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Cytosolic signalling and behaviour of oral neutrophils “Search for biochemical memory”

Elumalai, Geetha Lakshmi 2012. Cytosolic signalling and behaviour of oral neutrophils “Search for biochemical memory”. PhD Thesis, Cardiff University.
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Abstract

All inflammatory events are mark by infiltration by leukocytes including neutrophils, which cross the endothelium before following migratory cues to the site of infection, and phagocytosing the infectious microorganisms. Before crossing the endothelial wall, the neutrophils spread on the endothelium. It has been proposed that the necessary additional membrane for cell spreading results from unfolding of wrinkled cell membrane held in place by molecule like membrane linker protein (such as talin or ezrin). Both talin and ezrin are potential substrates for cleavage by the Ca2+ activated proteolytic enzyme, calpain-1. It is possible that this mechanism underlies the membrane unwrinkling events but it is yet to be proved. The major aim of this thesis was to look for evidence that proteolysis and redistribution of these proteins occurred during neutrophil shape change. This thesis provide confirmatory evidence that ezrin is cleaved during extravasation of neutrophils and also provide evidence that the subcellular location of talin and ezrin protein can serve as an biological marker to identify extravasted neutrophils. The subcellular locations of talin and ezrin were identified using immunocytochemistry. Both ezrin (87%) and talin (92%) were detected at the cell membrane of neutrophils. This pattern was lost in polarized neutrophils, as well as after an elevation of cytosolic calcium level and also after transmigration through endothelial monolayers in vitro. Under these conditions, the detected ezrin and talin was mainly cytosolic. The same translocation was observed in extravasated oral neutrophils and also neutrophils which had extravasted under pathological conditions (gingivitis and osteoarthritis). GFP-tagged ezrin was expressed in RAW cells, in order to investigate the mechanism behind the relocation of ezrin. It was found to be triggered by an elevation of cytosolic calcium and was irreversible. It was also triggered locally during phagocytosis at the site where the membrane expanded. Western blotting showed that ezrin (72-69 kDa intact) was cleaved under similar conditions with fragments at 55kDa, 51kDa and 49kDa being generated by elavated calcium and extravasation. This cleavage was sensitive to calcium and calpain inhibition. It was concluded that ezrin is present in the plasma membrane wrinkles of resting neutrophils, but that changes when the cytosolic calcium level changes, as occur during extravsation and phagocytosis. Addition to this, any dynamic change in the surface area of the plasma membrane (phagocytosis), cause a relocation of ezrin away from the plasma membrane. Evidence was also provided that ezrin can be uses as a biological marker to identify extravasated neutrophils

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Medicine
Subjects: R Medicine > R Medicine (General)
Date of First Compliant Deposit: 30 March 2016
Last Modified: 19 Mar 2016 23:13
URI: http://orca-mwe.cf.ac.uk/id/eprint/43089

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