Doutorado em Física (IF)
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Item Estudos de membranas modelo e efeitos de terpenos em membranas de leishmania por ressonância paramagnética eletrônica(Universidade Federal de Goiás, 2013-11-16) Camargos, Heverton Silva de; Alonso, Antônio; http://lattes.cnpq.br/5013069863616789; Alonso, Antônio; Nakamura, Celso Vataru; Ruggiero Neto, João; Dorta, Miriam Cristina Leandro; Anjos, Jorge Luiz Vieira dosElectron paramagnetic resonance (EPR) spectroscopy of spin labels was used to study the main structural accommodations of environment-sensitive probes in the bilayers of saturated phosphatidylcholines with acyl chains lengths ranging from 16 to 22 carbon atoms. The more detailed analysis were made on the spin probe 5-doxyl methyl stearate (5-DMS) whose EPR spectra allowed to identify two distinct spectral components in thermodynamic equilibrium at temperatures below and above the main phase transition. The EPR spectroscopy distinguishes two components associating lower motion with higher polarity (denoted component 1) and higher motion with lower polarity (component 2), which may be assigned to one shallow (more rigid structure) and one deep population of spin probe, respectively. At temperatures until 22◦C only one spectral component can be noted in the spectra whereas at 30◦C the component 1 coexists with an appreciable fraction of component 2. In the liquid-crystalline phase the 5-MSL showed two spectral components for all studied lipids in the entire range of measured temperatures. An accurate analysis of EPR spectra, performed using two fitting programs (NLLS and EPRSIM), allowed us to obtain the thermodynamic profile to these major probe accommodations. Focusing the analysis on two-component EPR spectra, it was studied the influences of cholesterol and a membrane permeation enhancer on the mobility and distribution of spin label on these two main bilayer environments. Parte II Cutaneous leishmaniasis is a neglected tropical disease that infects millions of people worldwide, representing a serious public health problem. The current treatment is based on chemotherapy, using pentavalent antimonials compounds, which cause serious side effects. Electron paramagnetic resonance (EPR) spectroscopy of the spin-label analog of stearic acid (5-DSA ) was used to monitor the effect of the terpenes α-terpineol, 1,8-cineole, III (+)-limonene and nerolidol on the plasma membrane fluidity of Leishmania amazonensis promastigotes. Cytotoxic effects on the parasite were also measured to investigate the relationship between the cytotoxic potential of terpenes and their ability to alter membrane fluidity. All terpenes increased the fluidity of the cellular membrane, without significant differences at higher concentrations. However, the minimum concentration required to cause a change in the membrane was very different between the terpenes and similar to that caused 50% growth inhibition (IC50) showing a correlation between membrane alterations and cytotoxicity. The IC50 values of terpenes analyzed showed the following relationship: nerolidol < (+)-limonene < α-terpineol < 1,8-cineole, with an IC50 of 8 μM for nerolidol and 4700 μM to 1,8- cineole. The EPR spectra of the maleimide derivative spin label (6-MSL) covalently attached to the Leishmania membrane proteins indicated that the terpenes essentially do not alter the dynamics of protein backbone and only increase the mobility of the nitroxide side chain. Cell lysis was not detected at cytotoxic concentrations, as measured by the presence of spin-labeled membrane fragments. Since the terpenes are considered potent skin permeation enhancers with low irritation potential, this work suggests checking the possibility of terpenes applications in the treatment of tegumentary leishmaniasis, where terpenes could perhaps perform a dual action of be an active principle and at the same time facilitate the penetration of other molecules with antileishmanial activity.Item Interações de terpenos com membranas de eritrócito, fibroblasto, estrato córneo e membrana modelo e interações de uma nanopartícula de ouro com membranas modelo(Universidade Federal de Goiás, 2014-04-25) Mendanha Neto, Sebastião Antônio; Goñi, Félix M.; Alonso, Antônio; http://lattes.cnpq.br/5013069863616789; Alonso, Antônio; Pagliuso, Pascoal José Giglio; Ito, Amando Siuiti; Avelar, Ardiley Torres; Carvalho, Jesiel FreitasThe interactions of terpenes with membranes of erythrocyte, fibroblasts, stratum corneum and the model membranes of 1,2-dipalmitoylsn -glycero-3-phosphocholine were investigated by using the the electron paramagnetic resonance and fluorescence spectroscopic of lipophilic probes. It has been shown that when added at high concentrations to systems having a high lipid/solvent ratio, terpenes such as 1,8-cineol, α-terpineol, (+)-limonene and nerolidol are able to self-stabilize in molecular aggregates which can extract the bilayers lipids. Studies on the hemolytic and cytotoxic potential of various terpenes showed that cell damage caused by these molecules are concentration dependent and that among the studied terpenes, nerolidol and α-terpineol are the most hemolytic and cytotoxic, while (+)-limonene and 1,8-cineole are the least hemolytic and cytotoxic. However, the low correlation between these two tests indicates that the processes involved in each case are not completely dependent. It was also shown that once embedded in the membrane, terpenes increase the fluidity of lipid bilayers and decrease the temperature of the main phase transition. Differences between increased fluidity promoted by sesquiterpene nerolidol and all monoterpenes studied were observed. Meanwhile, in a comparison of the effect of the monoterpenes studied, no significant differences in their ability to increase membrane fluidity were detected. Furthermore, it was demonstrated by using confocal and atomic force microscopy and fluorescence spectroscopy that the 1,2-distearoylsn -glycero-3-(Aurora nanoparticles) is better incorporated in lipid membranes under fluid phase and that the addition of 0.1% of these conjugated nanoparticles do not produces large variations in membrane fluidity and no causes substantial morphological changes of lipid bilayers.Item Interação da miltefosina com os componentes lipídicos e proteicos das membranas de eritrócito e Leishmania estudada por ressonância paramagnética eletrônica(Universidade Federal de Goiás, 2014-06-04) Moreira, Rodrigo Alves; Alonso, Antônio; http://lattes.cnpq.br/5013069863616789; Alonso, Antônio; Uliana, Silvia Reni Bortolin; Urbano, Ricardo Rodrigues; Loyola, Patrícia Resende Alo Nagib; Carvalho, Sheila Gonçalves do CoutoCutaneous leishmaniasis is a neglected tropical disease that infects millions of people worldwide, representing a serious public health problem. The miltefosine (MT) is an alkylphospholipid that has been approved for the treatment of breast cancer metastasis and visceral leishmaniasis, although the mechanism of action at the molecular level is poorly understood. Electron paramagnetic resonance (EPR) spectroscopy of the lipid spin lebel analog of stearic acid (5-DSA) and the maleimide derivative spin label (6-MSL) covalently bound to membrane proteins showed that the MT causes a large increase in the molecular dynamics of erythrocyte membranes (ghosts) and detergent resistant membranes (DRMs) prepared from erythrocyte membranes. In the vesicles of lipid raft constituents, it was shown that 20 mol% sphingomyelin could be replaced by 20 mol% MT with no change in the molecular dynamics. Furthermore, the effect of MT on DRMs was more pronounced than in erythrocyte ghosts, supporting the hypothesis that MT is a lipid raft modulator. At the reported MT-plasma concentrations found during the treatment of leishmaniasis (31-52μg/mL), our measurements in blood plasma indicated a hemolytic level of 2-5% and also showed that the MT concentration that changes the erythrocyte membrane fluidity to an extent that is detectable by EPR spectroscopy causes about 46% hemolysis. Subsequently, EPR studies performed with the same spin labels in the membrane of Leishmania (L.) amazonensis (promastigote) showed changes similar to those found in erythrocyte membranes. Cytotoxic effects on the parasites were also evaluated to investigate the relationships between the cytotoxic potential of MT and its ability to alter membrane fluidity. The EPR data showed that the minimum concentration of MT required to cause a change in the parasite membrane occurred near the values of MT concentration which inhibits 50 % of cell growth (IC50); thus, there is a correlation between the cytotoxicity and changes in the membrane. Although these III membrane alterations can be detected using a spin-labeled lipid, our experimental results indicated that MT interacts predominantly with the protein component of the membrane. Cell lysis was also detected by analyzing the supernatants of centrifuged samples for the presence of spin-labeled membrane fragments and cytoplasmic proteins. Using a method for the rapid incorporation of MT into the membrane, these effects were measured immediately after treatment under the same range of MT concentrations that cause cell growth inhibition. Cytotoxicity, estimated via microscopic counting of living and dead cells, indicated ∼ 70% cell death at the concentration of MT at which EPR spectroscopy detected a significant change in membrane dynamics. After this initial impact on the number of viable parasites, the processes of cell death and growth continued during the first 4 h of incubation. The EPR spectra of spin-labeled membrane-bound proteins were consistent with more expanded and solvent-exposed protein conformations, suggesting a detergent-like action. Thus, MT may form micelle-like structures around polypeptide chains, and proteins with a higher hydrophobicity may induce the penetration of hydrophilic groups of MT into the membrane, causing its rupture.