L’objectif de cette thèse est d’étudier expérimentalement les deformations de microcapsules dans un écoulement confiné. Les microcapsules sont composées d’albumine du sérum humain avec des concentrations de 5 à 20 [g/100mL]. Leur taille varie de 50 à 1000 [μm]. Les capsules sont injectées dans des écoulements de Poiseuille produits dans des canaux microfluidiques présentant deux sections différentes : circulaire ou carrée.La mesure des caractéristiques géométriques de microcapsules déformées couplée à des simulations numériques mène à la détermination du module de cisaillement surfacique. Cette caractéristique mécanique augmente fortement tant avec la taille qu’avec la concentration en protéine de la capsule, et plus précisément avec le produit de ces deux paramètres.Le fluide est ensemencé avec des microparticules pour mesurer l’écoulement induit par une capsule dans un capillaire cylindrique par la méthode de la vélocimétrie par suivi de particules. Les zones de recirculation et de perturbation sont alors déduites et comparées avec la simulation numérique d’un objet rigide dans un capillaire et présentant le profil donné par les expériences. Finalement un système original de visualisation optique est consacré à l’observation simultanée de la vue de côté et de la vue de face des capsules pour obtenir sa forme entière. Ceux-ci révèlent l’existence des plis tout autour de la membrane des capsules. Le seuil de formation et l’évolution de ces plis sont étudiés en fonction de la vitesse, de la taille et du confinement, dans des canaux de section circulaire ou carrée. / The objective of this thesis is to study experimentally microcapsule deformations in confined flows. The microcapsules are made of cross-linked proteins, the human serum albumin (HSA) with concentrations from 5 to 20 [g/100mL]. Their size vary from 50 to 1000 [μm]. Capsules are injected in Poiseuille flows generated within microfluidics channels with two different cross sections geometries : circular or square.The measurement of geometrical characteristics of deformed microcapsules coupled with numerical simulations leads to the determination of the surface shear modulus. This mechanical characteristic increases strongly with both the size and the protein concentration of the capsule, and more precisely with the product of these two parameters.The flow is seeded with microparticles to measure the induced flow of a capsule in a cylindrical capillary by particle tracking velocimetry. The recirculation and perturbation zones are then deduced and compared with numerical simulation of a rigid body flowing in a capillary. Finally an original system of optical visualization is dedicated to the simultaneous observation of the side and the front view of the capsules to get its whole shape. These reveal radial wrinkles all around capsules membrane. The formation threshold and the evolution of these wrinkles are studied as function of the capsule velocity and size and the confinement within capillaries with circular or square cross–section.

Cette thèse s’inscrit dans le contexte de l’ANR ISABEAU (Innovating for Structural Adhesive Bonding Evaluation and Analysis with Ultrasounds, associant des physico-chimistes et des acousticiens) et cherche à apporter des connaissances nouvelles sur l’évaluation par ultrasons du niveau d’adhésion d’un collage structural par ondes de Lamb. La première partie porte sur l’aspect cohésif et particulièrement sur l’influence du paramètre de réticulation de réseaux époxys sur les courbes de dispersion des ondes de Lamb. La deuxième partie est consacrée à la caractérisation de niveaux d’adhésion dans une structure bicouche Aluminium Epoxy en tenant compte aussi bien des aspects cohésifs qu’adhésifs. Des échantillons à niveau d’adhésion connus et maîtrisés sont réalisés avec l’aide de physico-chimistes, où l’époxy est partiellement ou totalement réticulé, et où l’interface substrat adhésif a subit différents traitements chimiques et/ou mécaniques. Les résultats expérimentaux sont confrontés à ceux issus du modèle rhéologique de Jones résolu par éléments finis. Enfin, la troisième partie porte sur l’étude d’un assemblage tricouche Aluminium/Epoxy/Aluminium et cherche à qualifier différents niveaux d’adhésion à l’aide du modèle de Jones par ondes guidées et également par l’étude des fréquences de coupure des modes verticaux de la structure. / This thesis is in the context of the ANR ISABEAU (Innovating for Structural Adhesive Bonding Evaluation and Analysis with Ultrasounds, with the association of physico-chemists and acousticians) and seeks to bring new knowledge of the ultrasonic evaluation of the level of adhesion of structural bonding using Lamb waves. The first part focuses on the cohesive aspect, in particular on the influence of the crosslinking parameter of the polymer on the Lamb waves dispersion curves. The second part is dedicated to the evaluation of the adhesion level of a bi-layer Aluminum Epoxy taking into account the both cohesive and adhesive aspects. Samples of controlled and known adhesion levels are manufactured with the help of physico-chemists, where the crosslinking of the epoxy is partial or total, and the interface substrate-adhesive have been treated chemically and/or mechanically. The experimental results are compared to those obtained from a predictive model based on the rheological model of Jones solved by finite elements method. Finally, the third part focuses on the study of tri-layers Aluminum/Epoxy/Aluminum, looking for the qualification of different levels of adhesion with the Jones model using guided waves as well as the cut-off frequencies of the structure.

Eukaryotic translation initiation is a complex and highly regulated process involving the ribosome, mRNA and proteins called eukaryotic initiation factors (eIFs). The overall aim of translation initiation is to position the ribosome at the initiation codon of the mRNA. eIF2, in its GTP-bound conformation, binds the initiator tRNA (Met-tRNAiMet) and delivers it to the 40S ribosomal subunit. When the anticodon of the tRNA is bound to the initiation codon, the GTP on eIF2 is hydrolysed to GDP. The guanine nucleotide exchange factor (GEF) eIF2B regenerates eIF2-GTP. eIF2 and eIF2B are multisubunit/multidomain protein complexes. Because information regarding the interface between each complex is limited, particularly the interface on the eIF2γ subunit, which binds the guanine-nucleotides and Met-tRNAiMet, interactions between the minimal GEF domain of eIF2Bε, εGEF, and eIF2 were mapped using mutagenesis and an in vitro cysteine cross-linking approach, with the cross-linker Mts-Atf-Biotin. Site-directed mutagenesis (SDM) was used to mutate five N-terminal and five C-terminal surface-exposed εGEF residues to cysteines. The mutant alleles were analysed in Saccharomyces cerevisiae and it was found that the gcd6-R574C allele was lethal and the gcd6-T572C was Gcd-. Further gcd6-R574 mutant alleles were also found to be lethal in yeast but expressed in vivo.εGEF-R574C has dramatically reduced GEF activity in vitro and binding assays showed that this mutant has significantly reduced affinity for eIF2. The εGEF-T572C and εGEF-S576C mutants also have severe and minor eIF2-binding defects respectively, while the C-terminal εGEF-Cys mutants have slightly reduced affinity for eIF2. The N-terminal εGEF-Cys mutants cross-link specifically to eIF2γ, while the C-terminal εGEF-Cys mutants interact predominantly with eIF2β. From the data obtained in this study, we propose a new model for eIF2B-mediated guanine-nucleotide exchange that reduces the importance of eIF2β and suggests εGEF resembles other GEFs in binding primarily to its G protein partner eIF2γ.

Orientador: Josimeri Hebling / Resumo: Objetivo: Investigar a citotoxicidade transdentinária da acroleína (ACR) sobre células pulpares e a resistência máxima à tração (RMT) da matriz dentinária e resistência da união (RU) resina-dentina após a biomodificação do colágeno dentinário por esse agente promotor de ligações cruzadas. Métodos: Discos de dentina (0,4 mm de espessura) foram obtidos de molares humanos hígidos e adaptados em câmaras pulpares artificiais. Células MDPC-23 foram semeadas na superfície pulpar desses discos e a superfície oclusal foi condicionada com ácido fosfórico por 15s. Sobre a dentina condicionada foi aplicado (n=9): água deionizada (controle), ACR 0,02%, 0,01%, 0,005%, glutaraldeído 5% (GD) ou peróxido de hidrogênio 3%. Após 60s, a superfície foi lavada e as câmaras foram incubadas por 24h. Foi avaliada a viabilidade das MDPC-23 (alamarBlue) aderidas na parede pulpar dos discos e os extratos foram aplicados em novas MDPC-23 e HDPCs (células da polpa dental humana) cultivadas em placas de cultura. Após 24h, essas células foram avaliadas quanto a viabilidade, atividade de fosfatase alcalina (ensaio da timolftaleína), presença de nódulos de mineralização (Alizarin red) e expressão gênica de ALPL, DSPP, MMP2, MMP9 e IL1B (PCRq). Vinte cinco molares adicionais foram seccionados para obter espécimes de dentina (n=50) que foram completamente desmineralizados com ácido fosfórico por 24h. Os espécimes de matriz dentinária foram tratados por 60s com: água, ACR 0,02%, 0,01%, 0,005% ou GD 5% e submetid... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Objective: To investigate the transdentinal cytotoxicity of acrolein (ACR) on pulp cells, as well as the ultimate tensile strength (UTS) of the dentin matrix, and the strength of resin-dentin bonds after dentin collagen biomodification with this cross-linker. Methods: Dentin disks (0.4 mm thick) were cut from sound human molars and adapted in artificial pulp chambers. MDPC-23 were seeded on the pulpal side of the disks and the occlusal surface was etched with phosphoric acid for 15s. The etched dentin was treated with (n=9): deionized water (control), 0.02%, 0.01%, 0.005% ACR, 5% glutaraldehyde (GD), or 3% hydrogen peroxide. After 60s, the surface was rinsed, and the chambers were incubated for 24h. The viability of MDPC-23 cells seeded on the disk was assessed (alamarBlue) and the extracts were applied on new MDPC-23 and HDPCs (human dental pulp cells) seeded in culture plates. After 24h, the viability of the cells was investigated as well as the activity of alkaline phosphatase, presence of mineralized nodules (Alizarin red) and ALPL, DSPP, MMP2, MMP9 and IL1b gene expression (qPCR). Additional twenty-five human molars were sectioned to obtain dentin specimens (n=50) which were completely demineralized in 10% phosphoric acid for 24h. The specimens were treated for 60s with: water, 0.02%, 0.01%, 0.005% ACR or 5% GD, and then submitted to a mechanical test to determine the UBS. Finally, flat dentin surfaces prepared in 40 human molars were etched with phosphoric acid and trea... (Complete abstract click electronic access below) / Mestre

Bacterial protein WrbA from E. coli is the founding member of a new family of FMN-dependent NAD(P)H oxidoreductases, forming a functional and structural bridge between bacterial flavodoxin and certain mammalian NAD(P)H:quinone oxidoreductase. For these reasons, protein WrbA is recently intensively studied using various analytical and computing methods. Protein WrbA participates in the protection of cells against oxidative stress, but precise function of the protein WrbA in vivo is still unknown. Protein WrbA forms multimers in solutions. In μM concentrations and at low temperature (4 řC) the protein is in the form of a dimer, with increasing temperature becomes tetrameric. Available three-dimensional crystal structure contains the information about the tetrameric form of the protein, the dimeric form has not been structurally characterized. This thesis was focused on the study of the dynamic behavior of protein WrbA in solution using methods of hydrogen-deuterium exchange and chemical cross-linking followed by mass spectrometric analysis with high resolution (FT-ICR). Behavior of the protein was monitored according to the presence of cofactor FMN. Effect of temperature and protein concentration was also studied. Hydrogen-deuterium exchange provided information about solvent accessibility and...

Transcription factors play crucial regulatory role within the cell and the entire multicellular organism. The important factor is its ability to interact with other regulatory proteins and DNA. Despite the fact that a large part of the interaction network is already documented, detailed information on the structure and dynamics of protein-protein and protein-DNA complexes is still scarce. In this thesis we focused on the possibility of studying conformational changes given by the transcription factor-DNA complex formation using the methods of structural mass spectrometry: hydrogen/deuterium exchange and chemical crosslinking. As a model, we chose a transcription factor FOXO4 which DNA binding domain is structurally well characterized both in free form and in the complex with DNA.

ABSTRACT Mixed function oxygenase system participates in biosynthesis of endogenous and metabolism of exogenous substances (e.g. drugs or chemical procarcinogens) in an organism. Substrates are biotransformed by terminal oxygenases - cytochromes P450 (P450). Catalytic properties of certain P450s (e.g. studied isoform 2B4) are altered in the presence of a redox partner - cytochrome b5 (cyb5). Both cytochromes are anchored by hydrophobic domains in a lipid membrane of endoplasmic reticulum whereas their catalytic domains are exposed to cytosol. Two zero-length cross-linking approaches were employed to extend present knowledge of P450 2B4 and cyb5 protein structure and protein-protein interactions: (1) interlinking of carboxylate and primary amine groups of amino acids by water soluble 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and (2) photo-initiated cross-linking by photo-labile methionine analog (pMet), which links to any amino acid after activation by UV-irradiation, either in hydrophilic or hydrophobic environment. pMet was incorporated to methionine site(s) of cyb5 during recombinant expression in E. coli, which was carried out in limit medium supplemented with amino acid analog. Optimization of experimental conditions led to ~20-30% substitution of the natural amino acid. Covalent...

Countless electron transport/transfer (ET) processes occur in living organisms every day. Therefore, their study is a crucial field of modern structural and functional proteomics. In many cases model proteins like azurin from P. aeruginosa are utilised in experiments. This blue copper protein is favoured due to a characteristic absorbance maximum at 630 nm in Cu(II) redox state of the central Cu atom. During its oxidation to Cu(I) state the A630 value decreases allowing UV-Vis detection of ET reaction progress. We have introduced a structural photoinducible analogue of canonical amino acid Met - L-2-amino-5,5-azihexanoic acid (photo-Met) - into azurin structure to study oligomerization in solution via photo-induced cross-linking (PIXL). Using previously optimised protocols for recombinant expression in E. coli B834 we have inserted photo-Met into azurin moieties: wild type azurin and Az2W mutant where two adjacent W residues with confirmed role in electron hopping across protein-protein interface are present. The incorporation percentage of photo-Met in analysed samples was determined after SDS-PAGE and in-gel protease digestion via MALDI-TOF MS. PIXL was employed to study azurin-azurin interaction and oligomerization under different total concentrations of protein (in range of 15-300 µM). The...

Block copolymers are commercially significant and fundamentally interesting class of polymeric materials. The ability to undergo interfacial thermodynamics-controlled microphase separation from a completely disordered state in the melt to a specifically defined ordered structure through self-organization makes the block copolymers based materials unique. Block copolymer are strongly replacing many of the commercially available polymers due to their unique microstructure and properties. The most practical interests of block copolymers lie in the area of thermoplastic elastomers (TPEs). The objective of the present thesis work is to developing novel roots for enhancing the physical and mechanical properties in block copolymer and multigraft copolymers. Initially the properties are tailored by controlling chemical architecture at synthesis level and by selective blending at production level. This gives an easy access for improvement of the material properties and this is one of my major tasks in the present research modules. Further the block copolymer based TPEs are cross-linked in presence of electron beam (EB) radiation for developing materials with superior properties. The electron beam radiation has the ability to alter material parameters at molecular level for enhancing the macroscopic properties. The desirable physical and chemical properties can be easily attained by varying the radiation beam parameters. In addition to that, controlling the material at nanometer scale is one of the greatest challenges for current nanocomposite research. In elastomeric materials it is very prominent to fill the rubber matrix with nano particles from carbon or silica by melt mixing technique for enhancing the material properties. Other than conventional melt mixing technique, sol–gel processing is also a versatile technique, which making it possible to produce a wide variety of materials and to provide existing materials with novel properties. A combination of in situ sol-gel reaction with electron beam cross-linking in TPEs from triblock copolymer has been demonstrated for the first time as one of the novel nanocomposite system in this work. The main advantage of this system lies in controlling the material behaviour by finely tuning the size of silica nano particle generated inside TPE during in situ sol-gel reaction. Finally, the various roots employed for enhancing the material behaviour in block copolymers in the above research module were secussfully employed on super elastic multigraft copolymers for improving their strength withour sacrificing the super elastic nature.

We investigated attachment and migration of human retinal pigment epithelial cells (primary, SV40-transfected and ARPE-19) on nanoscopically defined, two-dimensional matrices composed of parallel-aligned collagen type I fibrils. These matrices were used non-cross-linked (native) or after riboflavin/UV-A cross-linking to study cell attachment and migration by time-lapse video microscopy. Expression of collagen type I and IV, MMP-2 and of the collagen-binding integrin subunit α2 were examined by immunofluorescence and Western blotting. SV40-RPE cells quickly attached to the nanostructured collagen matrices and aligned along the collagen fibrils. However, they disrupted both native and cross-linked collagen matrices within 5 h. Primary RPE cells aligned more slowly without destroying either native or cross-linked substrates. Compared to primary RPE cells, ARPE-19 cells showed reduced alignment but partially disrupted the matrices within 20 h after seeding. Expression of the collagen type I-binding integrin subunit α2 was highest in SV40-RPE cells, lower in primary RPE cells and almost undetectable in ARPE-19 cells. Thus, integrin α2 expression levels directly correlated with the degree of cell alignment in all examined RPE cell types. Specific integrin subunit α2-mediated matrix binding was verified by preincubation with an α2-function-blocking antibody, which impaired cell adhesion and alignment to varying degrees in primary and SV40-RPE cells. Since native matrices supported extended and directed primary RPE cell growth, optimizing the matrix production procedure may in the future yield nanostructured collagen matrices serving as transferable cell sheet carriers. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.