UBC Theses and Dissertations

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UBC Theses and Dissertations

Biophysical investigation of leukocyte rolling adhesion at the single cell level Murad, Yousif


Cell rolling adhesion is a mode of passive directional migration under flow used by a variety of prokaryotic as well as eukaryotic cells. Leukocytes have been shown to make use of rolling adhesion in the inflammatory response by rolling along the endothelial cells covering blood vessel walls to find their target site. This process is mediated by a family of adhesion proteins called selectins. One selectin of interest to us, P-selectin, is expressed on the surface of endothelial cells and interacts with P-selectin glycoprotein ligand-1 (PSGL-1) on the surface of rolling leukocytes. This interaction is made of non-covalent interactions such as hydrogen bonds, van der Waals interactions and electrostatic interactions. Leukocyte rolling adhesion has been investigated by many groups in the literature using flow chambers. These studies have made use of a variety of methods to functionalize a glass surface with P-selectin (or any selectin of interest). Such methods have focused on passivating the surface with human vein endothelial cell (HUVECs) or with bovine serum albumin (BSA). Neither method has been shown to provide a homogenous surface of P-selectin of known density in the absence of any other protein interactions. These studies have also mainly focused on cell rolling at the population level and how it is affected by shear stress and receptor surface density amongst other factors. In this thesis, polyethylene glycol (PEG), a polymer used in surface passivation for single molecule studies, is adapted to create a well control surface of P-selectin and to exclude any other proteins from adsorbing to the surface. Our data has shown the many key features of cell rolling behavior are disguised or significantly distorted when looking at the overall population behavior. This leads us to direct our effort towards in-depth single cell biophysical studies thus eliminating the influence of cell-to-cell variability and different phenotypic traits. Here, we used precision-controlled syringe pumps to vary shear stress patterns affecting single cells in a rationally designed barrage of shear ramp and oscillation experiments with the purpose of unmasking previously unseen cell rolling behavior.

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