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.

A investigação dos mecanismos de interação dos vírus com as células do hospedeiro trazem informações relevantes para a identificação de alvos no desenvolvimento de drogas para impedir a penetração e/ou desenvolvimento dos vírus. Peptídeos desenhados a partir de proteínas virais foram desenvolvidos e testados quanto as suas capacidades de inibir o processo de fusão do vírus com a célula do hospedeiro. Alguns se encontram em fase de avaliação clínica. Anticorpos contra a proteína p24 do HIV-1 foram detectados no soro de pacientes HIV-positivos, e estes reconhecem pequenas seqüências peptídicas desta proteína. Neste trabalho foi analisada a interação entre uma seqüência peptídica correspondente aos aminoácidos 196-224 (AAMQMLKETINEEAAEWDRVHPVHAGPIA) da proteína p24, denominado p24- 1, com sistemas biomiméticos. Os sistemas utilizados foram filmes de Langmuir (monocamadas) de dipalmitoil fosfatidil colina (DPPC) e dipalmitoil fosfatidil glicerol (DPPG) e vesículas multilamelares (MLVs) de DPPC. O p24-1 encontra-se desorganizado em solução aquosa, mas com a interação com as MLVs de DPPC teve induzido uma conformação hélice ?, de acordo com o espectro de dicroísmo circular (CD). Esta característica foi confirmada pela predição de hélice a seguida por uma estrutura não ordenada contendo 11 resíduos do p24-1. As isotermas de pressão e potencial de superfície das monocamadas de DPPC foram afetadas com a presença de 0,05% mo1 de p24-1, com uma expansão de aproximadamente 5%. Para concentrações acima de 0,5% mo1 de p24-1 a expansão foi de 20%, com saturação do efeito da concentração. O efeito de expansão foi acompanhado por uma alteração na morfologia das monocamadas, estudados com microscopia no ângulo de Brewster (BAM). A incorporação do p24-1 impede a formação de grandes domínios de DPPC. O efeito cooperativo causado na monocamada de fosfolipídios pelo p24-1 sugere que esse tem um potencial na atividade antiviral por participar da expansão da membrana da célula hospedeira. / The investigation of the interaction mechanisms between the viruses and the host cells brings relevant information for the identification of targets on the development of drugs to prevent the penetration and/or development of the viruses. Peptides designed from viral proteins have been developed and tested on its capacities of inhibiting the merging process of the virus with the host cell. Some of them are in clinical evaluation. Antibodies against the protein p24 of the HIV-1 have been detected in the serum of HIV-positive patients, and they are able to recognize short peptide sequences of this protein. In this work, it was analyzed the interaction between a peptide sequence corresponding to amino acids 196-224 (AAMQMLKETINEEAAEWDRVHPVHAGPIA) of the protein p24, called p24- 1, and biomimetic systems. The systems used were Langmuir films (monolayers) of dipalmitoyl phosphatidyl choline (DPPC) and dipalmitoyl phosphatidyl glycerol (DPPG) and multilamelar vesicles (MLVs) of DPPC. p24-1 is found disorganized in watery solution, but with the interaction with the MLVs of DPPC it had induced a conformation ?-helix, according to the circular dichoism spectra (CD). This characteristic was confirmed by the prediction of ?-helix followed by an unordered structure with 11 residues of p24-1. The isotherms of pressure and potential of surface of the DPPC monolayers were affected by the presence of 0,05% mo1 of p24-1, with an expansion of approximately 5%. For concentrations above 0,5% mol of p24-1 the expansion was 20%, with saturation of the concentration effect. The expansion effect was followed by a morphologic alteration of the monolayers, studied with microscopy of the Brewster angle (BAM). The incorporation of p24-1 prevents the formation of large domains of DPPC. The cooperative effect caused in the phospholipid monolayer by p24-1 suggests that this peptide has a potential in the antiviral activity, once its participates on the expansion of the host cell membrane.

Eine wichtige Eigenschaft der Plasmamembran eukaryotischer Zellen ist die stabile transversale Asymmetrie der Phospholipide. Sie wird energieabhängig durch die Aktivität einer Aminophospholipidtranslokase aufrechterhalten und gilt als wichtige Voraussetzung für die Homöostasis der Zellen. Die Plasmamembran einiger Säugerzellen weist zudem laterale Lipiddomänen auf, denen eine wesentliche Bedeutung bei der Signaltransduktion zugeschrieben wird. Während der Genese durchlaufen die Membranen der Säugerspermien intensive Veränderungen. Um die Bedeutung der Phospholipidasymmetrie für die Funktion der Spermien zu untersuchen, wurde die Lokalisation und Dynamik von Phosphatidylserin in der Zell- und Akrosommembran von Eberspermien im Verlauf von Kapazitation und Akrosomreaktion betrachtet. Unter Ausnutzung der selektiven, kalziumabhängigen Bindung von AnnexinV an endogenes Phosphatidylserin konnte dessen Lokalisation an morphologisch differenzierten Zellen verfolgt werden. Eine Markierung der Zellen mit NBD-markierten Phospholipidanaloga lieferte zudem Informationen zur Dynamik der Phospholipide in der Plasmamembran. Die Differenzierung der Zellen erfolgte entweder am Durchflusszytometer oder fluoreszenz- bzw. elektronenmikroskopisch. Die Ergebnisse der vorliegenden Arbeit weisen sowohl auf eine transversale als auch laterale Ungleichverteilung der Lipide in der Zell- und Akrosommembran während der Genese der Spermien hin. Neben der stabilen transversalen Phospholipidasymmetrie der Plasmamembran konnte erstmals eine zytoplasmatische Lokalisation von Phosphatidylserin auf der äußeren Akrosommembran nachgewiesen werden. Somit akkumulieren die beiden einander zugewandten zytoplasmatischen Monolayer von Plasmamembran und äußerer Akrosommembran Phosphatidylserin. Kapazitationsbedingt kommt es zu einer engen Wechselwirkung zwischen Plasmamembran und äußerer Akrosommembran. Die Ausbildung lateraler Membrandomänen, in denen Phosphatidylserin zytoplasmatisch akkumuliert, wird als Voraussetzung für diese enge Assoziation diskutiert. Weitere Hinweise auf eine funktionelle Bedeutung lateraler Membrandomänen lieferten die Arbeiten zur Isolation Triton-unlöslicher Lipiddomänen aus der Plasmamembran von Forellenspermien. / One of the essential qualities of cell membranes in Eucaryotae is a stable transverse phospholipid asymmetry. It is regulated and maintained by ATP-dependent action of an aminophospholipid translocase and is a major prerequisite for cell homeostasis. The plasma membranes of several mammalian cells show moreover lateral lipid domains, which are imputed to play a significant role in signal transduction. The membranes of mammalian spermatozoa undergo significant changes during genesis. The localisation and dynamics of phosphatidylserine in the cell as well as acrosome membranes of boar sperm cells was studied during capacitation and acrosome reaction to assess the relevance of lipid asymmetry for sperm function. The localisation of endogenous phosphatidylserine in morphologically differentiated cells was followed using the selective calcium depending binding of annexinV. Information on the transverse dynamics of phospholipids in the plasma membrane was obtained by labelling the cells with a NBD-phospholipid analogues. The morphological status of the cells was assessed by flow cytometry, fluorescence and electron microscopy. The results of this study indicate both a transversal and lateral inhomogenous distribution of lipids in the cell membrane as well as in the outer acrosome membrane during sperm genesis. The plasma membrane of boar sperm shows a stable transversal lipid asymmetry characterised by an accumulation of phosphatidylserine in the cytoplasmic monolayer. Moreover a cytoplasmic localisation of phosphatidylserine on the outer acrosome membrane could be detected for the first time. Therefore the two facing cytoplasmic leaflets of the outer acrosome and cell membrane contain phosphatidylserine. Applying microscopy substantiated the hypothesis that there are close interactions between the cell membrane and the outer layer of the acrosome membrane because of capacitation. The cytoplasmic accumulation of phosphatidylserine in lateral lipid domains is probably essential for the strong association of plasma and outer acrosome membrane finally leading to local fusions of both membranes. An indication for the functional meaning of lateral membrane domains in sperm cells was futher deduced from the isolation of Triton-insoluble lipid domains from membranes of trout sperm cells.

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.

Les maladies métaboliques d’origine nutritionnelle sont caractérisées par un métabolisme des lipides perturbé et une inflammation métabolique. Les lipides polaires (LP) sont des agents émulsifiants utilisés dans l’industrie agroalimentaire. Le but de notre étude a été d’évaluer l’impact de l’utilisation de nouveaux LP issus du lait sur (i) la digestion et le métabolisme postprandial des lipides, et (ii) à plus long terme sur l’adiposité et l’inflammation dans le tissu adipeux (TA), en comparaison avec les LP de soja, actuellement les plus consommés.Chez la souris, les LP laitiers induisent une cinétique de lipémie postprandiale plus précoce en comparaison avec les LP de soja, associée à une augmentation de la lipolyse intestinale in vitro. De plus, les chylomicrons sécrétés durant le pic de lipémie sont plus petits avec les LP laitiers. D’autre part, la substitution d’une partie des lipides d’un régime hyper-lipidique par des LP laitiers ne modifie pas le stockage des lipides dans le foie et le TA contrairement aux LP de soja qui induisent une augmentation des lipides hépatiques, des adipocytes et des marqueurs inflammatoires dans le TA. Par ailleurs, Les LP laitiers induisent une diminution de l’expression génique de marqueurs de l’infiltration macrophagique dans le TA et du nombre de cellules à gobelet dans le côlon, suggérant une barrière intestinale renforcée.Ces travaux démontrent que les LP laitiers, par rapport aux LP de soja, stimulent la digestion des lipides et induisent une cinétique plus rapide de lipémie postprandiale. A long terme ils n’induisent pas les altérations métaboliques du TA observées en présence de LP de soja dans un régime hyper-lipidique / Metabolic diseases are characterized by an altered lipid metabolism and metabolic inflammation. Numerous food products contain polar lipid (PL) emulsifiers that could impact these risk factors. We evaluated the impact of using PL from milk (MPL) (i) acutely on lipid digestion and postprandial lipemia and (ii) in the longer term in addition to a high fat diet on adiposity and adipose tissue inflammation. We compared MPL to soybean PL (SPL) that is currently the main commercial source of PL.In mice, an emulsion stabilized by MPL results in a more rapid postprandial lipemia than an emulsion stabilized by SPL, with an early increase in lipemia and a faster clearance. Differences in lipemia can originate from differential kinetics of lipid hydrolysis in the mouse gut, as an increase intestinal TG hydrolysis is observed in vitro. Moreover, early MPL-derived chylomicrons are smaller than SPL-derived chylomicrons. In the longer term, compared with HF diet, HF-SPL diet increases hepatic lipids, white adipose tissue (WAT) mass, with larger and more numerous adipocytes and increases expression of pro-inflammatory adipokines. This is not observed with HF-MPL diet despite similar dietary intakes. HFP-MPL mice have a lower expression in WAT of marker of macrophage infiltration and more numerous goblet cells in the colon, suggesting an improved gut barrier function with this diet.Postprandial lipemia in mice can be modulated by emulsifying with MPL compared with SPL, partly through differences in chylomicron assembly, and intestinal TG hydrolysis rate. Moreover unlike SPL, MPL in a high fat diet do not induce WAT hypertrophy and inflammation

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.

The cytochrome P450 enzyme system is a multicomponent electron-transfer chain composed of a haem-containing monooxygenase cytochrome P450 (CYP) and one or more redox partners. Eukaryotic CYPs and their redox partner NADPH-dependent cytochrome P450 oxidoreductase (CPR) are involved in many biological processes. Each protein has one N- terminal membrane anchor domain for location within the endoplasmic reticulum (ER). In mammals, CYPs and CPR are especially abundant in liver cells, where they play important roles in the metabolism of steroids, fatty acids, and xenobiotic compounds including numerous drugs of pharmaceutical importance. Incorporation into lipid membranes is an important aspect of CYP and CPR function, influencing their kinetic properties and interactions. In this thesis, soluble nanometer-scale phospholipid bilayer membrane discs, "nanodiscs", were used as a reconstitution system to study the influence of lipid membrane composition on the activities of the abundant human CYP3A4 and human CPR. Both enzymes were expressed and purified from bacteria, and assembled into functionally active membrane-bound complexes in nanodiscs. Nanodisc assembly was assessed by a combination of native and denaturing gel electrophoresis, and a fluorimetric assay was developed to study CYP3A4 reaction kinetics using 7-benzyloxyquinoline as substrate. Kinetic properties were investigated with respect to different lipid membrane compositions: phosphatidyl choline; a synthetic lipid mixture resembling the ER; and natural lipids extracted from liver microsomes. Full activity of the CYP3A4 system, with electron transfer from NADPH via CPR, could only be reconstituted when both CYP3A4 and CPR were membrane-bound within the same nanodiscs. No activity was observed when CPR and CYP3A4 were each incorporated seperately into naodiscs then mixed together, or when soluble forms of CPR were mixed with pre-assembled CYP3A4-nanodiscs. Thus, assembly of the two proteins within the same membrane was shown to be essential for the function of the CPR-CYP3A4 electron transfer system. Comparison of the reaction kinetics in different membrane compositions revealed liver microsomal lipid to have an enhancing effect both on the activity of the assembled CPR-CYP3A4 nanodisc complex, and on the activity of CPR alone incorporated in nanodiscs, when compared either to the synthetic lipid mixture or to phosphatidyl choline alone. Thus, natural lipids appear to possess properties or include components important for the catalytic function of the CYP system, which are absent from synthetic lipid. Input of electrons, measured by NADPH consumption, exceeded product formation rate by the CPR-CYP3A4 complex in nanodiscs, indicating "leakage" in the electron flow, possibly due to uncoupling of the two enzymes. Uncoupling was shown to occur by developing a novel fluorimetric method using the dye MitSOX to detect superoxide production. The significance of this, and to what extent control of coupling could be a natural means of regulation of the CPR-CYP system, remains to be determined. Thus, phospholipid bilayer nanodiscs prove a powerful tool to enable detailed analysis of the reaction kinetics of membrane-reconstituted CPR-CYP systems, and to allow pertinent questions to be addressed concerning the integral significance of the membrane environment.

A investigação dos mecanismos de interação dos vírus com as células do hospedeiro trazem informações relevantes para a identificação de alvos no desenvolvimento de drogas para impedir a penetração e/ou desenvolvimento dos vírus. Peptídeos desenhados a partir de proteínas virais foram desenvolvidos e testados quanto as suas capacidades de inibir o processo de fusão do vírus com a célula do hospedeiro. Alguns se encontram em fase de avaliação clínica. Anticorpos contra a proteína p24 do HIV-1 foram detectados no soro de pacientes HIV-positivos, e estes reconhecem pequenas seqüências peptídicas desta proteína. Neste trabalho foi analisada a interação entre uma seqüência peptídica correspondente aos aminoácidos 196-224 (AAMQMLKETINEEAAEWDRVHPVHAGPIA) da proteína p24, denominado p24- 1, com sistemas biomiméticos. Os sistemas utilizados foram filmes de Langmuir (monocamadas) de dipalmitoil fosfatidil colina (DPPC) e dipalmitoil fosfatidil glicerol (DPPG) e vesículas multilamelares (MLVs) de DPPC. O p24-1 encontra-se desorganizado em solução aquosa, mas com a interação com as MLVs de DPPC teve induzido uma conformação hélice ?, de acordo com o espectro de dicroísmo circular (CD). Esta característica foi confirmada pela predição de hélice a seguida por uma estrutura não ordenada contendo 11 resíduos do p24-1. As isotermas de pressão e potencial de superfície das monocamadas de DPPC foram afetadas com a presença de 0,05% mo1 de p24-1, com uma expansão de aproximadamente 5%. Para concentrações acima de 0,5% mo1 de p24-1 a expansão foi de 20%, com saturação do efeito da concentração. O efeito de expansão foi acompanhado por uma alteração na morfologia das monocamadas, estudados com microscopia no ângulo de Brewster (BAM). A incorporação do p24-1 impede a formação de grandes domínios de DPPC. O efeito cooperativo causado na monocamada de fosfolipídios pelo p24-1 sugere que esse tem um potencial na atividade antiviral por participar da expansão da membrana da célula hospedeira. / The investigation of the interaction mechanisms between the viruses and the host cells brings relevant information for the identification of targets on the development of drugs to prevent the penetration and/or development of the viruses. Peptides designed from viral proteins have been developed and tested on its capacities of inhibiting the merging process of the virus with the host cell. Some of them are in clinical evaluation. Antibodies against the protein p24 of the HIV-1 have been detected in the serum of HIV-positive patients, and they are able to recognize short peptide sequences of this protein. In this work, it was analyzed the interaction between a peptide sequence corresponding to amino acids 196-224 (AAMQMLKETINEEAAEWDRVHPVHAGPIA) of the protein p24, called p24- 1, and biomimetic systems. The systems used were Langmuir films (monolayers) of dipalmitoyl phosphatidyl choline (DPPC) and dipalmitoyl phosphatidyl glycerol (DPPG) and multilamelar vesicles (MLVs) of DPPC. p24-1 is found disorganized in watery solution, but with the interaction with the MLVs of DPPC it had induced a conformation ?-helix, according to the circular dichoism spectra (CD). This characteristic was confirmed by the prediction of ?-helix followed by an unordered structure with 11 residues of p24-1. The isotherms of pressure and potential of surface of the DPPC monolayers were affected by the presence of 0,05% mo1 of p24-1, with an expansion of approximately 5%. For concentrations above 0,5% mol of p24-1 the expansion was 20%, with saturation of the concentration effect. The expansion effect was followed by a morphologic alteration of the monolayers, studied with microscopy of the Brewster angle (BAM). The incorporation of p24-1 prevents the formation of large domains of DPPC. The cooperative effect caused in the phospholipid monolayer by p24-1 suggests that this peptide has a potential in the antiviral activity, once its participates on the expansion of the host cell membrane.

Obesity-associated metabolic disorders are amongst the most prevalent causes of death worldwide. Understanding how obesity leads to the development of the Metabolic Syndrome (MetS) and cardiovascular disease (CVD) will enable the development of novel therapies that dissociate obesity from its cardiometabolic complications. Our laboratory views the functional capacity of white adipose tissue (WAT), the organ designed for safe lipid storage, as a key factor in the development of MetS and CVD. At a genetically-defined stage of the aberrant WAT expansion that occurs during obesity, adipocytes undergo a functional failure, resulting in an impaired control of serum free fatty acid (FFA) concentration. In such setting, FFAs and their metabolic derivatives accumulate in other organs, where they cause lipotoxicity, leading to the development of insulin resistance and CVD. We therefore aim to understand the pathophysiological mechanisms that induce adipocyte dysfunction. The past two decades of research have established the immune system as an important regulator of WAT function. The number of adipose tissue macrophages (ATMs), the most abundant immune cell type in WAT, increases during obesity, resulting in WAT inflammation. Multiple genetic and pharmacological intervention studies of murine models of obesity have assigned a causal link between ATM pro-inflammatory activation and WAT dysfunction. However, while the propagation of inflammation in ATMs during obesity has been extensively studied, factors triggering ATM inflammatory activation are less clear. Recently, our lab has observed lipid accumulation in the ATMs isolated from obese mice. Lipid-laden ATMs were pro-inflammatory, leading us to hypothesise that aberrant lipid build-up in macrophages triggers WAT inflammation during obesity. This thesis expands on the initial findings from our lab and describes two novel mechanisms that potentially contribute to lipid-induced inflammatory activation of ATMs. In chapter 3, the role of de novo phosphatidylcholine (PC) synthesis pathway during lipotoxicity in macrophages is addressed. The first part of the chapter demonstrates that lipotoxic environment increased de novo PC synthesis rate in bone marrow-derived macrophages (BMDMs) and ATMs, and that loss of rate-limiting enzyme in de novo PC synthesis pathway, CTP:phosphocholine cytidylyltransferase a (CCTa) diminished saturated FFA-induced inflammation in BMDMs. In the second part, I show that macrophage-specific CCTa deletion did not impact on the development of WAT inflammation or systemic insulin resistance, but had a minor benefitial effect on hepatic gene transcription during obesity. Chapter 4 develops on recent observations of interactions between sympathetic nerves and macrophages in WAT. In the first part of the chapter, I demonstrate that stimulating B2-adrenergic receptor (B2AR), the main receptor for sympathetic neurotransmitter norepinephrine in macrophages, enhanced intracellular triglyceride storage by up-regulating diacylglycerol O-acyltransferase 1 (Dgat1) gene expression in BMDMs. The second part of the chapter shows that macrophage-specific B2AR deletion did not modulate systemic glucose and lipid metabolism during obesity, but mice lacking B2ARs in macrophages demonstrated augmented hepatic glucose production on a chow diet. Furthermore, systemic B2AR blockade or macrophage-specific B2AR deletion in mice did not affect the thermogenic response to cold exposure. Chapter 5 includes the characterisation of B2AR stimulation-induced changes to the global cellular proteome of BMDMs, and a subsequent validation of the role of candidate transcription factors in regulating B2AR agonism-induced gene expression in BMDMs.

<p>In this study the formation of supported membranes onto planar solid supports has been investigated. The stability and activity of reconstituted membrane receptors has been studied. The potential use of such preparations in biosensor applications is discussed.</p><p>The lipid films were made by the Langmuir Blodgett and by the liposome fusion techniques. These supported films were characterised by ellipsometry, atomic force microscopy, surface plasmon resonance (SPR) and resonant mirror techniques. The thickness of the films was in agreement with that of a cell membrane. The kinetics of formation of the lipid films was studied and discussed.</p><p>The proteins, bacteriorhodopsin, cytochrome oxidase, acetylcholinesterase and the nicotinic acetylcholine receptor were reconstituted into the supported membrane. The subsequent analysis showed that the proteins were individually distributed and that the activity was retained, in some cases for several weeks after immobilisation.</p><p>The glycolipids, GM1, GM2, GD1b, asialo-GM1, globotriaosylceramide, lactosylceramide and galactosylceramide, were also reconstituted into the supported membranes. Their specific interaction with the toxin ricin or with its B-chain was examined using SPR. The affinity of intact toxin and of its B-chain differed markedly and was pH dependent. The carbohydrate chain length and charge density of the glycolipids also influenced the affinity.</p>