Biological membranes are actively involved in a multitude of processes in living cells; therefore, a detailed characterization of their structure, dynamics, and function is essential for an understanding of living organisms at the molecular level. In this work, we made use of the high spatial and temporal resolution offered by computer simulations to investigate the behavior of several molecular species which associate with cellular membranes. Using a combination of classical molecular dynamics simulations and ab initio electronic structure calculations, we were able to characterize nonlinear optical properties of membrane- embedded fluorescent probes and thus contribute to establishing two-photon polarization microscopy as a tool of structural biology. Moreover, our molecular dynamics simulations provided an atomistic picture of the reversible membrane binding of recoverin, a neuronal calcium-sensing protein involved in vision adaptation, and they also yielded an important insight into the mechanism of its calcium-induced myristoyl switch. In addition, we examined the biological role of cholesterol oxidation and compared two methods of representing transmembrane voltage in molecular dynamics simulations.

Les membranes biologiques sont impliquées dans divers mécanismes comme la reconnaissance moléculaire ou encore la fusion membranaire. Les lipides, principaux composants des membranes, sont inhérents à ces processus cellulaires, mais leur organisation et leur rôle fonctionnel au sein de ces systèmes sont très complexes. Dans ce travail, nous avons utilisé des modèles membranaires mimant ces systèmes biologiques pour identifier les interactions mises en jeu avec divers agents exogènes (AEs), en utilisant la microscopie à force atomique (AFM). Nous avons donc travaillé sur deux AEs différents qui interagissent potentiellement avec ces membranes. Le premier intervient directement dans le cadre de l'étude du paludisme. Le mécanisme moléculaire de cette maladie (impliquant probablement des structures lipidiques) n'étant pas clair, la compréhension de celui-ci faciliterait le développement de nouvelles molécules antipaludiques et/ou cibles thérapeutiques. Parallèlement notre étude s'est portée sur l'interaction des nanoparticules (NPs) de TiO2, notre second AE, avec les membranes. Très utilisées dans l'industrie, ces NPs de TiO2 pourraient avoir un impact sur la santé humaine, en interagissant notamment avec les membranes cellulaires. Des techniques biophysiques classiques ont tout d'abord été utilisées pour évaluer l'interaction de l'AE avec des systèmes biomimétiques. Ensuite, lorsque celle-ci est prouvée, l'AFM est utilisée pour visualiser les changements morphologiques des modèles membranaires en présence de ces AEs. Ainsi, pour chaque AE, nous avons finalement suggéré un mécanisme d'interaction afin de répondre aux problématiques soulevées. / Biological membranes play a crucial role as a biological barrier but, paradoxically, they were involved in various precesses : as a molecular recognition, enzymatic catalysis, or membrane fusion. Lipids, as a main component of membranes, are entailed in these process but composition, organization, and functional role in these biological systems are quite complex. Here, we use lipid models mimicking biological membrane to identify their interaction mechanisms with various exogenous agents (EAs) like peptides, nanoparticules, drugs, proteins, using atomic force microscopy (AFM). In this project, we worked on two different molecules which interact potentially with biological membrane. The first one was a molecule directly implicated in malaria disease. In fact, the molecular mechanism, probably involving lipid membranes, is still unclear. The understanding of this mechanism would participate to find new antimalarial drug or new therapeutic targets. The second EA studied was titanium dioxide nanoparticules (TiO2 NPs). Widely used in industry, this product from nanotechnology's development could have an impact on human health, whose potential toxicity mechanism still unknown. Biophysical techniques such as fluorescence spectroscopy, Langmuir monolayer were used to evaluate the potential interaction of the EAs with biomimetic membranes. Then, when the interaction was proved, AFM was used in order to visualize the effects inferred by these EAs. Using supported lipid bilayers, we imaged their behavior after injection of the EA concerned, at the nanometer scale. For each EA, we could suggest a mechanism of interaction and respond to the issues raised.

Neste trabalho foi utilizado um esquema multi-técnicas para estudar a base de interações moleculares protagonizadas por duas proteínas que possuem funções biológicas completamente distintas. A primeira delas, clorocatecol 1,2-dioxigenase (Pp 1,2-CCD), tem um apelo biotecnológico para área ambiental devido a sua capacidade de catalisar a degradação do composto clorocatecol, um intermediário comum no final da decomposição de diversos hidrocarbonetos aromáticos policíclicos. Essa característica pode promover a descontaminação de solos e águas poluídos revelando um grande potencial para aplicações em mecanismos de biorremediação. Além disso, a presença de moléculas anfipáticas junto à interface de ligação dos monômeros da CCD levantou a questão em relação à capacidade dessa família de enzimas de se ligar a membranas biológicas. Esse tipo de informação amplia o conhecimento acerca de mecanismos básicos de ação da enzima, aumentando a possibilidade de parceiros de interação, podendo levar a outras formas de controle da atividade biológica para uso em aplicações biotecnológicas como desenvolvimento de biossensores. O estudo dessa enzima está, portanto, voltado para a compreensão de suas interações com miméticos de membrana e a tentativas de imobilização da proteína nestas estruturas. Para isto, fazemos uso de técnicas biofísicas como dicroísmo circular, caloria diferencial de varredura e espectroscopias ópticas, e biomoleculares como desenvolvimento de oligonucleotídeos, reações de cadeia polimerase e análise de restrição. A outra vertente desta dissertação, tem como foco de estudo a proteína ligante de acil-CoA de Cryptococcus neoformans (CnACBP) clonada pela primeira vez em nosso laboratório. Homólogos de ACBP foram encontrados em todos os organismos distribuídos nos quatro reinos eucariotos, com alta similaridade sequencial (~48%). A sua presença ao longo dos reinos e seu envolvimento em diversos mecanismos metabólicos essenciais relacionados ao éster acil-CoA levaram à conclusão de que se trata de uma housekeeping protein, e não uma proteína específica, confinada a um tipo especializado de célula. Este trabalho traz uma caracterização inicial da CnACBP que busca esclarecer questões ainda em aberto e também aprofundar o conhecimento ainda muito vago de como cargas e a presença do ligante podem influenciar estrutura, estabilidade e função através de técnicas termodinâmicas e espectroscópicas antes de um aprofundamento de seu papel no interior da célula e interações com outras proteínas. / In this study, we used a multi technique approach to understand the basic molecular interactions of two proteins that have quite different biological functions. The first, chlorocatechol 1,2- dioxygenase (Pp 1,2-CCD), has an environmental appeal due to it ability to catalyze the degradation of chlorocatechol, a common intermediate in the end of the decomposition of many polycyclic aromatic hydrocarbons. This characteristic of decontaminating polluted soils and waters suggest a great potential for applications in bioremediation mechanisms. Moreover, the presence of amphipathic molecules at the interface of the CCD monomers raised issues related to the ability of this enzyme family of binding to biological membranes. Such information broadens the knowledge of the basic mechanisms of enzyme action, increasing the possibility of interaction partners and may lead to other forms of control of the biological activity for use in biotechnological applications, such as biosensors development. The study of this enzyme is therefore, aimed at understanding their interactions with mimetic membrane and immobilization attempts of the protein in these structures. For this purpose, we make use of biophysical techniques such as circular dichroism, differential scanning calorimetry and optical spectroscopies and biomolecular techniques, such as development of primers, polymerase chain reaction and restriction analysis. The other aspect of this dissertation is focused on the study of acyl-CoA binding protein of Cryptococcus neoformans (CnACBP) cloned for first time in our laboratory. Homologues of ACBP were found in all organisms distributed in the four kingdoms of eukaryotes, with high sequence similarity (~ 48%). Its widespread presence and their involvement in several key metabolic pathways related to the acyl-CoA ester led to the conclusion that ACBP is a housekeeping protein and not a specific protein contained a specialized cell type. Here we present an initial characterization of CnACBP that seeks to relevant issues regarding the proteins function. Our goal was to increase the still vague knowledge on how electrical charges and the presence of the binding partner may influence the structure, stability and function through thermodynamic and spectroscopic techniques. This is an initial step toward the full understanding of the role of protein in the cell.

No description available.

Neste trabalho foi utilizado um esquema multi-técnicas para estudar a base de interações moleculares protagonizadas por duas proteínas que possuem funções biológicas completamente distintas. A primeira delas, clorocatecol 1,2-dioxigenase (Pp 1,2-CCD), tem um apelo biotecnológico para área ambiental devido a sua capacidade de catalisar a degradação do composto clorocatecol, um intermediário comum no final da decomposição de diversos hidrocarbonetos aromáticos policíclicos. Essa característica pode promover a descontaminação de solos e águas poluídos revelando um grande potencial para aplicações em mecanismos de biorremediação. Além disso, a presença de moléculas anfipáticas junto à interface de ligação dos monômeros da CCD levantou a questão em relação à capacidade dessa família de enzimas de se ligar a membranas biológicas. Esse tipo de informação amplia o conhecimento acerca de mecanismos básicos de ação da enzima, aumentando a possibilidade de parceiros de interação, podendo levar a outras formas de controle da atividade biológica para uso em aplicações biotecnológicas como desenvolvimento de biossensores. O estudo dessa enzima está, portanto, voltado para a compreensão de suas interações com miméticos de membrana e a tentativas de imobilização da proteína nestas estruturas. Para isto, fazemos uso de técnicas biofísicas como dicroísmo circular, caloria diferencial de varredura e espectroscopias ópticas, e biomoleculares como desenvolvimento de oligonucleotídeos, reações de cadeia polimerase e análise de restrição. A outra vertente desta dissertação, tem como foco de estudo a proteína ligante de acil-CoA de Cryptococcus neoformans (CnACBP) clonada pela primeira vez em nosso laboratório. Homólogos de ACBP foram encontrados em todos os organismos distribuídos nos quatro reinos eucariotos, com alta similaridade sequencial (~48%). A sua presença ao longo dos reinos e seu envolvimento em diversos mecanismos metabólicos essenciais relacionados ao éster acil-CoA levaram à conclusão de que se trata de uma housekeeping protein, e não uma proteína específica, confinada a um tipo especializado de célula. Este trabalho traz uma caracterização inicial da CnACBP que busca esclarecer questões ainda em aberto e também aprofundar o conhecimento ainda muito vago de como cargas e a presença do ligante podem influenciar estrutura, estabilidade e função através de técnicas termodinâmicas e espectroscópicas antes de um aprofundamento de seu papel no interior da célula e interações com outras proteínas. / In this study, we used a multi technique approach to understand the basic molecular interactions of two proteins that have quite different biological functions. The first, chlorocatechol 1,2- dioxygenase (Pp 1,2-CCD), has an environmental appeal due to it ability to catalyze the degradation of chlorocatechol, a common intermediate in the end of the decomposition of many polycyclic aromatic hydrocarbons. This characteristic of decontaminating polluted soils and waters suggest a great potential for applications in bioremediation mechanisms. Moreover, the presence of amphipathic molecules at the interface of the CCD monomers raised issues related to the ability of this enzyme family of binding to biological membranes. Such information broadens the knowledge of the basic mechanisms of enzyme action, increasing the possibility of interaction partners and may lead to other forms of control of the biological activity for use in biotechnological applications, such as biosensors development. The study of this enzyme is therefore, aimed at understanding their interactions with mimetic membrane and immobilization attempts of the protein in these structures. For this purpose, we make use of biophysical techniques such as circular dichroism, differential scanning calorimetry and optical spectroscopies and biomolecular techniques, such as development of primers, polymerase chain reaction and restriction analysis. The other aspect of this dissertation is focused on the study of acyl-CoA binding protein of Cryptococcus neoformans (CnACBP) cloned for first time in our laboratory. Homologues of ACBP were found in all organisms distributed in the four kingdoms of eukaryotes, with high sequence similarity (~ 48%). Its widespread presence and their involvement in several key metabolic pathways related to the acyl-CoA ester led to the conclusion that ACBP is a housekeeping protein and not a specific protein contained a specialized cell type. Here we present an initial characterization of CnACBP that seeks to relevant issues regarding the proteins function. Our goal was to increase the still vague knowledge on how electrical charges and the presence of the binding partner may influence the structure, stability and function through thermodynamic and spectroscopic techniques. This is an initial step toward the full understanding of the role of protein in the cell.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2015-01-29T17:17:40Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Tese - Sebastião Antônio Mendanha Neto - 2014.pdf: 7092471 bytes, checksum: 12e06f6af9661e3416fd2248ee25333c (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2015-01-29T17:41:44Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Tese - Sebastião Antônio Mendanha Neto - 2014.pdf: 7092471 bytes, checksum: 12e06f6af9661e3416fd2248ee25333c (MD5) / Made available in DSpace on 2015-01-29T17:41:44Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Tese - Sebastião Antônio Mendanha Neto - 2014.pdf: 7092471 bytes, checksum: 12e06f6af9661e3416fd2248ee25333c (MD5) Previous issue date: 2014-04-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The interactions of terpenes with membranes of erythrocyte, fibroblasts, stratum corneum and the model membranes of 1,2-dipalmitoylsn -glycero-3-phosphocholine were investigated by using the the electron paramagnetic resonance and fluorescence spectroscopic of lipophilic probes. It has been shown that when added at high concentrations to systems having a high lipid/solvent ratio, terpenes such as 1,8-cineol, α-terpineol, (+)-limonene and nerolidol are able to self-stabilize in molecular aggregates which can extract the bilayers lipids. Studies on the hemolytic and cytotoxic potential of various terpenes showed that cell damage caused by these molecules are concentration dependent and that among the studied terpenes, nerolidol and α-terpineol are the most hemolytic and cytotoxic, while (+)-limonene and 1,8-cineole are the least hemolytic and cytotoxic. However, the low correlation between these two tests indicates that the processes involved in each case are not completely dependent. It was also shown that once embedded in the membrane, terpenes increase the fluidity of lipid bilayers and decrease the temperature of the main phase transition. Differences between increased fluidity promoted by sesquiterpene nerolidol and all monoterpenes studied were observed. Meanwhile, in a comparison of the effect of the monoterpenes studied, no significant differences in their ability to increase membrane fluidity were detected. Furthermore, it was demonstrated by using confocal and atomic force microscopy and fluorescence spectroscopy that the 1,2-distearoylsn -glycero-3-(Aurora nanoparticles) is better incorporated in lipid membranes under fluid phase and that the addition of 0.1% of these conjugated nanoparticles do not produces large variations in membrane fluidity and no causes substantial morphological changes of lipid bilayers. / As intera¸c˜oes de terpenos com membranas de eritr´ocito, fibroblastos, estrato c´orneo e membrana modelo composta de 1,2-dipalmitoil-sn -glicero-3-fosfocolina foram investigadas por meio das espectroscopias de ressonˆancia paramagn´etica eletrˆ onica e de fluorescˆencia por meio do uso de sondas lipof´ılicas. Foi poss´ıvel demonstrar que quando adicionados em altas concentra¸c˜oes `a sistemas que possuem uma alta rela¸c˜ao lip´ıdio/solvente, terpenos como o 1,8-cineol, α-terpineol, (+)-limoneno e nerolidol s˜ao capazes de se estabilizar em agregados moleculares capazes de extrair os lip´ıdios das bicamadas. Estudos sobre o potencial hemol´ıtico e citot´oxico de v´arios terpenos demostraram que os danos celulares causados por estas mol´eculas s˜ao dependentes da concentra¸c˜ao e que dentre os terpenos estudados, nerolidol e terpineol s˜ao os mais hemol´ıticos e citot´oxicos enquanto limoneno e cineol s˜ao os menos hemol´ıticos e citot´oxicos. Entretanto, a baixa correla¸c˜ao entre estes dois testes indica que os processos envolvidos em cada caso n˜ao s˜ao totalmente dependentes. Ficou demonstrado ainda que uma vez incorporados nas membranas, os terpenos aumentam a fluidez das bicamadas lip´ıdicas e diminuem a temperatura de sua transi¸c˜ao de fase principal. Diferen¸cas entre o aumento de fluidez promovido pelo sesquiterpeno nerolidol e por todos os monoterpenos estudados foram verificadas. Contudo, uma compara¸c˜ao entre o efeito dos monoterpenos estudados, n˜ao aponta para diferen¸cas significativas entre suas capacidades de aumento de fluidez. Al´em disso, foi demostrado atrav´es das microscopias confocal e de for¸ca atˆomica e da espectroscopia de fluorescˆencia que a 1,2-distearoil-sn -glicero-3-(Nanopart´ıculas Aurora) ´e melhor incorporada em membranas lip´ıdicas em fase fluida e que a adi¸c˜ao de 0,1% destas nanopart´ıculas conjugadas n˜ao produz grandes varia¸c˜oes na fluidez e n˜ao provoca mudan¸cas morfol´ogicas substanciais das bicamadas lip´ıdicas.

Advanced fluorescence techniques were used to explore tree distinct topics concerning biological membrane and their interactions. Following thesis is according to the topic divided into three parts: 1) Ionic effects were studied employing time dependent fluorescence shift experiments and molecular dynamic simulations. Combination of these two approaches are suitable to reveal characteristic like mobility and hydration of particular bilayer segment, lipid packing or ion binding sites. Halide anions were reported to adsorb to the cationic lipid bilayer specifically, altering membrane mobility and organization. Changes in observed parameters follows Hofmeister order. Their effect is mediated either by direct ionic interaction (soft, polarizable ions) as well as via alteration of water structure (hard, non-polarizable ions) in proximity of ion molecule. Further, divalent calcium was shown to bind strongly to neutral and negatively charged lipid bilayers. Several types of binding sites depending on calcium concentration were identified. 2) Two complementary lipopeptides, CPK and CPE, incorporated into distinct lipid bilayers serve as a minimal model inducing membrane fusion. Effectiveness of fusion event might be influenced by lipopeptide-membrane and lipopeptide-lipopeptide interaction. To reveal...

The diploma thesis deals with the topic of lipids and it is focused on the processing in the form of educational materials (educational text and educational animations) for chemistry and biology on secondary school. It is devided into two parts. The theoretical part defines the key concepts serving as theoretical background: interdisciplinarity, visualization, animation. There is also performed the analysis of RVP G and the representation of the topic of the diploma thesis in this curricular document. The final chapter of the theoretical part is dedicated to determing the criteria for the analysis of the educational materials and the characteristics of the animation program Adobe Animate CC. The analysis of available educational materials is performed according to predetermined criteria in the practical part, specifically animations available on the Internet. There are also introduced the educational materials so as the complex file of educational animations created in the program Adobe Animate CC and the educational text that serves as a guiding material to educational animation program. The materials include methodological guide for users. The created materials focus on the topic of the lipids and biological membranes, they emphasize interdisciplinarity of chemistry and biology and also connect...

Simulation of processes in cellular membranes Abstract Many important processes in cells involve ions, e.g., fusion of synaptic vesi- cles with neuronal cell membranes is controlled by a divalent cation Ca2+ ; and the exchange of Na+ and K+ drives the the fast electrical signal transmis- sion in neurons. We have investigated model phospholipid membranes and their interactions with these biologically relevant ions. Using state-of-the-art molecular dynamics simulations, we accurately quantified their respective affinites towards neutral and negatively charged phospholipid bilayers. In order to achieve that, we developed a new model of phospholipids termed ECC-lipids, which accounts for the electronic polarization via the electronic continuum correction implemented as charge rescaling. Our simulations with this new force field reach for the first time a quantitative agreement with the experimental lipid electrometer concept for POPC as well as for POPS with all the studied cations. We have also examined the effects of transmembrane voltage on phospholipid bilayers. The electric field induced by the voltage exists exclusively in the hydrophobic region of the membrane, where it has an almost constant strength. This field affects the structure of nearby water molecules highlighting its importance in electroporation. 1

In this thesis, phylogenomic and comparative structural analyses of several widespread energy converting enzymes were performed. The focus was on the major subfamilies of the enzymes that process nucleoside triphosphates (ATP and GTP) and on some key enzymes of the electron transfer chains. First, we analyzed the P-loop GTPases, RadA/RecA recombinases, chaperone GroEL, branched-chain α-ketoacid dehydrogenase kinases, chaperone Hsc70, actins, and membrane pyrophosphatases. In the each inspected family we could identify (1) members which were potassium-dependent and/or contained K+ ions in the active site, and (2) potassium-independent enzymes with lysine or arginine residues as catalytic groups that occupy the positions of potassium ions in the homologous, K+-dependent enzymes. Based on the results of our analyses, we suggest that the appearance of the K+-binding sites could precede in evolution the recruitment of positively charged residues (lysine or arginine "fingers") with the latter providing more possibilities to control the enzyme reactions. Second, we have described the distinctive features of a phylogenetically separated subfamily of rotary membrane ATPases which we named N-ATPases. The N-ATPases have a specific operon organization with two additional subunits, absent in other rotary ATPases, and a complete set of Na+-binding ligands in the membrane c-subunits. We made a prediction, which was later confirmed, that these enzymes are capable of Na+ translocation across the membrane and may confer salt tolerance on marine prokaryotes. Third, phylogenomic analysis of the cytochrome bc complexes suggests that these enzyme complexes initially emerged within the bacteria and were then transferred to archaea via lateral gene transfer on several independent occasions. Our analysis indicates that the ancestral form of the cytochrome bc complex was a b6f-type complex; the fusion of the cytochrome b6 and the subunit IV to a "long" cytochrome b of the cytochrome bc1 complexes could have happened in different lineages independently. Fourth, our phylogenomic and comparative structural analyses of the cytochrome bc1 complex and of cytochrome c allowed us to trace how these enzymes became involved in triggering of apoptosis in Metazoa. We could trace the emergence of a specific cardiolipin-binding site within the cytochrome bc complex and the evolution of structural traits that account for the involvement of the cytochrome c as a trigger of apoptosis in vertebrates.