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Title: Analysis of cell membrane lipids using Raman spectroscopy
Author: Czerwiec, Agnieszka
ISNI:       0000 0004 5369 6494
Awarding Body: Queen's University Belfast
Current Institution: Queen's University Belfast
Date of Award: 2014
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Ceramide accumulates in Cystic Fibrosis (CF) airways and ceramide reduction decreased inflammation, susceptibility to Pseudomonas aeruginosa (Pa) infection, mortality and mobility. The research undertaken here aimed to evaluate the anti-inflammatory effect of sphingolipid-modifying drugs in LPS-stimulated bronchial epithelial cells. Moreover, the use of Raman Spectroscopy to identify-the distribution of biomembrane lipids was evaluated. Firstly, Raman markers of selected lipids were determined and their specificity and changes of lipid-derived Raman peaks due to bio-compound interaction were confirmed in solution. Secondly, membrane lipids within cells were investigated using Surface-enhanced Raman Spectroscopy (SERS) employing silver nanoparticles (NPs) and self-assembled thiols monolayers I (SAMs). NPs and S03--terminated SAMs promote a weak SERS, compared to choline-, methyl- or OHterminated SAMs. However, cellular lipid signals were not detected, SERS combined with confocal Raman microscopy gave enriched Raman spectra, but the signal was unstable and was not suitable for analyses , Thirdly, the anti-inflammatory effect of amitriptyline and miglustat on Pa LPS-induced inflammation in CF and non-CF cell lines was investigated. LPS from a clinical Pa isolate failed to induce inflammation, compared to commercially available LPS and bacterial lysate. Neither of the drugs suppressed LPS-induced inflammation, in contrast to dexamethasone, and cell lines responded to treatments similarly. Finally, using Raman spectroscopy combined with principal component analysis, the distribution of cellular components (DNA, RNA, proteins), and lipids (phosphatidylcholine, sphingomyelin, phosphatidylinositol, cholesterol) in LPS-stimulated cells pretreated with amitriptyline or sphingomyelinase was semiquantitatively imaged: CF cells expressed more sphingomyelin than non-CF cells. Sphingomyelin was reduced by sphingomyelinase, but increased by amitriptyline and LPS, suggesting that sphingomyelin was not associated with inflammation. Overall Raman spectroscopy techniques have potential for the label-free study of the cellular lipid composition; however, further improvements in the sensitivity are needed. The observation that miglustat and amitriptyline target LPS-independent immune responses needs further investigations.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available