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| Author(s): |
Raffaela De Rosa
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Física (IF/SBI) |
| Defense date: | 2020-04-27 |
| Examining board members: |
Rosangela Itri;
Leandro Ramos Souza Barbosa;
Pietro Ciancaglini;
José Luiz de Souza Lopes;
Tayana Mazin Tsubone
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| Advisor: | Rosangela Itri |
| Abstract | |
Small angle x-ray scattering (SAXS) technique was here applied to (i) characterize model membranes (large unilamellar vesicles LUVs) composed of POPC (PC), PC:sphingomyelin (PC:SM, 1:1) and PC:SM:cholesterol (PC:SM:CO, 1:1:1), (ii) study structural changes in these membranes due to in situ photo-oxidation in the presence of four photosensitizers (FS): methylene blue (MB), azure A and B (AA and AB) and thionine (Ti), (iii) evaluate the influence of the oxidized lipids PC-OOH (hidroperodidated POPC) and 1-palmitoyl-2-azelaoyl- sn-glycero-3-phosphocholine (PAzPC) on the composition of the lipid membrane and (iv) investigate the interaction and aggregations mechanisms of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzyme on the surface of oxidized membranes and membranes containing lipid domains. The initial characterization of model membranes showed that the lipid composition affects structural parameters like membrane thickness and electron density distribution. Lipid bilayers composed only of POPC had a lipid bilayer thickness equal to 46(2)Å. Compared to the PC system, membrane composed of PC:SM (1:1) showed an increase of 6.6Å in the bilayer thickness. In the case of membranes composed by PC:SM:CO (1:1:1) the analysis of the SAXS curves indicated a coexistence of Ld-Lo phases, with differences in the bilayer thickness of 9.4Å. For membrane photosensitization study, we analyzed SAXS curves of LUVs composed of POPC, PC:SM (1:1) and PC:SM:CO (1:1:1) photo-irradiated for 2h in the presence of FS MB, AA, AB and Ti. Vesicles composed by PC:SM:CO presented lower structural alteration due to photosensitivity, suggesting that lipid domains of SM:CO hinder membrane damage. PC and PC:SM systems, the FS Ti promoted the greatest photo-damage in the membrane, resulting in lipid oxidation of about 31% for POPC membranes and 17% for PC:SM. It was observed an induction to formation of multilamellar structures for all lipid compositions with FSs even before exposure to photo- irradiation, suggesting that FS by itself promotes the multilamella formation. In general, the photosensitization effects observed in this study were not significant with regard to physical damage to membranes in terms of rupture. In terms of the impact of oxidized lipid on model membranes, it has been observed that the controlled addition of POPC-OOH to POPC bilayers leads to a decrease in the layer thickness of 46.4(6)Å for pure PC membranes to 37.6(4)Å when composed of 100% PC-OOH. For PC:PAzPC (67:33) we obtained bilayer thickness of 42.7(9)Å. For the ternary mixtures of PC:PC-OOH:PAzPC (60:25:15) and (47:25:28), the bilayer thickness was similar to that obtained for PC with 33% of oxidized lipid (PC-OOH or PAzPC). Analysis of PC:PC-OOH and PC:PAzPC systems taking into account the scattering of the chemical groups showed that the oxidized group OOH of the PC-OOH is placed preferably in the polar region of the membrane, close to the carbonyl and phosphate groups. In the case of PAzPC, the COO group presents a bimodal distribution with location in the hydrophobic and hydrophilic regions of the membrane. Finally, the GAPDH/PC:PC-OOH interaction study showed GAPDH aggregation regardless the membrane oxidation. Quantification of the enzymes oligomeric structure in the presence of membranes containing PC:PC-OOH showed dissociation from the tetrameric structure to monomers or dimers, without the possibility of distinguishing between the two structures. For measurements performed directly after mixing the solutions (t = 0h), the greatest enzyme dissociation was observed for lower oxidized membranes (PC-PC-OOH (67:33)). In systems composed of PC:SM:CO and PC-OOH:SM:CO under 2h of interaction the GAPDH kept the tetrameric structure, suggesting that lipid domains inhibit the enzyme dissociation in monomers or dimers. (AU) | |
| FAPESP's process: | 14/02511-5 - Physical damage on model membranes promoted by photosensitization |
| Grantee: | Raffaela de Rosa |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |