Orientador: João Ruggiero Neto / Resumo: Os peptídeos sintéticos L1A (IDGLKAIWKKVADLLKNT-NH2, Q = +3e) e seu análogo acetilado (acL1A, Q = +2e) utilizados neste estudo foram projetados para que tenham características estruturais semelhantes ao peptídeo Polybia-MP1 extraído do veneno da vespa Polybia paulista, em que um dos dois resíduos ácidos ocupa a segunda posição na região Nterminal, e resíduos básicos são terceiros e/ou quartos vizinhos dos resíduos ácidos. Esses peptídeos possuem significativa atividade bactericida seletiva para bactérias Gram-negativas, especialmente Escherichia coli, sem serem hemolíticos. Estudos anteriores, em sistemas modelo, demonstraram que a acetilação do N-terminal resultou no aumento da atividade lítica em vesículas aniônicas (8POPC/2POPG) em comparação com o L1A, o que sugeriu perturbação do empacotamento lipídico de modo mais eficaz para o análogo que é menos carregado. Considerando que a membrana plasmática de bactérias Gram-negativas contém majoritariamente fosfatidiletanolamina (PE) e fosfatidilglicerol (PG), o presente trabalho propôs investigar o impacto dos peptídeos L1A e acL1A em membranas modelo compostas por 3POPE/1DOPG utilizando uma variedade de técnicas experimentais. Os resultados demonstraram que ambos os peptídeos induziram segregação lipídica, sendo o análogo acetilado mais eficiente em recrutar PG e segregar PE. / Abstract: The synthetic peptides L1A (IDGLKAIWKKVADLLKNT-NH2, Q = +3e) and its acetylated analog (acL1A, Q = +2e) used in this study were designed to have some structural features similar to the peptide Polybia-MP1 extracted from the venom of the wasp Polybia paulista, in which one of the acidic residues occupies the second position on the N-terminus region and basic residues are third and/or fourth neighbors of the acidic residues. These peptides display significant bactericidal activity against Gram-negative bacteria, especially Escherichia coli, being non-hemolytic. Previous work performed in model membrane systems has shown that the N-terminal acetylation led to an increase on the lytic activity in anionic vesicles (8POPC/2POPG) compared with L1A, suggesting that the less charged peptide has higher ability to perturb the lipid-packing. Considering that the Gram-negative cell membranes contain mainly phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), the present work proposed to investigate the impact of L1A and acL1A on model membranes composed of 3POPE/1DOPG using a variety of experimental techniques. The results suggested that both peptides induced lipid segregation being the acetylated analog more efficient in recruiting PG and segregating PE. / Mestre

Des pathologies telles que la myopathie et certains types de cancer, peuvent être causées par une mauvaise formation de l’enveloppe nucléaire (EN), processus se produisant lors de chaque division cellulaire mais aussi lors de la formation du pronoyau mâle. Un modèle in vitro, dérivé de gamètes d’oursins, a été utilisé afin d’étudier les différentes étapes de formation de l’EN et a permis de révéler plusieurs informations essentielles. Un des points critiques est que des membranes fortement enrichies en phosphoinositides polyphosphorylés (PPIs) sont essentielles à la formation de l’EN, notamment lors des étapes de fusion membranaire. Ces membranes proviennent du cytoplasme de l’ovocyte fécondé (MV1) et des noyaux de spermatozoïdes (NERs). Nous avons construit des modèles membranaires mimant les compositions lipidiques de ces membranes, puis étudié leur structure, leur dynamique ainsi que leur morphologie par spectroscopie de RMN des solides et microscopie électronique. Nous avons montré que les PPIs induisent une courbure membranaire positive, conduisant à la formation de petites vésicules ou de micelles allongées. Plus important encore, dans le modèle « MV1», les membranes sont très fluides. Le modèle « NERs » est constitué de membranes globalement ordonnées, semblables aux phases dites « liquides ordonnées » avec une modulation apportée par la PPIs. Nous avons également construit un modèle membranaire minant la composition lipidique des vésicules MV2, membranes non-enrichies en PPIs mais représentant 90 % des vésicules participant à la formation de l’EN. Ce modèle membranaire présente une dynamique intermédiaire à celle observée pour les modèles MV1 et NERs. Ces propriétés nouvelles ont permis de proposer un mécanisme décrivant le rôle des PPIs lors de la fusion membranaire conduisant à la formation de l’enveloppe nucléaire. / Diseases, such as myopathies and some types of cancer, can be caused by abnormal nuclear envelope (NE) assembly, a process that takes place at each cell division and during male pronuclear formation. A cell-free assay from sea urchin gametes, that mimics the in vivo male pronucleus formation, has been used to dissect the various stages of NE assembly. This in vitro assay has revealed several novel features. One of the critical aspects is that membranes highly enriched in polyphosphorylated phosphoinositides (PPIs), are essential for NE formation, especially during the stage of membrane fusion. Theses membranes are extracted from the cytoplasm of the fertilised oocyte (MV1) and sperm nuclei (NERs). We made model membranes with similar lipid composition to MV1 and NERs and studied their structure, dynamics and morphologies by solid-state NMR spectroscopy and electron microscopy. We show that PPIs have a positive membrane curvature, inducing small vesicles and elongated micelles. More importantly, we illustrate that “MV1-like” membranes are very fluid. “NERs-like” membranes are globally ordered and belong to the family of liquid ordered phases. We also evidenced that PPIs can counterbalance in part the ordering effect of cholesterol. Moreover we made model membranes with similar lipid composition to MV2, non-enriched in PPIs membranes which constitute 90% of the vesicles forming the NE. This model membrane shows an in-between dynamics compared to MV1 and NERs. We therefore propose a mechanism describing the role of PPIs during membrane fusion leading to nuclear membrane assembly.

Influenza A Viren besitzen ein segmentiertes, einzelsträngiges RNA-Genom, welches in Form viraler Ribonukleoprotein (vRNP)-Komplexe verpackt ist. Während das virale Genom im Zellkern repliziert wird, finden Assemblierung und Knospung reifer Viruspartikel an der apikalen Plasmamembran statt. Für die Virusbildung müssen die einzelnen viralen Komponenten hierher gebracht werden. Während intrinsische apikale Signale der viralen Transmembranproteine bekannt sind, sind der zielgerichtete Transport und der Einbau des viralen Genoms in neuentstehende Virionen noch wenig verstanden. In dieser Arbeit wurden potentielle Mechanismen des vRNP-Transportes untersucht, wie die Fähigkeit der vRNPs mit Lipidmembranen zu assoziieren und die intrinsische subzellulären Lokalisation des viralen Nukleoproteins (NP), eines Hauptbestandteils der vRNPs. Es konnte gezeigt werden, dass vRNPs nicht mit Lipidmembranen assoziieren, was mittels Flotation aufgereinigter vRNPs mit Liposomen unterschiedlicher Zusammensetzung untersucht wurde. Die Ergebnisse deuten jedoch darauf hin, dass das virale M1 in der Lage ist, Bindung von vRNPs an negativ-geladene Lipidmembranen zu vermitteln. Subzelluläre Lokalisation von NP wurde des Weiteren durch Expression fluoreszierender NP-Fusionsproteine und Fluoreszenzphotoaktivierung untersucht. Es konnte gezeigt werden, dass NP allein nicht mit zytoplasmatischen Strukturen assoziiert, stattdessen aber umfangreiche Interaktionen im Zellkern eingeht und mit hoher Affinität mit bestimmten Kerndomänen assoziiert, und zwar den Nukleoli sowie kleinen Kerndomänen, welche häufig in der Nähe von Cajal-Körperchen und PML-Körperchen zu finden waren. Schließlich wurde ein experimenteller Ansatz etabliert, welcher erlaubt, den Transport vRNP-ähnlicher Komplexe mittels Fluoreszenzdetektion aufzuzeichnen und Einzelpartikelverfolgungsanalysen durchzuführen. Unterschiedliche Phasen des vRNP-Transportes konnten beobachtet werden und ein 3-Phasen-Transportmodell wird skizziert. / Influenza A viruses have a segmented single-stranded RNA genome, which is packed in form of viral ribonucleoprotein (vRNP) complexes. While the viral genome is replicated and transcribed in the host cell nucleus, assembly and budding of mature virus particles take place at the apical plasma membrane. Efficient virus formation requires delivery of all viral components to this site. While intrinsic apical targeting signals of the viral transmembrane proteins have been identified, it still remains poorly understood how the viral genome is transported and targeted into progeny virus particles. In this study, potential targeting mechanisms were investigated like the ability of vRNPs to associate with lipid membranes and the intrinsic ability of the viral nucleoprotein (NP) – which is the major protein component of vRNPs – for subcellular targeting. It could be shown that vRNPs are not able to associate with model membranes in vitro, which was demonstrated by flotation of purified vRNPs with liposomes of different lipid compositions. Results indicated, however, that the matrix protein M1 can mediate binding of vRNPs to negatively charged lipid bilayers. Intrinsic subcellular targeting of NP was further investigated by expression of fluorescent NP fusion protein and fluorescence photoactivation, revealing that NP by itself does not target cytoplasmic structures. It was found to interact extensively with the nuclear compartment instead and to target specific nuclear domains with high affinity, in particular nucleoli and small interchromatin domains that frequently localized in close proximity to Cajal bodies and PML bodies. An experimental approach was finally established that allowed monitoring the transport of vRNP-like complexes in living infected cells by fluorescence detection. It was possible to perform single particle tracking and to describe different stages of vRNP transport between the nucleus and the plasma membrane. A model of three-stage transport is suggested.

Ionic Liquids (ILs) has been attracting attention, both from academia and industry, given the numerous applications of these systems. ILs are salts, usually composed by an organic ion, and a counterion which could be organic or inorganic, and, interestingly they are found at liquid state at room temperature. Our interest in studying ILs comes from its low toxicity. Some recent studies have shown that the toxicity of the ILs ishigher than believed, in particular for biologically relevant systems. The main goal of this research is to study the influence of the ionic liquid 1-tetradecyl-3-methylimidazolium chloride ([C14MIM][Cl]) with membrane systems. To do so, we made use of different lipids: POPC, Sphingomyelin, Cholesterol, POPG, DPPC, DPPG and DMPC. For each of these systems, the influence of ILs concentration were elucidated by means of a systematic study through different experimental techniques: Small Angle X-ray scattering (SAXS), dynamic light scattering (DLS), fluorescence anisotropy, optical microscopy and z-potential. Since [C14MIM][Cl] has a positive charge on the imidazolium group, the superficial charge of all vesicles increased. For zwitterionic vesicles no significant change in size and melting temperature were noticed. The imidazolium-based ionic liquid diminished the gel-fluid transition temperature for negatively charged lipids. For DPPC:DPPG (1:1), for instance, the transition temperature decreased from 42.50±0.13oC to 25.27±0.33oC and for DPPG from 46.12±0.22 oC to 36.6±0.38 oC. For DPPG, the vesicle hydrodynamic diameter increased from 84±0.1nm to 176±0.1nm, whereas for DPPC:DPPG it increased from 95±0.1nm to 196±0.1nm. The electronic density profile, obtained by SAXS, supported the penetration of the [C14MIM][Cl] into the negative bilayer structure. 15 mol% of [C14MIM][Cl] increased the polar head thickness of DPPC vesicles from 11.1±0.6 Å to 18.0±0.7 Å, without alter significantly the inner region of the membrane.Qualitative results obtained with optical microscopy showed that the IL incorporation destabilize the membrane asymmetry (between the leaflets) leading to the formation of pores (evidenced by optical contrast lost) and the presence of buds. We believe that this work could improve the understanding of the effects of ILs in the presence of biological relevant systems / Os líquidos iônicos (LI) tem atraído grande atenção, tanto da academia quanto da indústria, devido às suas numerosas aplicações. LI são sais, normalmente compostos por um íon orgânico, e um contra-íon que pode ser orgânico ou inorgânico, mas que tem como característica ser encontrado no estado líquido à temperaturas próximas a ambiente. Nosso interesse em estudar LIs vem de sua baixa toxicidade, atribuída a sua baixa volatilidade. Entretanto, alguns estudos recentes mostraram que a toxicidade dos LI é maior do que se acreditava, em particular com sistemas de relevância biológica.O objetivo principal desta dissertação é estudar a influência do líquido iônico 1-tetradecil-3-metilimidazólio cloreto ([C14MIM][Cl]) com sistemas de membrana. Para isso, utilizamos diferentes lipídios, como o POPC, esfingomielina, colesterol, POPG, DPPC,DPPG e o DMPC. Para cada um destes sistemas, a influência da concentração de LI foi elucidada por meio de um estudo sistemático através de diferentes técnicas experimentais, tais como: espalhamento de raio-X a baixos ângulos (SAXS), espalhamento dinâmico de luz (DLS), anisotropia de fluorescência, microscopia óptica e potencial-z. Uma vez que o componente iônico de [C14MIM][Cl] tem uma carga positiva no grupo imidazólio, a carga superficial de todas as vesículas estudadas aqui aumentou. Entretanto, para asvesículas compostas pelos lipídeos zwitteriónicos, não tenha sido observada qualquer alteração significativa no tamanho e na temperatura de transição de fase gel-fluido. O [C14MIM][Cl] altera a organização interna entre as moléculas de lipídio com carga negativa. Consequentemente, à medida que a quantidade de LI aumenta, a temperatura de transição de fase diminui e o tamanho médio das vesículas aumenta. Para o sistema DPPC:DPPG (1:1) a temperatura de transição de fase caiu de 42.50 ± 0.13 oC para 25.27 ± 0.33 oC e para as vesículas de DPPG de 46.12±0.22 oC para 36.6±0.38 oC. Quanto ao diâmetro hidrodinâmico médio, no caso do DPPG este valor aumentou de 84±0.1 nm para 176±0.1 nm, enquanto que para a mistura DPPC:DPPG (1:1) ele passou de 95±0.1nm para 196±0.1nm. Indicando assim que o LI incorpora na bicamada lipídica negativamente carregada. O perfil de densidade eletrônica, obtido por SAXS, confirma a penetração do [C14MIM][Cl] na bicamada lipídica. Diferentemente, para a membrana lipídica zwitteriónica o LI tende a se situar perto da região da cabeça polar sem afetar significativamente a região do interior da bicamada lipídica. Por outro lado, a presença de15 mol% de [C14MIM][Cl] aumenta a espessura da região polar das bicamadas das vesículas de DPPC de ~ 11.1±0.6 Å para ~ 18.0±0.7 Å. Os resultados qualitativos da microscopia óptica mostraram que a incorporação da LI desestabiliza a assimetria da membrana entre as camadas interna e externa, além de sugerir o aparecimento de poros (evidenciado pela perda do contraste ótico das vesículas) e estruturas chamadas de buds. Esperamos que este trabalho melhore a compreensão dos efeitos do LI na presença de organismos biológicos.

Biological membranes are heterogeneous two-dimensional fluids composed of lipids, sterols and proteins that act as complex gateways and define the cell boundary. The functions of these interfaces are diverse and specific to individual organisms, cell types, and tissues. Membranes must take up nutrients and small molecules, release waste products, bind ligands, transmit signals, convert energy, sense the environment, maintain cell adhesion, control cell migration, and much more while forming a tight barrier around the cell. The molecular mechanisms and structural details responsible for this diverse set of functions of biological membranes are still poorly understood, however. Developing new tools capable of probing and determining the local molecular organization, structure, and dynamics of membranes and their components is critical for furthering our knowledge about these important cellular processes that are often linked to health and diseases. Combinatorial microscopy takes advantage of the rich properties of light (intensity, wavelength, polarization, etc.) to create new forms of imaging that quantify the motions, orientations, and binding kinetics of the sample’s biomolecular constituents. These new optical imaging modalities can also be further combined with other types of microscopy to produce spatially correlated micrographs that provide complementary pieces of information about the sample under investigation that would otherwise remain hidden from the observer if the two imaging techniques were applied independently. The first part of this thesis provides a detailed account of the construction of a specialized hybrid microscopy platform that combines polarized total internal reflection fluorescence microscopy (pTIRFM) with atomic force microscopy (AFM) for the purpose of studying fundamental sterol-lipid and antimicrobial peptide-lipid interactions in model membranes. The second half describes a combined pTIRFM and Förster resonance energy transfer (FRET) imaging method to elucidate the oligomeric state and spatial distribution of carcinoembryonic-antigen-related cell-adhesion molecules (CEACAMs) in the membranes of living cells.

Biological membranes are heterogeneous two-dimensional fluids composed of lipids, sterols and proteins that act as complex gateways and define the cell boundary. The functions of these interfaces are diverse and specific to individual organisms, cell types, and tissues. Membranes must take up nutrients and small molecules, release waste products, bind ligands, transmit signals, convert energy, sense the environment, maintain cell adhesion, control cell migration, and much more while forming a tight barrier around the cell. The molecular mechanisms and structural details responsible for this diverse set of functions of biological membranes are still poorly understood, however. Developing new tools capable of probing and determining the local molecular organization, structure, and dynamics of membranes and their components is critical for furthering our knowledge about these important cellular processes that are often linked to health and diseases. Combinatorial microscopy takes advantage of the rich properties of light (intensity, wavelength, polarization, etc.) to create new forms of imaging that quantify the motions, orientations, and binding kinetics of the sample’s biomolecular constituents. These new optical imaging modalities can also be further combined with other types of microscopy to produce spatially correlated micrographs that provide complementary pieces of information about the sample under investigation that would otherwise remain hidden from the observer if the two imaging techniques were applied independently. The first part of this thesis provides a detailed account of the construction of a specialized hybrid microscopy platform that combines polarized total internal reflection fluorescence microscopy (pTIRFM) with atomic force microscopy (AFM) for the purpose of studying fundamental sterol-lipid and antimicrobial peptide-lipid interactions in model membranes. The second half describes a combined pTIRFM and Förster resonance energy transfer (FRET) imaging method to elucidate the oligomeric state and spatial distribution of carcinoembryonic-antigen-related cell-adhesion molecules (CEACAMs) in the membranes of living cells.

Ionic Liquids (ILs) has been attracting attention, both from academia and industry, given the numerous applications of these systems. ILs are salts, usually composed by an organic ion, and a counterion which could be organic or inorganic, and, interestingly they are found at liquid state at room temperature. Our interest in studying ILs comes from its low toxicity. Some recent studies have shown that the toxicity of the ILs ishigher than believed, in particular for biologically relevant systems. The main goal of this research is to study the influence of the ionic liquid 1-tetradecyl-3-methylimidazolium chloride ([C14MIM][Cl]) with membrane systems. To do so, we made use of different lipids: POPC, Sphingomyelin, Cholesterol, POPG, DPPC, DPPG and DMPC. For each of these systems, the influence of ILs concentration were elucidated by means of a systematic study through different experimental techniques: Small Angle X-ray scattering (SAXS), dynamic light scattering (DLS), fluorescence anisotropy, optical microscopy and z-potential. Since [C14MIM][Cl] has a positive charge on the imidazolium group, the superficial charge of all vesicles increased. For zwitterionic vesicles no significant change in size and melting temperature were noticed. The imidazolium-based ionic liquid diminished the gel-fluid transition temperature for negatively charged lipids. For DPPC:DPPG (1:1), for instance, the transition temperature decreased from 42.50±0.13oC to 25.27±0.33oC and for DPPG from 46.12±0.22 oC to 36.6±0.38 oC. For DPPG, the vesicle hydrodynamic diameter increased from 84±0.1nm to 176±0.1nm, whereas for DPPC:DPPG it increased from 95±0.1nm to 196±0.1nm. The electronic density profile, obtained by SAXS, supported the penetration of the [C14MIM][Cl] into the negative bilayer structure. 15 mol% of [C14MIM][Cl] increased the polar head thickness of DPPC vesicles from 11.1±0.6 Å to 18.0±0.7 Å, without alter significantly the inner region of the membrane.Qualitative results obtained with optical microscopy showed that the IL incorporation destabilize the membrane asymmetry (between the leaflets) leading to the formation of pores (evidenced by optical contrast lost) and the presence of buds. We believe that this work could improve the understanding of the effects of ILs in the presence of biological relevant systems / Os líquidos iônicos (LI) tem atraído grande atenção, tanto da academia quanto da indústria, devido às suas numerosas aplicações. LI são sais, normalmente compostos por um íon orgânico, e um contra-íon que pode ser orgânico ou inorgânico, mas que tem como característica ser encontrado no estado líquido à temperaturas próximas a ambiente. Nosso interesse em estudar LIs vem de sua baixa toxicidade, atribuída a sua baixa volatilidade. Entretanto, alguns estudos recentes mostraram que a toxicidade dos LI é maior do que se acreditava, em particular com sistemas de relevância biológica.O objetivo principal desta dissertação é estudar a influência do líquido iônico 1-tetradecil-3-metilimidazólio cloreto ([C14MIM][Cl]) com sistemas de membrana. Para isso, utilizamos diferentes lipídios, como o POPC, esfingomielina, colesterol, POPG, DPPC,DPPG e o DMPC. Para cada um destes sistemas, a influência da concentração de LI foi elucidada por meio de um estudo sistemático através de diferentes técnicas experimentais, tais como: espalhamento de raio-X a baixos ângulos (SAXS), espalhamento dinâmico de luz (DLS), anisotropia de fluorescência, microscopia óptica e potencial-z. Uma vez que o componente iônico de [C14MIM][Cl] tem uma carga positiva no grupo imidazólio, a carga superficial de todas as vesículas estudadas aqui aumentou. Entretanto, para asvesículas compostas pelos lipídeos zwitteriónicos, não tenha sido observada qualquer alteração significativa no tamanho e na temperatura de transição de fase gel-fluido. O [C14MIM][Cl] altera a organização interna entre as moléculas de lipídio com carga negativa. Consequentemente, à medida que a quantidade de LI aumenta, a temperatura de transição de fase diminui e o tamanho médio das vesículas aumenta. Para o sistema DPPC:DPPG (1:1) a temperatura de transição de fase caiu de 42.50 ± 0.13 oC para 25.27 ± 0.33 oC e para as vesículas de DPPG de 46.12±0.22 oC para 36.6±0.38 oC. Quanto ao diâmetro hidrodinâmico médio, no caso do DPPG este valor aumentou de 84±0.1 nm para 176±0.1 nm, enquanto que para a mistura DPPC:DPPG (1:1) ele passou de 95±0.1nm para 196±0.1nm. Indicando assim que o LI incorpora na bicamada lipídica negativamente carregada. O perfil de densidade eletrônica, obtido por SAXS, confirma a penetração do [C14MIM][Cl] na bicamada lipídica. Diferentemente, para a membrana lipídica zwitteriónica o LI tende a se situar perto da região da cabeça polar sem afetar significativamente a região do interior da bicamada lipídica. Por outro lado, a presença de15 mol% de [C14MIM][Cl] aumenta a espessura da região polar das bicamadas das vesículas de DPPC de ~ 11.1±0.6 Å para ~ 18.0±0.7 Å. Os resultados qualitativos da microscopia óptica mostraram que a incorporação da LI desestabiliza a assimetria da membrana entre as camadas interna e externa, além de sugerir o aparecimento de poros (evidenciado pela perda do contraste ótico das vesículas) e estruturas chamadas de buds. Esperamos que este trabalho melhore a compreensão dos efeitos do LI na presença de organismos biológicos.

La BSP1, principale protéine du plasma séminal bovin, interagit avec les membranes des spermatozoïdes et joue un rôle crucial dans les événements qui conduisent à la fécondité des spermatozoïdes, lors du phénomène de la capacitation. Le but de cette recherche est d’investiguer la nature de ces interactions. Ce travail vise à démontrer l’influence des lipides qui composent les membranes sur l’action de la protéine BSP1. À l’aide de la fluorescence intrinsèque de la protéine, l’affinité de la protéine a été caractérisée pour quatre systèmes lipidiques. Les résultats montrent que la composition lipidique affecte significativement l'affinité de la protéine pour les membranes. Nous avons observé l'ordre suivant : 1-palmitoyl-2-oléoyl-sn-glycéro-3-phosphocholine (POPC) > POPC/1-palmitoyl-2-oléoyl-sn-glycéro-3-phosphoéthanolamine (POPE) ≈ POPC/1-palmitoyl-2-oléoyl-sn-glycéro-3-phospho-L-sérine (POPS) > POPC/cholestérol. La protéine interagit préférentiellement avec POPC. La présence de POPE, POPS, ou cholestérol dans la membrane diminue systématiquement l’affinité. Il est connu que la présence de POPE ou cholestérol augmente l’empilement des lipides dans les membranes. Cet effet de condensation des chaînes pourrait être défavorable à l’insertion de la partie hydrophobe de la protéine dans les membranes et réduire ainsi l'affinité. La diminution de l’affinité de la protéine induite par la présence de POPS, un lipide chargé négativement, pourrait être associée aux interactions électrostatiques répulsives car la protéine porte une charge globale négative. La littérature mentionne que la BSP1 extrait sélectivement les phospholipides de type choline et le cholestérol lors de son association avec les membranes de spermatozoïdes. Un efflux lipidique est aussi observé avec des membranes modèles. Nous avons désiré caractériser la « solubilisation » des membranes par la BSP1, par diffusion dynamique de la lumière. Comme étape préliminaire, nous avons étudié comment le détergent Triton X-100 solubilise les membranes en utilisant cette technique. Les mesures démontrent que la composition lipidique des membranes (POPC, POPC/POPE, POPC/1-palmitoyl-2-oléoyl-sn-glycéro-3- [phospho-rac-(1-glycérol)] (POPG)) n’affecte pas le mécanisme général de solubilisation/reconstitution des membranes modèles. Il a été montré qu'il existe trois régions lors des processus de solubilisation pour les différents systèmes lipidiques : i) le détergent se distribue dans les membranes, ii) une coexistence de membranes saturées en détergents et de micelles mixtes de phospholipides/Triton X-100 et iii) exclusivement des micelles mixtes de phospholipides/Triton X-100. Nos résultats montrent que la forme conique de POPE augmente la résistance des membranes à la solubilisation. La présence de POPG, apportant une charge négative à l’interface des membranes, n’induit aucun changement aux processus de solubilisation/reconstitution des membranes par Triton X-100. La diffusion dynamique de la lumière a également permis d’observer si la protéine BSP1 induit des modifications morphologiques des membranes suite à son interaction avec les membranes de POPC. Nos observations n'ont montré aucune variation significative de la taille des particules lors du titrage des vésicules de POPC par la protéine, sur une gamme de rapport molaire de POPC/BSP1 variant de 20 à 0.6. Avec des compositions aussi différentes, on suppose une transition des vésicules saturées en protéine à des complexes de protéine avec un peu de lipides. Cependant, il semble impossible avec la diffusion dynamique de la lumière de différencier ces particules. / BSP1, the main protein in bovine seminal plasma, interacts with sperm membranes and plays a crucial role in events that lead to sperm fertility, during the capacitation. The purpose of this research is to investigate the nature of these interactions. This work aims to demonstrate the influence of the lipids that compose membranes on the action of the BSP1 protein. Using the intrinsic fluorescence of the protein, the affinity of the protein was characterized for four lipid systems. The results show that the lipid composition significantly affects the affinity of the protein for membranes. We observed the following order: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) > POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) ≈ POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) > POPC/cholesterol. The protein interacts preferentially with POPC. The presence of POPE, POPS, or cholesterol in membranes decreases systematically the affinity. It is established that the presence of POPE or cholesterol increases the packing of lipids in membranes. This condensation effect could be detrimental to the insertion of the hydrophobic part of the protein into the membranes and reduces, as a consequence, the affinity. The decrease in protein affinity induced by the presence of POPS, a negatively charged lipid, could be associated with repulsive electrostatic interactions as the protein global charge is negative. The literature mentions that BSP1 selectively extracts choline phospholipids and cholesterol when combined with sperm membranes. A lipid efflux is also observed with model membranes. We characterized membrane "solubilisation" by BSP1, using dynamic light scattering. As a preliminary step, we studied how Triton X-100 detergent solubilizes membranes using this technique. The measurements showed that the lipid composition of the membranes does not affect the general solubilization/reconstitution mechanism of the model membranes (POPC, POPC/POPE, POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1-rac-glycerol) (POPG)). It is known that three different regions exist during the solubilization process for the different lipid systems: i) the detergent is distributed in the membranes, ii) a coexistence of membranes saturated with detergents and mixed phospholipid/Triton X-100 micelles and iii) exclusively mixed phospholipid/Triton X-100 micelles. Our results show that the conical shape of POPE increases the resistance of the membranes to solubilization. The presence of POPG, bringing a negative charge at the membrane interface, does not induce any change in solubilization/reconstitution processes. Dynamic light scattering also made it possible to observe if the BSP1 protein induces morphological changes in the membranes following its interaction with POPC membranes. Our observations showed no significant variation in particle size during the titration of POPC vesicles by the protein, over a molar ratio range of POPC/BSP1 from 20 to 0.6. Considering such different compositions, a transition from vesicles saturated with protein to protein complexes with some lipids is assumed. However, it appeared impossible with dynamic light scattering to differentiate these particles.

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Les principes actifs, dans leur chemin vers leur cible thérapeutique, rencontrent une ou plusieurs membranes biologiques (plasmique, intracellulaire). Les interactions entre un principe actif et ces membranes sont importantes : d’une part les propriétés pharmacocinétiques de la molécule active (transport, distribution, accumulation) en dépendent, d’autre part le principe actif peut modifier les propriétés structurales des membranes. L’étude de ces interactions est rendue difficile par la complexité des membranes en termes de composition (lipidique et protéique) et de structure (hétérogénéité de l’organisation). Par conséquent, l’utilisation de systèmes modèles simplifiés est nécessaire. Au cours de ce travail de thèse nous avons cherché à réaliser des bicouches lipidiques modèles dont les caractéristiques se rapprochaient de celles des membranes biologiques en complexifiant progressivement leur composition lipidique. Nous avons ensuite étudié l’interaction d’une molécule anti-VIH squalénisée, le ddC-SQ, avec nos modèles de membrane.Un des rôles essentiels des membranes biologiques étant de séparer deux milieux aqueux de composition ionique différente, nous avons étudié dans un premier temps l’influence de la nature du milieu d’hydratation sur les propriétés thermiques et structurales des bicouches lipidiques. A pH physiologique, nous avons mis en évidence que seuls les ions divalents (à faibles concentrations) induisaient de profondes modifications structurales en provoquant la formation de vésicules unilamellaires dans les systèmes simples. Une seconde partie de nos travaux a consisté à étudier l’interaction d’un antiviral squalénisé, le ddC-squalène (ddC-SQ), avec nos différentes bicouches modèles. Cet analogue nucléosidique a été associé de manière covalente à une chaîne de squalène afin d’améliorer ses propriétés pharmacocinétiques. Cette squalénisation confère à la molécule la capacité de s’auto-assembler en nanoparticules présentant une structure cubique bicontinue. Les résultats obtenus ont révélé que le principe actif squalénisé interagissait fortement avec les membranes à l’inverse de la molécule native. L’organisation structurale des systèmes modèles est profondément modifiée par l’insertion du ddC-SQ, ce qui pourrait influer sur l’activité du composé. / Drugs must cross one or more biological membranes (plasma membrane, intracellular membrane) to reach their intracellular target. Interactions between drug and membranes play a significant role in the pharmacokinetic properties of drug such as transport, distribution, accumulation. Moreover, drugs may alter membrane properties. The complexity of the composition (protein and lipid) and the structural properties (heterogeneity) of membranes leads to a difficult investigation of these interactions. Consequently, use of simplified model membranes is needed. In this work, model lipid bilayer systems in which the lipid organization mimics the arrangement of lipids in natural membrane have been developed. In this way, the complexity of lipid composition mixtures has been progressively increased. The primary function of membrane is to physically separate aqueous compartments from their surroundings. The intracellular and extracellular fluids differ in ionic composition. This study firstly consists to estimate the influence of aqueous medium nature on the thermodynamic and structural properties of these model membranes.In physiological conditions (pH 7.4, ionic strength 150 mM), the most significant change was obtained in the presence of divalent ions. Markedly change in lipid organization was observed and the formation of unilamellar vesicles has been evidenced (at low concentrations) in simple model bilayers. Interactions of an antiretroviral nucleoside analogue, the SQddC, with lipid systems constitute the second part of our work. Squalene has been covalently coupled to ddC, in order to improve its therapeutic index. Squalenoylation leads to amphiphilic prodrugs which self-organize as nanoparticles. ddC weakly interacts with lipid membranes while SQddC-SQ can insert into membranes between hydrophobic alkyl chains and induce disruption of lipid organization. Consequently, the efficacy and/or toxicity of this drug could change.