Lateral diffusion of membrane components (lipids and proteins) is an important membrane property to measure since the essential process of absorption of anti-cancer and other drugs -some of which are not soluble in lipids and therefore would not be able to penetrate the cell membrane through passive diffusion- lies on it. In particular, the procedure of diffusion into the cell cytoplasm is reliant on free volumes in the membrane (passive diffusion) as well as carrier proteins (facilitated diffusion). By enhancing the mobility of lipids and/or proteins, the possibility of the carrier protein to "encapsulate" pharmacological components maxim- izes, as a "scanning" of the proteins gets performed due to the fluid phase of a biological membrane. At the same time, the increased mobility of the lipids facilitates the passage of lipid-soluble molecules into the cell. Thus, given that the success of anticancer treatments heavily depends on their absorption by the cell, a significant enhancement of the cell mem- brane permeability (permeabilisation) is rendered vital to the applicability of the technique. For this reason, there is augmented interest in combined methods such as Nanotechnology based drug delivery that is focused on the development of optimally designed therapeutic agents along with the application of shock waves to enhance the membrane permeability to the agents. This study examines the impact of shock waves on a numerical model of a biological membrane. Cont/d.

Interações de peptídeos antimicrobianos com modelos de membranas biológicas têm sido extensivamente estudadas para entender as funções destes peptídeos e para elucidar seus mecanismos de ação. Muitos esforços têm sido realizados para aumentar a potência e a especificidade desses peptídeos com o propósito de serem mais seletivos aos organismos patogênicos do que às células dos hospedeiros, como também para um melhor entendimento desta classe de processo biológico. Os mecanismos geralmente propostos para a atividade antimicrobiana envolvem a permeabilização das membranas celulares pela formação de poros ou por outras mudanças nas membranas. Um ponto fundamental no entendimento da atividade é que a composição de lipídeos das membranas dos patógenos e dos hospedeiros é diferente, observação que é entendida como a chave principal da seletividade dos peptídeos antimicrobianos. O objetivo do presente trabalho é contribuir para o entendimento da ação de peptídeos antimicrobianos pelo estudo, por simulação molecular, do peptídeo antimicrobiano indolicidina e de alguns de seus mutantes. A indolicidina é um peptídeo constituído por 13 resíduos de aminoácidos que foi isolada dos neutrófilos de bovinos cuja função é ingerir e matar bactérias. Apesar de numerosos estudos experimentais, não se sabe ainda como a indolicidina atua. Este conhecimento é importante tanto no entendimento dos processos de defesa dos organismos multicelulares como no desenvolvimento de novos antibióticos. Estas questões foram abordadas através do estudo do comportamento da indolicidina e de alguns dos seus mutantes em solução aquosa e em interação com modelos de membranas celulares. / Interactions of the antimicrobial peptides with biological membrane models have been broadly studied to understand the function and the action mechanism of this class of peptides. Many efforts have been realized to increase the potency and the specificity of these peptides with the purpose of obtain more selective pathogen antimicrobials with decrease of the toxic effects and for a better explanation of the biological process concerned in the peptide action. The action mechanism approached to antimicrobial peptides concern the cellular membrane permeation by pore formation or other type of membrane disruption. A fundamental point in the knowledge of the activity is the distinct lipid composition of pathogen and host cells that is conceived as the principal key point in the selectivity of the antimicrobial peptides. The aim of the present work is to contribute for the knowledge of the action of the antimicrobial peptide indolicidin and some of its mutants by molecular dynamics simulation. The indolicidin is a short 13 amino acid residues antimicrobial peptide that was isolated from bovine neutrofils that have the function of ingest and kill pathogens. The action mechanism of the indolicidin is not yet known despite of numerous experimental studies realized with this peptide. The interaction of the indolicidin with the membrane models is important both for the knowledge of the defense machinery of the live organisms and for the development of new antimicrobials. These questions were approached by the study of the indolicidin and some of its mutants in solution and in interaction with cell membrane models.

Lateral diffusion of membrane components (lipids and proteins) is an important membrane property to measure since the essential process of absorption of anti-cancer and other drugs -some of which are not soluble in lipids and therefore would not be able to penetrate the cell membrane through passive diffusion- lies on it. In particular, the procedure of diffusion into the cell cytoplasm is reliant on free volumes in the membrane (passive diffusion) as well as carrier proteins (facilitated diffusion). By enhancing the mobility of lipids and/or proteins, the possibility of the carrier protein to "encapsulate" pharmacological components maxim- izes, as a "scanning" of the proteins gets performed due to the fluid phase of a biological membrane. At the same time, the increased mobility of the lipids facilitates the passage of lipid-soluble molecules into the cell. Thus, given that the success of anticancer treatments heavily depends on their absorption by the cell, a significant enhancement of the cell mem- brane permeability (permeabilisation) is rendered vital to the applicability of the technique. For this reason, there is augmented interest in combined methods such as Nanotechnology based drug delivery that is focused on the development of optimally designed therapeutic agents along with the application of shock waves to enhance the membrane permeability to the agents. This study examines the impact of shock waves on a numerical model of a biological membrane. Cont/d.

Submitted by (edna.saturno@ufrpe.br) on 2016-08-25T14:25:58Z No. of bitstreams: 1 Ernesto Rodriguez-Salas.pdf: 2263672 bytes, checksum: e3402ae3eb8a78aa7240c1d83859157e (MD5) / Made available in DSpace on 2016-08-25T14:25:59Z (GMT). No. of bitstreams: 1 Ernesto Rodriguez-Salas.pdf: 2263672 bytes, checksum: e3402ae3eb8a78aa7240c1d83859157e (MD5) Previous issue date: 2006-02-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Search for materials, methods and means of conservation of membranes and tissues has been accomplished either for preservation of anatomic pieces or for utilization in homologue and heterologue transplants. In the present work, a bibliographical review about materials and means of conservation of biological membranes utilized in surgery procedures was realized. Three experiments aimed at: (I) – Determine concentration of the antibiotics benzilpeniciline, sulphate of estreptomicine and anfotericine B in the environment of conservation ERS®-04-09-11-16-21; (II) – Characterize morphology and histopathology of the BPMs (III) – Evaluate biomechanical characteristics of BPMs conserved in the environment proposed. Initially, 10 BPMs were collected, which registered an average weight of 21g after processed. In order to determine concentration of peniciline, a dosage taking weight into account weight was used, whereas for sulphate of estreptomicine and anfotericine B, procedure used in cell cultivation was adapted. Final results indicated dosages of 2,0 mg/ml; 0,035 mg/ml e 0,030 mg/ml respectively. In experiment II – four samples were collected and sectioned in 10 fragments of 2 cm2, determining weight, size and thickness, according to treatments T-0 - In natura; T-30 -, T-60 e T-90, respectively at 30, 60 and 90 days after collecting. Morphometrical results demonstrated augmentation of thickness, size and weight of BPMs in the several timings in relation to in natura state and histopathological studies (optic and electronic microscopy) brought to evidence maintenance of BPMs structure, although an enlargement of spacing between collagen fibers took place. Fibroblasts presented varied degrees of vacuolization and necrosis at all timings. In the third experiment, same procedure of collecting and conservation as in experiment II were obeyed. Size of samples was of 13 cm x 20 cm for evaluation of thickness, maximum force, tension and expansion through computerized dynamometer EMIC. Taking all results into account, we come to the conclusion is the environment proposed is capable of maintaining the BPMs free of contaminants for a period of 90 days with the same morphological pattern and collagen fibers arrangement. The BPMs conserved in the environment and maintained under refrigeration temperature 12±2ºC can be used in surgery procedures in an ideal period of between 13 and 69 days given conservation of capacity of expasion and augmentation of maximum force and thickness. A reduction of tension to which BMP was submitted because of timing was also observed, which does not endanger use of BPMs in surgery procedures such as reconstitution of articular capsules, herniorafies, reparation surgeries of thick traumatisms with loss of conjunctive tissue, and so forth. / A busca de materiais, métodos e meios de conservação de membranas e tecidos vêm sendo realizada seja para preservação de peças anatômicas ou para uso nos transplantes homólogos e heterólogos. No presente trabalho foi realizada uma revisão bibliográfica sobre os materiais e meios de conservação de membranas biológicas utilizadas em procedimentos cirúrgicos. Três experimentos objetivaram: (I) - Determinar a concentração dos antibióticos benzilpenicilina, sulfato de estreptomicina e anfotericina B no meio de conservação ERS®04-09-11-16-21; (II) - Caracterizar a morfologia e histopatologia das MPBs (III) - Avaliar as características biomecânicas das MPBs, conservadas no meio proposto. Inicialmente, foram coletadas 10 MPBs, que após processadas, registraram peso médio de 21 g. Para determinação da concentração de penicilina, utilizou-se a dosagem atendendo ao peso, enquanto que para o sulfato de estreptomicina e anfotericina B, adaptou-se o procedimento utilizado em cultivo celular. Os resultados finais indicaram dosagens de 2,0 mg/ml; 0,035 mg/ml e 0,030 mg/ml nesta ordem respectivamente. No experimento II – quatro amostras foram coletadas e seccionadas em 10 fragmentos de 2 cm2, determinando-se peso, tamanho e espessura, segundo os tratamentos T-0 - In natura; T-30 -, T-60 e T-90, respectivamente aos 30, 60 e 90 dias da coleta. Os resultados morfofométricos demonstraram aumento de espessura, tamanho e peso das MPBs nos diversos tempos em relação ao estado in natura e estudos histopatológicos (microscopia óptica e eletrônica) evidenciaram a manutenção da estrutura das MPBs, apesar de haver um maior espaçamento entre as fibras colágenas. Os fibroblastos apresentaram graus variados de vacuolização e necrose em todos os tempos. No terceiro experimento, obedeceram-se aos mesmos procedimentos de coleta e conservação do experimento II. Já, o tamanho das amostras foi de 13 cm x 20 cm para avaliação da espessura, força máxima, tensão e alongamento por meio de dinamômetro computadorizado EMIC. Tendo em vista todos os resultados, conclui-se que o meio proposto é capaz de manter as MPBs livres de contaminantes por um de 90 dias com o mesmo padrão morfológico e arranjo das fibras colágenas. As MPBs conservadas no meio e mantidas em temperatura de refrigeração 12±2ºC podem ser utilizadas em procedimentos cirúrgicos num período ideal entre 13 e 69 dias dado pela conservação da capacidade de alongamento e pelo aumento da força máxima e espessura. Houve também uma redução da tensão a que foi submetida a MPB, em função do tempo, o que não compromete o uso das MPBs em procedimentos cirúrgicos como reconstituição de cápsulas articulares, herniorafias, cirurgia reparadora de extensos traumatismos com perda de tecido conjuntivo, entre outros.

Interações de peptídeos antimicrobianos com modelos de membranas biológicas têm sido extensivamente estudadas para entender as funções destes peptídeos e para elucidar seus mecanismos de ação. Muitos esforços têm sido realizados para aumentar a potência e a especificidade desses peptídeos com o propósito de serem mais seletivos aos organismos patogênicos do que às células dos hospedeiros, como também para um melhor entendimento desta classe de processo biológico. Os mecanismos geralmente propostos para a atividade antimicrobiana envolvem a permeabilização das membranas celulares pela formação de poros ou por outras mudanças nas membranas. Um ponto fundamental no entendimento da atividade é que a composição de lipídeos das membranas dos patógenos e dos hospedeiros é diferente, observação que é entendida como a chave principal da seletividade dos peptídeos antimicrobianos. O objetivo do presente trabalho é contribuir para o entendimento da ação de peptídeos antimicrobianos pelo estudo, por simulação molecular, do peptídeo antimicrobiano indolicidina e de alguns de seus mutantes. A indolicidina é um peptídeo constituído por 13 resíduos de aminoácidos que foi isolada dos neutrófilos de bovinos cuja função é ingerir e matar bactérias. Apesar de numerosos estudos experimentais, não se sabe ainda como a indolicidina atua. Este conhecimento é importante tanto no entendimento dos processos de defesa dos organismos multicelulares como no desenvolvimento de novos antibióticos. Estas questões foram abordadas através do estudo do comportamento da indolicidina e de alguns dos seus mutantes em solução aquosa e em interação com modelos de membranas celulares. / Interactions of the antimicrobial peptides with biological membrane models have been broadly studied to understand the function and the action mechanism of this class of peptides. Many efforts have been realized to increase the potency and the specificity of these peptides with the purpose of obtain more selective pathogen antimicrobials with decrease of the toxic effects and for a better explanation of the biological process concerned in the peptide action. The action mechanism approached to antimicrobial peptides concern the cellular membrane permeation by pore formation or other type of membrane disruption. A fundamental point in the knowledge of the activity is the distinct lipid composition of pathogen and host cells that is conceived as the principal key point in the selectivity of the antimicrobial peptides. The aim of the present work is to contribute for the knowledge of the action of the antimicrobial peptide indolicidin and some of its mutants by molecular dynamics simulation. The indolicidin is a short 13 amino acid residues antimicrobial peptide that was isolated from bovine neutrofils that have the function of ingest and kill pathogens. The action mechanism of the indolicidin is not yet known despite of numerous experimental studies realized with this peptide. The interaction of the indolicidin with the membrane models is important both for the knowledge of the defense machinery of the live organisms and for the development of new antimicrobials. These questions were approached by the study of the indolicidin and some of its mutants in solution and in interaction with cell membrane models.

A range of simulations have been conducted to investigate the behaviour of a diverse set of complex biological membrane systems. The processes of interest have required simulations over extended time and length scales, but without sacrifice of molecular detail. For this reason, the primary technique used has been coarse-grained molecular dynamics (CG MD) simulations, in which small groups of atoms are combined into lower-resolution CG particles. The increased computational efficiency of this technique has allowed simulations with time scales of microseconds, and length scales of hundreds of nm. The membrane-permeabilizing action of the antimicrobial peptide maculatin 1.1 was investigated. This short α-helical peptide is thought to kill bacteria by permeabilizing the plasma membrane, but the exact mechanism has not been confirmed. Multiscale (CG and atomistic) simulations show that maculatin can insert into membranes to form disordered, water-permeable aggregates, while CG simulations of large numbers of peptides resulted in substantial deformation of lipid vesicles. The simulations imply that both pore-forming and lytic mechanisms are available to maculatin 1.1, and that the predominance of either depends on conditions such as peptide concentration and membrane composition. A generalized study of membrane protein aggregation was conducted via CG simulations of lipid bilayers containing multiple copies of model transmembrane proteins: either α-helical bundles or β-barrels. By varying the lipid tail length and the membrane type (planar bilayer or spherical vesicle), the simulations display protein aggregation ranging from negligible to extensive; they show how this biologically important process is modulated by hydrophobic mismatch, membrane curvature, and the structural class or orientation of the protein. The association of influenza hemagglutinin (HA) with putative lipid rafts was investigated by simulating aggregates of HA in a domain-forming membrane. The CG MD study addressed an important limitation of model membrane experiments by investigating the influence of high local protein concentration on membrane phase behaviour. The simulations showed attenuated diffusion of unsaturated lipids within HA aggregates, leading to spontaneous accumulation of raft-type lipids (saturated lipids and cholesterol). A CG model of the entire influenza viral envelope was constructed in realistic dimensions, comprising the three types of viral envelope protein (HA, neuraminidase and M2) inserted into a large lipid vesicle. The study represents one of the largest near-atomistic simulations of a biological membrane to date. It shows how the high concentration of proteins found in the viral envelope can attenuate formation of lipid domains, which may help to explain why lipid rafts do not form on large scales in vivo.

Orientador: Marcia Rita Fernandes Machado / Banca: Maria Aparecida de Alcântara / Banca: Hélio Takachi Okuda / Banca: Gilson Hélio Toniollo / Banca: Marcos Lania de Araújo / Resumo: O objetivo desta investigação foi descrever o comportamento mecânico e as características histológicas de peritônio da paca (Agouti paca) a fresco e conservado em glicerina a 98%. Amostras frescas e conservadas em glicerina por períodos de 30, 60 e 90 dias foram submetidas a testes mecânicos de tração, e também caracterizadas pelos seus aspectos histológicos e microscópicos , utilizando-se microscopias de luz e eletrônica de varredura. Quatro animais adultos, machos e fêmeas, com peso corporal médio de 8 quilogramas, foram utilizadas para colheita de amostras de peritônio. Não foram observadas diferenças significativas na integridade estrutural e ultraestrutural dos elementos que constituem as amostras a fresco e conservadas em glicerina. Evidenciou-se, portanto, células mesoteliais e tecido conjuntivo (ou conectivo) sem grandes alterações. Todos os tecidos conservados em glicerina a 98% apresentaram diminuição na rigidez e aumento na ductibilidade e tenacidade, similarmente às amostra de bovinos. Pode-se concluir que a glicerina é uma substância eficiente para a conservação de membranas biológicas, melhorando as propriedades mecânicas e permitindo que as membranas suportem maiores forças de deformação / Abstract: The purpose of this investigation is to describe the mechanical behavior and histological characteristics of fresh and 98% glycerin conserved Agouti paca peritoneum. Fresh samples and those conserved in glycerin for periods of 30, 60 and 90 days were submitted to mechanical traction tests, as well as characterized by its histological and microscopic aspects by optical and scanning electron microscopy. Four adult animals, males and females, about eight kilograms each, were used to collect peritoneum samples. There was no significant difference in structural and ultrastructural integrity of the elements that constitutes the samples of fresh and 98% glycerin conserved material, evidencing mesothelial cells and connecting tissue without large alterations. All the evaluated tissues conserved in 98% glycerin showed a decrease in their rigidness and also an increase in the ductility and tenacity similar to bovine. It can be concluded, that glycerin is efficient for the conservation of biological membranes.improving the mechanical properties allowing the membranes to handle larger forces and deformation / Doutor

Made available in DSpace on 2014-06-11T19:31:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-10-30Bitstream added on 2014-06-13T19:41:02Z : No. of bitstreams: 1 camargo_ad_dr_jabo.pdf: 1540231 bytes, checksum: 34d93ffe1666cbbcc625963f119dd1a6 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo desta investigação foi descrever o comportamento mecânico e as características histológicas de peritônio da paca (Agouti paca) a fresco e conservado em glicerina a 98%. Amostras frescas e conservadas em glicerina por períodos de 30, 60 e 90 dias foram submetidas a testes mecânicos de tração, e também caracterizadas pelos seus aspectos histológicos e microscópicos , utilizando-se microscopias de luz e eletrônica de varredura. Quatro animais adultos, machos e fêmeas, com peso corporal médio de 8 quilogramas, foram utilizadas para colheita de amostras de peritônio. Não foram observadas diferenças significativas na integridade estrutural e ultraestrutural dos elementos que constituem as amostras a fresco e conservadas em glicerina. Evidenciou-se, portanto, células mesoteliais e tecido conjuntivo (ou conectivo) sem grandes alterações. Todos os tecidos conservados em glicerina a 98% apresentaram diminuição na rigidez e aumento na ductibilidade e tenacidade, similarmente às amostra de bovinos. Pode-se concluir que a glicerina é uma substância eficiente para a conservação de membranas biológicas, melhorando as propriedades mecânicas e permitindo que as membranas suportem maiores forças de deformação / The purpose of this investigation is to describe the mechanical behavior and histological characteristics of fresh and 98% glycerin conserved Agouti paca peritoneum. Fresh samples and those conserved in glycerin for periods of 30, 60 and 90 days were submitted to mechanical traction tests, as well as characterized by its histological and microscopic aspects by optical and scanning electron microscopy. Four adult animals, males and females, about eight kilograms each, were used to collect peritoneum samples. There was no significant difference in structural and ultrastructural integrity of the elements that constitutes the samples of fresh and 98% glycerin conserved material, evidencing mesothelial cells and connecting tissue without large alterations. All the evaluated tissues conserved in 98% glycerin showed a decrease in their rigidness and also an increase in the ductility and tenacity similar to bovine. It can be concluded, that glycerin is efficient for the conservation of biological membranes.improving the mechanical properties allowing the membranes to handle larger forces and deformation

The very fundamental properties of biological membranes can be understood by studying their formation. This sets a good foundation for research related to how the membranes interact with organic molecules and ions; something of great value in the quest of explaining transport phenomena through cell membranes. It is furthermore of growing interest within the pharmacological research and contributes to the apprehension of life at the molecular level. In this thesis Molecular Dynamics has been used to simulate how evenly distributed phospholipids solvated in water leads to the formation of monolayers. An automation program has been written in Python for performing these simulations and is to be used as the foundation for performing simulations in further studies. The program was used to simulate model systems of high- and low concentrations of DPPC lipids. The DPPC lipid, like most other lipids, consist of a hydrophilic "head" part and two lipophilic "tails", which is the main cause of the lipids interacting in such a manner that forms membranes. The low concentration system was simulated for a total of 3 ns with all lipids having reached the surface at 1.5 ns, and the all lipids in the high concentration system had risen at 41 ns with a total simulation time of 43 ns. / De mest grundläggande egenskaperna hos cellmembran kan förstås genom att studera hur dessa bildas. Detta skapar en bra grund för forskning relaterad till hur membranen interagerar med organiska molekyler och joner; något av stort värde i bemödandet att förklara transportfenomen genom cellmembran. Dessutom är det av växande intresse inom den farmakologiska forskningen och bidrar till kunskapen om liv på den molekylära nivån. I denna avhandling har Molekylär Dynamik använts för att simulera hur jämnt fördelade fosfolipider lösta i vatten leder till bildandet av monoskiktade membran. Ett automatiseringsprogram har skrivits i Python för att utföra dessa simuleringar och ska komma att användas som grund för genomförandet av simuleringar i vidare studier. Programmet användes för att simulera modellsystem med höga och låga koncentrationer av DPPC lipider. DPPC lipiden, liksom de flesta andra lipider, består av en hydrofil ''huvud'' -del och två lipofila ''svansar'', vilket är den huvudsakliga orsaken till att lipiderna interagerar på ett sådant sätt som driver bildandet av ett membran. Lågkoncentrationssystemet simulerades i totalt 3 ns, varav 1,5 ns behövdes för att alla lipider skulle nå vattenytan. Alla lipider i högkoncentrationssystemet hade kommit upp till ytan efter 41 ns och för detta system utfördes simuleringen under en total tid på 43 ns.