Développement du cartilage articulaire équin du fœtus à l’adulte : imagerie par résonance magnétique et microscopie en lumière polarisée

La structure du cartilage articulaire adulte est caractérisée par la présence de couches créées par l’orientation des fibres de collagène (Benninghoff, 1925). Avant de présenter la structure adulte classique en arcades “de Benninghoff”, le cartilage subit une série de changements au cours de sa maturation (Julkunen et al., 2010; Lecocq et al., 2008). Toutefois, un faible nombre d’études s’est intéressé à la structure du collagène du cartilage articulaire in utero. Notre objectif était d’étudier la maturation de la surface articulaire de l’épiphyse fémorale distale chez le cheval, en employant à la fois l’imagerie par résonance magnétique (IRM) et la microscopie en lumière polarisée après coloration au rouge picrosirius, au niveau de sites utilisés dans les études de réparation tissulaire et de sites prédisposés à l’ostéochondrose (OC). Le but était de décrire le développement normal du réseau de collagène et la relation entre les images IRM et la structure histologique. Des sections provenant de cinq sites de l’épiphyse fémorale distale de 14 fœtus et 10 poulains et adultes ont été colorées au rouge picrosirius, après que le grasset ait été imagé par IRM, dans l’optique de visualiser l’agencement des fibres de collagène de type II. Les deux modalités utilisées, IRM et microscopie en lumière polarisée, ont démontré la mise en place progressive d’une structure en couches du réseau de collagène, avant la naissance et la mise en charge de l’articulation.Adult articular cartilage has a zonal or layered structure, created by the predominant collagen fibre orientation (Benninghoff, 1925). Before reaching the classical “Benninghoff structure”, major changes take place with maturation from juvenile to adult cartilage (Julkunen et al., 2010; Lecocq et al., 2008). However, there have been few studies addressing the in utero collagen structure of articular cartilage. Our objective was to study the maturation of the distal femoral epiphysis articular surface, employing both magnetic resonance imaging and polarized light microscopy with picrosirius red staining, at sites employed for cartilage repair studies or susceptible to osteochondrosis to describe normal development of the spatial architecture of the collagen network at these sites and the relationship between magnetic resonance images and histology. Samples were harvested from five sites from the distal femoral epiphysis of 14 fetuses and 10 foals and adults, after the stifle was imaged with magnetic resonance imaging. Sections were stained with picrosirius red to determine the structural arrangement of the type II collagen fibres. Both magnetic resonance imaging and polarized light microscopy revealed an early progressive structural laminar/zonal organization of the collagen network, prior to birth and postnatal load-bearing

A non-rigid registration approach for quantifying myocardial contraction in tagged MRI using generalized information measures.

International audienceWe address the problem of quantitatively assessing myocardial function from tagged MRI sequences. We develop a two-step method comprising (i) a motion estimation step using a novel variational non-rigid registration technique based on generalized information measures, and (ii) a measurement step, yielding local and segmental deformation parameters over the whole myocardium. Experiments on healthy and pathological data demonstrate that this method delivers, within a reasonable computation time and in a fully unsupervised way, reliable measurements for normal subjects and quantitative pathology-specific information. Beyond cardiac MRI, this work redefines the foundations of variational non-rigid registration for information-theoretic similarity criteria with potential interest in multimodal medical imaging

The mechanisms and dynamics of αvβ3 integrin clustering in living cells

During cell migration, the physical link between the extracellular substrate and the actin cytoskeleton mediated by receptors of the integrin family is constantly modified. We analyzed the mechanisms that regulate the clustering and incorporation of activated αvβ3 integrins into focal adhesions. Manganese (Mn2+) or mutational activation of integrins induced the formation of de novo F-actin–independent integrin clusters. These clusters recruited talin, but not other focal adhesion adapters, and overexpression of the integrin-binding head domain of talin increased clustering. Integrin clustering required immobilized ligand and was prevented by the sequestration of phosphoinositole-4,5-bisphosphate (PI(4,5)P2). Fluorescence recovery after photobleaching analysis of Mn2+-induced integrin clusters revealed increased integrin turnover compared with mature focal contacts, whereas stabilization of the open conformation of the integrin ectodomain by mutagenesis reduced integrin turnover in focal contacts. Thus, integrin clustering requires the formation of the ternary complex consisting of activated integrins, immobilized ligands, talin, and PI(4,5)P2. The dynamic remodeling of this ternary complex controls cell motility

The Tyrosine-Autokinase UbK Is Required for Proper Cell Growth and Cell Morphology of Streptococcus pneumoniae

International audienceProtein phosphorylation is a key post-translational modification required for many cellular functions of the bacterial cell. Recently, we identified a new protein-kinase, named UbK, in Bacillus subtilis that belongs to a new family of protein-kinases widespread in bacteria. In this study, we analyze the function of UbK in Streptococcus pneumoniae. We show that UbK displays a tyrosine-kinase activity and autophosphorylates on a unique tyrosine in vivo. To get insights into its cellular role, we constructed a set of pneumococcal ubk mutants. Using conventional and electron microscopy, we show that the ubk deficient strain, as well as an ubk catalytic dead mutant, display both severe cell-growth and cell-morphology defects. The same defects are observed with a mutant mimicking permanent phosphorylation of UbK whereas they are not detected for a mutant mimicking defective autophosphorylation of UbK. Moreover, we find that UbK phosphorylation promotes its ability to hydrolyze ATP. These observations show that the hydrolysis of ATP by UbK serves not only for its autophosphorylation but also for a distinct purpose essential for the optimal cell growth and cell-morphogenesis of the pneumococcus. We thus propose a model in which the autophosphorylation/dephosphorylation of UbK regulates its cellular function through a negative feedback loop

The Fibrillar Collagen Family

Collagens, or more precisely collagen-based extracellular matrices, are often considered as a metazoan hallmark. Among the collagens, fibrillar collagens are present from sponges to humans, and are involved in the formation of the well-known striated fibrils. In this review we discuss the different steps in the evolution of this protein family, from the formation of an ancestral fibrillar collagen gene to the formation of different clades. Genomic data from the choanoflagellate (sister group of Metazoa) Monosiga brevicollis, and from diploblast animals, have suggested that the formation of an ancestral α chain occurred before the metazoan radiation. Phylogenetic studies have suggested an early emergence of the three clades that were first described in mammals. Hence the duplication events leading to the formation of the A, B and C clades occurred before the eumetazoan radiation. Another important event has been the two rounds of “whole genome duplication” leading to the amplification of fibrillar collagen gene numbers, and the importance of this diversification in developmental processes. We will also discuss some other aspects of fibrillar collagen evolution such as the development of the molecular mechanisms involved in the formation of procollagen molecules and of striated fibrils

Les dominantes en pathologie locomotrice du cheval de concours de saut d'obstacles

L épreuve de concours de saut d obstacles requiert de la part de l athlète cheval des qualités techniques et mentales, ingrédients essentiels au succès. Cette épreuve se distingue par des sollicitations intenses de l appareil locomoteur, très spécifiques des gestes imposés au cours des phases successives du saut. Cette thèse a pour but de recenser les principales pathologies de l appareil locomoteur auxquelles le cheval de concours de saut d obstacles doit faire face. En premier lieu sont présentées les caractéristiques de l épreuve ainsi que du cheval athlète. Enfin, la dernière partie vise à proposer aux professionnels qui gravitent autour du cheval de saut d obstacles, un plan de suivi pour cet athlète, en soulignant le rôle capital du vétérinaire.NANTES-Ecole Nat.Vétérinaire (441092302) / SudocTOULOUSE-EN Vétérinaire (315552301) / SudocSudocFranceF

Rôle d une protéine de la matrice extracellulaire, la ténascine-X, sur l étalement des cellules sur un substrat de collagène I.

La ténascine-X (TNX) est une glycoprotéine de la matrice extracellulaire capable d interagir avec certains collagènes ainsi que des protéoglycanes. Elle possède la structure modulaire caractéristique des membres de la famille des ténascines avec 3 types de domaines : des motifs de type EGF, des domaines similaires au FNIII et, enfin, une extrémité globulaire similaire au fibrinogène. La déficience en TNX chez l homme conduit à une forme récessive du syndrome d Ehlers-Danlos caractérisée par une hypermobilité articulaire et une peau fragile très extensible. Elle est également corrélée à un phénotype invasif plus important dans un modèle de tumeur injectée chez la souris knock-out pour la TNX. Ces données suggèrent un rôle de la TNX à la fois dans l assemblage matriciel et dans la régulation du comportement cellulaire. Ce travail de thèse a permis de caractériser l existence d une interaction directe entre la TNX et le collagène de type I, dépendante de la conformation en triple hélice de ce dernier. L étude de l interaction entre différents types de cellules (fibrosarcome, fibroblastes primaires ) et la TNX a confirmé que la TNX recombinante permet une faible adhérence des cellules en utilisant un récepteur de la famille des intégrines. De plus, il a été démontré par des études morphométriques que la TNX est capable de moduler l étalement cellulaire sur un substrat de collagène I. Cette modulation passe par une modification des structures mises en place par les cellules pour interagir avec leur environnement. Ainsi, la formation des filopodes est augmentée en présence de TNX. Par ailleurs, les adhésions focales adoptent une composition et une localisation singulières en présence de TNX par rapport à notre condition contrôle sur collagène I. La signalisation associée à ces structures est également modulée par la TNX puisqu une diminution de l activité de FAK et de Rac ainsi qu une diminution de la phosphorylation de la paxilline ont pu être mesurées. Ce travail de thèse constitue la première étude des effets cellulaires de cette protéine de la matrice extracellulaire encore méconnue. Il apporte les premières évidences attribuant à la TNX le rôle de protéine matricellulaire capable de moduler les interactions cellule-matrice. Ces propriétés pourraient favoriser le phénotype prométastatique observé chez les souris déficientes en TNX.Tenascin-X (TNX) is a large glycoprotein from the extracellular matrix associated with collagen fibrils and proteoglycans. TNX shares the typical modular structure of the TN family members with EGF, FNIII and Fbg domains. TNX deficiency leads to a recessive human form of Ehlers Danlos syndrome characterized by joint hypermobility, skin fragility and hyperextensible skin. It is also correlated with a more invasive phenotype in tumour-induced TNX-/- mice, suggesting a role in the extracellular matrix assembly and in the modulation of cell behaviour. In this thesis, we identify a direct interaction between TNX and collagen I in a triple-helical conformation. Concerning the interaction between cells and TNX, we demonstrate that TNX allows weak adhesion of numerous cell lines (fibrosarcoma cells, primary fibroblasts...), mediated by at least one integrin receptor. Moreover, using morphometrical analysis, we show that recombinant TNX can modulate cell spreading on collagen I. This modulation occurs through the modification of the cellular structures responsible for the interaction between cells and their surrounding matrix. For example, the formation of filopodia is enhanced when TNX is added to collagen I. In addition, focal adhesions display a modified composition and localisation. The signalling pathways initiated from these adhesion sites are modulated by TNX, i.e. FAK and small GTPase Rac activities are downregulated and paxillin phosphorylation is impaired. Together, the results of this thesis demonstrate that TNX regulates cell-matrix induced signalisation and modifies cell spreading. Hence, TNX can be considered as an authentic matricellular protein. These data constitute promising foundations to understand the prometastatic phenotype observed in TNX-deficient miceLYON1-BU.Sciences (692662101) / SudocSudocFranceF

A model of tenascin-X integration within the collagenous network

AbstractTenascin-X is an extracellular matrix protein whose absence leads to an Ehlers-Danlos syndrome in humans, characterized mainly by disorganisation of collagen and elastic fibril networks. After producing recombinant full-length tenascin-X in mammalian cells, we find that this protein assembled into disulfide-linked oligomers. Trimers were the predominant form observed using rotary shadowing. By solid phase interaction studies, we demonstrate that tenascin-X interacts with types I, III and V fibrillar collagen molecules when they are in native conformation. The use of tenascin-X variants with large regions deleted indicated that both epidermal growth factor repeats and the fibrinogen-like domain are involved in this interaction. Moreover, we demonstrate that tenascin-X binds to the fibril-associated types XII and XIV collagens. We thus suggest that tenascin-X, via trimerization and multiple interactions with components of collagenous fibrils, plays a crucial role in the organisation of extracellular matrices

Competency framework to support need seeker innovation training

International audienceEmpowering key competencies to support a specific innovation strategy becomes a critical issue to the long term survival of many today's firms. This imperative reflects a challenge for design and innovation education, in properly preparing students. In this article, we present a competency framework to guide the implementation of a competency-based learning of design and innovation. In particular, we focus on skills required to implement an innovation strategy derived by a 'superior end-user understanding' to get first to market, namely the Need-Seeker Strategy. Our methodology combines an extensive literature review, an empirical study on project-based learning and expert interviews to result in an original competency framework supporting the need-seeker strategy. We believe that this is the first competency framework specifying conjointly individual and collective competencies as well as leadership competencies supporting a need-seeker innovation process