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Polyelectrolyte Building Blocks for Nanotechnology: Atomic Force Microscopy Investigations of Polyelectrolyte-Lipid Interactions, Polyelectrolyte Brushes and Viral CagesCuéllar Camacho, José Luis 26 July 2013 (has links) (PDF)
The work presented here has a multidisciplinary character, having as a common factor the characterization of self-assembled nanostructures through force spectroscopy. Exploring AFM as a tool for characterizing self-assembly and interaction forces in soft matter nanostructures, three different Bio and nonbiological systems where investigated, all of them share the common characteristic of being soft matter molecular structures at the nanoscale. The studied systems in question are: a) Polyelectrolyte – lipid nanocomposites. Single polyelectrolyte adsorption-desorption from supported lipid bilayers, b) Polyelectrolyte brushes and c) Virus-Like particles (VLPs). The scientific interest and industrial applications for each of these different nanostructures is broad, and their potential uses in the near future ranges from smart nanocontainers for drug and gene delivery, surface platforms for molecular recognition to the development of new nanodevices with ultrasensitive external stimuli responsiveness. These nano-structures are constructed following assembly of smaller subunits and belong to representative examples of soft matter in modern nanotechnology.
The stability, behavior, properties and long term durability of these self-organized structures depends strongly on the environmental conditions to which they are exposed since their building mechanism is a balance between attractive noncovalent interactions and momentum transmitted collisions due Brownian motion of the solvent molecules. For example a set of long chain molecules firmly attached to one end to a surface will alter their conformation as the space between them is reduced or the environmental conditions are modified (i.e. ionic strength, pH or temperature). For a highly packed condition, this fuzzy surface known as a polyelectrolyte brush will then behave as a responsive material with tunable responsiveness.
Thus the objective in the present case was to investigate the change in morphology and the mechanical response of a polyelectrolyte brush to external forces by application of AFM nanoindentations under different ionic strength conditions. The degree of penetration of the AFM tip through the brush will provide insights into the forces exerted by the brush against the tip. Compressions on the brush should aid to characterize its changes in compressibility for different salt concentrations.
For the second chosen system, the interaction between two assembled interfaces was investigated at the single molecular level. A multilayered film formed by the consecutive assembly of oppositely charged polyelectrolytes and subsequently coated with a lipid membrane represents a fascinating soft composite material resembling more than a few structural components emerging in living organisms. The fluid bilayer, thus provide a biocompatible interface where additional functionalities can further be integrated (fusion peptides for instance). The smooth polymer cushion confers not only structural flexibility but also adaptability of the chosen substrate properties to be coated. This type of interface could be useful in the development of novel molecular biosensors with single molecule recognition capacities or in the fabrication of assays against pathogenic agents. The aim of this project was to study the molecular binding mechanism between the last polyelectrolyte layer and the lipid head group of the lower lipid leaflet. Understanding this adsorption mechanism between both interfaces, should likewise contribute to improve the fabrication of lipid coated polymeric nano/micro capsules with targeting properties. For example this could be critical in the field of nonviral gene therapy, where the improvement in the design of condensates of nucleic acids and other polymers with lipids (lipoplexes) are of main interest for its posterior use as delivery vectors.
Finally, viral capsids were investigated. These naturally occurring assembled nanocontainers within living organisms stand for a remarkable example of nature’s morphological designs. These structures self-assemble from a small number of different proteins occurring in identical copies. The capsid as a self-assembled structure carries multiple functions: compaction of the genome, protection against external chemical threats, target recognition, structural support and finally facilitating the release of the genome into the host cell. It is highly interesting how these different functions are organized within the capsid which consists, for example, in the case of the norovirus of 180 identical copies of one single protein.
Therefore, the mechanical stability and elastic properties of virus-like particles of Rubella and Norovirus were investigated by external application of loading forces with an AFM tip. The measurements were performed under conditions relevant for the virus infection mechanism. The applied compressions on these protein shells at pH values mimicking the virus life cycle will aid to learn about possible internal transitions among proteins which may be important for switching between the various functions of the capsid. The choice of two unrelated viral systems with different entry pathways into the cell and with different morphological architectures is expected to reveal crucial information about the stability and mechanical resistance to deformation of these empty membrane-coated and bare viral capsids. This last might provide clues on the stage of particle disassembly and cargo release during the final step of the infection process.
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Norovirus translation and replicationLu, Jia January 2018 (has links)
Human norovirus (HuNoV) is the leading cause of gastroenteritis worldwide. Despite the significant disease and economic burden, currently there are no licensed vaccines or antivirals. The understanding of norovirus biology has been hampered by the inability to cultivate HuNoV in cell culture. To establish a tissue culture system, infectious HuNoVs were purified from clinical stool samples. HuNoV replication was tested in different cell types. The B-cell and intestinal organoids culture systems were validated. In addition, using organoids culture a DNA-based reverse genetic system was shown to recover infectious HuNoV. Due to the challenges associated with cultivating HuNoV, murine norovirus (MNV) was used as a surrogate system to understand the role of eIF4E phosphorylation in norovirus pathogenesis, and VP1-RdRp interaction in regulating viral genome replication. MNV infection results in the phosphorylation of the translation initiation factor eIF4E, re-programming host-cell translation during infection. Inhibiting eIF4E phosphorylation reduces MNV replication in cell culture suggesting a role in viral replication. A mouse model with eIF4E S209A, a phosphor-ablative mutation, was established to understand the role of eIF4E phosphorylation in MNV pathogenesis. In vitro and in vivo characterisations demonstrated that eIF4E phosphorylation may have multiple roles in norovirus-host interactions, but overall has little impact on MNV pathogenesis. The shell domain (SD) of norovirus major capsid protein VP1 interacts with viral RNA-dependent RNA polymerase (RdRp) in a genogroup-specific manner to enhance de novo initiation of RdRp, and to promote negative-strand RNA synthesis. To understand how VP1 regulates norovirus genome replication, chimeric MNVs with genogroup-specific residues mutagenised were characterised in vitro and in vivo. A single amino acid mutation was shown to destabilise viral capsid. SDs with reduced VP1-RdRp interaction showed less capacity to stimulate RdRp, resulting in delayed virus replication. In vivo, the replication of an MNV-3 with homologous mutations was abolished, highlighting the crucial role of this interaction.
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Polyelectrolyte Building Blocks for Nanotechnology: Atomic Force Microscopy Investigations of Polyelectrolyte-Lipid Interactions, Polyelectrolyte Brushes and Viral CagesCuéllar Camacho, José Luis 30 January 2013 (has links)
The work presented here has a multidisciplinary character, having as a common factor the characterization of self-assembled nanostructures through force spectroscopy. Exploring AFM as a tool for characterizing self-assembly and interaction forces in soft matter nanostructures, three different Bio and nonbiological systems where investigated, all of them share the common characteristic of being soft matter molecular structures at the nanoscale. The studied systems in question are: a) Polyelectrolyte – lipid nanocomposites. Single polyelectrolyte adsorption-desorption from supported lipid bilayers, b) Polyelectrolyte brushes and c) Virus-Like particles (VLPs). The scientific interest and industrial applications for each of these different nanostructures is broad, and their potential uses in the near future ranges from smart nanocontainers for drug and gene delivery, surface platforms for molecular recognition to the development of new nanodevices with ultrasensitive external stimuli responsiveness. These nano-structures are constructed following assembly of smaller subunits and belong to representative examples of soft matter in modern nanotechnology.
The stability, behavior, properties and long term durability of these self-organized structures depends strongly on the environmental conditions to which they are exposed since their building mechanism is a balance between attractive noncovalent interactions and momentum transmitted collisions due Brownian motion of the solvent molecules. For example a set of long chain molecules firmly attached to one end to a surface will alter their conformation as the space between them is reduced or the environmental conditions are modified (i.e. ionic strength, pH or temperature). For a highly packed condition, this fuzzy surface known as a polyelectrolyte brush will then behave as a responsive material with tunable responsiveness.
Thus the objective in the present case was to investigate the change in morphology and the mechanical response of a polyelectrolyte brush to external forces by application of AFM nanoindentations under different ionic strength conditions. The degree of penetration of the AFM tip through the brush will provide insights into the forces exerted by the brush against the tip. Compressions on the brush should aid to characterize its changes in compressibility for different salt concentrations.
For the second chosen system, the interaction between two assembled interfaces was investigated at the single molecular level. A multilayered film formed by the consecutive assembly of oppositely charged polyelectrolytes and subsequently coated with a lipid membrane represents a fascinating soft composite material resembling more than a few structural components emerging in living organisms. The fluid bilayer, thus provide a biocompatible interface where additional functionalities can further be integrated (fusion peptides for instance). The smooth polymer cushion confers not only structural flexibility but also adaptability of the chosen substrate properties to be coated. This type of interface could be useful in the development of novel molecular biosensors with single molecule recognition capacities or in the fabrication of assays against pathogenic agents. The aim of this project was to study the molecular binding mechanism between the last polyelectrolyte layer and the lipid head group of the lower lipid leaflet. Understanding this adsorption mechanism between both interfaces, should likewise contribute to improve the fabrication of lipid coated polymeric nano/micro capsules with targeting properties. For example this could be critical in the field of nonviral gene therapy, where the improvement in the design of condensates of nucleic acids and other polymers with lipids (lipoplexes) are of main interest for its posterior use as delivery vectors.
Finally, viral capsids were investigated. These naturally occurring assembled nanocontainers within living organisms stand for a remarkable example of nature’s morphological designs. These structures self-assemble from a small number of different proteins occurring in identical copies. The capsid as a self-assembled structure carries multiple functions: compaction of the genome, protection against external chemical threats, target recognition, structural support and finally facilitating the release of the genome into the host cell. It is highly interesting how these different functions are organized within the capsid which consists, for example, in the case of the norovirus of 180 identical copies of one single protein.
Therefore, the mechanical stability and elastic properties of virus-like particles of Rubella and Norovirus were investigated by external application of loading forces with an AFM tip. The measurements were performed under conditions relevant for the virus infection mechanism. The applied compressions on these protein shells at pH values mimicking the virus life cycle will aid to learn about possible internal transitions among proteins which may be important for switching between the various functions of the capsid. The choice of two unrelated viral systems with different entry pathways into the cell and with different morphological architectures is expected to reveal crucial information about the stability and mechanical resistance to deformation of these empty membrane-coated and bare viral capsids. This last might provide clues on the stage of particle disassembly and cargo release during the final step of the infection process.
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Théorie de Landau de cristallisation et l'approche d'ondes de densité dans les systèmes complexes / Landau theory of crystallization and density waves approach in complex systemsKonevtsova, Olga 29 November 2013 (has links)
Le nombre croissant de nanostructures physiques et biologiques sont caractérisées par l'ordre non-cristallin et par les propriétés physiques et biologiques non-conventionnels. Parmi ses systèmes il faut distinguer les capsides virales. Ces coquilles solides qui sont formées par un certain nombre dec opies de la même protéine protègent le virus des agressions et facilitent le processus d'infection de la cellule hôte. La distribution des positions de protéines dans une capside est très régulière et montre un degré très élevé d'ordre, aussi bien orientationnel que positionnel. Les capsides virales de topologie sphérique possèdent la symétrie icosaédrique compatible avec l'ordre cristallin local, mais incompatible avec la symétrie cristalline globale et interdite dans les structures périodiques.Ici, sur l'exemple des Papovavirus, nous montrons l'existence d'un nouveau type d'organisation qui résulte dans l'ordre quasicristallin pentagonal chiral de protéines dans des capsides de topologie sphérique et géométrie dodécaédrique. La formation de cet ordre est décrite dans le cadre de la théorie de Landau de cristallisation. Les particularités de la structure sont élucidées grâce à la théorie d'élasticité des quasicristaux comme le résultat de la déformation phason nonlinéaire.La généralisation de la théorie de Landau de cristallisation que nous proposons permet également de décrire des structures quasicristallines octogonales et décagonales grâce à la minimisation contrainte de l'énergie libre, et donne un nouveau sens physique à la notion de « fenêtre de projection » utilisée dans la cristallographie multidimensionnelle. / A growing number of physical and biological nanostructures are characterized by non-crystallineorder and by unconventional physical and biological properties. Among these systems one can distinguish viral capsids. These solid shells formed by a certain number of copies of the same protein protect viruses from aggressions and facilitate infection of the host cell. Protein distributionin a capsid is quite regular and shows high degree of order, both orientational and positional. Viral capsids with spherical topology have icosahedral symmetry compatible with local crystalline orderbut incompatible with the global one and forbidden in periodic structures.Here, on the example of Papovaviruses we show the existence of a new type of organization whichresults in the chiral pentagonal quasicrystalline order of proteins in capsids with spherical topology and dodecahedral geometry. The formation of this order is described in the frame of the Landau theory of crystallization. The theory of elasticity of quasicrystals is used to show that the structure peculiarities result from the non-linear phason strain.Generalization of the proposed Landau theory of crystallization allows us to describe octagonal and decagonal quasicrystalline structures using constrained minimization of the free energy, thus giving a new physical sense to the « projection window » notion used in multi-dimensionalcrystallography.
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