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International audience; Diabetes is a major concern of our society as it affects one person out of 11 around the world. Elastic fiber alterations due to diabetes increase the stiffness of large arteries, but the structural effects of these alterations are poorly known. To address this issue, we used synchrotron X-ray microcomputed tomography with in-line phase contrast to image in three dimensions C57Bl6J (control) and db/db (diabetic) mice with a resolution of 650 nm/voxel and a field size of 1.3 mm3. Having previously shown in younger WT and db/db mouse cohorts that elastic lamellae contain an internal supporting lattice, here we show that in older db/db mice the elastic lamellae lose this scaffold. We coupled this label-free method with automated image analysis to demonstrate that the elastic lamellae from the arterial wall are structurally altered and become 11% smoother (286,665 measurements). This alteration suggests a link between the loss of the 3D lattice-like network and the waviness of the elastic lamellae. Therefore, waviness measurement appears to be a measurable elasticity indicator and the 3D lattice-like network appears to be at the origin of the existence of this waviness. Both could be suitable indicators of the overall elasticity of the aorta.

Simple Summary This work aimed to investigate the interactions of lumican-derived peptides and MMP-14. An in silico approach unraveled key residues in the amino acid sequence of MMP-14 interacting with the Small Leucine-Rich Proteoglycan (SLRP) lumican-derived peptides. The in silico docking analysis demonstrated that the interaction of a cyclic lumican-derived peptide (L9Mc, 12 amino acids) with MMP-14 was preferential with the MT-Loop domain of MMP-14 while the linear lumican-derived peptide (lumcorin, 17 amino acids) interacted more with the catalytic site. L9Mc significantly inhibited the migration of murine B16F1 but not human HT-144 melanoma cells and the activity of MMP-14 but with less efficacy than lumican and lumcorin. This result led us to investigate the effect of L9Mc on cell proliferation, which is independent of MMP-14 activity. L9Mc significantly inhibited the proliferation of B16F1 but not HT-144 melanoma cells in vitro and primary melanoma tumor growth. Altogether, the biological assays validated the prediction of the in silico study. Abstract Lumican, a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM), displays anti-tumor properties through its direct interaction with MMP-14. Lumican-derived peptides, such as lumcorin (17 amino acids) or L9M (10 amino acids), are able to inhibit the proteolytic activity of MMP-14 and melanoma progression. This work aimed to visualize the interactions of lumican-derived peptides and MMP-14. Molecular modeling was used to characterize the interactions between lumican-derived peptides, such as lumcorin, L9M, and cyclic L9M (L9Mc, 12 amino acids), and MMP-14. The interaction of L9Mc with MMP-14 was preferential with the MT-Loop domain while lumcorin interacted more with the catalytic site. Key residues in the MMP-14 amino acid sequence were highlighted for the interaction between the inhibitory SLRP-derived peptides and MMP-14. In order to validate the in silico data, MMP-14 activity and migration assays were performed using murine B16F1 and human HT-144 melanoma cells. In contrast to the HT-144 melanoma cell line, L9Mc significantly inhibited the migration of B16F1 cells and the activity of MMP-14 but with less efficacy than lumican and lumcorin. L9Mc significantly inhibited the proliferation of B16F1 but not of HT-144 cells in vitro and primary melanoma tumor growth in vivo. Thus, the site of interaction between the domains of MMP-14 and lumcorin or L9Mc were different, which might explain the differences in the inhibitory effect of MMP-14 activity. Altogether, the biological assays validated the prediction of the in silico study. Possible and feasible improvements include molecular dynamics results.

Sialidases, or neuraminidases, are involved in several human disorders such as neurodegenerative, infectious and cardiovascular diseases, and cancers. Accumulative data have shown that inhibition of neuraminidases, such as NEU1 sialidase, may be a promising pharmacological target, and selective inhibitors of NEU1 are therefore needed to better understand the biological functions of this sialidase. In the present study, we designed interfering peptides (IntPep) that target a transmembrane dimerization interface previously identified in human NEU1 that controls its membrane dimerization and sialidase activity. Two complementary strategies were used to deliver the IntPep into cells, either flanked to a TAT sequence or non-tagged for solubilization in detergent micelles. Combined with molecular dynamics simulations and heteronuclear nuclear magnetic resonance (NMR) studies in membrane-mimicking environments, our results show that these IntPep are able to interact with the dimerization interface of human NEU1, to disrupt membrane NEU1 dimerization and to strongly decrease its sialidase activity at the plasma membrane. In conclusion, we report here new selective inhibitors of human NEU1 of strong interest to elucidate the biological functions of this sialidase.

International audience; Angiogenesis is defined as the formation of new capillaries by sprouting from the pre-existing microvasculature. It occurs in physiological and pathological processes particularly in tumor growth and metastasis. α1, α2, α3, and α6 NC1 domains from type IV collagen were reported to inhibit tumor angiogenesis. We previously demonstrated that the α4 NC1 domain from type IV collagen, named Tetrastatin, inhibited tumor growth in a mouse melanoma model. The inhibitory activity was located in a 13 amino acid sequence named QS-13. In the present paper, we demonstrate that QS-13 decreases VEGF-induced-angiogenesis in vivo using the Matrigel plug model. Fluorescence molecular tomography allows the measurement of a 65% decrease in Matrigel plug angiogenesis following QS-13 administration. The results are confirmed by CD31 microvessel density analysis on Matrigel plug slices. QS-13 peptide decreases Human Umbilical Vein Endothelial Cells (HUVEC) migration and pseudotube formation in vitro. Relevant QS-13 conformations were obtained from molecular dynamics simulations and docking. A putative interaction of QS-13 with α 5 β 1 integrin was investigated. The interaction was confirmed by affinity chromatography, solid phase assay, and surface plasmon resonance. QS-13 binding site on α 5 β 1 integrin is located in close vicinity to the RGD binding site, as demonstrated by competition assays. Collectively, our results suggest that QS-13 exhibits a mighty anti-angiogenic activity that could be used in cancer treatment and other pathologies with excessive angiogenesis such as hemangioma, psoriasis or diabetes.

Among the various post-translational modifications, N-glycosylations are particularly important. They are linked to asparagine residues and their function as well as the one of the protein can be altered by modifications such as sialic acid hydrolysis. Since in vitro studies of N-glycans can be a challenging process (glycosylation chains have a great diversity and contain many reactive groups), in silico characterization using molecular dynamics simulation seems to be a good tool capable of overcoming experimental shortcomings thanks to exhaustive conformational samplings. In this paper, the Umbrella Visualization, a recent implementation into the molecular viewer UnityMol, is presented. This new and original visualization method is offering the possibility to follow and decipher the dynamics of very flexible sugar chains and enable the identification of the protein surface covered and potentially impacted by glycans. The latest module, described here and integrated within the Umbrella Visualization, complements the original statistical approach and allows for a meaningful description of glycan/protein interplay by combining, with shadow mapping, labelling, and hydrophobic properties of the surrounding aminoacids., Workshop on Molecular Graphics and Visual Analysis of Molecular Data, Session 2, 17, 21, Camille Besançon, Hua Wong, Rajas Rao, Manuel Dauchez, Nicolas Belloy, Jessica Prévoteau-Jonquet, and Stéphanie Baud, CCS Concepts: Computing methodologies --> Graphics file formats; Scientific visualization; Modeling methodologies; Texturing

International audience

Mammalian glycosaminoglycans are linear complex polysaccharides comprising heparan sulfate, heparin, dermatan sulfate, chondroitin sulfate, keratan sulfate and hyaluronic acid. They bind to numerous proteins and these interactions mediate their biological activities. GAG–protein interaction data reported in the literature are curated mostly in MatrixDB database (http://matrixdb. univ-lyon1.fr/). However, a standard nomenclature and a machine-readable format of GAGs together with bioinformatics tools for mining these interaction data are lacking. We report here the building of an automated pipeline to (i) standardize the format of GAG sequences interacting with proteins manually curated from the literature, (ii) translate them into the machine-readable GlycoCT format and into SNFG (Symbol Nomenclature For Glycan) images and (iii) convert their sequences into a format processed by a builder generating three-dimensional structures of polysaccharides based on a repertoire of conformations experimentally validated by data extracted from crystallized GAG–protein complexes. We have developed for this purpose a converter (the CT23D converter) to automatically translate the GlycoCT code of a GAG sequence into the input file required to construct a three-dimensional model.

Rapporteurs: Stefan BLUGEL, Professeur à l'IFF KFA (Jülich, Allemagne) Cyrille BARRETEAU, Ingénieur-Chercheur CEA (Saclay) Autres membres du jury: Peter ZEPPENFELD, Professeur l'Université de Linz (Autriche) Guy TREGLIA, Directeur de Recherche au CRMCN-CNRS (Marseille) Christophe RAMSEYER, Professeur à l'Université de Franche-Comté Claude GIRARDET, Professeur à l'Université de Franche-Comté; This PhD concerns the growth and the study of physical properties of nanometric structures that can be obtained on stepped surfaces. Previous experimental and theoretical studies showed that with self-organized methods it is possible to obtain a large number of nanostructures, but in general the quality of the nanowires depends strongly on the preparation and on the deposition statistic. The original idea of this work consist in assisting the growth using a STM tip. In order to characterize the specific properties of the structures, it is necessary to describe the substrate and its interactions with the adsorbates very precisely. The DFT code FLEUR is used to characterize energetically and structurally different platinum surfaces. In the continuity of this work, the determination of magnetic properties associated with Co chain (free or supported on vicinal Pt surfaces) is investigated. In the first part, a study related to growth phenomenon conducted numerically with a Kinetic Monte Carlo (KMC) model is presented. In the case of the Xe/Cu(110) system, we show that the presence of a fixed STM tip, depending on its height relative to the substrate can locally modify the atomic diffusion and allow the measure of diffusion coefficients related to the motions of the atoms. In a more general frame, it was shown that it is possible to improve the profile of nanowire deposited on vicinal surfaces using a moving STM tip in a repulsive or in an attractive mode. The perfection of the wires depends on the number, the type of the sweepings and also on the considerered temperature range. Then, the electronic properties of platinum surfaces were investigated. After the study of the three flat platinum surfaces namely (111), (100) and (110) surfaces, we focused on the (233) vicinal surface. This one presents the same geometry as the steps of the experimental Pt(997) surfaces. Surface energies, step energies, local density of states or bandstructures have been calculated. The calculations of STM pictures were done in order to interpret the STM pictures of the Pt(997) surface obtained at the EPFL (Lausanne, team of K. Kern and H. Brune). Neither the relaxation (internal) nor the local density of states near the step edge can explain the protusion observed at this location on some of the experimental images. But, this does not mean that our calculations are wrong. The protusion can be due to the experimental set up which does not follow fast enough in time the evolution of the LDOS. A second application of the calculation of STM pictures is relative to the adsorption of Ir atoms on a Ir(111) substrate. FLAPW calculations and the experiments of Thomas Michely's team show that the tip can distinguish unequivocally two different stackings (hcp or fcc) of comparable energie but with different density of states around the Fermi level. Finally, the study of the magnetic properties of Co nanowires unsupported or adsorbed at the foot of Pt step edges is approached. For the unsupported system, the magnetic moment of the cobalt atom decreases from 3 uB in the case of an isolated atom to 2.33 uB for the chain, 2.09 uB for the monolayer and 1.65 uB in the bulk. Calculations were also conducted on periodic arrays of Co wires supported on Pt(233) and eventually relaxed systems. It is shown that the influence of the substrate on the magnetic moment of the Co atom is not very big. On the other hand, a strong quenching of the orbital moments is observed. The magnetic and orbitals moments are equal respectively to 2.105 uB and [0.058-0.091] uB depending on the orientation of the spin quantization axis. The determination of the magnetic anisotropy shows that the easy axis is along the chain direction when the system is not relaxed. Only the relaxation can explain the fact that the easy axis goes out of the chain axis.; Le travail de cette thèse concerne la croissance et l'étude des propriétés physiques de structures nanométriques susceptibles de se former sur des surfaces à marches. De précédentes études tant expérimentales que théoriques ont montré que même si les méthodes de croissances auto organisées permettent d'obtenir un grand nombre de nanostructures, la qualité des fils est très dépendante des conditions de préparation et de la statistique du dépôt. L'idée originale de ce travail consiste à assister la croissance en utilisant une pointe de microscope STM. Afin de caractériser les propriétés spécifiques des structures alors formées, il convient d'avoir préalablement décrit de façon exhaustive le substrat et son interaction avec les atomes adsorbés. Le code de calcul DFT FLEUR est utilisé afin de caractériser diverses surfaces de platine tant du point de vue structurel qu'énergétique. Dans le prolongement de ce travail, nous abordons également la caractérisation de propriétés magnétiques de chaines de cobalt non supportées ou bien déposées sur des surfaces de platine vicinales. Dans la première partie, nous présentons une étude concernant des phénomènes de croissance menée numériquement à l'aide d'un modèle KMC (Kinetic Monte Carlo). Pour le système Xe/Cu(110), nous montrons que la présence d'une pointe STM fixe peut, suivant la hauteur à laquelle elle est placée au dessus de la surface modifier localement la diffusion des adatomes et également permettre la mesure des coefficients de diffusion caractéristiques du déplacement des atomes. Nous nous sommes ensuite placés dans un cadre plus général et nous avons montré qu'il est possible d'améliorer les profils de nanofils déposés sur des surfaces vicinales à l'aide d'une pointe STM mobile utilisée en mode répulsif ou attractif. La perfection du fil est fonction du nombre et du type de balayages effectués, ainsi que de l'intervalle de température considéré. Nous nous sommes ensuite intéressés aux propriétés électroniques des surfaces de platine. Après avoir considéré en premier lieu les trois surfaces plates classiques (111), (100) et (110) notre attention s'est portée sur une surface vicinale (233) présentant la même géométrie de marche que la surface expérimentale (997) mais possédant une largeur de terrasses inférieure. Nous avons mené des calculs d'énergie de surface ou de marche, de densité d'états, ou encore de structures de bande. Les calculs des images STM ont été réalisés pour interpréter les images STM de la surface Pt(997) obtenues à l'EPFL (Lausanne, équipe de K. Kern et H. Brune). Ni la relaxation (vers l'intérieur), ni la densité d'état locale au voisinage de la marche ne permettent de comprendre la protubérance observée à cet endroit sur certains clichés expérimentaux. Ceci ne veut pas pour autant dire que nos calculs sont erronés. La protubérance observée peut être due à une erreur expérimentale et notamment au temps de réponse de la pointe. Une deuxième application du calcul d'images STM est relative à l'adsorption d'atomes d'Iridium sur un substrat (111) de même espèce. Les calculs FLAPW et les expériences du groupe de T. Michely (RWTH, Aachen) montrent sans ambiguïté que la pointe peut distinguer deux types d'empilements (hcp et fcc) d'énergie comparable mais présentant des densités d'états différentes au niveau de Fermi. Pour finir, l'étude des propriétés magnétiques de nanofils de cobalt non supportés ou adsorbés au pied des marches de platine est abordée. Pour le système non supporté, le moment magnétique passe de 3 uB pour l'atome de cobalt isolé, à 2.33 uB pour la chaîne, à 2.09 uB pour la monocouche et 1.65 uB pour le volume. Le calcul a ensuite été fait pour une succession périodique de fils de cobalt supportés par le platine (233) et relaxés. Les calculs montrent que le substrat a peu d'influence sur le moment de spin. Par contre, on observe un blocage fort du moment magnétique orbital par le champ cristallin créé par les atomes de platine. Les moments magnétiques de spin et orbital sont égaux respectivement à 2.105 uB et [0.058-0.091]uB selon la direction de l'aimantation. Le calcul de l'anisotropie magnétique semble montrer que l'axe de facile aimantation est dirigé selon les fils lorsque le système n'est pas relaxé. Seule la relaxation permet d'expliquer le fait que l'axe de facile aimantation sort de la chaîne.

Rapporteurs: Stefan BLUGEL, Professeur à l'IFF KFA (Jülich, Allemagne) Cyrille BARRETEAU, Ingénieur-Chercheur CEA (Saclay) Autres membres du jury: Peter ZEPPENFELD, Professeur l'Université de Linz (Autriche) Guy TREGLIA, Directeur de Recherche au CRMCN-CNRS (Marseille) Christophe RAMSEYER, Professeur à l'Université de Franche-Comté Claude GIRARDET, Professeur à l'Université de Franche-Comté; This PhD concerns the growth and the study of physical properties of nanometric structures that can be obtained on stepped surfaces. Previous experimental and theoretical studies showed that with self-organized methods it is possible to obtain a large number of nanostructures, but in general the quality of the nanowires depends strongly on the preparation and on the deposition statistic. The original idea of this work consist in assisting the growth using a STM tip. In order to characterize the specific properties of the structures, it is necessary to describe the substrate and its interactions with the adsorbates very precisely. The DFT code FLEUR is used to characterize energetically and structurally different platinum surfaces. In the continuity of this work, the determination of magnetic properties associated with Co chain (free or supported on vicinal Pt surfaces) is investigated. In the first part, a study related to growth phenomenon conducted numerically with a Kinetic Monte Carlo (KMC) model is presented. In the case of the Xe/Cu(110) system, we show that the presence of a fixed STM tip, depending on its height relative to the substrate can locally modify the atomic diffusion and allow the measure of diffusion coefficients related to the motions of the atoms. In a more general frame, it was shown that it is possible to improve the profile of nanowire deposited on vicinal surfaces using a moving STM tip in a repulsive or in an attractive mode. The perfection of the wires depends on the number, the type of the sweepings and also on the considerered temperature range. Then, the electronic properties of platinum surfaces were investigated. After the study of the three flat platinum surfaces namely (111), (100) and (110) surfaces, we focused on the (233) vicinal surface. This one presents the same geometry as the steps of the experimental Pt(997) surfaces. Surface energies, step energies, local density of states or bandstructures have been calculated. The calculations of STM pictures were done in order to interpret the STM pictures of the Pt(997) surface obtained at the EPFL (Lausanne, team of K. Kern and H. Brune). Neither the relaxation (internal) nor the local density of states near the step edge can explain the protusion observed at this location on some of the experimental images. But, this does not mean that our calculations are wrong. The protusion can be due to the experimental set up which does not follow fast enough in time the evolution of the LDOS. A second application of the calculation of STM pictures is relative to the adsorption of Ir atoms on a Ir(111) substrate. FLAPW calculations and the experiments of Thomas Michely's team show that the tip can distinguish unequivocally two different stackings (hcp or fcc) of comparable energie but with different density of states around the Fermi level. Finally, the study of the magnetic properties of Co nanowires unsupported or adsorbed at the foot of Pt step edges is approached. For the unsupported system, the magnetic moment of the cobalt atom decreases from 3 uB in the case of an isolated atom to 2.33 uB for the chain, 2.09 uB for the monolayer and 1.65 uB in the bulk. Calculations were also conducted on periodic arrays of Co wires supported on Pt(233) and eventually relaxed systems. It is shown that the influence of the substrate on the magnetic moment of the Co atom is not very big. On the other hand, a strong quenching of the orbital moments is observed. The magnetic and orbitals moments are equal respectively to 2.105 uB and [0.058-0.091] uB depending on the orientation of the spin quantization axis. The determination of the magnetic anisotropy shows that the easy axis is along the chain direction when the system is not relaxed. Only the relaxation can explain the fact that the easy axis goes out of the chain axis.; Le travail de cette thèse concerne la croissance et l'étude des propriétés physiques de structures nanométriques susceptibles de se former sur des surfaces à marches. De précédentes études tant expérimentales que théoriques ont montré que même si les méthodes de croissances auto organisées permettent d'obtenir un grand nombre de nanostructures, la qualité des fils est très dépendante des conditions de préparation et de la statistique du dépôt. L'idée originale de ce travail consiste à assister la croissance en utilisant une pointe de microscope STM. Afin de caractériser les propriétés spécifiques des structures alors formées, il convient d'avoir préalablement décrit de façon exhaustive le substrat et son interaction avec les atomes adsorbés. Le code de calcul DFT FLEUR est utilisé afin de caractériser diverses surfaces de platine tant du point de vue structurel qu'énergétique. Dans le prolongement de ce travail, nous abordons également la caractérisation de propriétés magnétiques de chaines de cobalt non supportées ou bien déposées sur des surfaces de platine vicinales. Dans la première partie, nous présentons une étude concernant des phénomènes de croissance menée numériquement à l'aide d'un modèle KMC (Kinetic Monte Carlo). Pour le système Xe/Cu(110), nous montrons que la présence d'une pointe STM fixe peut, suivant la hauteur à laquelle elle est placée au dessus de la surface modifier localement la diffusion des adatomes et également permettre la mesure des coefficients de diffusion caractéristiques du déplacement des atomes. Nous nous sommes ensuite placés dans un cadre plus général et nous avons montré qu'il est possible d'améliorer les profils de nanofils déposés sur des surfaces vicinales à l'aide d'une pointe STM mobile utilisée en mode répulsif ou attractif. La perfection du fil est fonction du nombre et du type de balayages effectués, ainsi que de l'intervalle de température considéré. Nous nous sommes ensuite intéressés aux propriétés électroniques des surfaces de platine. Après avoir considéré en premier lieu les trois surfaces plates classiques (111), (100) et (110) notre attention s'est portée sur une surface vicinale (233) présentant la même géométrie de marche que la surface expérimentale (997) mais possédant une largeur de terrasses inférieure. Nous avons mené des calculs d'énergie de surface ou de marche, de densité d'états, ou encore de structures de bande. Les calculs des images STM ont été réalisés pour interpréter les images STM de la surface Pt(997) obtenues à l'EPFL (Lausanne, équipe de K. Kern et H. Brune). Ni la relaxation (vers l'intérieur), ni la densité d'état locale au voisinage de la marche ne permettent de comprendre la protubérance observée à cet endroit sur certains clichés expérimentaux. Ceci ne veut pas pour autant dire que nos calculs sont erronés. La protubérance observée peut être due à une erreur expérimentale et notamment au temps de réponse de la pointe. Une deuxième application du calcul d'images STM est relative à l'adsorption d'atomes d'Iridium sur un substrat (111) de même espèce. Les calculs FLAPW et les expériences du groupe de T. Michely (RWTH, Aachen) montrent sans ambiguïté que la pointe peut distinguer deux types d'empilements (hcp et fcc) d'énergie comparable mais présentant des densités d'états différentes au niveau de Fermi. Pour finir, l'étude des propriétés magnétiques de nanofils de cobalt non supportés ou adsorbés au pied des marches de platine est abordée. Pour le système non supporté, le moment magnétique passe de 3 uB pour l'atome de cobalt isolé, à 2.33 uB pour la chaîne, à 2.09 uB pour la monocouche et 1.65 uB pour le volume. Le calcul a ensuite été fait pour une succession périodique de fils de cobalt supportés par le platine (233) et relaxés. Les calculs montrent que le substrat a peu d'influence sur le moment de spin. Par contre, on observe un blocage fort du moment magnétique orbital par le champ cristallin créé par les atomes de platine. Les moments magnétiques de spin et orbital sont égaux respectivement à 2.105 uB et [0.058-0.091]uB selon la direction de l'aimantation. Le calcul de l'anisotropie magnétique semble montrer que l'axe de facile aimantation est dirigé selon les fils lorsque le système n'est pas relaxé. Seule la relaxation permet d'expliquer le fait que l'axe de facile aimantation sort de la chaîne.