Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli O157:H7 (EHEC) form characteristic lesions on infected mammalian cells called actin pedestals. Each of these two pathogens injects its own translocated intimin receptor (Tir) molecule into the plasma membranes of host cells. Interaction of translocated Tir with the bacterial outer membrane protein intimin is required to trigger the assembly of actin into focused pedestals beneath bound bacteria. Despite similarities between the Tir molecules and the host components that associate with pedestals, recent work indicates that EPEC and EHEC Tir are not functionally interchangeable. For EPEC, Tir-mediated binding of Nck, a host adaptor protein implicated in actin signaling, is both necessary and sufficient to initiate actin assembly. In contrast, for EHEC, pedestals are formed independently of Nck, and require translocation of bacterial factors in addition to Tir to trigger actin signaling.

It has been well documented that mRNA is associated with the cytoskeleton, and that this relationship is involved in translation and mRNA sorting. The molecular components involved in the attachment of mRNA to the cytoskeleton are only poorly understood. The objective of this thesis was to directly visualize the interaction of mRNA with the cytoskeleton, with sufficient resolution to identify the filament systems and structures involved. This work required the development of novel in situ hybridization methods for use with electron microscopy. This allowed resolution to visualize single mRNA molecules and individual filaments. The development of a silver enhancement methodology for both the light and electron microscopic detection of biotinated oligo-dT probes permitted a synoptic view of the intracellular distribution of poly(A) mRNA. At the light microscope, the distribution of poly(A) mRNA did not resemble the individual distribution patterns of microfilaments, intermediate filaments or microtubules. Ultrastructural examination revealed that poly(A) mRNA was not uniformly distributed along cytoskeletal filaments, but clustered at their intersections. The composition of these mRNA containing structures was investigated by both morphologic and in situ hybridization analysis using antibodies to cytoskeletal proteins. In thin sections, polysomes were observed attached to both microfilaments and intermediate filaments. To permit the simultaneous detection of oligo-dT hybridization and specific cytoskeletal proteins, a double labelling method using colloidal gold conjugated antibodies was developed. The majority of poly(A) mRNA was associated with the actin cytoskeleton, with 72% of the hybridization localized within 5nm of a labelled microfilament. Within the actin cytoskeleton, poly(A) mRNA was localized to intersections of orthogonal networks. Greater than 50% of poly(A) colocalized with the actin crosslinking proteins, filamin and α-actinin, but not vinculin. A significant amount of poly(A) mRNA was found to be associated with intermediate filaments. The double label gold analysis demonstrated that 33% of the hybridization signal localized within 5nm of labelled vimentin filaments. Prior disorganization of the actin cytoskeleton using cytochalasin did not disrupt the association of mRNA with vimentin. These observations are consistent with our morphologic results of polysome-intermediate filament associations, and indicate that microfilaments are not the only filament system to which mRNA is bound. Furthermore, a small amount of hybridization signal (12%) consistently was observed along microtubules, providing an additional cytoskeletal network to distribute mRNA. To further characterize the spatial organization of mRNA within the cytoskeleton, ultrastructural methods were developed to directly visualize individual mRNA molecules. First, oligonucleotide probes chemically modified with a single hapten and directly conjugated primary reagents were used to permit detection of an individual hybridized probe molecule by a single gold particle. Second, biotin and digoxigenin labelled oligonucleotide probes were used to simultaneously visualize the intermolecular and intramolecular relationships of two nucleic acid sequences. Third, reverse transcriptase was used to extend hybridized primers in situ which permitted visualization of the poly(A) sequence concomittant with the conformation of an mRNA molecule. These methods have permitted analysis of how single mRNA molecules may be positioned with respect to each other within the cytoskeleton. The ultrastructural visualization of mRNA within its structural environment has demonstrated heterogeneous interactions with the cytoskeleton. Future work will be needed to further characterize the mechanism of mRNA attachment. The proteins which bridge nucleic acid sequences to specific intersections can be identified. It will be interesting to learn how the identified mRNA-cytoskeletal interactions might be involved in the regulation of both mRNA translation and intracellular location. Lastly, and perhaps the most challenging goal, is to investigate whether the identified mRNA-cytoskeletal interactions are used by the cell to influence its own shape, polarity and architecture.

Enteropathogenic Escherichia coli (EPEC) is one of many bacterial and viral pathogens that can exploit the eukaryotic actin cytoskeleton for its own purposes. EPEC injects its own receptor, Tir, into the host cell plasma membrane where, upon binding the bacterial adhesin, intimin, can trigger actin assembly beneath bound bacteria resulting in characteristic actin "pedestals". The formation of these lesions is thought to be critical for bacterial colonization; and can also provide insight into actin dynamics of mammalian cells. EPEC Tir stimulates multiple signaling pathways converging on a central actin nucleation promoting factor, N-WASP. The best-characterized pathway of actin pedestal formation also involves the eukaroytic adaptor protein, Nck, but at least two Nck-independent signaling cascades have also been identified. Multiple aspects of Tir-mediated signaling cascades remain unclear. For example, although Nck can directly bind and activate N-WASP, current models of Tir-mediated, Nck-dependent actin signaling postulate an indirect interaction between Nck and N-WASP mediated by one or more unidentified host factors. Additionally, the relationship of this pathway to the Nck-independent pathways is unknown. To better understand Tir-mediated actin assembly, a detailed and quantitative analysis of the domain requirements of Nck and N-WASP for pedestal formation was conducted. The results indicate that, contrary to previously favored models, Nck is unlikely to require additional host factors to bind N-WASP during pedestal formation, but apparently directly stimulates this nucleation promoting factor. In addition, the results show that the Nck-dependent and -independent pathways target distinct regulatory domains of N-WASP.

Deux types de matériaux membranaires ont été développés pour la séparation du CO2. Les premiers associent un matériau polymère de référence (Pebax®) à de nouveaux additifs pseudopeptidiques bioconjugués. Ces bioconjugués pseudopeptide-polymère ont été obtenus par une stratégie de synthèse de type « grafting-to » à partir de pseudopeptides 1:1[α/α-Nα-Bn-hydrazino] dimère et tétramère fonctionnalisés par une fonction alcyne. La partie oligomérique poly(acrylate de diéthylène glycol) (PEDEGA) a été synthétisée de façon contrôlée par « Single Electron Transfer Living Radical Polymerization » (SET-LRP) à partir d’un amorceur azido fonctionnel permettant un couplage direct par chimie « click » CuAAC. L'influence de ces additifs sur les propriétés de sorption et de séparation du CO2 a été analysée en termes de relations propriétés-morphologie-structure. Ces additifs originaux ont permis d’augmenter les performances de séparation du CO2 de la membrane référence ; en améliorant de 46% la perméabilité au CO2, tout en maintenant de bonnes sélectivités αCO2/N2 = 44 et αCO2/CH4 = 13. Par ailleurs, deux familles de poly(uréthane-imide)s (PUIs) à architecture contrôlée ont été développées dans l'objectif d'obtenir des matériaux avec des proportions très élevées en unités oxyde d'éthylène tout en évitant leur cristallisation. Des PUIs multi-blocs linéaires ont été synthétisés par polycondensation avec différentes tailles de bloc souple polyéther Jeffamine, correspondant à des taux massiques de phase souple variant entre 40% et 70%. Afin d'augmenter encore la proportion de phase souple jusqu'à la valeur très élevée de 85%, des PUIs multi-blocs greffés ont ensuite obtenus par une stratégie de "grafting-to" à partir d'un précurseur PUI comportant des fonctions alcyne latérales et d'oligomères azido-PEDEGA de différentes masses molaires. L’évolution de leurs performances de séparation du CO2 a été corrélée à leur proportion de phase souple et morphologie et à leur capacité d’interaction avec le CO2. Au taux maximal de phase souple (85%), on obtient des performances élevées (PCO2 = 196 Barrer ; αCO2/N2 = 39 et αCO2/CH4 = 12). Ainsi, par rapport au PUI greffable précurseur, le greffage d'oligomères PEDEGA a permis de multiplier la perméabilité jusqu’à 17 fois en maintenant la valeur élevée de αCO2/N2 constante et avec une diminution de seulement 30% de αCO2/CH4. En permettant d'obtenir une proportion très élevée de phase souple non cristalline, la stratégie de greffage s'est donc révélée exceptionnellement efficace pour améliorer les performances des copolymères multi-blocs PUI pour la capture du CO2. / In this PhD thesis, two types of membrane materials were developed for CO2 separation. The first ones associate a reference polymer material (Pebax®) with new pseudopeptidic bioconjugates additives. These pseudopeptide-polymer bioconjugates were obtained by a "grafting-to" synthetical pathway from alkyne-functionalized 1:1[α/α-Nα-Bn-hydrazino] dimer and tetramer pseudopeptides. Poly(diethylene glycol acrylate) (PEDEGA) oligomeric part was synthesized under controlled conditions using Single Electron Transfer Living Radical Polymerization (SET-LRP) from an azido-functionalized initiator allowing direct coupling via CuAAC "click" chemistry. The influence of these additives on CO2 sorption and separation properties was analyzed in terms of properties-morphology-structure relationships. These original additives allowed to enhance CO2 separation performances of the reference membrane, increasing CO2 permeability by 46%, and maintaining good selectivities αCO2/N2 = 44 et αCO2/CH4 = 13. In another work, two families of poly(urethane-imide)s (PUIs) with controlled architecture were developed for obtaining membrane materials with high content in ethylene-oxide units while avoiding their crystallization. Linear multi-blocks PUIs were first synthesized by polycondensation with different sizes of Jeffamine polyether soft block, corresponding to soft block contents varying from 40 to 70%wt. To further increase the soft phase content until a very high level (85%wt), grafted multi-blocks PUIs were obtained by a "grafting-to" strategy from an alkyne-functionalized precursor PUI and azido-PEDEGA oligomers with different molar weights. The evolution of their CO2 separation performances were correlated to their soft phase content, morphology and CO2 sorption ability. For the maximum soft phase content (85%wt), high performances were obtained for CO2 separation (PCO2 = 196 Barrer ; αCO2/N2 = 39 et αCO2/CH4 = 12). Compared to the precursor PUI, the grafting strategy allowed to increase CO2 permeability 17-fold. At the same time, the good selectivity αCO2/N2 was maintained with a slight decrease (-30%) of the selectivity αCO2/CH4. By allowing very high non-crystalline soft phase contents, the grafting strategy was exceptionally efficient in improving multi-block copolymer performances for CO2 capture

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2015-12-02T22:22:38Z No. of bitstreams: 1 RobertaTalitaStopilha_DISSERT.pdf: 9026948 bytes, checksum: 15e7a4c81ad84759d46e4d3ed6775c19 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2015-12-09T19:28:21Z (GMT) No. of bitstreams: 1 RobertaTalitaStopilha_DISSERT.pdf: 9026948 bytes, checksum: 15e7a4c81ad84759d46e4d3ed6775c19 (MD5) / Made available in DSpace on 2015-12-09T19:28:21Z (GMT). No. of bitstreams: 1 RobertaTalitaStopilha_DISSERT.pdf: 9026948 bytes, checksum: 15e7a4c81ad84759d46e4d3ed6775c19 (MD5) Previous issue date: 2014-09-05 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / Os complexos polieletrol?ticos nanoparticulados foram obtidos usando quitosana e poli(metacrilato de s?dio). A forma??o dos complexos foi avaliada por turbidimetria, condutometria, viscosimetria e espalhamento din?mico da luz. A presen?a de excesso de cargas positivas pode ser evidenciada atrav?s da an?lise do potencial zeta. O di?metro das part?culas foi caracterizado pela t?cnica de espalhamento din?mico da luz e a morfologia por microscopia de for?a at?mica. Em todos os experimentos uma mudan?a abrupta de comportamento foi evidenciada no intervalo de raz?o molar (grupos carboxilato/amino) entre 0,7-0,8. Essas mudan?as de comportamento puderam ser relacionadas com uma proposta de mecanismo de forma??o dos complexos polieletrol?ticos, baseada na redu??o das dimens?es macromoleculares dos aglomerados de complexos polieletrol?ticos sol?veis seguidos de segrega??o de fase. / Polyelectrolyte complexes (PECs) nanoparticles were prepared using chitosan and sodium polymethacrylate. The complex formation was investigated using turbidimetry, conductometry, viscometry, and dynamic light scattering. The presence of excess positive charges was evidenced by zeta potential measurements. The particle diameter was characterized by dynamic light scattering and the morphology by atomic force microscopy. In all experiments an abrupt change in behavior was observed at a carboxyl:amino molar ratio around 0.7?0.8. Those changes in behavior were related to a proposed mechanism of complex formation based on the decrease of macromolecular dimensions of soluble polyelectrolyte complex clusters, followed by phase segregation

Hydroxyapatite (HAP) coatings are used in orthopaedic surgery for bone regeneration. Current methods of phase quantification of HAP coatings suffer from drawbacks. A novel methodology of quantitative phase analysis of HAP coatings has been devised and validated. This method, based on whole pattern fitting with a fundamental parameters approach, incorporates amorphous calcium phosphate (ACP) and apatite phases into structural refinements. A comparison of the structural and chemical properties of plasma sprayed (PS) and novel electrodeposited (ED) HAP coatings has been conducted. ED coatings contained less ACP and more preferred orientation than the PS coatings, although the stoichiometry was similar. In vitro investigations of PS and ED coatings in simulated body fluid and foetal calf serum revealed that both are bioactive. A carbonated apatite layer produced on the ED coatings was -0.7μm thick with a stoichiometry and chemical constituents similar to that of natural bone apatite. PS coatings produced a nanocrystalline carbonated apatite layer (-4μm). For the first time it has been possible to model crystalline HAP and nanocrystalline apatite as independent phases and obtain accurate lattice parameters for each. A positive linear correlation has been made between microstrain and the solubility of HAP and carbonated apatites. Dissolution studies have shown that the behaviour of HAP and carbonated apatite is dominated by crystallite size at low undersaturation and by crystallite size and microstrain at high undersaturation for crystallites between -30OA- 1000A. Metastable equilibrium occurred for crystallites <_400A at low undersaturation. Carbonate content did not affect the solubility or dissolution behaviour. A novel technology for coating polymeric tape with HAP for potential use in anterior cruciate ligament reconstruction has been devised. Mechanical tests have demonstrated that no adverse properties are induced by the coating technology. Cell culture studies have shown that the HAP layer is capable of enhanced attachment, proliferation and differentiation of osteoblast cells compared to uncoated tape.

Surface engineering with polymer brushes has become one of the most versatile techniques to tailor surface properties of substrates for a broad variety of (bio-) technological applications. We report on a new facile approach to prepare defined and dense polymer brushes on planar substrates by surface-initiated Cu(0) mediated controlled radical polymerization (SI-CuCRP) of numerous vinyl monomers using a copper plate at room temperature. The fabrication of a variety of homo-, block, gradient and patterned polymer brushes as well as polymer brush arrays is demonstrated. The SI-CuCRP was found to be strictly surface-confined, of highly living character, proceeds remarkably fast and results in polymer brushes of very high grafting densities. The brush layer thickness can be modulated by the polymerization time or by the distance of the copper plate to the modified substrate. As the copper plate can be reused multiple times, no additional copper salts are added and only minimal amount of chemicals is needed, the simple and low-cost experimental conditions allows researchers from various fields to prepare tailored polymer brush surfaces for their needs.

Molecular brushes of poly(2-oxazoline)s (POx) are an intriguing class of polymers as they combine a unique architecture with the properties of POx as a biomaterial. Here, the synthesis of several POx macromonomers with methacrylate end groups and consecutive grafting through polymerization by aqueous atom transfer radical polymerization (ATRP) at room temperature is reported. 1H-NMR spectroscopy and size exclusion chromatography (SEC) confirmed the synthesis of POx molecular brushes with maximum side chain grafting densities, narrow molar mass distributions (Đ ≤ 1.16) and final molar masses corresponding to the initial macromonomer : initiator ratio. Chain extension experiments show high end group fidelity and formation of block copolymer molecular brushes, and kinetic studies revealed a polymerization behavior of oligo(2-methyl-2-oxazoline) methacrylate very similar to the frequently used oligo(ethylene glycol) methacrylate (OEGMA475). Aqueous solutions of POx molecular brushes with poly(2-ethyl- and 2-isopropyl-2-oxazoline) side chains exhibit the typically defined thermoresponsive behavior with a tunable, very narrow and reversible phase transition

Content: Nowadays, protein-based nanoparticle as a biodegradable, biocompatible product attracts considerable interest for new uses in specialized technical areas. Gelatin is a denatured, biodegradable, and nonimmunogenic protein obtained by controlled hydrolysis of the triple-helix structure of collagen into single-strain molecules. As an amphiphilic biopolymer, gelatin can easily assemble into different kinds of aggregates under the defined pH and temperature and the resulting gelatin nanoparticles have been developed to be applied in the food industry and biomedical fields. Herein we report a novel macromolecular microsphere composites (MMC) hydrogels with the use of prepared methacrylated gelatin nanoparticles (MA-GNP) as the cross-linker. MA-GNP have the ability of chemical crosslinking by the polymerization of C=C bonds, such that the composite hydrogels can be formed by radical polymerization of acrylamide (AAm) on the surface of MA-GNP. The smooth spherical particles with an average size of ~100 nm have been synthesized through a modified two-step desolvation method as proved by atomic force microscopy (AFM). The results of nuclear magnetic resonance and dynamic light scattering further confirm the presence of reactive groups (C=C bonds) in the particles and its narrow sizes distribution. The resulting composite hydrogels (MA-GNP/PAAm) are porous materials with tunable pore sizes and exhibit enhanced compressive resistance and elasticity as well. Increasing appropriately the dosage of MA-GNP reduces the equilibrium swelling ratio and improves thermal stability of the gels. Moreover, all the hydrogels exhibit prolonged blood-clotting time, nonhemolytic nature and strong suitability for cell proliferation, indicating the improved antithrombogenicity and excellent cyto-compatibility. It suggests that the novel MA-GNP/PAAm hydrogels have potential application as tissue engineer scaffold materials, and the MA-GNP can be a promising macromolecular microsphere cross-linker for application in biomedical materials. The present work not only exploits new strategies to fabricate MMC hydrogels but also advance the potential application of biodegradable gelatin-based nanoparticles in biomedical fields. Take-Away: 1. A well-dispersed methacrylated gelatin nanoparticle (MA-GNP) with an average size of ~100 nm is presented by a modified two-step desolvation method. 2. MA-GNP is readily introduced into the polyacrylamide (PAAm) system as a cross-linker to prepare macromolecular microsphere composites (MMC) hydrogels via a free radical polymerization reaction. 3. MA-GNP is an effective cross-linker, improving both the compressive resistance and elasticity of MMC hydrogels as well as the biocompatibility.

Currently, human norovirus is the leading cause of acute gastroenteritis and accounts for most cases of foodborne illnesses in the United States each year. Due to its tissue culture inefficiency, studies of human norovirus have been crippled for more than forty years.Tulane virus (TV) stands out as a suitable surrogate of human norovirus given its high amino acid identity with human norovirus and its well-established cell culture system. It was first isolated from rhesus macaques (Macaca mulatta) in 2008 and identified as a novel Calicivirusrepresenting a new genus, Recovirus genus (Farkas et al., 2008). However, there are still unanswered questions about its infectious cycle and the essential factors for its infection. In this study, we have obtained a TV variant (the 9-6-17 strain) that has lost the binding ability to the B-type histo-blood group antigen (HBGA), which was proposed to be the receptor of both TV and human norovirus. In the first chapter, we outline how the sequence analysis,structural biology studies, and mutagenesis studies of the 9-6-17 TV strain have shed light on the interaction with its host cell receptor. To investigate the key residues for HBGA binding, we established the full-length infectious clone of the 9-6-17 TV strain. We present a highly selective transformation of serine 367, located in the predicted HBGA binding site, into a lysine residu e. Our results advance the understanding of genetic changes in TV required for adaptation to cell culture environments. Cryo-EM is an awarding winning technique that has been the greatest scientific breakthrough in recent years. It was awarded the Nobel Prize in Chemistry in 2017. Despite the technological advances of the direct electron detector and image processing software, several major roadblocks remain for high-resolution structure determination with cryo-EM. In the later chapters, we explored the most efficient way of using VPP to enhance image contrast, how to tackle the airwater interface problem by encapsulating target protein, how to reach a higher resolution by refining high order parameters, and the helical indexing problem in real space. These technical advances would benefit the whole cryo-EM community by providing convenient tools or insights for future directions.