: FGL2 Expression in HUVEC Controls: RT-PCR Amplification Plot ..........................................................................................29 3. Figure 3: FGL2 Expression in HUVEC Controls: Relative Quantity Chart..................................................................................................29 4. Figure 4: Effects of IL-2 on FGL2 Expression in HUVEC cells, Female Donor...............................................................................................................30 5. Figure 5: Effects of IL-2 on FGL2 Expression in HUVEC cells, Female Donor...............................................................................................................30 6. Figure 6: Effects of IL-2 on FGL2 Expression in HUVEC cells, Male Donor...............................................................................................................31 7. Figure 7: Effects of IL-2 on FGL2 Expression in HUVEC cells, Male Donor...............................................................................................................31 8. Figure 8: Effects of IFN-γ on FGL2 Expression in HUVEC cells , Female Donor...............................................................................................................32 9. Figure 9: Effects of IFN-γ on FGL2 Expression in HUVEC cells, Female Donor...............................................................................................................32 10. Figure 10: Effects of IFN-γ on FGL2 Expression in HUVEC cells, Male Donor..............................................................................................................33 11. Figure 11: Effects of IFN-γ on FGL2 Expression in HUVEC cells, Male Donor..............................................................................................................33 12. Figure 12: Effects of TNF-α on FGL2 Expression in HUVEC cells, Female Donor..............................................................................................................34 13. Figure 13: Effects of TNF-α on FGL2 Expression in HUVEC cells, Female Donor..............................................................................................................34 14. Figure 14: Effects of TNF-α on FGL2 Expression in HUVEC cells, Male Donor......................................................................................................... ....35 ix 15. Figure 15: Effects of TNF-α on FGL2 Expression in HUVEC cells, Male Donor...............................................................................................................35 16. Figure 16: GAPDH Expression in Control HUVEC Cell Samples.............................................................................................................36 17. Figure 17: Effects of IL-2 on GAPDH expression: HUVEC cells, Female Donor................................................................................................................36 18. Figure 18: Effects of IL-2 on GAPDH expression: HUVEC cells, Male Donor................................................................................................................37 19. Figure 19: Effects
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