Atualmente, a demanda por alimentos com apelo funcional tem se tornado cada vez mais recorrente dentre os consumidores devido a uma crescente busca por hábitos de vida mais saudáveis. Sendo assim, o desenvolvimento de técnicas que possibilitem uma adição mais efetiva de ingredientes funcionais em matrizes alimentícias se torna uma necessidade. Essas técnicas devem possibilitar principalmente (i) a incorporação de mecanismos de liberação sustentada na formulação; (ii) o aumento da bioacessibilidade e biodisponibilidade aos ingredientes, a partir do controle da microestrutura do alimento. Esse projeto visa contemplar essas duas premissas, ao propor a encapsulação de dois bioativos hidrofóbicos, a curcumina e a vitamina D3, conhecidos pelas suas propriedades antioxidantes e nutracêuticas, em carreadores de origem lipídica, os lipossomas, estabilizando-os com diferentes hidrocoloides - goma xantana, goma guar e inulina. Os lipossomas foram produzidos por hidratação de prolipossomas e suas propriedades físico-químicas foram caracterizadas ao longo de 42 dias de armazenagem, a partir de análises de diâmetro médio hidrodinâmico, potencial zeta, colorimetria instrumental e quantificação de bioativos encapsulados. Análises que permitiram a caracterização da microestrutura das dispersões produzidas também foram realizadas, sendo elas: calorimetria diferencial de varredura (DSC), espalhamento de raios-X a baixos ângulos (SAXS) e ensaios reológicos. As análises de SAXS mostraram que lipossomas produzidos na presença de curcumina são mais estáveis que àqueles produzidos na ausência da mesma e que não houve mudança na estrutura da bicamada lipídica das vesículas após a adição de vitamina D3, mesmo quando uma alta concentração foi incorporada ao sistema (80.000 UI). Por fim, verificou-se que a coencapsulação foi possível em lipossomas multilamelares estabilizados apenas com gomas guar e xantana, resultado que pode ser comprovado pelo alto teor de retenção dos bioativos ao longo do tempo de armazenagem. / Currently, the demand for food with functional appeal has become increasingly recurrent among the consumers due to a growing search for healthier living habits. Therefore, the development of techniques that allow a more effective addition of functional ingredients in food matrices becomes a necessity. These techniques should mainly enable to (i) incorporate a sustained release mechanisms into the formulation; (ii) increase the bioaccessibility and bioavailability to these ingredients, from the control of the food microstructure. This project aims to contemplate these two premises by proposing the encapsulation of two hydrophobic bioactives, curcumin and vitamin D3, known for their antioxidant and nutraceutical properties, in liposomes - lipid carriers - stabilizing them with different hydrocolloids - xanthan gum, guar gum and inulin. Liposomes were produced by proliposomes hydration and their physicochemical properties were characterized during 42 days of storage, including analyzes of hydrodynamic average diameter, zeta potential, instrumental colorimetry and quantification of encapsulated bioactives. Analyzes that allowed the microstructure characterization of the produced dispersions were also performed, including: differential scanning calorimetry (DSC), small-angle X-ray scattering and rheological tests. The SAXS analysis showed that liposomes produced in the presence of curcumin were more stable when compared to the empty ones and that there was no change in the lipid bilayer of the vesicles after the addition of vitamin D3, even when a high concentration was incorporated into the system (80,000 IU). Finally, it was concluded that the coencapsulation was possible in multilamellar liposomes stabilized with guar and xanthan gums, a result that can be evidenced by the high content of bioactives retained throughout the storage time.

Atualmente, a demanda por alimentos com apelo funcional tem se tornado cada vez mais recorrente dentre os consumidores devido a uma crescente busca por hábitos de vida mais saudáveis. Sendo assim, o desenvolvimento de técnicas que possibilitem uma adição mais efetiva de ingredientes funcionais em matrizes alimentícias se torna uma necessidade. Essas técnicas devem possibilitar principalmente (i) a incorporação de mecanismos de liberação sustentada na formulação; (ii) o aumento da bioacessibilidade e biodisponibilidade aos ingredientes, a partir do controle da microestrutura do alimento. Esse projeto visa contemplar essas duas premissas, ao propor a encapsulação de dois bioativos hidrofóbicos, a curcumina e a vitamina D3, conhecidos pelas suas propriedades antioxidantes e nutracêuticas, em carreadores de origem lipídica, os lipossomas, estabilizando-os com diferentes hidrocoloides - goma xantana, goma guar e inulina. Os lipossomas foram produzidos por hidratação de prolipossomas e suas propriedades físico-químicas foram caracterizadas ao longo de 42 dias de armazenagem, a partir de análises de diâmetro médio hidrodinâmico, potencial zeta, colorimetria instrumental e quantificação de bioativos encapsulados. Análises que permitiram a caracterização da microestrutura das dispersões produzidas também foram realizadas, sendo elas: calorimetria diferencial de varredura (DSC), espalhamento de raios-X a baixos ângulos (SAXS) e ensaios reológicos. As análises de SAXS mostraram que lipossomas produzidos na presença de curcumina são mais estáveis que àqueles produzidos na ausência da mesma e que não houve mudança na estrutura da bicamada lipídica das vesículas após a adição de vitamina D3, mesmo quando uma alta concentração foi incorporada ao sistema (80.000 UI). Por fim, verificou-se que a coencapsulação foi possível em lipossomas multilamelares estabilizados apenas com gomas guar e xantana, resultado que pode ser comprovado pelo alto teor de retenção dos bioativos ao longo do tempo de armazenagem. / Currently, the demand for food with functional appeal has become increasingly recurrent among the consumers due to a growing search for healthier living habits. Therefore, the development of techniques that allow a more effective addition of functional ingredients in food matrices becomes a necessity. These techniques should mainly enable to (i) incorporate a sustained release mechanisms into the formulation; (ii) increase the bioaccessibility and bioavailability to these ingredients, from the control of the food microstructure. This project aims to contemplate these two premises by proposing the encapsulation of two hydrophobic bioactives, curcumin and vitamin D3, known for their antioxidant and nutraceutical properties, in liposomes - lipid carriers - stabilizing them with different hydrocolloids - xanthan gum, guar gum and inulin. Liposomes were produced by proliposomes hydration and their physicochemical properties were characterized during 42 days of storage, including analyzes of hydrodynamic average diameter, zeta potential, instrumental colorimetry and quantification of encapsulated bioactives. Analyzes that allowed the microstructure characterization of the produced dispersions were also performed, including: differential scanning calorimetry (DSC), small-angle X-ray scattering and rheological tests. The SAXS analysis showed that liposomes produced in the presence of curcumin were more stable when compared to the empty ones and that there was no change in the lipid bilayer of the vesicles after the addition of vitamin D3, even when a high concentration was incorporated into the system (80,000 IU). Finally, it was concluded that the coencapsulation was possible in multilamellar liposomes stabilized with guar and xanthan gums, a result that can be evidenced by the high content of bioactives retained throughout the storage time.

<p>Prothrombinase is composed of a proteinase, factor Xa (Xa), its cofactor Va (Va), Ca²⁺ and a zymogen, prothrombin (II), assembled on a phospholipid surface. During coagulation, prothrombinase accelerates II to thrombin conversion; but during anticoagulation, it protects the proteinase from inhibition by antithrombin (AT) ± unfractionated heparin (UFH). Although the degree of Xa protection by prothrombinase varies according to the reports in literature, moderate to significant protective effects have been consistently reported by most investigators. To overcome the limitations of UFH, our laboratory has developed a covalent complex of AT and UFH (ATH) with superior anticoagulant responses. To further understand the mechanisms of enhanced anticoagulant activity of ATH, we proceeded to study inhibition of the prothrombinase complex<em> </em>on synthetic vesicles, activated platelets and red blood cells (RBCs). Using discontinuous inhibition assays, we determined the rate of inhibition of prothrombinase-complexed Xa compared to control Xa. With synthetic vesicles, Xa was protected from inhibition by AT+UFH when in prothrombinase, while only a mild protective effect was observed with ATH. Omission of various components of the prothrombinase led to a reduction in Xa protection for AT+UFH. However, an increased Xa protection against ATH was observed when II was omitted from the prothrombinase. In comparison to the synthetic vesicle system, activated platelets showed a similar trend for protection of Xa in reactions involving prothrombinase ± components, while no protection of Xa was observed for ATH reactions. Alternatively, RBCs showed differences relative to vesicles in that increased protection of Xa occurred with omission of II and Va for AT+UFH, whereas omission of Va increased protection against ATH inhibition. In addition, ATH had improved inhibition of thrombin generation, fibrin formation and plasma coagulation compared to AT+UFH. Studies of fluorescently labelled Xa and inhibitors detailed binding interactions with prothrombinase subunits. Overall, the results suggest that a covalent linkage between AT and heparin improves inactivation of prothrombinase complexed-Xa leading to down-regulation of prothrombinase function.</p> / Doctor of Philosophy (Medical Science)

Interactions between biomolecules and cellulose films at solid/liquid interfaces was studied by surface plasmon resonance spectroscopy (SPR), quartz crystal microbalance with dissipation monitoring (QCM-D) and in situ atomic force microscopy (AFM) measurements. This dissertation shows the porous character of nanocrystalline cellulose films as the key feature for enhanced adsorption of chemically modified polysaccharides and provides quantitative analysis of polymer supported phospholipid structures as a stable platform for studying membrane-related processes. Smooth cellulose I films were prepared by spincoating cellulose nanocrystal suspensions onto positively charged self-assembled monolayers on gold. The adsorption of pullulan cinnamate (PC) onto cellulose surfaces increased with increasing degree of cinnamate substitution. The interactions between PCs with higher degree of substitution (DS) and porous nanocrystalline cellulose (NC) films presumably generated looped multilayer PC structures that adsorbed more than twice as much onto NC films than onto regenerated cellulose (RC) films. PC chains not only covered the NC surface but also penetrated into the porous film. The porous features of NC film are responsible for the greater adsorption of polymer chains relative to tightly packed RC films. Adsorption of phospholipid vesicles onto RC and NC films was also studied. Aggregates of intact vesicle were observed on NC surfaces with high water content ~ 84 % by mass. Phospholipid patches with smooth features were found to assemble onto RC surfaces with a lower degree of hydration ~ 30 % by mass. Vesicle membrane breakage was triggered by a destabilizing agent, LysoPC. The great mass decrease, and changes in dissipation and degree of hydration for phospholipid structures after exposure to LysoPC corresponded to the transformation from vesicles to layered structures. Initial binding of LysoPC micelles to unruptured vesicles was clearly resolved in SPR, whereas the huge mass decrease associated with bound water hides the initial adsorption of LysoPC onto vesicles in QCM-D experiments. The intitial binding of LysoPC micelles onto vesicle membranes lasted for 200 seconds with a maximal increase of 14 % by mass prior to vesicle collapse. The role of cholesterol in phospholipid interactions with model cellulose surfaces was also considered. Supported vesicle layers over RC surfaces were observed for vesicle membranes containing ≥ 6.3 % by mole cholesterol, whereas phospholipid or phospholipid with lower cholesterol content formed disconnected lipid islands on RC surfaces. Meanwhile, intact vesicles were always observed on NC surfaces for phospholipid/cholesterol blends regardless of the cholesterol content. The intact vesicles on cellulose surfaces were attributed to the ability of cholesterol to accommodate vesicle deformation. These studies showed the impact of mesoscale structure of cellulose films on adsorbates. It sheds light on the role of the lignin-carbonhydrate-complex in plant cell wall structure and will inform the next generation of biomimetic nanocomposites. The designed polymer supported biomimetic membranes provide a perfect platform to develop intact and ruptured protoplast systems for the study of plant cell wall self-assembly. / Ph. D.

L'obésité est une maladie définie par une accumulation de masse grasse dans le tissu adipeux ayant des conséquences néfastes pour la santé. Les causes de l’obésité sont multiples. Dans un travail récent, il y a été démontré le rôle de la pollution environnementale dans la prise de poids. Dans ce travail, les hypothèses selon lesquelles les récepteurs adrénergiques situés à la surface des cellules adipeuses seraient le siège de l’action des polluants aromatiques polycycliques ont été vérifiées par le dosage de plusieurs agonistes et antagonistes spécifiques et non spécifiques en présence ou non du benzo[a]pyrène sur des récepteurs humains et de cellules d’hamster chinois (CHO). Les quantités d’AMPc obtenues montrent que les HAP ne se déposent pas sur les récepteurs β1, β2, β3 adrénergiques.Cette accumulation se fait au niveau des phospholipides de la membrane cytoplasmique des cellules. Ce qui cause une rigidité des membranes.Cette observation tend à renforcer l'hypothèse selon laquelle le benzo[a]pyrène induirait une inhibition de la lipolyse par l'accumulation au niveau de la bicouche de phospholipides et des changements de conformation de la bicouche de phospholipides dans les environs des récepteurs à sept domaines transmembranaires qui sont β-adrénergiques.La liaison de la bicouche phospholipidique avec les HAP utilisés est une réaction exothermi-que avec un faible dégagement de chaleur / Obesity is a disease defined by an accumulation of fat in adipose tissue with adverse consequences for health. The causes of obesity are many.In recent work, there was demonstrated the role of environmental pollution in weight gain.In this work, the assumptions that the adrenergic receptors on the surface of fat cells would home to the accumulation of polycyclic aromatic pollutants have been verified by measurement of several agonists and antagonists specific and non-specific in the presence or absence of benzo[a]pyrene receptors on human cells and Chinese hamster (CHO). The amounts of cAMP obtained showed that PAHs are not deposited on β-receptors, β1, β2, β3 adrenergic receptors.This accumulation occurs at the cytoplasmic membrane phospholipids of the cells. What cau-ses stiffness of the membranes. This observation tends to reinforce the hypothesis that benzo [a]pyrene induce an inhibition of lipolysis by the accumulation in the phospholipid bilayer and conformational changes of the bilayer phospholipids in the vicinity of receptors seven transmembrane domains which are β-adrenergic receptors

Apresentamos os resultados de estudos sobre a utilização de técnicas baseadas no fenômeno de fluorescência para a investigação de processos relacionados a membranas modelo. Nessa investigação, estão envolvidas medidas de propriedades espectrais de absorção e emissão de luz por cromóforos adequados, determinação xperimental de perfis de decaimento temporal da fluorescência e correlação temporal de emissão fluorescente, bem como a utilização apropriada de metodologias para análise e interpretação dos dados experimentais. Foram utilizados diversos compostos que apresentam absorção e emissão na região ultravioleta/visível, como as sondas lipofílicas 2-Amino-N-hexadecil-benzamida (Ahba), 6-lauryl-2-dimethylaminonaphthalene (Laurdan), N-(7-nitrobenz-2-oxa-1,3- diazol-4-yl) (NBD), em diferentes condições: meio aquoso homogêneo, suspensões de micelas de Sodium Dodecyl Sulfate (SDS), Cetyl-trimethyl-ammonium-bromide (CTAB) e 3-(Dodecyl-Dimethyl-Ammonio)-propane-sulfonate (DPS) e vesículas de fosfolipídios, como o 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC), o 1,2-Dimyristoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)](Sodium Salt) (DMPG) e o 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC). Supressores alquilpiridínios de diferentes comprimentos da cadeia alquila e, portanto, diferentes afinidades por agregados anfifílicos, foram utilizados em experimentos de supressão da fluorescência da sonda Ahba. Usando o formalismo que descreve fenômenos de supressão dependente de colisões entre fluoróforo e supressor, observamos que as taxas de supressão são maiores em presença de agregados anfifílicos carregados negativamente: micelas de SDS e vesículas de DMPG; em micelas zwiteriônicas o processo é mais eficiente quando a hidrofobicidade do supressor é grande, o que ocorre quando a cadeia alquila é mais longa. Realizamos experimentos de transferência de energia por ressonância de Förster (FRET) onde o grupo fluorescente da sonda lipofílica Ahba atuou como doador. Como aceitadores utilizamos os compostos Acridina Laranja, -(2,4,dinitrofenil)-etilenodiamina (Eddnp) e o NBD ligado a fosfolipídios. Fizemos uso do programa CONTIN para análise de dados experimentais de perfis de decaimento da fluorescência em sistemas em que ocorre transferência de energia e obtivemos distribuições de distâncias para os pares Ahba/Eddnp e Ahba/NBD-fosfolipídios na presença de vesículas de fosfolipídios. Para este último par, verificou-se que a distribuição de distâncias depende da temperatura do sistema, ou seja, da fase da bicamada, da concentração de aceitador e da posição onde o NBD está ligado ao fosfolipídio. Analisamos a utilização da sonda Laurdan em presença de vesículas de DMPC e POPC, em experimentos de espectroscopia de correlação de fluorescência. Embora tenha apresentado sinal elevado de fluorescência, a sonda é fotodegradável. Os mesmos experimentos de correlação de fluorescência foram realizados com o Ahba que, apesar de ter se mostrado bastante fotoestável, revelou não ser uma sonda adequada para uso em tal técnica. O espectro de excitação a dois fótons foi obtido para esta sonda, com máximo de absorção em 695 nm. Em experimentos de microscopia de fluorescência, o Ahba mostrou ser um bom marcador fluorescente para membranas lipídicas, ao possibilitar a aquisição de imagens de fluorescência de vesículas gigantes marcadas. / In this work we showed results from studies about the use of fluorescence spectroscopy techniques as a tool to investigate amphiphilic aggregates, used as a model of the cell membrane. We performed measurements on the spectral properties of light absorption and emission of adequate chromophors, registered the experimental timeresolved decay of fluorescence and time correlated fluorescence emission of the probes and used also adequate methodologies for the analysis and interpretation of experimental data. Several compounds presenting absorption and emission in the UV/visible spectral range were employed: the lipophilic probes 2-Amino-N-hexadecil-benzamida (Ahba), 6-lauryl-2-dimethylaminonaphthalene (Laurdan), N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD), in different environment:homogeneous aqueous medium, micelles of surfactants like Sodium Dodecyl Sulfate (SDS), Cetyl-trimethyl-ammonium-bromide (CTAB) and 3- (Dodecyl-Dimethyl-Ammonio)-propane-sulfonate (DPS) and phospholipid vesicles of 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC), 1,2-Dimyristoyl-sn-Glycero-3- [Phospho-rac-(1-glycerol)](Sodium Salt) (DMPG) and 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC). Alkyilpyridinium halides with different alkyl chain length were employed fluorescence quenchers of the Ahba probe. Using the Stern-Volmer model to describe the quenching phenomena dependent on fluorophor/quencher collision, we observed that higher quenching rates were obtained in the presence of negatively charged amphiphilic agreggates: SDS micelles and DMPG vesicles; in the presence of zwitterionic vesicles the quenching efficiency was more efficient when the quencher hydrophobicity was high (long alkyl chain). We performed Förster resonance energy transfer (FRET) experiments where the fluorescent moiety of the probe Ahba was the energy donor. As acceptors molecules we used Acridine Orange, Ethylene-diamine-dinitrophenyl (Eddnp) and NBD-labeled phospholipids. The computational package CONTIN was adapted to analyze the experimentally obtained fluorescence decay profiles of the donor in the presence of the acceptor, in order to determine the distance distribution between the Ahba/Eddnp and Ahba/NBD-phospholipids pairs in the presence of lipid vesicles. For the Ahba/NBD pair, the distances were dependent on the emperature of the system (or the phase bilayer behavior), the acceptor concentration and the NBD position in the phospholipid. We observed that the Laurdan probe can be used in studies about DMPC vesicles diffusion using fluorescence correlation spectroscopy techniques. Investigation about the use of the probe Ahba with this technique had shown that its maximum absorption for two photon excitation occurs near to 695 nm, but it is not an appropriated probe to FCS experiments due to its very low brightness. On the other hand, Ahba can be used as a membrane fluorescent label in membrane fluorescence microscopy, as we can see in the fluorescence imaging experiments with giant vesicles labeled with Ahba.

Apresentamos os resultados de estudos sobre a utilização de técnicas baseadas no fenômeno de fluorescência para a investigação de processos relacionados a membranas modelo. Nessa investigação, estão envolvidas medidas de propriedades espectrais de absorção e emissão de luz por cromóforos adequados, determinação xperimental de perfis de decaimento temporal da fluorescência e correlação temporal de emissão fluorescente, bem como a utilização apropriada de metodologias para análise e interpretação dos dados experimentais. Foram utilizados diversos compostos que apresentam absorção e emissão na região ultravioleta/visível, como as sondas lipofílicas 2-Amino-N-hexadecil-benzamida (Ahba), 6-lauryl-2-dimethylaminonaphthalene (Laurdan), N-(7-nitrobenz-2-oxa-1,3- diazol-4-yl) (NBD), em diferentes condições: meio aquoso homogêneo, suspensões de micelas de Sodium Dodecyl Sulfate (SDS), Cetyl-trimethyl-ammonium-bromide (CTAB) e 3-(Dodecyl-Dimethyl-Ammonio)-propane-sulfonate (DPS) e vesículas de fosfolipídios, como o 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC), o 1,2-Dimyristoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)](Sodium Salt) (DMPG) e o 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC). Supressores alquilpiridínios de diferentes comprimentos da cadeia alquila e, portanto, diferentes afinidades por agregados anfifílicos, foram utilizados em experimentos de supressão da fluorescência da sonda Ahba. Usando o formalismo que descreve fenômenos de supressão dependente de colisões entre fluoróforo e supressor, observamos que as taxas de supressão são maiores em presença de agregados anfifílicos carregados negativamente: micelas de SDS e vesículas de DMPG; em micelas zwiteriônicas o processo é mais eficiente quando a hidrofobicidade do supressor é grande, o que ocorre quando a cadeia alquila é mais longa. Realizamos experimentos de transferência de energia por ressonância de Förster (FRET) onde o grupo fluorescente da sonda lipofílica Ahba atuou como doador. Como aceitadores utilizamos os compostos Acridina Laranja, -(2,4,dinitrofenil)-etilenodiamina (Eddnp) e o NBD ligado a fosfolipídios. Fizemos uso do programa CONTIN para análise de dados experimentais de perfis de decaimento da fluorescência em sistemas em que ocorre transferência de energia e obtivemos distribuições de distâncias para os pares Ahba/Eddnp e Ahba/NBD-fosfolipídios na presença de vesículas de fosfolipídios. Para este último par, verificou-se que a distribuição de distâncias depende da temperatura do sistema, ou seja, da fase da bicamada, da concentração de aceitador e da posição onde o NBD está ligado ao fosfolipídio. Analisamos a utilização da sonda Laurdan em presença de vesículas de DMPC e POPC, em experimentos de espectroscopia de correlação de fluorescência. Embora tenha apresentado sinal elevado de fluorescência, a sonda é fotodegradável. Os mesmos experimentos de correlação de fluorescência foram realizados com o Ahba que, apesar de ter se mostrado bastante fotoestável, revelou não ser uma sonda adequada para uso em tal técnica. O espectro de excitação a dois fótons foi obtido para esta sonda, com máximo de absorção em 695 nm. Em experimentos de microscopia de fluorescência, o Ahba mostrou ser um bom marcador fluorescente para membranas lipídicas, ao possibilitar a aquisição de imagens de fluorescência de vesículas gigantes marcadas. / In this work we showed results from studies about the use of fluorescence spectroscopy techniques as a tool to investigate amphiphilic aggregates, used as a model of the cell membrane. We performed measurements on the spectral properties of light absorption and emission of adequate chromophors, registered the experimental timeresolved decay of fluorescence and time correlated fluorescence emission of the probes and used also adequate methodologies for the analysis and interpretation of experimental data. Several compounds presenting absorption and emission in the UV/visible spectral range were employed: the lipophilic probes 2-Amino-N-hexadecil-benzamida (Ahba), 6-lauryl-2-dimethylaminonaphthalene (Laurdan), N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD), in different environment:homogeneous aqueous medium, micelles of surfactants like Sodium Dodecyl Sulfate (SDS), Cetyl-trimethyl-ammonium-bromide (CTAB) and 3- (Dodecyl-Dimethyl-Ammonio)-propane-sulfonate (DPS) and phospholipid vesicles of 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC), 1,2-Dimyristoyl-sn-Glycero-3- [Phospho-rac-(1-glycerol)](Sodium Salt) (DMPG) and 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC). Alkyilpyridinium halides with different alkyl chain length were employed fluorescence quenchers of the Ahba probe. Using the Stern-Volmer model to describe the quenching phenomena dependent on fluorophor/quencher collision, we observed that higher quenching rates were obtained in the presence of negatively charged amphiphilic agreggates: SDS micelles and DMPG vesicles; in the presence of zwitterionic vesicles the quenching efficiency was more efficient when the quencher hydrophobicity was high (long alkyl chain). We performed Förster resonance energy transfer (FRET) experiments where the fluorescent moiety of the probe Ahba was the energy donor. As acceptors molecules we used Acridine Orange, Ethylene-diamine-dinitrophenyl (Eddnp) and NBD-labeled phospholipids. The computational package CONTIN was adapted to analyze the experimentally obtained fluorescence decay profiles of the donor in the presence of the acceptor, in order to determine the distance distribution between the Ahba/Eddnp and Ahba/NBD-phospholipids pairs in the presence of lipid vesicles. For the Ahba/NBD pair, the distances were dependent on the emperature of the system (or the phase bilayer behavior), the acceptor concentration and the NBD position in the phospholipid. We observed that the Laurdan probe can be used in studies about DMPC vesicles diffusion using fluorescence correlation spectroscopy techniques. Investigation about the use of the probe Ahba with this technique had shown that its maximum absorption for two photon excitation occurs near to 695 nm, but it is not an appropriated probe to FCS experiments due to its very low brightness. On the other hand, Ahba can be used as a membrane fluorescent label in membrane fluorescence microscopy, as we can see in the fluorescence imaging experiments with giant vesicles labeled with Ahba.

Este estudo reúne os principais resultados de fluorescência estática e resolvida no tempo sobre a interação da porfirina meso-tetrakis (4-metilpiridil), na forma de base livre (TMPyP) e complexada com Zn2+ (ZnTMPyP), com vesículas de fosfolipídio. Adicionalmente foram utilizadas as técnicas de potencial zeta e espalhamento de luz dinâmico (DLS, do inglês \"dynamic light scattering\"). As vesículas de fosfolipídio foram formadas por dois conjuntos de fosfolipídios: saturados e insaturados. O primeiro grupo é formado pela mistura dos fosfolipídios zwiteriônico 1,2-dipalmitoil-sn-glicero-3-fosfocolina (DPPC) e aniônico 1,2-dipalmitoil-sn-3-glicero-[fosfo-rac-(1- glicerol)] (DPPG), a diferentes razões molares. Os estudos utilizando tais sistemas foram realizados abaixo (25oC) e acima (50oC) da temperatura de transição de fase gel-líquido cristalino destes fosfolipídios (~ 41oC). O segundo grupo é formado pela mistura dos fosfolipídios zwiteriônico 1-palmitoil-2-oleoil-sn-glicero-3-fosfocolina (POPC) e aniônico 1-palmitoil-2-oleoil-sn-glicero-3-fosfo(1-rac-glicerol) (POPG). Como a transição de fase destes dois fosfolipídios ocorre a temperaturas negativas, todos os experimentos foram realizados a 25oC (vesículas no estado líquido cristalino). Todos os sistemas foram preparados através do método de extrusão para a obtenção de vesículas grandes unilamelares (LUV, do inglês \"large unilamellar vesicles\"). As análises dos dados de fluorescência indicaram que a atração eletrostática entre os substituíntes (positivamente carregados) das porfirinas TMPyP e ZnTMPyP e o grupo das cabeças polares (camada de Stern) das vesículas de fosfolipídio desempenha um papel fundamental na associação da porfirina. A distribuição da TMPyP entre o meio aquoso (tampão) e as vesículas de fosfolipídio foi evidenciada pela coexistência de um tempo de vida de fluorescência mais curto (~ 5 ns) e outro mais longo (~ 9-11 ns), respectivamente. Baseado nos valores das constantes pré-exponenciais, estudos adicionais mostram que a distribuição acima é afetada pela concentração de sal na solução. Os resultados de supressão de fluorescência com o supressor iodeto de potássio (KI) indicaram que ambas porfirinas estão localizadas, preferencialmente, na região da camada de Stern. Este resultado foi confirmado pelos estudos de potencial zeta e de DLS, os quais mostraram uma neutralização parcial das cargas negativas na superfície das vesículas devido à associação da porfirina. / This study presents time-resolved and steady-state fluorescence results on the interaction of the meso-tetrakis (4-methylpyridil) porphyrin, in free base form (TMPyP), and complexed with Zn2+ (ZnTMPyP), with phospholipid vesicles. Zeta potential and dynamic light scattering (DLS) techniques were also used. Phospholipid vesicles were formed by two phospholipid systems: saturated and unsaturated. The first group is a mixture of zwiterionic dipalmitoyl-sn-glycero-3-phosphocoline (DPPC) and anionic 1,2-dipalmitoyl-sn-3-glycero-[phospho-rac-(1-glycerol)] (DPPG) phospholipids, at different molar ratios. Measurements were performed bellow (25oC) and above (50oC) the main gel-liquid crystalline phase transition temperature (~ 41oC). The second group is constituted by a mixture of zwiterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline (POPC) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1-rac-glycerol) (POPG) phospholipids, at different molar ratios. Since the gel-liquid crystalline phase transition of these phospholipids occurs at a very low temperature value, all experiments were performed at 25oC (liquid crystalline state vesicles). All phospholipid systems were prepared through the extrusion method in order to obtain large unilamellar vesicles (LUV). The fluorescence data analyses indicated that the electrostatic attraction between the porphyrin substituents (positively charged) and the polar head groups of the phospholipid vesicles (Stern layer) plays an important role on the porphyrin binding affinity. The distribution of TMPyP between the aqueous medium (buffer) and the phospholipid vesicles was characterized by the coexistence of a shorter (~ 5 ns) and a longer (~ 9-11 ns) fluorescence lifetimes, respectively. Based on the pre- exponential values, additional time-resolved experiments showed a redistribution of the porphyrin at increasing salt concentration. The quenching studies, using potassium iodide (KI) as quencher, indicated that both TMPyP and ZnTMPyP are preferentially located at the Stern layer region. This result is in agreement with the zeta potential and DLS findings, which demonstrated a partial neutralization of the negative charges at the vesicle surface due to the porphyrin association.

Este estudo reúne os principais resultados de fluorescência estática e resolvida no tempo sobre a interação da porfirina meso-tetrakis (4-metilpiridil), na forma de base livre (TMPyP) e complexada com Zn2+ (ZnTMPyP), com vesículas de fosfolipídio. Adicionalmente foram utilizadas as técnicas de potencial zeta e espalhamento de luz dinâmico (DLS, do inglês \"dynamic light scattering\"). As vesículas de fosfolipídio foram formadas por dois conjuntos de fosfolipídios: saturados e insaturados. O primeiro grupo é formado pela mistura dos fosfolipídios zwiteriônico 1,2-dipalmitoil-sn-glicero-3-fosfocolina (DPPC) e aniônico 1,2-dipalmitoil-sn-3-glicero-[fosfo-rac-(1- glicerol)] (DPPG), a diferentes razões molares. Os estudos utilizando tais sistemas foram realizados abaixo (25oC) e acima (50oC) da temperatura de transição de fase gel-líquido cristalino destes fosfolipídios (~ 41oC). O segundo grupo é formado pela mistura dos fosfolipídios zwiteriônico 1-palmitoil-2-oleoil-sn-glicero-3-fosfocolina (POPC) e aniônico 1-palmitoil-2-oleoil-sn-glicero-3-fosfo(1-rac-glicerol) (POPG). Como a transição de fase destes dois fosfolipídios ocorre a temperaturas negativas, todos os experimentos foram realizados a 25oC (vesículas no estado líquido cristalino). Todos os sistemas foram preparados através do método de extrusão para a obtenção de vesículas grandes unilamelares (LUV, do inglês \"large unilamellar vesicles\"). As análises dos dados de fluorescência indicaram que a atração eletrostática entre os substituíntes (positivamente carregados) das porfirinas TMPyP e ZnTMPyP e o grupo das cabeças polares (camada de Stern) das vesículas de fosfolipídio desempenha um papel fundamental na associação da porfirina. A distribuição da TMPyP entre o meio aquoso (tampão) e as vesículas de fosfolipídio foi evidenciada pela coexistência de um tempo de vida de fluorescência mais curto (~ 5 ns) e outro mais longo (~ 9-11 ns), respectivamente. Baseado nos valores das constantes pré-exponenciais, estudos adicionais mostram que a distribuição acima é afetada pela concentração de sal na solução. Os resultados de supressão de fluorescência com o supressor iodeto de potássio (KI) indicaram que ambas porfirinas estão localizadas, preferencialmente, na região da camada de Stern. Este resultado foi confirmado pelos estudos de potencial zeta e de DLS, os quais mostraram uma neutralização parcial das cargas negativas na superfície das vesículas devido à associação da porfirina. / This study presents time-resolved and steady-state fluorescence results on the interaction of the meso-tetrakis (4-methylpyridil) porphyrin, in free base form (TMPyP), and complexed with Zn2+ (ZnTMPyP), with phospholipid vesicles. Zeta potential and dynamic light scattering (DLS) techniques were also used. Phospholipid vesicles were formed by two phospholipid systems: saturated and unsaturated. The first group is a mixture of zwiterionic dipalmitoyl-sn-glycero-3-phosphocoline (DPPC) and anionic 1,2-dipalmitoyl-sn-3-glycero-[phospho-rac-(1-glycerol)] (DPPG) phospholipids, at different molar ratios. Measurements were performed bellow (25oC) and above (50oC) the main gel-liquid crystalline phase transition temperature (~ 41oC). The second group is constituted by a mixture of zwiterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline (POPC) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1-rac-glycerol) (POPG) phospholipids, at different molar ratios. Since the gel-liquid crystalline phase transition of these phospholipids occurs at a very low temperature value, all experiments were performed at 25oC (liquid crystalline state vesicles). All phospholipid systems were prepared through the extrusion method in order to obtain large unilamellar vesicles (LUV). The fluorescence data analyses indicated that the electrostatic attraction between the porphyrin substituents (positively charged) and the polar head groups of the phospholipid vesicles (Stern layer) plays an important role on the porphyrin binding affinity. The distribution of TMPyP between the aqueous medium (buffer) and the phospholipid vesicles was characterized by the coexistence of a shorter (~ 5 ns) and a longer (~ 9-11 ns) fluorescence lifetimes, respectively. Based on the pre- exponential values, additional time-resolved experiments showed a redistribution of the porphyrin at increasing salt concentration. The quenching studies, using potassium iodide (KI) as quencher, indicated that both TMPyP and ZnTMPyP are preferentially located at the Stern layer region. This result is in agreement with the zeta potential and DLS findings, which demonstrated a partial neutralization of the negative charges at the vesicle surface due to the porphyrin association.

Phospholipid vesicles are common model systems for cell membranes. Important aspects of the membrane function relate to its mechanical properties. Here we have investigated the deformation behaviour of phospholipid vesicles in a dual-beam laser trap, also called an optical stretcher. This study explicitly makes use of the inherent heating present in such traps to investigate the dependence of vesicle deformation on temperature. By using lasers with different wavelengths, optically induced mechanical stresses and temperature increase can be tuned fairly independently with a single setup. The phase transition temperature of vesicles can be clearly identified by an increase in deformation. In the case of no heating effects, a minimal model for drop deformation in an optical stretcher and a more specific model for vesicle deformation that takes explicitly into account the angular dependence of the optical stress are presented to account for the experimental results. Elastic constants are extracted from the fitting procedures, which agree with literature data. This study demonstrates the utility of optical stretching, which is easily combined with microfluidic delivery, for the future serial, high-throughput study of the mechanical and thermodynamic properties of phospholipid vesicles.