| dc.description.abstract | This work presents an investigation into the physiological state of the red blood cell (RBC) membrane. Particular focus is given to the properties of the composite membrane and their implication in cellular signalling resulting from increased mechanical deformation. Intense synchrotron x-ray radiation was used to probe Langmuir monolayers akin to both leaflets of the RBC plasma membrane. Complex phospholipid mixtures were spread at the air-subphase interface of a Langmuir trough, and investigated at a surface pressure of 30 mN/m and temperature of 20°C. Three geometries were used to probe the monolayers: grazing incidence x-ray diffraction (GIXD) measured lateral ordering and crystalline structure within the monolayer, and both grazing incidence x-ray off-specular scattering (GIXOS) and x-ray reflectivity (XRR) measured the monolayer thickness and electron density profiles perpendicular to the air-subphase interface. On the pure water subphase GIXD results demonstrated a hexagonal packing structure for both leaflets, although there were dissimilarities in the parameters of this packing. The inner leaflet (IL) monolayer demonstrated larger domains of crystalline order than the outer leaflet (OL) monolayer, most likely a result of the IL's high phosphatidylethanolamine (PE) content in comparison to the OL's high sphingomyelin (SM) content. The two monolayers were of comparable thickness overall, however, a greater electron density profile was measured for the IL monolayer. This was suggestive of differences in the phospholipid tilt angle, relative to the normal of the air-subphase interface, in the two monolayers. The effect of oxidative stress on the mechanical properties of the RBC membrane was extensively measured using thermal fluctuation spectroscopy (TFS). The spectra of healthy RBCs were measured before and after treatment with three different oxidising agents, chosen for their known ability to target specific components of the membrane. Changes in each of the elastic moduli of the cell can be attributed to oxidative damage to a particular component of the membrane, since the lipid bilayer dominates the contribution to the cell's bending modulus and the cytoskeleton governs the shear modulus. In good agreement with the literature, the results demonstrated that hydrogen peroxide (H₂O₂) preferentially targeted the cytoskeleton and cumene hydroperoxide (cumOOH) targeted the lipid bilayer to a greater extent than H₂O₂, whilst diamide was less specific, affecting both the lipid bilayer and cytoskeleton to a varying degree. In parallel, a novel method for measuring the mechanically stimulated adenosine triphosphate (ATP) release from RBCs was developed. Cells were subjected to an externally applied increased shear rate, using a cannula attached to a syringe pump which drove the flow. This novel approach allowed for acute control of experimental parameters, such as the shear rate. Sample ATP was measured using a standard luciferin based assay, which demonstrated that oxidative modification of the RBC membrane leads to an altered ability to release ATP. Moreover, the change in ability of the RBCs to release ATP upon mechanical stimulation was most likely a result of underlying biological processes associated with the oxidative damage, rather than a change to the cell's mechanical properties. In all stages of this work the molecular action of dimethyl sulphoxide (DMSO) on the RBC membrane was investigated. This posed an interesting question since DMSO has far reaching applications, but the true effects of the solvent are poorly understood. At 10 vol-%, the x-ray monolayer studies demonstrated a strong reorientating effect of the aqueous-DMSO subphase on both the IL and OL monolayers, with the creation of DMSO rich/lipid poor domains. In the TFS study, DMSO treatment at concentrations of 1, 5 and 10 vol-% were shown to weakly affect the mechanical properties of the cell but importantly, recovery was observed. However, the membrane became significantly permeable to ATP upon DMSO treatment, even without mechanical stimulation. This result offers experimental evidence of the pore forming nature of DMSO, as suggested by molecular dynamics simulations. Together, these results provide clarity over the molecular action of DMSO on the RBC and, through applicability to other cell types, offer justification for the many uses of the solvent. | en_GB |
