Almeida, Rodrigo Freire Martins deMarquês, Joaquim Manuel Trigo, 1986-Leitão, Helena Monteirinho2024-03-192024-03-1920242023http://hdl.handle.net/10451/63499Tese de mestrado, Bioquímica e Biomedicina , 2023, Universidade de Lisboa, Faculdade de CiênciasAmidst a growing global trend of fungal infections and diminishing antifungal drug efficacy, the fungal plasma membrane emerges as a potential target for enhanced antifungal treatments. By employing innovative experimental approaches, the concomitant use of fluorescent membrane probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and its derivatives, cationic TMA-DPH and anionic PA-DPH, this study aimed to i) develop a new methodology to study sphingolipid-enrich domains (SLEDs) in gel phase in Saccharomyces cerevisiae plasma membrane and ii) further characterize the biophysical properties and organization of the plasma membrane of Saccharomyces cerevisiae cells. All probes were shown to not compromise cellular viability. In terms of cellular distribution, DPH was confirmed to distribute across both the plasma and internal membranes, while TMA-DPH and PA-DPH localized predominantly in the plasma membrane. Notably, fluorescence anisotropy assays over time revealed that, at short incubation times, TMA-DPH and PA-DPH label different regions of the membrane, most likely due to their opposite charges. It is hypothesized that TMA-DPH, owing to its positive charge, will firstly incorporate into SLEDs due to electrostatic interactions with negative charges present in sphingolipids polar head group. This trend was observed in both wild-type and sur2∆ cells lacking C4 - OH group in the sphingoid base. Trans-parinaric acid (t-PnA) was also employed to assess the properties of gel SLEDs, which were identified in sur2∆ cells for the first time. Considering the long lifetime component of t-PnA in these cells (~44 ns), SLEDs are more compact compared to wt cells. This is in line with previous results obtained in other yeast strains with altered sphingolipid profile, namely scs7∆ and ipt1∆, where SLEDs also exhibited alterations in their compactness. This study reinforces that lipid composition, especially the hydroxylation pattern of sphingolipids, plays a pivotal role in defining membrane properties, thus SLEDs may constitute an explored target for antifungal therapies.engMembrana plasmática fúngicaSaccharomyces cerevisiaeDomínios ricos em esfingolípidos (SLEDs)Hidroxilação de esfingolípidosSonda fluorescente de membranaTeses de mestrado - 2024master thesis203606507