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Title: The expression of potential molecular candidates for chloride ion channels in primary human granulocytes and granulocytic cell lines
Author: Kirk, Kirsty-Anne
ISNI:       0000 0004 5352 0501
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2014
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Introduction and hypothesis: The project aims to identify potential candidates for chloride (Cl-) ion channels in granulocytes, and granulocytic cell lines. It is hypothesised that Cl- ion channels, in particular hBest1, are implicated in the role of granulocytes in response to inflammation. Methodology: Two main methodologies were used; laboratory techniques and systematic review. Laboratory techniques included RT-PCR, flow cytometry and western blot analysis to characterise the expression of hANO1, hBest1 and hCLCA1 as potential chloride ion channels in granulocytes and granulocytic cell lines. Systematic review was performed to identify whether chloride ion channels are up-regulated in COPD and asthma. Results: RT-PCR demonstrated hCLCA1 expression in granulocytes and eosinophils but not HL60. hBest1 and hBest3 was expressed in all 3 cell types. In granulocytes, flow cytometry demonstrated greater hCLCA1 protein expression intracellularly, compared to hBest1 protein, and greater hBest1 plasma membrane expression compared to hCLCA1 (P<0.05). There was a negative correlation between hBest1, and hCLCA1 but also a weak negative correlation between hBest1 and hANO1 (P<0.05). Granulocytes stimulated with IL-13 over 24 hours, had a greater protein expression both intracellularly and at the plasma membrane. There was increased migration of HL60s when transfected with hBest1, in response to fMLP (P<0.05). Systematic review did not support the project due to limitations. Conclusions: There is a complex relationship between hBest1, hCLCA1 and hANO1 which may contribute to the function of granulocytes. HBest1 protein expression peaked 24 hours after continuous stimulation with IL-13. This correlates with peak symptom expression in diseases such as COPD and asthma. It is suggested that hBest1 has a role in migration and activation of granulocytes, through regulation of cell shape and volume. It is concluded that hBest1 is a novel therapeutic target in the control of symptoms in chronic inflammatory lung diseases.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available