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- All Subjects: Cellular Adhesion
- Creators: Ugarova, Tatiana
Description
Platelets are specialized blood cells that play crucial role in normal physiologic and pathologic processes such as hemostasis, inflammation, wound healing, and host defense. Activation of platelets is essential for platelet function and it includes a complex interplay of adhesion and intracellular signaling molecules. Platelets are known to be activated during vessel injury by a complex interaction of soluble agonists and once activated, they adhere to sub-endothelial matrix to aggregate and secrete granules leading to the formation of platelet aggregate that is necessary for thrombus formation. Platelet integrin plays a central role in platelet adhesive reactions by serving as a receptor for fibrinogen involved in bidirectional transmembrane signaling. In order to elucidate the interaction of integrin with cytoplasmic signaling molecules during inside-out and outside-in signaling, we have studied the kinetics of the recruitment of talin, kindling, filmin-A, skelemin, Scr and syk to the B3 cytoplasmic tails. Platelets were isolated from human blood and activated with ADP/Epinephrine for different times. The complexes of *** with signaling proteins were obtained by immunoprecipitation of platelet lysates with anit-*** monoclonal antibody and then analyzed by Western blotting using antibodies directed against selected signaling proteins. Our results show different kinetics in recruitment of signaling molecules to the B3 integrin cytoplasmic tail during inside-out and outside in signaling.
ContributorsYantas, Alexa Susan (Author) / Ugarova, Tatiana (Thesis director) / Podolnikova, Nataly (Committee member) / Turaga, Ramya (Committee member) / Barrett, The Honors College (Contributor)
Created2012-05
Description
Cell adhesion is an important aspect of many biological processes. The atomic force microscope (AFM) has made it possible to quantify the forces involved in cellular adhesion using a technique called single cell force spectroscopy (SCFS). AFM based SCFS offers versatile control over experimental conditions for probing directly the interaction between specific cell types and specific proteins, surfaces, or other cells. Transmembrane integrins are the primary proteins involved in cellular adhesion to the extra cellular matix (ECM). One of the chief integrins involved in the adhesion of leukocyte cells is αMβ2 (Mac-1). The experiments in this dissertation quantify the adhesion of Mac-1 expressing human embryonic kidney (HEK Mac-1), platelets, and neutrophils cells on substrates with different concentrations of fibrinogen and on fibrin gels and multi-layered fibrinogen coated fibrin gels. It was shown that multi-layered fibrinogen reduces the adhesion force of these cells considerably. A novel method was developed as part of this research combining total internal reflection microscopy (TIRFM) with SCFS allowing for optical microscopy of HEK Mac-1 cells interacting with bovine serum albumin (BSA) coated glass after interacting with multi-layered fibrinogen. HEK Mac-1 cells are able to remove fibrinogen molecules from the multi-layered fibrinogen matrix. An analysis methodology for quantifying the kinetic parameters of integrin-ligand interactions from SCFS experiments is proposed, and the kinetic parameters of the Mac-1 fibrinogen bond are quantified. Additional SCFS experiments quantify the adhesion of macrophages and HEK Mac-1 cells on functionalized glass surfaces and normal glass surfaces. Both cell types show highest adhesion on a novel functionalized glass surface that was prepared to induce macrophage fusion. These experiments demonstrate the versatility of AFM based SCFS, and how it can be applied to address many questions in cellular biology offering quantitative insights.
ContributorsChristenson, Wayne B (Author) / Ros, Robert (Thesis advisor) / Beckstein, Oliver (Committee member) / Lindsay, Stuart (Committee member) / Ugarova, Tatiana (Committee member) / Arizona State University (Publisher)
Created2016