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Open Vwf Thesis Wtemplate Maya2.Pdf THE PENNSYLVANIA STATE UNIVERSITY SCHREYER HONORS COLLEGE DEPARTMENT OF BIOMEDICAL ENGINEERING A CONTROL STUDY USING DNA TO TEST THE EFFECTS OF SHEAR STRESS ON VON WILLEBRAND FACTOR MAYA ANN-MARIE JANKOWSKA SPRING 2017 A thesis submitted in partial fulfillment of the requirements for a baccalaureate degree in Biomedical Engineering with honors in Biomedical Engineering Reviewed and approved* by the following: Keefe B. Manning Associate Professor of Biomedical Engineering Schreyer Associate Dean of Academic Affairs Thesis Supervisor William O. Hancock Professor of Biomedical Engineering Honors Advisor Peter J. Butler Engineering Associate Dean for Education Professor of Biomedical Engineering Faculty Reader * Signatures are on file in the Schreyer Honors College. i ABSTRACT In an effort to understand how the von Willebrand Factor (vWF), a blood clotting glycoprotein, and applied shear forces are related, this project focuses on a positive control study using plasmid DNA. Elevated shear stress levels, commonly introduced after ventricular assist device (VAD) implantation, are believed to cause unfurling and subsequent enzymatic cleavage of vWF. This makes vWF impotent and subjects the patient to high risks of bleeding. However, due to vWF's large and complex structure, a positive control of plasmid DNA will produce a more predictable experiment. Through attaching enzymatically-cut and treated plasmids around the entire exterior of biotinylated polystyrene beads, an effective radius of the bead plus the DNA length is assumed. The hypothesis is the effective radius of DNA is independent of shear rates. Within this study, the aims are to (1) determine 2 plasmid-enzyme pairs with one of comparable length to unfurled vWF and one of shorter length, (2) attach the treated enzymatically-cut plasmid to beads, (3) subject the beads to shear forces in an optical trap to compare effective radii of DNA-coated beads to non-coated beads, and (4) determine the effect of shear rates on DNA-coated and non-coated bead effective radii. Thus, the control study post- processing techniques and data can be reproduced and applied to vWF. A quantifiable difference in bead effective radius is observed between the DNA-coated beads and the non-coated beads. This presumably confirms that DNA was coated onto the beads. Effective radii was not significantly affected by shear rates when comparing the beads on each slide. ii TABLE OF CONTENTS LIST OF FIGURES ..................................................................................................... iii LIST OF TABLES ....................................................................................................... iv ACKNOWLEDGEMENTS ......................................................................................... v Chapter 1 Introduction ................................................................................................. 1 Von Willebrand Factor ..................................................................................................... 2 Von Willebrand Disease .................................................................................................. 6 Shear Stress Fluid Mechanics Theory .............................................................................. 8 Ventricular Assist Devices ............................................................................................... 11 Chapter 2 Optical Trap Theory .................................................................................... 14 Chapter 3 Parallels between DNA and vWF ............................................................... 17 Effective Radius ............................................................................................................... 18 Objectives and Hypothesis ............................................................................................... 19 Chapter 4 Materials and Methods ................................................................................ 20 Preparing the DNA-Coated Beads ................................................................................... 20 Running the Bead on the Optical Trap ............................................................................. 26 Chapter 5 Results ......................................................................................................... 38 Spring Constant ................................................................................................................ 38 Bead Displacement........................................................................................................... 41 Effective Radius Values ................................................................................................... 43 Chapter 6 Discussion ................................................................................................... 47 DNA-Coated Beads Analysis ........................................................................................... 47 Optical Trap Analysis ...................................................................................................... 50 Chapter 7 Future Work ................................................................................................ 55 Appendix A Displacement and Phase Raw Data - pACYC177 ................................. 58 Appendix B Displacement and Phase Raw Data – pUC19......................................... 59 BIBLIOGRAPHY ........................................................................................................ 60 iii LIST OF FIGURES Figure 1: Elongation of vWF under high shear (a) cartoon depiction of vWF below and above critical shear rate (b) fluorescence image of the relaxation of vWF after reducing shear rate to 0 s-1; adapted from Siediecki et al.8 .............................................................................. 2 Figure 2: vWF facilitating the adhesion between exposed collagen and its platelet receptors (GPIb/IX) to assist blood clotting at wound site, adapted from Geisen et al.1 ................. 4 Figure 3: Graphic representation of vWF A-D domains and main binding sites, including ADAMTS13 cleavage site. Regions where mutations in types of VWD have been found are also indicated; adapted from De Meyer et al.15 ................................................................ 5 Figure 4: (a) A healthy aortic valve in comparison to a severely stenotic valve; adapted from Medtronic19 (b) the decrease in high-molecular weight (ultra-large) vWF with increasing pressure gradient. Increasing pressure gradient is directly correlated with increasing valve stenosis; adapted from Vincentelli et al.18 ........................................................................ 7 Figure 5: Laminar, or smooth fluid movement in orderly layers, Couette flow between two parallel plates with a stationary lower plate and an upper plate moving at some arbitrary velocity; adapted from Cengel et al.22 .............................................................................. 9 Figure 6: Electrophoresis of vWF multimers in a healthy and VAD-implant patient, adapted from Geisen, et al.1 Smaller multimers can be seen lower in the gel as they pass faster through the gel. It is evident that the VAD patient is missing large vWF bands in the top portion of the gel. The densitometry curve on the right indicates this phenomenon. ............................. 12 Figure 7: Correlation between increasing VAD operating speed and decreasing percentage of high molecular weight vWF multimers; adapted from Meyer et al.24 ...................................... 13 Figure 8: Optical trap representation with bead. Beads are attracted to the center of the beam and slightly above the beam waist. Hooke’s law is at play with the spring force in the trap; adapted from Candela26 .................................................................................................... 15 Figure 9: A single beam trap; adapted from Svoboda et al.27 The light focus is seen at the top indicated by the darkest region. The black arrows indicate the light focusing through the circular bead. The gray arrows pointing relatively upwards indicate reaction forces pulling the bead upwards. ............................................................................................................. 16 Figure 10: Cartoon depiction of “effective radius” extending from the center of the bead to the edge of the DNA/vWF. DNA effective radii will resemble the ‘r3’ bead, adapted from Corsetti.31 ......................................................................................................................... 18 Figure 11: Depiction of plasmid with multi-cloning site (MCS), a DNA segment with several unique sites for restriction enzymes to cut. These restriction sites are not present anywhere else on the plasmid. .......................................................................................................... 21 Figure 12: Cartoon depiction of cut plasmid with biotinylated 3’ ends ................................... 23 iv Figure 13: Flow chamber sealed on all four sides with the right chamber containing control beads and the left chamber containing DNA-coated beads. Two slides were constructed for the 2 DNA-coated bead groups. ................................................................................................ 27 Figure 14: The optical trap consists of the IX71 Olympus inverted microscope with QPD, condenser, objective, stage, laser, and CCD camera. ......................................................
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