The appropriate lipid environment is crucial for the proper function of membrane proteins. There is a tremendous variety of lipid molecules in the membrane and so far it is often unclear which component of the lipid matrix is essential for the function of a respective protein. Lipid molecules and proteins mutually influence each other; parameters such as acyl chain order, membrane thickness, membrane elasticity, permeability, lipid-domain and annulus formation are strongly modulated by proteins. More recent data also indicates that the influence of proteins goes beyond a single annulus of next-neighbor boundary lipids. Therefore, a mesoscopic approach to membrane lipid-protein interactions in terms of elastic membrane deformations has been developed. Solid-state NMR has greatly contributed to the understanding of lipid-protein interactions and the modern view of biological membranes. Methods that detect the influence of proteins on the membrane as well as direct lipid-protein interactions have been developed and are reviewed here. Examples for solid-state NMR studies on the interaction of Ras proteins, the antimicrobial peptide protegrin-1, the G protein-coupled receptor rhodopsin, and the K+ channel KcsA are discussed.

No description available.

The pulsed field gradient fourier transform nuclear magnetic resonance (PFG-FTNMR) method to measure translational diffusion coefficients in multicomponent systems has been applied to amphiphilic molecules forming liquid crystalline phases. By analyzing the concentration dependence of the diffusion coefficients of water and amphiphile in a micellar system of N,N-dimethyldodecy lamine oxide (DDAO) in water it was possible to conclude that the micelles formed were polydisperse in size and shape. It was also shown that solubilization of small amounts of hydrophobic molecules into the micelles induces spherical micelles of a narrow size distribution. From the magnitude of the lateral diffusion coefficient in the cubic phase of DDAO/water it was concluded that this phase is built up of bicontinous aggregates. The lipid lateral diffusion in the cubic phase of monooleoylglycerol (MO)/water has been measured. The decrease in the lateral diffusion of MO in this phase, when the water was replaced by glycerol, was ascribed to changes in viscosity in the polar region. Measurements by electron spin resonance and time-resolved fluorescence spectroscopy showed that changes in viscosity of the solvent also affected the motions in the hydrocarbon region. The diffusion coefficients of all three components in the cubic phase located in the lowwater region of the ternary system of diacylglycerol (DAG)/soybean phosphatidylcholine (SPC)/water have been determined. Conclusive evidence was provided for that this cubic phase is built up of reversed micelles containing mainly SPC in a continous matrix of mainly DAG. The effect on the phase properties of DDAO upon incorporation of the peptide gramicidin D has been investigated. It was shown that gramicidin D induces a lamellar phase at all water contents. The change in the order parameter profile of the C-2H bonds in perdeuterated DDAO upon incorporation of gramicidin D is compatible with theoretical calculations for proteins exhibiting a positive hydrophobic mismatch. A method for using the PFG FTNMR technique in measurements of the transmembrane exchange rate of small molecules in vesicular suspensions is discussed and some preliminary data is shown. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1994, härtill 4 uppsatser</p> / digitalisering@umu

Many biological processes, pathogens, and pharmaceuticals act upon, cellular membranes. Accordingly, cell membrane mimics are attractive targets for biosensing, with research, pathology, and pharmacology applications. Lipid bilayers represent a versatile sensor functionalization platform providing antifouling properties, and many receptor integration options, uniquely including transmembrane proteins. Bilayer-coated sensors enable the kinetic characterization of membrane/analyte interactions. Addressed theoretically and experimentally is the self-assembly of model membranes on plasmonic sensors. Two categories of plasmonic sensors are studied in two parts. Part I aims to deposit raft-forming bilayers on planar nanoaperture arrays suitable for multiplexing and device integration. By vesicle fusion, planar bilayers are self-assembled on thiol-acid modified flame-annealed gold without the need for specific lipid head-group requirements. Identification of coexisting lipid phases is accomplished by AFM imaging and force spectroscopy mapping. These methods are successfully extended to metallic, plasmon-active nanohole arrays, nanoslit arrays and annular aperture arrays, with coexisting phases observed among the holes. Vis-NIR transmission spectra of the arrays are measured before and after deposition, indicating bilayer detection. Finally, the extraction of membrane proteins from cell cultures and incorporation into model supported bilayers is demonstrated. These natural membrane proteins potentially act as lipid-bound surface receptors. Part II aims to encapsulate in model lipid bilayers, metallic nanoparticles, which are used as probes in surface enhanced Raman spectroscopy. Three strategies of encapsulating particles, and incorporating Raman-active dyes are demonstrated, each using a different dye: malachite green, rhodamine-PE, and Tryptophan. Dye incorporation is verified by SERS and the bilayer is visualized and measured by TEM, with support from DLS and UV-Vis spectroscopy. In both parts, lipid-coated sensors are successfully fabricated and characterized. These results represent important and novel solutions to the functionalization of plasmonic surfaces with biologically relevant cell membrane mimics.

No description available.

abstract: Biological membranes are critical to cell sustainability by selectively permeating polar molecules into the intracellular space and providing protection to the interior organelles. Biomimetic membranes (model cell membranes) are often used to fundamentally study the lipid bilayer backbone structure of the biological membrane. Lipid bilayer membranes are often supported using inorganic materials in an effort to improve membrane stability and for application to novel biosensing platforms. Published literature has shown that a variety of dense inorganic materials with various surface properties have been investigated for the study of biomimetic membranes. However, literature does not adequately address the effect of porous materials or supports with varying macroscopic geometries on lipid bilayer membrane behavior. The objective of this dissertation is to present a fundamental study on the synthesis of lipid bilayer membranes supported by novel inorganic supports in an effort to expand the number of available supports for biosensing technology. There are two fundamental areas covered including: (1) synthesis of lipid bilayer membranes on porous inorganic materials and (2) synthesis and characterization of cylindrically supported lipid bilayer membranes. The lipid bilayer membrane formation behavior on various porous supports was studied via direct mass adsorption using a quartz crystal microbalance. Experimental results demonstrate significantly different membrane formation behaviors on the porous inorganic supports. A lipid bilayer membrane structure was formed only on SiO2 based surfaces (dense SiO2 and silicalite, basic conditions) and gamma-alumina (acidic conditions). Vesicle monolayer adsorption was observed on gamma-alumina (basic conditions), and yttria stabilized zirconia (YSZ) of varying roughness. Parameters such as buffer pH, surface chemistry and surface roughness were found to have a significant impact on the vesicle adsorption kinetics. Experimental and modeling work was conducted to study formation and characterization of cylindrically supported lipid bilayer membranes. A novel sensing technique (long-period fiber grating refractometry) was utilized to measure the formation mechanism of lipid bilayer membranes on an optical fiber. It was found that the membrane formation kinetics on the fiber was similar to its planar SiO2 counterpart. Fluorescence measurements verified membrane transport behavior and found that characterization artifacts affected the measured transport behavior. / Dissertation/Thesis / Ph.D. Chemical Engineering 2011

Neste estudo investigamos os efeitos dos ácidos graxos sobre a função e sinalização intracelular de linfócitos humanos. Os ácidos oléico (OA) e linoléico (LA), em baixas concentrações, estimularam a proliferação celular induzida pela IL-2 através do aumento da fosforilação da proteína PKC-<font face=\"symbol\">Z que levou a um aumento da fosforilação de ERK 1/2. Já os ácidos palmítico (PA), esteárico (SA), DHA e EPA diminuíram a proliferação destas células e inibiram a fosforilação de JAK1 e 3, STAT5, ERK e Akt. Os resultados obtidos são sugestivos de que o efeito inibitório promovido por PA, SA, DHA e EPA sobre a proliferação de linfócitos ocorreu devido à diminuição da fosforilação de proteínas fundamentais para a proliferação celular. Por outro lado, OA e LA estimularam a proliferação de linfócitos aumentando a fosforilação de ERK 1/2 através da ativação de PKC-<font face=\"symbol\">Z, efeito dependente da PI3K. O efeito inibitório promovido pelo DHA está associado a uma alteração na quantidade de lipid rafts na membrana plasmática nos quais o receptor de IL-2 está localizado. / The effect of fatty acids (FA) on interleukin -2 (IL-2) signaling pathway in human lymphocytes was investigated. Docosahexaenoic (DHA), eicosapentaenoic (EPA), palmitic (PA) and stearic (SA) acids decreased lymphocyte proliferation in concentrations above 50 <font face=\"symbol\">mM. However, oleic (OA) and linoleic (LA) acids increase lymphocyte proliferation at 25 <font face=\"symbol\">mM. PA, SA, DHA and EPA decreased JAK 1, JAK 3, STAT 5 and AKT phosphorylation induced by IL-2 but OA and LA did not cause any effect. OA and LA increased ERK1/2 phosphorylation whereas the other FA caused a marked decrease. PKC-<font face=\"symbol\">Z phosphorylation was decreased by OA and LA only. In conclusion, the inhibitory effect of PA, SA, DHA and EPA on lymphocyte proliferation observed in our previous study was due to a decrease in protein phosphorylation activated by IL-2. Probably, OA and LA stimulated lymphocyte proliferation by increasing ERK 1/2 phosphorylation throught PKC-<font face=\"symbol\">Z activation. The inhibition of JAK 1, JAK3, STAT 5, ERK1/2 and Akt phosphorylation caused by DHA is associated to a decrease in membrane lipid rafts contend.

Orientador: Francisco Benedito Teixeira Pessine / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-23T23:52:35Z (GMT). No. of bitstreams: 1 Sousa_Marcelode_D.pdf: 3494030 bytes, checksum: 462ff3b363ece6e2ff2f83e5d1028485 (MD5) Previous issue date: 2013 / Resumo: Nos dias atuais não há cura e nem vacina para as pessoas portadoras de HIV. A erradicação do vírus não é possível porque a maioria está depositado em órgãos que são de difícil acesso aos fármacos antirretrovirais. Uma alternativa seria direcionar os fármacos à estes órgãos por intermédio de nanocarreadores. Os fármacos utilizados neste trabalho foram a Nevirapina, o Saquinavir e o Efavirenz e o nanocarregador foi as Nanopartículas Lipídicas Sólidas (NLS). As NLS foram produzidas utilizando ácido esteárico, ácido mirístico e Compritol 888 ATO como matrizes dos nanocarregadores, brometo de cetiltrimetilamônio, Lipoid-S75 e Tween 80 como surfatantes e n-butanol como co-surfatante. A formulação otimizada foi obtida através de um planejamento experimental 2 com ponto central utilizando os surfatantes como variáveis. A molécula direcionadora adicionada na superfície das nanopartículas foi a d-manose, a qual estava ligada na octadecilamina. Este açúcar foi escolhido por que é receptor dos macrófagos/monócitos. A estratégia é que estas células capturem os nanocarregadores carregados com fármacos antirretrovirais e os enviem aos órgãos depósitos de HIV. Estas NLS foram caracterizados pelas técnicas de espalhamento dinâmico de luz, potencial Zeta, Calorimetria Diferencial de Varredura (DSC), Difratometria de raios-X e Microscopia Eletrônica de Varredura (MEV), além dos estudos de liberação in vitro. / Abstract: Nowadays there is neither cure nor vaccine for people living with HIV. However, virus eradication is not possible because most are deposited in organs that are difficult access to antiretroviral drugs. An alternative would be to target the drugs to these organs via nanocarriers. The drugs used in this work were Nevirapine, Saquinavir and Efavirenz and the nanocarriers were Solid Lipid Nanoparticles (SLN). The SLN were produced using stearic acid, myristic acid and Compritol 888 ATO as matrix of nanocarriers, cetyltrimethyl ammonium bromide, Lipoid-S75 and Tween 80 as surfactants and n-butanol as cosurfactant. The optimized formulation was obtained through an 2 experimental design with central point using surfactants as variable. The target molecule added in the nanocarriers surface was d-mannose, which was linked in the octadecylamine. This sugar was chosen because it is receptor of macrophages/monocytes. The strategy is that these cells capture the nanocarriers loaded antiretroviral drugs and it send deposit organs of HIV. These SLN were characterized by techniques dynamic light scattering (DLS), zeta Potential, Differential Scanning Calorimetry (DSC), X-ray diffraction and Scanning Electron Microscopy (MEV), beyond of in vitro released studies. / Doutorado / Físico-Química / Doutor em Ciências

Orientadores: Eneida de Paula, Daniele Ribeiro de Araújo / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T00:52:12Z (GMT). No. of bitstreams: 1 Barbosa_RaqueldeMelo_D.pdf: 5424104 bytes, checksum: a7f0888c7fb32138387068083d5bf74f (MD5) Previous issue date: 2013 / Resumo: Nanopartículas lipídicas sólidas (SLN) e carreadores lipídicos nanoestruturados (NLC) têm sido utilizados com sucesso como sistemas de liberação modificada. O anestésico local dibucaína (DBC) foi encapsulado em SLN e NLC objetivando aplicação tópica, para melhora de sua disponibilidade redução de efeitos adversos. As nanopartículas lipídicas foram preparadas pelas técnicas de sonicação (Son) ou homogeinização à alta pressão (HP), sendo utilizados palmitato de cetila (CP) ou miristato de miristila (MM) como matrizes lipídicas sólidas, acrescidos (NLC) ou não (SLN) de uma mistura de triglicerídeos de ácido cáprico e caprílico; poloxamer 188 foi usado como tensoativo. A DBC encapsulada foi quantificada por metodologia validada por cromatografia líquida de alta eficiência. As análises físico-químicas compreenderam diâmetro médio, potencial zeta, distribuição de tamanhos e morfologia das nanopartículas, percentual de encapsulação além de medidas de calorimetria exploratória de varredura (DSC), espectroscopia de infravermelho (FTIR), ressonância paramagnética eletrônica (RPE) e difração de raios X à baixo ângulo (SAXS). Medidas in vitro do perfil de liberação do fármaco, da estimativa de fluxo, deformação e elasticidade das partículas através de membranas artificiais e de toxicidade em cultura de células (fibloblastos 3T3 e queratinócitos HaCat) foram feitas. A estabilidade das amostras foi avaliada em função do tempo e testes de antinocicepção (tail flick, em ratos Wistar) foram usados para avaliar a atividade terapêutica in vivo. O diâmetro médio das partículas de SLN e NLC produzidas foi similar (ca. 200nm). A estabilidade física das nanopartículas foi satisfatória por até 240 dias de armazenamento a 4 ºC, principalmente para NLCMM/HP com e sem DBC, sugerindo que a metodologia de HP produz partículas mais estáveis. Todas as formulações apresentaram eficiência de encapsulação maior que 70%, sendo que NLCMMDBC/HP apresentou a maior encapsulação (90,54 ± 0,95%). Medidas de FTIR e DSC revelaram a DBC molecularmente dispersa na matriz lipídica das nanopartículas. Quanto à organização molecular das SLN e NLC, resultados de SAXS indicaram a existência de arranjos lipídicos lamelares no interior das SLN, não alterados pela adição da DBC; as medidas de RPE com marcadores de spin doxil-estearato revelaram espectros compatíveis com bicamadas, com maior organização molecular dos lipídios das SLN e NLC, após inserção da DBC. Ensaios in vitro confirmaram a liberação modificada da dibucaína associada às partículas, governada por difusão de Fick. Tanto a elasticidade quanto o fluxo das partículas in vitro apresentaram baixos valores evidenciando deposição das mesmas nas membranas com poros de 30 nm. A citotoxicidade intrínsica da DBC sobre ambos os tipos celulares foi reduzida após encapsulação nas SLN e NLC. O efeito analgésico in vivo da DBC a 0,05% aplicada topicamente (dispersa em gel de carbopol) aumentou significativamente após encapsulação nas formulações, em particular para SLNCPDBC liofilizada com o crioprotetor maltose. Assim, formulações de dibucaína em SLN ou NLC, preparadas com MM ou CP mostraram-se promissoras como bases para produtos farmacêuticos de liberação modificada, para anestesia dérmica / Abstract: Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC), intended for topical application, were successfully prepared as sustained release systems for the encapsulation of the local anesthetic dibucaine (DBC), aiming to reduce its toxic effects and to improve its availability. The particles were prepared by two differents procedures: sonication (Son) or high pressure homogenization (HP), employing either cetyl palmitate (CP) or myristyl myristate (MM) as the solid lipid matrix, in the presence (NLC) or absence (SLN) of a mixture of capric and caprylic acids; poloxamer 188 was used as surfactant. DBC was quantified through a validated HPLC procedure. Physico-chemical analysis of the nanoparticles included measurements of size distribution, zeta potential, morphology, DBC encapsulation efficiency, as well as exploratory scanning calorimetry (DSC), infrared spectroscopy (FTIR), electron paramagnetic resonance (EPR) and small angle X-ray scattering (SAXS) tests. In vitro analysis of the release profile, flow and elasticity of the particles were performed through artificial membranes while toxicity was tested in 3T3 fibroblasts and HaCaT keratinocytes in culture. Stability of the formulations as a function of time was also measured. The therapeutic activity of the formulations was determined using antinociception tests (tail flick) in Wistar rats. SLN and NLC produced by both methodologies were similar (~200 nm), but HP produced more stable nanoparticles. The physical stability of the nanoparticles was satisfactory during a storage period of 240 days, especially for NLCMM/HP with or without DBC. All formulations showed encapsulation efficiencies higher than 70%, the greatest being assigned for NLCMMDBC/HP (90.54 ± 0.95%). FTIR and DSC revealed that DBC was molecularly dispersed in the lipid matrix of the nanoparticles. As for the SLN and NLC molecular packing, SAXS diffractrograms indicated the existence of lamellar repeats in SLN core region, which were not disturbed by the addition of DBC while EPR data with doxyl stearate probes revealed spectra compatible with bilayers, with higher molecular order in the presence of DBC. In vitro assays confirmed the prolonged release of dibucaine from the nanoparticles, by Fickian diffusion. Nanoparticles's elasticity and flow were low showing deposition on the surface of 30 nm pore membranes. The intrinsic cytotoxicity of DBC against both cell types was decreased, when encapsulated in SLN and NLC. The in vivo analgesic effect of 0.05% DBC topically applied (dispersed in carbopol gel) was significantly prolonged in the nanoparticle formulations, largely for SLNCPDBC lyophilized with maltosis as crioprotector. In conclusion, dibucaine formulations in SLN or NLC prepared with MM or CP are promising for the development of pharmaceutical products intended for prolonged dermal anesthesia / Doutorado / Bioquimica / Doutora em Biologia Funcional e Molecular

Orientador: Francisco Benedito Teixeira Pessine / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-24T01:26:17Z (GMT). No. of bitstreams: 1 Marques_LeticiaPaifer_M.pdf: 4333961 bytes, checksum: dd287508ba192ad95556c2094f8d9d2a (MD5) Previous issue date: 2013 / Resumo: Através de modificações na estrutura química da molécula de pectina cítrica, polissacarídeo utilizado nas indústrias alimentícias como agente gelificante/espessante, foi desenvolvido um Sistema Depot para entrega subcutânea de nanopartículas lipídicas sólidas (NLS) e carreadores lipídicos nanoestruturados (CLN) contendo, respectivamente, Dexametasona (DXM) e Valerato de Betametasona (BTM). Os fármacos foram encapsulados nesses sistemas devido à necessidade de modificar seus perfis de liberação, diminuindo o número de aplicações e sua dosagem, reduzindo ocorrência de efeitos adversos. O produto da reação de oxidação da pectina cítrica foi caracterizado através de Espectroscopia da região do Infravermelho, Análise Termogravimétrica e Calorimetria Diferencial de Varredura. Os resultados indicaram o sucesso da reação, confirmado através da gelificação do hidrogel de pectina cítrica oxidada. As NLS e os CLN apresentaram valores de diâmetro médio em torno de 80nm, alta eficiência de encapsulação e perfis de liberação prolongada; para os CLN o fármaco BTM foi liberado ao longo de 144 horas e para as NLS o fármaco DXM foi liberado ao longo de 24 horas. Estes resultados mostram que as NLS de DXM poderiam ser utilizadas para tratar processos inflamatórios agudos e que os CLN de BTM seriam úteis no tratamento de processos inflamatórios crônicos / Abstract: A modified chemical structure of citrus pectin was used to develop a Depot system for subcutaneuous delivery of solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) containing, respectively, Dexamethasone (DXM) and Betamethasone Valerate (BTM). Citrus pectin is a polysaccharide used in food industry as a gelling/thickener agent. The drugs were encapsulated in this system with the aim to modify their release profiles. This would result in the reduction of the number of applications and dosage. As a consequence, it would also reduce adverse side effects that these drugs may cause. The citrus pectin oxidation product was characterized by Infrared Spectroscopy, Thermogravimetric Analysis and Differential Scanning Calorimetry. The results indicated that the reaction occurred. This was confirmed by gelation of the citrus pectin oxidation product. SLN and CLN showed values with diameters around 80nm, high encapsulation efficiency and sustained release profiles. BTM was released from the CLN over 144 hours and DXM was released from the NLS over 24 hours. These results showed that the DXM-NLS would be useful in the treatment of acute inflammatory processes. BTM-CLN could be applied in the treatment of chronic inflammatory processes / Mestrado / Físico-Química / Mestra em Química