Navegando por Palavras-chave "Eletroporação"

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    Eletroporação e estabilidade de membranas aniônicas com diferentes níveis de complexidade
    (Universidade Federal de São Paulo, 2022-07-19) Leomil, Fernanda dos Santos Costa [UNIFESP]; Riske, Karin do Amaral [UNIFESP]; Dimova, Rumiana; http://lattes.cnpq.br/6408828276550407; http://lattes.cnpq.br/9178927522709552; http://lattes.cnpq.br/4382156853241028
    A estabilidade das membranas celulares é de importância vital para as células. Após estímulos de alta intensidade, como os causados pela aplicação de pulsos elétricos, poros são abertos na membrana (eletroporação) e se fecham em poucos ms, permitindo que a integridade da membrana seja preservada, conforme observado em vesículas unilamelares gigantes (GUVs) neutras de PC (fosfatidilcolina). No entanto, a adição do lipídio negativo PG (fosfatidilglicerol) mostrou desempenhar um papel importante na desestabilização das membranas após eletroporação: (i) alguns poros se abrem indefinidamente, levando à completa perda da integridade da vesícula (bursting) e (ii) GUVs que sobreviveram à poração exibem permeabilidade de longa duração, revelando a permanência de poros submicroscópicos após o fechamento dos macroporos. Nesta tese, foi investigada a resposta mecânica de GUVs a campos elétricos, com foco na estabilidade de membranas contendo frações crescentes de lipídios aniônicos de conhecida relevância biológica, como o fosfatidilinositol (PI), fosfatidilinositol 4,5-bisfosfato (PIP2), cardiolipina (CL), fosfatigilglicerol (PG) e extrato lipídico de E. coli, bem como foi examinada a relevância da assimetria dos lipídios negativos entre as monocamadas na estabilidade dessas membranas. Também foram explorados sistemas mais complexos, como vesículas gigantes de membrana plasmática (GPMVs), que representam um passo intermediário entre GUVs lipídicas e células, apresentando as características lipídicas e proteicas das células que as originaram. Métodos experimentais e computacionais foram desenvolvidos e aperfeiçoados ao longo do trabalho para a realização e análise dos experimentos executados. Em GUVs com distribuição simétrica de lipídios aniônicos, medidas da ocorrência de eventos desestabilizantes, como bursting e permeabilidade duradoura, mostraram que as membranas contendo frações mais altas de lipídios negativos (50 mol%) são mais instáveis e propensas à desestabilização. Esses efeitos, que se mostraram independentes do tipo de lipídio negativo utilizado, foram relacionados a uma redução de cerca de 50% na tensão de linha do poro, propriedade que governa o seu fechamento e que foi calculada através de um software desenvolvido durante o projeto (PoET). GUVs com distribuição assimétrica de lipídio negativo entre as monocamadas da membrana foram obtidas por dois métodos diferentes: transferência de fase e alterações do pH do meio externo das vesículas. A assimetria foi verificada por meio de um ensaio de supressão de fluorescência aperfeiçoado ao longo do estudo, em combinação com um algoritmo desenvolvido para medidas de fluorescência em GUVs. Eventos desestabilizantes foram observados já para pequenos graus de assimetria de carga (5 mol%), que potencializam a ocorrência de eventos mais extremos, como o bursting, devido à geração de curvatura espontânea acentuada. Quando consideramos modelos ainda mais relevantes, resultados preliminares mostram que existem diferenças na forma com que GPMVs e GUVs respondem a pulsos elétricos, provavelmente devido à maior complexidade exibida pelas membranas das GPMVs. Os resultados apresentados aqui evidenciam que os lipídios aniônicos possuem um papel importante na desestabilização das membranas após eletroporação, sendo os efeitos realçados no caso em que estes estão assimetricamente distribuídos entre as monocamadas das membranas, como observado nas membranas celulares. Acreditamos que os achados possam contribuir para o desenvolvimento de sistemas de entrega intracelular de drogas baseados em eletroporação mais eficientes. Ainda, julgamos que o aperfeiçoamento do ensaio de supressão de fluorescência e o desenvolvimento do software para cálculo da tensão de linha e do algoritmo para medidas de fluorescência serão de grande contribuição para a comunidade científica, permitindo a realização de experimentos e obtenção de dados de forma mais confiável e reprodutível.
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    Estudo comparativo de protocolos de imunização gênica: eletroporação aumenta consistentemente a resposta imune humoral
    (Universidade Federal de São Paulo (UNIFESP), 2008-03-26) Parise, Carolina Bellini [UNIFESP]; Han, Sang Won [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Gene immunization may bypass some difficulties found with protein immunogen, such as the obtainment of purified antigen and the immune response reactivity to its native conformation. Thus, antigen encoding DNA inserted into a vector is inoculated into animal and the protein expressed in vivo with the appropriate three-dimensional structure stimulates the production of specific antibodies. However, the gene immunization methods may determine the efficacy of DNA vaccine. The present study compared four protocols of DNA immunization, using three different antigens: two of them phylogenetically conservative, human vascular endothelial growth factor 165 (hVEGF165) and human fibroblast growth factor-2 (hFGF-2), and other from vegetal origin, Kunitz-type serine protease inhibitor from Bauhinia bauhinioides (BbKi). For this, Balb/c mice were immunized with plasmid DNA encoding hVEGF165, hFGF-2 or BbKi genes. In the first protocol, animals were immunized by intrasplenic (i.s.) pathway; in the second, intramuscularly (i.m.); in the third, i.m. injections were followed by electroporation (ep); and in the fourth, i.m. injections followed by ep were performed in animals pre-immunized with antigen-transfected cells. Sera were analyzed by ELISA to detect the presence of anti-hVEGF165, -hFGF-2 or -BbKi antibodies. Results showed that i.s. immunization did not elicit detectable humoral immune response in our conditions. On the other hand, statistical analyses revealed that the ep improved the immune response to i.m. immunization and that three DNA immunizations followed by ep elicited the same effect obtained by two i.m. immunizations followed by ep in pre-immunized animals with antigentransfected cells. Our results showed that all protocols worked similarly for the three studied antigens and that i.m. gene immunization followed by ep was the more advantageous protocol. In addition, the immune response to i.m. immunization followed by ep was comparable to that obtained by protein immunization. Finally, these data allowed us select an efficient DNA immunization protocol that makes possible the antibody obtainment in the lack of purified protein.
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    Estudo de propriedades biofísicas de vesículas unilamelares gigantes como modelo para entrega intracelular de materiais: eletroporação e fusão de membranas
    (Universidade Federal de São Paulo (UNIFESP), 2016-11-30) Lira, Rafael Bezerra de [UNIFESP]; Riske, Karin Do Amaral [UNIFESP]; http://lattes.cnpq.br/9178927522709552; http://lattes.cnpq.br/9253308829387337; Universidade Federal de São Paulo (UNIFESP)
    Biological membranes are the structures that define the boundaries of cells, acting as a selective barrier, regulating the intra and extracellular transport. Many biological substances have their target in the cell?s interior, but are not able to cross the membrane to reach them. Therefore, many methods for the intracellular delivery of materials have been developed; among them, electroporation and fusogenic liposomes. During electroporation, one or more intense electrical pulses are applied and the membrane becomes transiently permeable, allowing the entry of the substances into the cell. The method is efficient but has high toxicity. The fusion between liposomes and cells results in the delivery of the liposome-encapsulated substance, but the underlying mechanisms are not well known and the efficiency is limited. Biomimetic models are generally employed to mimic real systems in a controlled way. One of the most common biomimetic system are the giant unilamellar vesicles (GUVs). In this thesis, we use GUVs with mixed the zwitterionic (PC) and anionic (PG) lipids to study the processes of electroporation and fusion with fusogenic liposomes, and both processes are studied by a series of microscopy-based techniques. Electroformed pores in electrically neutral GUVs are transient, with a pore lifetime in the order of a few milliseconds. In contrast, electroformed pores in some negatively charged GUVs open indefinitely, leading to a total vesicle collapse, in a process termed bursting. The results presented here show that the bursting fraction depends on the fraction of PG in the membrane, lipid insaturation (lipids with insaturation in one or both hydrophobic tails) e certain additives in the medium, especially Ca+2. Bursting propensity is related to the reduction n pore edge tension (?), from 57,9±26,1 e 53,7±11,5 pN in neutral POPC and DOPE membranes, respectively, to values lower than 20 pN in membranes containing PG ? 20 mol%. Such a reduction also depends on the presence of médium additives, and the ? values are higher for vesicles in the presence of Ca+2 compared to Na+, but only in the presence of PG ? 20 mol%. Destabilization propensity is, therefore, a result of a reduction in ?. Formed pores in charged membranes have a composition similar to that in intact membranes. Reminiscent pores after GUV electroporation have a higher permeability to macromolecules. Negative membranes also display differences in other membrane properties compared to neutral ones, such as reduction in lipid mobility, increase in membrane order and spontaneous curvature. The fusion of GUVs with different fractions of PG in PC membranes, with fusogenic liposomes, was studied in a new LUV-GUV fusogenic system, in which fusion was observed in real time and its effects directly observed and quantified under the microscope. In this system, many fusion intermediates (adhesion, hemifusion and full-fusion) can be detected and the morphological transformations directly observed. Förster resonance energy transfer (FRET) assays allow to measure the amount of lipids transferred from the LUVs to the GUVs, and show that the transition hemifusion6 full fusion occurs in the range of 10-20 mol% PG. At low PG mol fractions (? 10 mol%), the interaction leads mainly to adhesion and hemifusion and increases membrane tension, whereas at high PG mol fractions (? 20 mol%), the interaction results in full fusion, leading to GUV increase in area. The construction of a FRET calibration curve allows the determination of the final membrane composition after fusion in every GUV. During hemifusion, the amount of cationic lipids transferred fro LUVs is < 5 mol%, whereas upon full fusion, the final GUV composition contains up to 20 mol% DOTAP. In every case, fusion occurs by a charge neutralization mechanism. The results presented here (i) clarifies the responsible mechanisms of bursting of negatively charged GUVs, (ii) define the membrane charge determinants of fusion in a system of membranes with opposite charges and, (iii) show that many properties of electrically membranes are distinct from their neutral counterparts. We believe that the findings presented here may help on the development of better delivery systems, with better efficiency and lower side effects.
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    Fator de crescimento do endotélio vascular na viabilidade do retalho musculofasciocutâneo abdominal transverso do músculo reto do abdome, após abdominoplastia, em ratos
    (Universidade Federal de São Paulo (UNIFESP), 2009-03-25) Freitas, André Luiz Pires de [UNIFESP]; Ferreira, Lydia Masako [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Introduction: Transverse rectus abdominis musculocutaneous (TRAM) flap may present necrosis, in pacients with risk factors. Abdominoplasty is a risk factor due to disruption and transection of vascular perforators. Perspectives on naked plasmid DNA encoding vascular endothelial growth factor (VEGF) as gene therapy for angiogenesis and neovascularization of TRAM flap harvesting after abdominoplasty, originated this study. Objective: The objective of this study was to determine the effect of VEGF plasmid DNA in TRAM flap’s rat model viability after abdominoplasty. Methods: Thirty-two Wistar rats were distributed into four groups (n=8). The caudal pedicled TRAM flap was harvested in all animals and it was the only procedure in the group I (TRAM). In groups II (Abdominoplasty) and III (Plasmid), an abdominoplasty procedure was performed with intramuscularly injection of saline solution and plasmid DNA without VEGF gene, respectively. In Group IV (VEGF) 100 micrograms of plasmid encoding VEGF, was injected into the rectus muscle. TRAM flaps were harvested thirty days after abdominoplasty. Results: TRAM group, Abdominoplasty group, Plasmid group and VEGF group showed a mean percentage of necrosis of 24.65%, 62.49%, 57.80% and 18,33%, respectively (p=0,001). TRAM flap’s immunohistochemical analysis with antibody HHF-35 showed a significant increase in number of vessels in the group IV. Conclusion: The plasmid vector including VEGF after abdominoplasty improved TRAM flap viability and the number of the vessels in rats.
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    Fator de crescimento do endotélio vascular na viabilidade do retalho musculofasciocutâneo transverso do reto do abdome, em ratos submetidos à nicotina
    (Universidade Federal de São Paulo (UNIFESP), 2009-07-30) Silveira, Tiago Santos [UNIFESP]; Ferreira, Lydia Masako [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Introduction: Several factors can reduce the viability of the TRAM flap, among them the nicotine has been made responsible by the loss partial or total of these flaps. Objective: To evaluate the action of the Vascular endothelial Growth Factor in the viability of the Transverse Rectus Abdominis Musculocutaneous, in rats submitted to the nicotine. Methods: Sixty Wistar EPM-1 rats were used, adult males, weighing from 230 to 300g, randomized in 4 groups of 15 animals each: Group Control composed by animals that were submitted to the TRAM flap; Group Nicotine composed by animals that were nicotine exposed and submitted of TRAM flap; Group VEGF composed by animals submitted to the administration of VEGF plasmidial before the TRAM flap; and Group Nicotina+VEGF composed by animals that were exposed to the nicotine, trated with administration of VEGF and submitted to the TRAM flap. For analysis of the results they were done necrosis area and vascular density. Results: There was estatistic significant differentiates in the comparison among all of the groups, regarding the variables necrosis area and vascular density (p <0,05). Conclusion: The Vascular Endothelial Growth Factor increased the viability of the Transverse Rectus Abdominis Musculocutaneous, in rats submitted to the nicotine.