763 research outputs found
Smart hybrid nanomaterials for biomimetic membranes
This thesis focuses on the preparation of nanomaterials made of proteins and polymers. Even though the technology has advanced in the last decades to design new devices at the atomic scale, researchers are still inspired by what Nature has produced and optimized for millions of years. Following this concept, this work uses proteins forming water-filled channels, called porins, which regulate the flow of ions and biomolecules in cellular life. Two proteins were studied: Omp2a and VDAC36. The first part of the dissertation is the thermomechanical properties study of the latest hybrid membrane developed by the IMEM group: an thin nanoperforated poly(lactic acid) (PLA) film with the Omp2a porin immobilized onto the surface . For this purpose, a new equipment based on the microcantilever technology was used. The SCAnning LAser analyzer (SCALA) characterizes the coated cantilevers which allows the following of the cantilever bending induced by the compression/expansion of the sample coating (i.e. proteins or polymers). In this study, the intermolecular reorganization of Omp2a aggregates was evidenced as well as the protein secondary structure stability against temperature. The same method was employed to study the impact of nanofeatures (perforations and drugs domains) on films of PLA. They affected the glass transition and the cold crystallization temperatures. The changes were dependent on the size and abundance of the nanofeatures, which can modulate the properties of future materials. Moreover, this work established a protocol for the study of biomolecules and polymers attached to microcantilevers, allowing an accurate study of the thermomechanical properties using very low amounts of sample.
The second part of the thesis is the development of new hybrid nanomaterials composed of VDAC36, PLA and poly(3,4-ethylenedioxythiophene) (PEDOT). An efficient protocol was established for the production of VDAC36 and its subsequent refolding was achieved. The beta-barrel nature of the protein was revealed and its tendency to form oligomers was demonstrated. Finally, the size of the protein inner channel could be determined. The VDAC36 was added to the polymer material made of three alternating layers of PLA and PEDOT. The electrical properties of the material were modified by the addition of the protein: the overall resistance was reduced and the supercapacitive behaviour was enhanced. The description of the electrical equivalent circuit also revealed that the protein induced the diffusion of ions. To improve the material, the number of layers was increased and the conducting polymer was modified by incorporating a monomer bearing a dodecyl chain. The modifications were proved useful as the protein content and the electrical properties increased. Finally, the new hybrid material could provide an adaptive electrical response according to the concentration of biomolecules.Esta tesis se centra en la preparación de nanomateriales basados en proteínas y polímeros. A pesar de los avances realizados en las últimas décadas en el diseño de nuevos dispositivos a escala nanométrica, los investigadores aún se inspiran en lo que la Naturaleza ha producido y ha optimizado durante millones de años. A partir de esta premisa, en este trabajo se han usado proteínas, que constituyen canales de agua y cuya función es regular el paso de iones y biomoléculas en organismos celulares. Las proteínas involucradas son Omp2a y VDAC36. La primera parte de esta disertación se centra en el estudio de las propiedades termo-mecánicas de los componentes una novedosa membrana híbrida desarrollada per el grupo IMEM: una película ultra-delgada de ácido poli(láctico) (PLA) nano-perforada y funcionalizada en la superficie con moléculas de Omp2a. Para su caracterización se usó un nuevo equipo basado en la tecnología de micro-palancas. Un analizador laser de barrido (SCALA, el acrónimo de dicho aparato en inglés) permite caracterizar palancas recubiertas de muestra polimérica mediante la reflexión de un rayo de luz láser sobre la superficie del soporte revestido. Mediante su acoplado a una cámara termo-controlada, SCALA permite seguir la deformación del soporte inducida per la compresión/expansión de la muestra en forma de recubrimiento (ya sean polímeros como proteínas). Mediante esta técnica se evidenció la reorganización intermolecular en agregados de la proteína Omp2a, así como la alta estabilidad de su estructura secundaria en frente de la temperatura. El mismo método fue usado para estudiar el impacto de las nano-características sobre las películas de PLA. Nano-poros, nano-perforaciones y nano-dominios fueron añadidos a los films de PLA. Dichas modificaciones afectan tanto a su transición vítrea como a la cristalización en frío de dichas películas. Los cambios observados dependen del tamaño y la abundancia de las nano-modificaciones, lo cual va a permitir modular las propiedades de futuros nano-materiales. Más aún, este trabajo ha establecido las bases para un protocolo general de uso de micro-palancas para estudiar proteínas y polímeros unidos a ellas, permitiendo la caracterización de sus propiedades termo-mecánicas usando cantidades ínfimas de material. Se pudo establecer un protocolo eficiente para la producción de VDAC36 i su subsecuente re-naturalización por medio de una combinación de detergentes y alcoholes. Per medio de experimentos de dicroísmo circular se puso de manifiesto su naturaleza de barril beta y se mostró su tendencia a formar oligómeros mediante entrecruzamientos químicos. El tamaño del poro se pudo determinar mediante ensayos de hinchado. A continuación, VDAC36 se incorporó al material polimérico constituido por tres capas de polímero, alternando PLA y PEDOT. Las propiedades eléctricas de este material quedaron visiblemente modificadas por la adición de la proteína sobre los films de polímero: se redujo su resistancia mientras que su comportamiento como supercondensador, consecuencia la presencia de PEDOT, aumentó. La descripción del circuito eléctrico equivalente reveló a su vez que la proteína inducía la difusión de iones. Para mejorar la retención de proteínas y la integridad mecánica del material, las capas de polímero de la membrana se aumentaron hasta cinco. A su vez, el monómero de EDOT se modificó para incorporar una cadena de dodecilo y poder así imitar una membrana celular. Estas últimas modificaciones se mostraron de gran utilidad puesto que el contenido en proteína aumentó y los cambios eléctricos se hicieron más pronunciados. Finalmente, este nuevo material híbrido fue capaz de proporcionar una respuesta eléctrica adaptativa como respuesta a cambios en la concentración de biomoléculas.Postprint (published version
3D structure of a Brucella melitensis porin: molecular modelling in lipid membranes
Brucella melitensis is a pathogenic bacterium responsible for brucellosis in mammals and humans. Its outer membrane proteins (Omp) control the diffusion of solutes through the membrane, and they consequently have a crucial role in the design of diagnostics and vaccines. Moreover, such proteins have recently revealed their potential for protein-based biomaterials. In the present contribution, the structure of the B. melitensis porin Omp2a is built using the RaptorX threading method. This is a 16-stranded ß-barrel with an a-helix on the third loop folding inside the barrel and forming the constriction zone of the channel, a typical feature of general porins such as PhoE and OmpF. The preferential diffusion of cations over anions experimentally observed in anterior studies is evidenced by the presence of distinct clusters of charges in the extracellular loops and in the inner pore. Docking studies support the previously reported hypothesis of Omp2a ability to aid maltotetraose diffusion. The monomer model is then assembled into a homotrimer, stabilized by the L2 loop involved in most of the interface interactions. The stability of the trimer is evaluated in three bilayers: pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and a mixture of 1:1 of POPC/POPE. All-atom molecular dynamics simulations demonstrate the ß-barrel-structural stability over time even though a breathing-like motion is observed. Compared to the pure bilayers, the POPC/POPE better preserves the integrity of the protein and its channel. Overall, this work demonstrates the relevancy of the Omp2a model and will help to design new therapeutic agents and bioinspired nanomaterialsPeer ReviewedPostprint (author's final draft
Free-standing flexible and biomimetic hybrid membranes for ions and ATP transport
The transport of metabolites across robust, flexible and free-standing biomimetic membranes made of three perforated poly (lactic acid) (pPLA) layers, separated by two anodically polymerized conducting layers of poly (3,4-ethylenedioxythiophene-co-3-dodecylthiophene), and functionalized on the external pPLA layers with a voltage dependent anion channel (VDAC) protein, has been demonstrated. The three pPLA layers offer robustness and flexibility to the bioactive platform and the possibility of obtaining conducing polymer layers by in situ anodic polymerization. The incorporation of dodecylthiophene units, which bear a 12 carbon atoms long linear alkyl chain, to the conducting layers allows mimicking the amphiphilic environment offered by lipids in cells, increasing 32% the efficiency of the functionalization. Electrochemical impedance measurements in NaCl and adenosine triphosphate (ATP) solutions prove that the integration of the VDAC porin inside the PLA perforations considerably increases the membrane conductivity and is crucial for the electrolyte diffusion. Such results open the door for the development of advanced sensing devices for a broad panel of biomedical applicationsPeer ReviewedPostprint (author's final draft
Structural and functional characterization of Solanum tuberosum VDAC36
As it forms water-filled channel in the mitochondria outer membrane and diffuses essential metabolites such as NADH and ATP, the voltage-dependent anion channel (VDAC) protein family plays a central role in all eukaryotic cells. In comparison with their mammalian homologues, little is known about the structural and functional properties of plant VDACs. In the present contribution, one of the two VDACs isoforms of Solanum tuberosum , stVDAC36, has been successfully overexpressed and refolded by an in-house method, as demonstrated by the information on its secondary and tertiary structure gathered from circular dichroism and intrinsic fluorescence. Cross-linking and molecular modeling studies have evidenced the presence of dimers and tetramers, and they suggest the formation of an intermolecular disulfide bond between two stVDAC36 monomers. The pore-forming activity was also assessed by liposome swelling assays, indicating a typical pore diameter between 2.0 and 2.7 nm. Finally, insights about the ATP binding inside the pore are given by docking studies and electrostatic calculations.Peer ReviewedPostprint (author's final draft
Nanofeatures affect the thermal transitions of polymer thin films:a microcantilever-based investigation
Nanofeatures affect the thermal transitions of polymer thin films:a microcantilever-based investigation
Thermomechanical Response of a Representative Porin for Biomimetics
The thermomechanical response of Omp2a, a representative porin used for the fabrication of smart biomimetic nanomembranes, has been characterized using microcantilever technology and compared with standard proteins. For this purpose, thermally induced transitions involving the conversion of stable trimers to bigger aggregates, local reorganizations based on the strengthening or weakening of intermolecular interactions, and protein denaturation have been detected by the microcantilever resonance frequency and deflection as a function of the temperature. Measurements have been carried out on arrays of 8-microcantilevers functionalized with proteins (Omp2a, lysozyme and bovine serum albumin). To interpret the measured nanofeatures, the response of proteins to temperature has been also examined using other characterization techniques, including real time wide angle X-ray diffraction. Results not only demonstrate the complex behavior of porins, which exhibit multiple local thermal transitions before undergoing denaturation at temperatures higher than 105 °C, but also suggest a posttreatment to control the orientation of immobilized Omp2a molecules in functionalized biomimetic nanomembranes and, thus, increase their efficacy in ion transport.Peer Reviewe
Self-standing, conducting and capacitive biomimetic hybrid nanomembranes for selective molecular ion separation
Hybrid free-standing biomimetic materials are developed by integrating the VDAC36 ß-barrel protein into robust and flexible three-layered polymer nanomembranes. The first and third layers are prepared by spin-coating a mixture of poly(lactic acid) (PLA) and poly(vinyl alcohol) (PVA). PVA nanofeatures are transformed into controlled nanoperforations by solvent-etching. The two nanoperforated PLA layers are separated by an electroactive layer, which is successfully electropolymerized by introducing a conducting sacrificial substrate under the first PLA nanosheet. Finally, the nanomaterial is consolidated by immobilizing the VDAC36 protein, active as an ion channel, into the nanoperforations of the upper layer. The integration of the protein causes a significant reduction of the material resistance, which decreases from 21.9 to 3.9 kO cm2. Electrochemical impedance spectroscopy studies using inorganic ions and molecular metabolites (i.e.L-lysine and ATP) not only reveal that the hybrid films behave as electrochemical supercapacitors but also indicate the most appropriate conditions to obtain selective responses against molecular ions as a function of their charge. The combination of polymers and proteins is promising for the development of new devices for engineering, biotechnological and biomedical applications.Hybrid free-standing biomimetic materials are developed by integrating the VDAC36 ß-barrel protein into robust and flexible three-layered polymer nanomembranes. The first and third layers are prepared by spin-coating a mixture of poly(lactic acid) (PLA) and poly(vinyl alcohol) (PVA). PVA nanofeatures are transformed into controlled nanoperforations by solvent-etching. The two nanoperforated PLA layers are separated by an electroactive layer, which is successfully electropolymerized by introducing a conducting sacrificial substrate under the first PLA nanosheet. Finally, the nanomaterial is consolidated by immobilizing the VDAC36 protein, active as an ion channel, into the nanoperforations of the upper layer. The integration of the protein causes a significant reduction of the material resistance, which decreases from 21.9 to 3.9 kO cm2. Electrochemical impedance spectroscopy studies using inorganic ions and molecular metabolites (i.e.L-lysine and ATP) not only reveal that the hybrid films behave as electrochemical supercapacitors but also indicate the most appropriate conditions to obtain selective responses against molecular ions as a function of their charge. The combination of polymers and proteins is promising for the development of new devices for engineering, biotechnological and biomedical applications.Peer ReviewedPostprint (author's final draft
Les droits disciplinaires des fonctions publiques : « unification », « harmonisation » ou « distanciation ». A propos de la loi du 26 avril 2016 relative à la déontologie et aux droits et obligations des fonctionnaires
The production of tt‾ , W+bb‾ and W+cc‾ is studied in the forward region of proton–proton collisions collected at a centre-of-mass energy of 8 TeV by the LHCb experiment, corresponding to an integrated luminosity of 1.98±0.02 fb−1 . The W bosons are reconstructed in the decays W→ℓν , where ℓ denotes muon or electron, while the b and c quarks are reconstructed as jets. All measured cross-sections are in agreement with next-to-leading-order Standard Model predictions.The production of , and is studied in the forward region of proton-proton collisions collected at a centre-of-mass energy of 8 TeV by the LHCb experiment, corresponding to an integrated luminosity of 1.98 0.02 \mbox{fb}^{-1}. The bosons are reconstructed in the decays , where denotes muon or electron, while the and quarks are reconstructed as jets. All measured cross-sections are in agreement with next-to-leading-order Standard Model predictions
Physics case for an LHCb Upgrade II - Opportunities in flavour physics, and beyond, in the HL-LHC era
The LHCb Upgrade II will fully exploit the flavour-physics opportunities of the HL-LHC, and study additional physics topics that take advantage of the forward acceptance of the LHCb spectrometer. The LHCb Upgrade I will begin operation in 2020. Consolidation will occur, and modest enhancements of the Upgrade I detector will be installed, in Long Shutdown 3 of the LHC (2025) and these are discussed here. The main Upgrade II detector will be installed in long shutdown 4 of the LHC (2030) and will build on the strengths of the current LHCb experiment and the Upgrade I. It will operate at a luminosity up to 2×1034
cm−2s−1, ten times that of the Upgrade I detector. New detector components will improve the intrinsic performance of the experiment in certain key areas. An Expression Of Interest proposing Upgrade II was submitted in February 2017. The physics case for the Upgrade II is presented here in more depth. CP-violating phases will be measured with precisions unattainable at any other envisaged facility. The experiment will probe b → sl+l−and b → dl+l− transitions in both muon and electron decays in modes not accessible at Upgrade I. Minimal flavour violation will be tested with a precision measurement of the ratio of B(B0 → μ+μ−)/B(Bs → μ+μ−). Probing charm CP violation at the 10−5 level may result in its long sought discovery. Major advances in hadron spectroscopy will be possible, which will be powerful probes of low energy QCD. Upgrade II potentially will have the highest sensitivity of all the LHC experiments on the Higgs to charm-quark couplings. Generically, the new physics mass scale probed, for fixed couplings, will almost double compared with the pre-HL-LHC era; this extended reach for flavour physics is similar to that which would be achieved by the HE-LHC proposal for the energy frontier
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