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This thesis examines the dynamics of the formation of tethers, which are tubes of lipids 20 - 200 nm in diameter. In particular, this work investigates how the loading rate affects the observed threshold force at which a tether forms from a vesicle membrane. Tether dynamics are important to a myriad of biological processes such as signaling when white blood cells adhere to the walls of healthy and diseased blood vessels, or in the transport of intracellular material between neighboring cells. To understand the dynamics of tether formation in such systems more fully, the studies presented in this thesis focus on the dependence of the force needed to create a tether on the rate of force change. To conduct these experiments, I combined, for the first time, a microfabricated magnetic force transducer, or a microscale device that generates well-controlled and localized magnetic fields, and microaspiration, a technique to apply known tension to a lipid membrane. Using the combined global and local mechanical control of the joint system, I discovered a strong correlation between the threshold force of tether formation and the applied force ramp. An energy model, based upon that used to describe membrane rupture, characterized the observed dependencies and provided a mechanism to examine physically relevant quantities within the system. The usefulness of this combined approach was further substantiated by determining the influence of membrane modulators, including cholesterol, tension, adhesion site concentration, and phosphatidylserine, on the dependence seen between force threshold and force rates. Additionally, application of the experimental technique developed in this thesis led to the calculation of the inter-layer drag coefficient between membrane leaflets and to the first measurements of the thermal expansivity in aspirated 1-stearoy1-2-oleoyl- sn -glycero-3-phosphatidylcholine vesicles. This new tool for dynamic studies of membrane mechanics may further be extended to study how tethers form off of flowing cells or how phase regimes, induced by the presence of cholesterol, influence membrane dynamics.

This thesis presents the experimental work performed on the spectrin protein. The aim of the work was to study the direct interactions of spectrin, the cytoskeleton of RBCs, with membrane lipid to determine its effects on the mechanical properties of the lipid bilayer. Motivation for this work came from a lack of unanimity in the field of spectrin, and the hypothesized potential of the protein to perforate giant unilamellar vesicles. The work aimed to investigate and determine how spectrin-lipid interactions influence membrane mesoscopic morphology and biophysics in ways that could ultimately be important to cellular function. For this purpose, a protocol was implemented to take into account the different aspects of the binding. Direct visualisation of the spectrin-lipid interaction and distribution was achieved using confocal fluorescence microscopy. Changes in the mechanical properties of the membrane were investigated using the micropipette aspiration technique. Finally the thermodynamics of the interaction were considered with isothermal titration calorimetry experiments. This allowed evaluation of the protein-lipid interaction in a complete and coherent manner. Experiments were also performed on another elastic protein, alpha-elastin, for comparison. In addition to its similarities with spectrin (both possess hydrophobic domains and entropy elasticity), elastin is auto-fluorescent which makes it an attractive model protein. Elastin was also used as a sample model to implement new techniques using nonlinear optics microscopy.

A acne vulgar é uma das doenças cutâneas mais comuns, apresentando como um de seus fatores fisiopatológicos primários a colonização pelo microrganismo Propionibacterium acnes. Atualmente, têm-se buscado terapias alternativas para o combate ao P. acnes, destacando-se alguns ácidos graxos, como o ácido laúrico (LA). O LA é uma molécula pouco solúvel em água, sendo possível sua incorporação em lipossomas. Os lipossomas apresentam capacidade de encapsulação/ liberação de ativos e impedem a desidratação da pele, tornando-se ingredientes inovadores na área de cosméticos. Foram preparados lipossomas de dipalmitoilfosfatidilcolina (DPPC) contendo diferentes concentrações de LA, que variaram de 0 a 50% da concentração total em mol, em quatro pHs: 9,0, 7,4, 5,0 e 3,0. Nestes pHs o estado de protonação do LA muda variando de 0 a -1. Os lipossomas foram extrusados por filtros com poros de 100 nm de diâmetro visando à obtenção de vesículas unilamelares grandes (LUV). As LUV foram caracterizadas quanto a sua estabilidade em condições de prateleira, temperatura de transição de fase da bicamada, encapsulamento no interior aquoso, liberação do LA, difusão das vesículas na pele e seus aspectos morfológicos foram caracterizados por espalhamento de raios-X a baixo ângulo (SAXS) e crio-microscopia eletrônica de transmissão. Estudos de estabilidade mostraram que independentemente da concentração de LA, as formulações são mais estáveis em pHs mais altos, quando LA está em sua maioria na forma de laurato. Os experimentos de DSC revelaram que em pHs 3,0 e 5,0 e concentrações maiores de LA, a interação deste ácido graxo com as bicamadas é favorecida, havendo um aumento da temperatura de transição de fase (Tm) e diminuição da cooperatividade. Análises de taxa de incorporação de sondas hidrofílicas confirmaram a presença de um compartimento aquoso interno para as vesículas de DPPC:LA. O LA conseguiu permear a pele no período avaliado e pouco LA foi liberado das vesículas em condições de temperatura ambiente. A morfologia das LUV se mostrou bem diferente da esperada e se observaram vesículas com mais de uma bicamada e outros formatos que não o esférico. Estes resultados podem auxiliar na otimização das condições para uma formulação que poderá ser usada no tratamento da acne, aumentando a eficácia do LA no sítio alvo. / Acne vulgaris is one of the most common skin diseases, presenting as one of its causes the microorganism Propionibacterium acnes colonization. Currently, it has been sought alternatives therapies against P. acnes, especially some fatty acids, like the lauric acid (LA). LA is a molecule with low water solubility, allowing its incorporation in liposomes. Liposomes have encapsulation/release capacity of drugs and promote skin lipids regeneration, becoming an innovative ingredient in cosmetics area. Dipalmitoylphosphatidylcholine (DPPC) vesicles containing different LA concentrations were prepared at pHs: 9.0, 7.4, 5.0 and 3.0. At these pHs the LA protonation changes and its charge varies from 0 to -1. The vesicles were extruded through filters containing pores of 100 nm to obtain large unilamellar vesicles (LUV) that were characterized for stability in shelf conditions, bilayer phase transition temperature, aqueous internal compartment encapsulation, LA release and in vitro skin permeation. Its morphological features were characterized by small angle X-ray scattering (SAXS) and cryo-electron transmission microscopy. Stability assays showed that, regardless LA concentration, formulations were more long-term stable in higher pHs, when LA is mostly in the form of laurate. The differential scanning calorimetry, DSC, experiments showed that, at pHs 3.0 and 5.0 and higher LA concentrations, the interaction between the bilayer and LA is favored, increasing phase transition temperature (Tm) and reducing cooperativity. Incorporation of hydrophilic probes confirmed the presence of an internal aqueous compartment in DPPC:LA vesicles. The LA managed to permeate the skin on the period evaluated and, in ambient conditions, low LA concentration was released from the vesicles. LUV containing more than one bilayer and non-spherical structures were observed. The obtained results may help in the optimization of the conditions for a formulation that can be used in the treatment of acne and improving the effectiveness of LA delivery to the target site.

Esta tese apresenta resultados do estudo de interações entre biomoléculas e membranas compostas de lipídio. Duas classes de biomoléculas foram utilizadas, em primeiro lugar fragmentos de DNA que se inserem na camada aquosa entre as membranas, e peptídeos, que se inserem na parte hidrofóbica da membrana. As membranas de lipídio são constituídas basicamente por lecitina de soja, organizada em vesículas ou fases lamelares. Foram preparados complexos de lipídios e DNA com fragmentos de 150 pares de base, onde variou-se a composição das membranas na fase lamelar, com a adição de um co-surfactante comercial composto de uma mistura de ácidos graxos. Com essa abordagem foi possível alterar as propriedades elásticas da fase lamelar onde os fragmentos de DNA se inserem. Técnicas de espalhamento de raios-X foram aplicadas para a caracterização estrutural de duas composições distintas da fase lamelar hospedeira, revelando uma variedade de polimorfismos, como já havia sido observado para outras composições. Foi demonstrado que a transição entre os diferentes arranjos dos fragmentos de DNA está correlacionada a mudanças nas propriedades elásticas da fase lamelar hospedeira. No estudo da interação entre peptídeos e membranas, foram utilizadas vesículas e fases lamelares compostas apenas de lecitina. Os peptídeos utilizados foram duas sequências curtas de aminoácidos: L,L-difenilalanina (FF) e a cisteína-difenilalanina (CFF). A propriedade de auto-organização da FF tem sido reconhecida como elemento chave para a formação de fibras amilóides envolvidas em doenças neurodegenerativas como Alzheimer e Parkinson. Experimentos de FTIR demonstraram que os peptídeos se situam na interface da bicamada interagem mais fortemente com os grupos fosfato do lipídio. A incorporação do peptídeo FF promove alterações nas propriedades elásticas das membranas e, no caso das vesículas, observa-se uma transição multilamelar-unilamelar induzida pelo aumento da concentração de peptídeo. Com a presença do peptídeo CFF essa transição ocorre para concentrações baixas, provavelmente devido a ligações de hidrogênio entre o peptídeo e a membrana, alterando assim a entropia local da membrana. Observou-se ainda, com técnicas de dicroísmo circular e espalhamento de raios-X (WAXS) que, mesmo para essas sequências curtas, os peptídeos se agregam formando estruturas cristalinas que contribuem para a alteração das interações entre membranas e desestabilização da estrutura lamelar. / This thesis presents results of the study of interactions between biomolecules and membranes composed of lipid. Two classes of biomolecules were studied, first fragments of DNA inserted into the aqueous layer between membranes and second, peptides that are inserted into the hydrophobic part of the membrane. Lipid membranes are basically constituted by soy lecithin, which is a biocompatible lipid, organized into vesicles or lamellar phases. Lipid and DNA complexes were prepared with 150 bp DNA fragments, varying the composition of the membranes in the lamellar phase, with the addition of a commercial commercial co-surfactant composed of a mixture of fatty acids. With this approach, it was possible to change the elastic properties of the lamellar phase where the DNA fragments are inserted. X-ray scattering techniques were applied for the structural characterization of two distinct compositions of the host lamellar phase, revealing a variety of polymorphisms, as had already been observed for other compositions. It has been shown that transitions between the different arrangements of the DNA fragments are correlated to changes in the elastic properties of the host lamellar phase. In the study of the interaction between peptides and membranes, vesicles and lamellar phases composed only of lecithin were used. The peptides used were two short amino acid sequences; L, L-diphenylalanine (FF) and cysteine diphenylalanine (CFF). The self-association property of FF has been recognized as a key element for the formation of amyloid fibers involved in neurodegenerative pathologies such as Alzheimer\'s and Parkinson\'s disease. FTIR experiments have shown that the peptides are at the interface of the bilayer and interact more strongly with the phosphate groups of the lipid. The incorporation of FF peptide promotes changes in the elastic properties of the membranes and in the case of the vesicles, a multilamellar-unilamellar transition is observed induced by the increase of the peptide concentration. With the presence of CFF peptide this transition occurs at low concentration, probably due to hydrogen bonds between peptide and membrane which change the local entropy of the membrane. It is also observed with circular dichroism and X-ray scattering (WAXS) techniques that, even for these short sequences, the peptides aggregate forming crystalline structures that contribute to the modification of membrane interactions and destabilization of the lamellar structure.

The permeability of lipid bilayer membranes to glucose and carboxyfluorescein has been studied in model membranes. Using an enzyme assay, the permeability of glucose was monitored spectrometrically with both large and giant unilamellar vesicles (LUVs and GUVs). The permeability of carboxyfluorescein was studied by entrapping the dye and monitoring its leakage over time from a single GUV. Permeability study using GUVs may provide new information that cannot be obtained from LUVs.The stability of lipid membranes was enhanced by incorporating polymer scaffold. LUVs were prepared with hydrophobic monomers partitioned and then polymerized inside the hydrophobic interior of the lipid bilayers. The sizes of the formed polymers were characterized using gel permeation chromatography and mass spectrometry. This study suggests that large molecular weight polymers were formed inside the lipid bilayers and that the stability of the membranes is related to the size of the polymers.

Esta tese apresenta resultados do estudo de interações entre biomoléculas e membranas compostas de lipídio. Duas classes de biomoléculas foram utilizadas, em primeiro lugar fragmentos de DNA que se inserem na camada aquosa entre as membranas, e peptídeos, que se inserem na parte hidrofóbica da membrana. As membranas de lipídio são constituídas basicamente por lecitina de soja, organizada em vesículas ou fases lamelares. Foram preparados complexos de lipídios e DNA com fragmentos de 150 pares de base, onde variou-se a composição das membranas na fase lamelar, com a adição de um co-surfactante comercial composto de uma mistura de ácidos graxos. Com essa abordagem foi possível alterar as propriedades elásticas da fase lamelar onde os fragmentos de DNA se inserem. Técnicas de espalhamento de raios-X foram aplicadas para a caracterização estrutural de duas composições distintas da fase lamelar hospedeira, revelando uma variedade de polimorfismos, como já havia sido observado para outras composições. Foi demonstrado que a transição entre os diferentes arranjos dos fragmentos de DNA está correlacionada a mudanças nas propriedades elásticas da fase lamelar hospedeira. No estudo da interação entre peptídeos e membranas, foram utilizadas vesículas e fases lamelares compostas apenas de lecitina. Os peptídeos utilizados foram duas sequências curtas de aminoácidos: L,L-difenilalanina (FF) e a cisteína-difenilalanina (CFF). A propriedade de auto-organização da FF tem sido reconhecida como elemento chave para a formação de fibras amilóides envolvidas em doenças neurodegenerativas como Alzheimer e Parkinson. Experimentos de FTIR demonstraram que os peptídeos se situam na interface da bicamada interagem mais fortemente com os grupos fosfato do lipídio. A incorporação do peptídeo FF promove alterações nas propriedades elásticas das membranas e, no caso das vesículas, observa-se uma transição multilamelar-unilamelar induzida pelo aumento da concentração de peptídeo. Com a presença do peptídeo CFF essa transição ocorre para concentrações baixas, provavelmente devido a ligações de hidrogênio entre o peptídeo e a membrana, alterando assim a entropia local da membrana. Observou-se ainda, com técnicas de dicroísmo circular e espalhamento de raios-X (WAXS) que, mesmo para essas sequências curtas, os peptídeos se agregam formando estruturas cristalinas que contribuem para a alteração das interações entre membranas e desestabilização da estrutura lamelar. / This thesis presents results of the study of interactions between biomolecules and membranes composed of lipid. Two classes of biomolecules were studied, first fragments of DNA inserted into the aqueous layer between membranes and second, peptides that are inserted into the hydrophobic part of the membrane. Lipid membranes are basically constituted by soy lecithin, which is a biocompatible lipid, organized into vesicles or lamellar phases. Lipid and DNA complexes were prepared with 150 bp DNA fragments, varying the composition of the membranes in the lamellar phase, with the addition of a commercial commercial co-surfactant composed of a mixture of fatty acids. With this approach, it was possible to change the elastic properties of the lamellar phase where the DNA fragments are inserted. X-ray scattering techniques were applied for the structural characterization of two distinct compositions of the host lamellar phase, revealing a variety of polymorphisms, as had already been observed for other compositions. It has been shown that transitions between the different arrangements of the DNA fragments are correlated to changes in the elastic properties of the host lamellar phase. In the study of the interaction between peptides and membranes, vesicles and lamellar phases composed only of lecithin were used. The peptides used were two short amino acid sequences; L, L-diphenylalanine (FF) and cysteine diphenylalanine (CFF). The self-association property of FF has been recognized as a key element for the formation of amyloid fibers involved in neurodegenerative pathologies such as Alzheimer\'s and Parkinson\'s disease. FTIR experiments have shown that the peptides are at the interface of the bilayer and interact more strongly with the phosphate groups of the lipid. The incorporation of FF peptide promotes changes in the elastic properties of the membranes and in the case of the vesicles, a multilamellar-unilamellar transition is observed induced by the increase of the peptide concentration. With the presence of CFF peptide this transition occurs at low concentration, probably due to hydrogen bonds between peptide and membrane which change the local entropy of the membrane. It is also observed with circular dichroism and X-ray scattering (WAXS) techniques that, even for these short sequences, the peptides aggregate forming crystalline structures that contribute to the modification of membrane interactions and destabilization of the lamellar structure.

Cellular membranes are important targets for many membrane-active peptides and drug compounds. Here we are interested in deciphering how lipid membranes are perturbed by several membrane-active molecules, including the transmembrane domain of the influenza M2 protein (M2TM), aggregates formed by a synthetic polyglutamine peptide, and three polyphenol compounds (i.e., tamoxifen, genistein, and verapamil). We employ phase-separated ternary lipid model membranes in the form of giant unilamellar vesicles (GUVs) to simulate raft-like structures that have been proposed to govern many important processes in plasma membranes (e.g., intracellular singling and trafficking). Specifically, we use fluorescent microscopy to interrogate how those membrane additives modulate the phase behavior of free-standing GUVs, as well as the miscibility transition temperature (Tm). We find that M2TM increases Tm and causes vesicle budding; polyglutamine aggregates disrupt lipid membranes; and the three polyphenol compounds exert disparate effects on GUV Tm.

A acne vulgar é uma das doenças cutâneas mais comuns, apresentando como um de seus fatores fisiopatológicos primários a colonização pelo microrganismo Propionibacterium acnes. Atualmente, têm-se buscado terapias alternativas para o combate ao P. acnes, destacando-se alguns ácidos graxos, como o ácido laúrico (LA). O LA é uma molécula pouco solúvel em água, sendo possível sua incorporação em lipossomas. Os lipossomas apresentam capacidade de encapsulação/ liberação de ativos e impedem a desidratação da pele, tornando-se ingredientes inovadores na área de cosméticos. Foram preparados lipossomas de dipalmitoilfosfatidilcolina (DPPC) contendo diferentes concentrações de LA, que variaram de 0 a 50% da concentração total em mol, em quatro pHs: 9,0, 7,4, 5,0 e 3,0. Nestes pHs o estado de protonação do LA muda variando de 0 a -1. Os lipossomas foram extrusados por filtros com poros de 100 nm de diâmetro visando à obtenção de vesículas unilamelares grandes (LUV). As LUV foram caracterizadas quanto a sua estabilidade em condições de prateleira, temperatura de transição de fase da bicamada, encapsulamento no interior aquoso, liberação do LA, difusão das vesículas na pele e seus aspectos morfológicos foram caracterizados por espalhamento de raios-X a baixo ângulo (SAXS) e crio-microscopia eletrônica de transmissão. Estudos de estabilidade mostraram que independentemente da concentração de LA, as formulações são mais estáveis em pHs mais altos, quando LA está em sua maioria na forma de laurato. Os experimentos de DSC revelaram que em pHs 3,0 e 5,0 e concentrações maiores de LA, a interação deste ácido graxo com as bicamadas é favorecida, havendo um aumento da temperatura de transição de fase (Tm) e diminuição da cooperatividade. Análises de taxa de incorporação de sondas hidrofílicas confirmaram a presença de um compartimento aquoso interno para as vesículas de DPPC:LA. O LA conseguiu permear a pele no período avaliado e pouco LA foi liberado das vesículas em condições de temperatura ambiente. A morfologia das LUV se mostrou bem diferente da esperada e se observaram vesículas com mais de uma bicamada e outros formatos que não o esférico. Estes resultados podem auxiliar na otimização das condições para uma formulação que poderá ser usada no tratamento da acne, aumentando a eficácia do LA no sítio alvo. / Acne vulgaris is one of the most common skin diseases, presenting as one of its causes the microorganism Propionibacterium acnes colonization. Currently, it has been sought alternatives therapies against P. acnes, especially some fatty acids, like the lauric acid (LA). LA is a molecule with low water solubility, allowing its incorporation in liposomes. Liposomes have encapsulation/release capacity of drugs and promote skin lipids regeneration, becoming an innovative ingredient in cosmetics area. Dipalmitoylphosphatidylcholine (DPPC) vesicles containing different LA concentrations were prepared at pHs: 9.0, 7.4, 5.0 and 3.0. At these pHs the LA protonation changes and its charge varies from 0 to -1. The vesicles were extruded through filters containing pores of 100 nm to obtain large unilamellar vesicles (LUV) that were characterized for stability in shelf conditions, bilayer phase transition temperature, aqueous internal compartment encapsulation, LA release and in vitro skin permeation. Its morphological features were characterized by small angle X-ray scattering (SAXS) and cryo-electron transmission microscopy. Stability assays showed that, regardless LA concentration, formulations were more long-term stable in higher pHs, when LA is mostly in the form of laurate. The differential scanning calorimetry, DSC, experiments showed that, at pHs 3.0 and 5.0 and higher LA concentrations, the interaction between the bilayer and LA is favored, increasing phase transition temperature (Tm) and reducing cooperativity. Incorporation of hydrophilic probes confirmed the presence of an internal aqueous compartment in DPPC:LA vesicles. The LA managed to permeate the skin on the period evaluated and, in ambient conditions, low LA concentration was released from the vesicles. LUV containing more than one bilayer and non-spherical structures were observed. The obtained results may help in the optimization of the conditions for a formulation that can be used in the treatment of acne and improving the effectiveness of LA delivery to the target site.

Vesicles are a type of biological or biomimetic particle consisting of one or more often spherical bilayers made up of amphipathic molecules, creating a closed system. They can function as an encapsulating device, holding hydrophilic molecules on the inside of the bilayer membrane(s) or hydrophobic molecules in the non-polar interstitial space in the middle of the bilayers. Because of this capacity to carry molecules, vesicles are a premier system for drug delivery and even theranostics in vivo. A peptide-based approach to release of encapsulated molecules has previously been developed but since drug delivery vesicles are in the size range of nanometers, the mechanisms have not been visualized. This project aims to produce giant unilamellar vesicles as a model system used to visualize membrane interactions vital to the understanding and further development of smaller vesicle-based systems for drug delivery. Giant unilamellar vesicles were produced successfully and a preparation protocol was established. Additionally, some membrane interactions were investigated using fluorescence microscopy.