Navegando por Palavras-chave "Calorimetria"

Agora exibindo 1 - 4 de 4
  • Imagem de Miniatura
    Item
    Acesso aberto (Open Access)
    O efeito da composição lipídica da membrana no estudo do mecanismo de ação do peptídeo antimicrobiano esculentina 1b (1-18)
    (Universidade Federal de São Paulo (UNIFESP), 2019-09-26) Silva, Isabela Moreira [UNIFESP]; Perez, Katia Regina [UNIFESP]; http://lattes.cnpq.br/2250091739407083; http://lattes.cnpq.br/7641887893666670; Universidade Federal de São Paulo (UNIFESP)
    The antimicrobial peptides are part of innate immune system of several organisms. They are characterized by the presence of cationic and hydrophobic amino acids that assists in the interaction with plasma membranes. Esculentin 1b (1-18) is an antimicrobial peptide containing 46 amino acids, whose region composed of the first 18 amino acids residues presents bactericidal activity as the whole peptide, without hemolytic activity. The mode of action of antimicrobial peptide Esc 1b (1-18) was studied using mimetic membranes composed of zwitterionic and anionic phospholipids in different physical states. Measurements of carboxyfluorescein leakage, isothermal titration calorimetry and interaction measures using Lagmuir monolayers were made to verify the effect of the peptide on model membranes. To observe the influence of Esc 1b (1-18) in the bilayer, measurements of differential scanning calorimetry and electron paramagnetic resonance were made, and the effect of aggregation caused by the peptide in the membranes was verified using measures of static and dynamic light scattering and Zeta potential. Measurements of circular dichroism and polarization modulation infrared reflection-absorption spectroscopy were made to determine the secondary structure of Esc 1b (1-18) in the presence of vesicles and monolayers, respectively. In general, the results of carboxyfluorescein leakage, isothermal titration calorimetry and maximum insertion pressure show that the peptide interacts mainly with negatively charged and fluid state membranes. Furthermore, it was observed that Esc 1b (1-18) causes a larger influence in the phase transition on DPPG membranes and the peptide inserts deeply on negatively charged membranes. Lastly, it was observed that the peptide presents α-helical secondary structure in the presence of charged membranes. Therefore, the antimicrobial peptide Esculentin 1b (1-18) interacts selectively with negatively charged membranes.
  • Imagem de Miniatura
    Item
    Acesso aberto (Open Access)
    Estudo do papel da interação eletrostática no processo de solubilização de membranas miméticas por detergentes com cargas diferentes
    (Universidade Federal de São Paulo, 2023-10-16) Oliveira, Maria Clara Araújo [UNIFESP]; Riske, Karin do Amaral [UNIFESP]; http://lattes.cnpq.br/9178927522709552; http://lattes.cnpq.br/2314524859774065
    Introdução: Detergentes são moléculas anfifílicas capazes de solubilizar membranas biológicas, separando seus componentes. Estudos com sistemas biomiméticos são importantes para a compreensão dos aspectos físico-químicos do processo de solubilização de membranas. A maioria desses estudos se limita ao uso de lipídios neutros, como a fosfatidilcolina (PC). No entanto, é sabido que biomembranas possuem uma fração de lipídios aniônicos, como o fosfatidilglicerol (PG), e vários detergentes comumente empregados são iônicos. Objetivo: Estudar o processo de solubilização de bicamadas lipídicas neutras e aniônicas por detergentes com cargas nula, positiva e negativa. Materiais e métodos: Foram utilizadas vesículas unilamelares grandes (LUVs) e gigantes (GUVs) compostas por PC (zwiteriônico), PG (aniônico) e mistura PC:PG 1:1, e os detergentes aniônico dodecil sulfato de sódio (SDS), catiônico brometo de cetil trimetil amônio (CTAB) e não-iônico octil glicopiranosídeo (OG). O processo de solubilização foi estudado por medidas de turbidez, microscopia ótica e técnicas calorimétricas. Resultados: Medidas de turbidez de LUVs ao longo da titulação com os detergentes mostraram que as membranas aniônicas foram mais suscetíveis à solubilização que as zwiteriônicas, já que foi necessária uma concentração menor de todos os detergentes para causar a solubilização completa. A partir da microscopia ótica de GUVs observamos que o detergente OG, não-iônico, causa um aumento da superfície da vesícula devido a um rápido flip-flop, seguido da solubilização completa da membrana. Por outro lado, os detergentes iônicos geraram uma explosão súbita das GUVs, pois se inserem apenas na monocamada externa, o que leva a um aumento da tensão da membrana. A técnica de calorimetria de titulação isotérmica (ITC) permitiu a obtenção do calor associado à partição dos detergentes em LUVs antes do início do processo de solubilização. O processo de incorporação do detergente OG foi sempre endotérmico e semelhante para as três composições de membrana, mas apresentou uma afinidade um pouco maior pelas membranas aniônicas. Já os detergentes iônicos, SDS e CTAB, apresentaram resultados sensíveis à interação eletrostática. O CTAB apresentou um processo de incorporação exotérmico, com uma intensidade muito maior para membranas aniônicas, devido à atração eletrostática. O processo de inserção do SDS passou de exotérmico em membranas zwiteriônicas para endotérmico em membranas aniônicas. A técnica de calorimetria diferencial de varredura (DSC) permitiu avaliar o efeito dos detergentes na transição gel-fluido das bicamadas. Os detergentes OG e SDS levaram a uma diminuição no valor da temperatura de transição com um alargamento dos picos. Foi necessária uma concentração menor desses detergentes para causar diminuição na entalpia da transição de membranas aniônicas. Por outro lado, a temperatura de transição de fase das membranas ficou praticamente inalterada na presença de altas concentrações de CTAB, mostrando um processo de solubilização incompleto. Conclusão: A interação eletrostática entre detergente e membrana não é preponderante no processo de solubilização e as membranas aniônicas (contendo PG) se apresentaram mais suscetíveis ao processo de solubilização, independentemente da carga do detergente.
  • Imagem de Miniatura
    Item
    Acesso aberto (Open Access)
    Interação de moléculas sintéticas anfifílicas catiônicas com modelos miméticos de biomembranas
    (Universidade Federal de São Paulo (UNIFESP), 2018-09-27) Almeida, Marcio Moreira de [UNIFESP]; Riske, Karin do Amaral [UNIFESP]; http://lattes.cnpq.br/9178927522709552; Universidade Federal de São Paulo (UNIFESP)
    Bacterial infections represent a serious health problem, aggravated by the resistance of these microorganisms to conventional antibiotic agents. Therefore, alternative treatments, such as those based on antimicrobial peptides and synthetic molecules with similar characteristics, have been recently sought. Antimicrobial peptides are amphiphilic and cationic molecules present in the immune system of plants and animals that cause the death of microorganisms through the destruction of their membrane. In this work, two classes of synthetic cationic amphiphilic molecules with possible antimicrobial action were studied. These molecules consist of two hydrocarbon chains and two ammonium groups, located at the ends of the hydrophobic chains (Bolalike) or polar region of the molecule (Geminilike). In addition, different lengths of amphiphilic hydrocarbon chains were tested. The molecules studied were B7, B11, G7 and G11, where the letter identifies the class (Bola or Gemini) and the numbers the length of the acyl chain. To understand the mechanism of action of these molecules, membrane mimetic models were used: large (LUVs) and giant (GUVs) unilamellar vesicles composed of POPC, a zwitterionic lipid, and POPG, an anionic lipid. The composition of POPC:POPG 7:3 was chosen to mimic bacterial membranes, whereas pure POPC bilayers were used as biomimetic model of eukaryotic membranes. Different techniques were used to evaluate the interaction between the amphiphilic molecules and the LUVs: isothermal titration calorimetry (ITC), to obtain information about the thermodynamics of the interaction, dynamic scattering (DLS) and zeta potential of the vesicles to measure the size and surface charge of the vesicles as the cationic molecules were added, and leakage assays of a fluorescence probe encapsulated in the LUVs to quantify the lytic activity of the molecules. In addition, GUV optical microscopy experiments were performed to visualize the effects caused by amphiphilic molecules on the membrane. The results show that both the hydrophilic and hydrophobic balance of the molecules and the positions of the charges (Bola or Gemini) have an effect on the activity and mechanism of action of these molecules. Geminilike molecules showed greater affinity for the membrane, but this affinity did not result in a considerable membrane permeabilization. On the other hand, even though Bolalike molecules did not exhibit such high membrane affinity, they caused considerable membrane permeabilization. Among the amphiphilic molecules tested, the molecule B11 presented a lytic activity comparable to that of antimicrobial peptides and is, therefore, the most promising molecule as a possible antimicrobial agent.
  • Imagem de Miniatura
    Item
    Acesso aberto (Open Access)
    Solubilização de membranas modelo de diferentes composições pelo detergente triton X-100
    (Universidade Federal de São Paulo (UNIFESP), 2017-03-29) Mattei, Bruno [UNIFESP]; Riske, Karin do Amaral [UNIFESP]; http://lattes.cnpq.br/9178927522709552; http://lattes.cnpq.br/4260343698344529; Universidade Federal de São Paulo (UNIFESP)
    Detergents are amphiphilic molecules widely used as solubilizing agents Biological membranes. However, biological membranes treated with detergents Ac-ese insoluble fragments rich in sphingolipids and cholesterol. Due to similarities of These features have been associated with lipid rafts and the liquid phase The. Here the study of the physical-chemical aspects that govern the process of UUilllCATiOn of membrane mimetic models: lipid vesicles of controlled composition Pure or mixtures, mainly POPC, SM and cholesterol). Many different Techniques were used to study the solubilization process of lipid vesicles by Detergent TX-100, one of the most used detergents in Biological membranes. The general phenomenon and the size of the structures formed during the Solubilization was obtained by turbidity, static and / or dynamic light scattering; The aspects Thermodynamic properties of the detergent-lipid interaction were investigated by titration calorimetry And the possible interference of the detergent in the phase transition of lipids was investigated Differential scanning calorimetry; The ability of detergents to permeabilize the The membrane was investigated by kinetics of leakage of a fluorescent probe and microscopy Confocal; The process of solubilization of giant vesicles was evaluated by optical microscopy of Phase contrast and fluorescence. In the work, different lipid compositions were investigated (Pure phospholipids, binary lipid mixtures and ternary lipid mixtures) in order to obtain Information on the factors that influence the obtaining of insoluble membrane fragments. Through the experiments it was possible to conclude that pure cell membranes (POPC and SM), reaching the liquid-disordered phase and gel are totally soluble to the action of Detergent, whereas the addition of cholesterol, inducing a higher degree of Lipids leads to conditions in which the membranes are partially or totally insoluble; The factor of Insolubility is conditioned to cholesterol concentration in vesicles with binary mixtures Of lipids, reaching total insolubility in situations in which the membrane tends or reaches the Liquid-ordered phase; This is also reflected in the detergent affinity constant for these Membranes. In addition, vesicles in the gel phase are solubilized with lower concentrations of TX-100 than vesicles in the liquid-disordered phase, possibly due to the lower Ability of the membrane to accommodate detergents; Added to this, the addition of detergent Also appears to induce a phase transition in vesicles in the gel phase and a coexistence of Fluid phases in POPC: cholesterol vesicles. In ternary lipid mixtures, the situations in Higher solubility resistance is observed in regions with Greater fraction of SM and cotesterol. Finally, the membranes become permeable in TX-100 / lipid molars lower than the solubilization process, showing that even before To reach the bi-co-existent phase of the membrane, the detergent already changes the membrane structure.