Functional glycosylation of dystroglycan is crucial for thymocyte development in the mouse.

BACKGROUND: Alpha-dystroglycan (alpha-DG) is a cell surface receptor providing a molecular link between the extracellular matrix (ECM) and the actin-based cytoskeleton. During its biosynthesis, alpha-DG undergoes specific and unusual O-glycosylation crucial for its function as a high-affinity cellular receptor for ECM proteins. METHODOLOGY/PRINCIPAL FINDINGS: We report that expression of functionally glycosylated alpha-DG during thymic development is tightly regulated in developing T cells and largely confined to CD4(-)CD8(-) double negative (DN) thymocytes. Ablation of DG in T cells had no effect on proliferation, migration or effector function but did reduce the size of the thymus due to a significant loss in absolute numbers of thymocytes. While numbers of DN thymocytes appeared normal, a marked reduction in CD4(+)CD8(+) double positive (DP) thymocytes occurred. In the periphery mature naïve T cells deficient in DG showed both normal proliferation in response to allogeneic cells and normal migration, effector and memory T cell function when tested in acute infection of mice with either lymphocytic choriomeningitis virus (LCMV) or influenza virus. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates that DG function is modulated by glycosylation during T cell development in vivo and that DG is essential for normal development and differentiation of T cells.

Vesicular transport of d-serine in astrocytes

The concept of tripartite synapse suggests that astrocytes make up a functional synapse with pre- and postsynaptic neuronal elements to modulate synaptic transmission through the regulated release of neuromodulators called gliotransmitters. Release of gliotransmitters such as glutamate or D-serine has been shown to depend on Ca21-dependent exocytosis. However, the origin (cytosolic versus vesicular) of the released gliotransmitter is still a matter of debate. The existence of Ca21-regulated exocytosis in astrocytes has been questioned mostly because the nature of secretory organelles which are loaded with gliotransmitters is unknown. Here we show the existence of a population of vesicles that uptakes and stores glutamate and D-serine in astrocytes which are present in situ. Immunoisolated glial organelles expressing synaptobrevin 2 (Sb2) display morphological and biochemical features very similar to synaptic vesicles. We demonstrate that these organelles not only contain and uptake glutamate but also display a glia-specific transport activity for D-serine. Furthermore, we report that the uptake of D-serine is energized by a H1-ATPase present on the immunoisolated vesicles and that cytosolic chloride ions modulate the uptake of D-serine. Finally, we show that serine racemase (SR), the synthesizing enzyme for D-serine, is anchored to the membrane of glial organelles allowing a local and efficient concentration of the gliotransmitter to be transported. We conclude that vesicles in astrocytes do exist with the goal to store and release D-serine, glutamate and most likely other neuromodulators.

Reversal of activity-mediated spine dynamics and learning impairment in a mouse model of Fragile X syndrome.

Fragile X syndrome (FXS) is characterized by intellectual disability and autistic traits, and results from the silencing of the FMR1 gene coding for a protein implicated in the regulation of protein synthesis at synapses. The lack of functional Fragile X mental retardation protein has been proposed to result in an excessive signaling of synaptic metabotropic glutamate receptors, leading to alterations of synapse maturation and plasticity. It remains, however, unclear how mechanisms of activity-dependent spine dynamics are affected in Fmr knockout (Fmr1-KO) mice and whether they can be reversed. Here we used a repetitive imaging approach in hippocampal slice cultures to investigate properties of structural plasticity and their modulation by signaling pathways. We found that basal spine turnover was significantly reduced in Fmr1-KO mice, but markedly enhanced by activity. Additionally, activity-mediated spine stabilization was lost in Fmr1-KO mice. Application of the metabotropic glutamate receptor antagonist α-Methyl-4-carboxyphenylglycine (MCPG) enhanced basal turnover, improved spine stability, but failed to reinstate activity-mediated spine stabilization. In contrast, enhancing phosphoinositide-3 kinase (PI3K) signaling, a pathway implicated in various aspects of synaptic plasticity, reversed both basal turnover and activity-mediated spine stabilization. It also restored defective long-term potentiation mechanisms in slices and improved reversal learning in Fmr1-KO mice. These results suggest that modulation of PI3K signaling could contribute to improve the cognitive deficits associated with FXS.

Regulation of nonphosphorylated and phosphorylated forms of neurofilament proteins in the prefrontal cortex of human opioid addicts.

The neurofilament (NF) proteins (NF-H, NF-M, and NF-L for high, medium, and low molecular weights) play a crucial role in the organization of neuronal shape and function. In a preliminary study, the abundance of total NF-L was shown to be decreased in brains of opioid addicts. Because of the potential relevance of NF abnormalities in opioid addiction, we quantitated nonphosphorylated and phosphorylated NF in postmortem brains from 12 well-defined opioid abusers who had died of an opiate overdose (heroin or methadone). Levels of NF were assessed by immunoblotting techniques using phospho-independent and phospho-dependent antibodies, and the relative (% changes in immunoreactivity) and absolute (changes in ng NF/microg total protein) amounts of NF were calculated. Decreased levels of nonphosphorylated NF-H (42-32%), NF-M (14-9%) and NF-L (30-29%) were found in the prefrontal cortex of opioid addicts compared with sex, age, and postmortem delay-matched controls. In contrast, increased levels of phosphorylated NF-H (58-41%) and NF-M (56-28%) were found in the same brains of opioid addicts. The ratio of phosphorylated to nonphosphorylated NF-H in opioid addicts (3.4) was greater than that in control subjects (1.6). In the same brains of opioid addicts, the levels of protein phosphatase of the type 2A were found unchanged, which indicated that the hyperphosphorylation of NF-H is not the result of a reduced dephosphorylation process. The immunodensities of GFAP (the specific glial cytoskeletol protein), alpha-internexin (a neuronal filament related to NF-L) and synaptophysin (a synapse-specific protein) were found unchanged, suggesting a lack of gross changes in glial reaction, other intermediate filaments of the neuronal cytoskeletol, and synaptic density in the prefrontal cortex of opioid addicts. These marked reductions in total NF proteins and the aberrant hyperphosphorylation of NF-H in brains of opioid addicts may play a significant role in the cellular mechanisms of opioid addiction.

Regulation of the integration of newly generated neurons in the adult hippocampus

Hippocampal adult neurogenesis results in the continuous formation of new neurons in the adult hippocampus, which participate to learning and memory. Manipulations increasing adult neurogenesis have a huge clinical potential in pathologies involving memory loss. Intringuingly, most of the newborn neurons die during their maturation. Thus, increasing newborn neuron survival during their maturation may be a powerful way to increase overall adult neurogenesis. The factors governing this neuronal death are yet poorly known. In my PhD project, we made the hypothesis that synaptogenesis and synaptic activity play a role in the survival of newborn hippocampal neurons. We studied three factors potentially involved in the regulation of the synaptic integration of adult-born neurons. First, we used propofol anesthesia to provoke a global increase in GABAergic activity of the network, and we evaluated the outcome on newborn neuron synaptic integration, morphological development and survival. Propofol anesthesia impaired the dendritic maturation and survival of adult-born neurons in an age-dependent manner. Next, we examined the development of astrocytic ensheathment on the synapses formed by newborn neurons, as we hypothesized that astrocytes are involved in their synaptic integration. Astrocytic processes ensheathed the synapses of newborn neurons very early in their development, and the processes modulated synaptic transmission on these cells. Finally, we studied the cell-autonomous effects of the overexpression of synaptic adhesion molecules on the development, synaptic integration and survival of newborn neurons, and we found that manipulating of a single adhesion molecule was sufficient to modify synaptogenesis and/or synapse function, and to modify newborn neuron survival. Together, these results suggest that the activity of the neuronal network, the modulation of glutamate transport by astrocytes, and the synapse formation and activity of the neuron itself may regulate the survival of newborn neurons. Thus, the survival of newborn neurons may depend on their ability to communicate with the network. This knowledge is crucial for finding ways to increase neurogenesis in patients. More generally, understanding how the neurogenic niche works and which factors are important for the generation, maturation and survival of neurons is fundamental to be able to maybe, one day, replace neurons in any region of the brain.

Upper Triassic to Cretaceous radiolaria from Nicaragua and northern Costa Rica - The Mesquito composite oceanic terrane

We propose a new terrane subdivision of Nicaragua and Northern Costa Rica, based on Upper Triassic to Upper Cretaceous radiolarian biochronology of ribbon radiolarites, the newly studied Siuna Serpentinite Mélange, and published 40Ar/39Ar dating and geochemistry of mafic and ultramafic igneous rock units of the area. The new Mesquito Composite Oceanic Terrane (MCOT) comprises the southern half of the Chortis Block, that was assumed to be a continental fragment of N-America. The MCOT is defined by 4 corner localities characterized by ultramafic and mafic oceanic rocks and radiolarites of Late Triassic, Jurassic and Early Cretaceous age: 1. The Siuna Serpentinite Mélange (NE-Nicaragua), 2. The El Castillo Mélange (Nicaragua/Costa Rica border), 3.The Santa Elena Ultramafics (N-Costa Rica) and, 4. DSDP Legs 67/84. 1. The Siuna Serpentinite Mélange contains, high pressure metamorphic mafics and Middle Jurassic (Bajocian-Bathonian) radiolarites in original, sedimentary contact with arc-metandesites. The Siuna Mélange also contains Upper Jurassic black detrital chert formed in a marginal (fore-arc?) basin shortly before subduction. A phengite 40Ar/39Ar -cooling age dates the exhumation of the high pressure rocks as 139 Ma (earliest Cretaceous). 2. The El Castillo Mélange comprises a radiolarite block tectonically embedded in serpentinite that yielded a diverse Rhaetian (latest Triassic) radiolarian assemblage, the oldest fossils recovered so far from S-Central America. 3. The Santa Elena Ultramafics of N-Costa Rica together with the serpentinite outcrops near El Castillo (2) in Southern Nicaragua, are the southernmost outcrops of the MCOT. The Santa Elena Unit (3) itself is still undated, but it is thrust onto the middle Cretaceous Santa Rosa Accretionary Complex (SRAC), that contains Lower to Upper Jurassic, highly deformed radiolarite blocks, probably reworked from the MCOT, which was the upper plate with respect to the SRAC. 4. Serpentinites, metagabbros and basalts have long been known from DSDP Leg 67/84 (3), drilled off Guatemala in the Nicaragua-Guatemala forearc basement. They have been restudied and reveal 40Ar/39Ar dated Upper Triassic to middle Cretaceous enriched Ocean Island Basalts and Jurassic to Lower Cretaceous depleted Island arc rocks of probable Pacific origin. The area between localities 1-4 is largely covered by Tertiary to Recent arcs, but we suspect that its basement is made of oceanic/accreted terranes. Earthquake seismic studies indicate an ill-defined, shallow Moho in this area. The MCOT covers most of Nicaragua and could extend to Guatemala to the W and form the Lower (southern) Nicaragua Rise to the NE. Some basement complexes of Jamaica, Hispaniola and Puerto Rico may also belong to the MCOT. The Nicoya Complex s. str. has been regarded as an example of Caribbean crust and the Caribbean Large Igneous Province (CLIP). However, 40Ar/39Ar - dates on basalts and intrusives indicate ages as old as Early Cretaceous. Highly deformed Jurassic and Lower Cretaceous radiolarites occur as blocks within younger intrusives and basalts. Our interpretation is that radiolarites became first accreted to the MCOT, then became reworked into the Nicoya Plateau in Late Cretaceous times. This implies that the Nicoya Plateau formed along the Pacific edge of the MCOT, independent form the CLIP and most probably unrelated with he Galapagos hotspot. No Jurassic radiolarite, no older sediment age than Coniacian-Santonian, and no older 40Ar/39Ar age than 95 Ma is known from S-Central America between SE of Nicoya and Colombia. For us this area represents the trailing edge of the CLIP s. str.

Stereologic estimates of total spinophilin-immunoreactive spine number in area 9 and the CA1 field: Relationship with the progression of Alzheimer's disease

The loss of presynaptic markers is thought to represent a strong pathologic correlate of cognitive decline in Alzheimer's disease (AD). Spinophilin is a postsynaptic marker mainly located to the heads of dendritic spines. We assessed total numbers of spinophilin-immunoreactive puncta. in the CA I and CA3 fields of hippocampus and area 9 in 18 elderly individuals with various degrees of cognitive decline. The decrease in spinophilin-immunoreactivity was significantly related to both Braak neurofibrillary tangle (NFT) staging and clinical severity but not A beta deposition staging. The total number of spinophilin-immunoreactive puncta in CA I field and area 9 were significantly related to MMSE scores and predicted 23.5 and 61.9% of its variability. The relationship between total number of spinophilin-immunoreactive puncta in CA I field and MMSE scores did not persist when adjusting for Braak NFT staging. In contrast, the total number of spinophilin-immunoreactive puncta in area 9 was still significantly related to the cognitive outcome explaining an extra 9.6% of MMSE and 25.6% of the Clinical Dementia Rating scores variability. Our data suggest that neocortical dendritic spine loss is an independent parameter to consider in AD clinicopathologic correlations.

Plate tectonics of the Altaids

Abstract: The Altaids consist in a huge accretionary-type belt extending from Siberia through Mon-golia, northern China, Kyrgyzstan and Kazakhstan. They were formed from the Vendian through the Jurassic by the accretion of numerous displaced and exotic terranes (e.g. island arc, ribbon microcontinent, seamount, basaltic plateau, back-arc basin). The number, nature and origin of the terranes differ according to the palaeotectonic models of the different authors. Thanks to a geo- dynamic study (i.e. definition of tectonic settings and elaboration of geodynamic scenarios) and plate tectonics modelling, this work aims to present an alternative model explaining the Palaeozoic palaeotectonic evolution of the Altaids. Based on a large set of compiled geological data related to palaeogeography and geodyna¬mic (e.g. sedimentology, stratigraphy, palaeobiogeography, palaeomagnetism, magmatism, me- tamorphism, tectonic...), a partly new classification of the terranes and sutures implicated in the formation of the Altaids is proposed. In the aim to elaborate plate tectonics reconstructions, it is necessary to fragment the present arrangement of continents into consistent geological units. To avoid confusion with existing terminology (e.g. tectonic units, tectono-stratigraphic units, micro- continents, terranes, blocks...), the new concept of "Geodynamic Units (GDU)" was introduced. A terrane may be formed by a set of GDUs. It consists of a continental and/or oceanic fragment which has its own kinematic and geodynamic evolution for a given period. With the same ap-proach, the life span and type of the disappeared oceans is inferred thanks to the study of the mate-rial contained in suture zones. The interpretation of the tectonic settings within the GDUs comple-ted by the restoration of oceans leads to the elaboration of geodynamic scenarios. Since the Wilson cycle was presented in 1967, numerous works demonstrated that the continental growth is more complex and results from diverse geodynamic scenarios. The identification of these scenarios and their exploitation enable to elaborate plate tectonics models. The models are self-constraining (i.e. space and time constraints) and contest or confirm in turn the geodynamic scenarios which were initially proposed. The Altaids can be divided into three domains: (1) the Peri-Siberian, (2) the Kazakhstan, and (3) the Tarim-North China domains. The Peri-Siberian Domain consists of displaced (i.e. Sayan Terrane Tuva-Mongolian, Lake-Khamsara Terrane) and exotic terranes (i.e. Altai-Mongolian and Khangai-Argunsky Terrane) accreted to Siberia from the Vendian through the Ordovician. Fol-lowing the accretion of these terranes, the newly formed Siberia active margin remained active un-til its part collision with the Kazakhstan Superterrane in the Carboniferous. The eastern part of the active margin (i.e. East Mongolia) continued to act until the Permian when the North-China Tarim Superterrane collided with it. The geodynamic evolution of the eastern part of the Peri-Siberian Domain (i.e. Eastern Mongolia and Siberia) is complicated by the opening of the Mongol-Okhotsk Ocean in the Silurian. The Kazakhstan Domain is composed of several continental terranes of East Gondwana origin amalgamated together during the Ordovician-Silurian time. After these different orogenic events, the Kazakhstan Superterrane evolved as a single superterrane until its collision with a Tarim-North China related-terrane (i.e. Tianshan-Hanshan Terrane) and Siberian Continent during the Devonian. This new organisation of the continents imply a continued active margin from Siberia, to North China through the Kazakhstan Superterrane and the closure of the Junggar- Balkash Ocean which implied the oroclinal bending of the Kazakhstan Superterrane during the entire Carboniferous. The formation history of the Tarim-North China Domain is less complex. The Cambrian northern passive margin became active in the Ordovician. In the Silurian, the South Tianshan back-arc Ocean was open and led to the formation of the Tianshan-Hanshan Terrane which collided with the Kazakhstan Superterrane during the Devonian. The collision between Siberia and the eastern part of the Tarim-North China continents (i.e. Inner Mongolia), implied by the closure of the Solonker Ocean, took place in the Permian. Since this time, the major part of the Altaids was formed, the Mongol-Okhotsk Ocean only was still open and closed during the Jurassic. Résumé: La chaîne des Altaïdes est une importante chaîne d'accrétion qui s'étend en Sibérie, Mon-golie, Chine du Nord, Kirghizstan et Kazakhstan. Elle s'est formée durant la période du Vendian au Jurassique par l'accrétion de nombreux terranes déplacés ou exotiques (par exemple arc océa-nique, microcontinent, guyot, plateau basaltique, basin d'arrière-arc...). Le nombre, la nature ou encore l'origine diffèrent selon les modèles paléo-tectoniques proposés par les différents auteurs. Grâce à une étude géodynamique (c'est-à-dire définition des environnements tectoniques et éla-boration de scénarios géodynamiques) et à la modélisation de la tectonique des plaques, ce travail propose un modèle alternatif expliquant l'évolution paléo-tectonique des Altaïdes. Basé sur une large compilation de données géologiques pertinentes en termes de paléo-géographie et de géodynamique (par exemple sédimentologie, stratigraphie, paléo-biogéographie, paléomagnétisme, magmatisme, métamorphisme, tectonique...), une nouvelle classification des terranes et des sutures impliqués dans la formation des Altaïdes est proposée. Dans le but d'élabo¬rer des reconstructions de plaques tectoniques, il est nécessaire de fragmenter l'arrangement actuel des continents en unités tectoniques cohérentes. Afin d'éviter les confusions avec la terminolo¬gie existante (par exemple unité tectonique, unité tectono-stratigraphique, microcontinent, block, terrane...), le nouveau concept d' "Unité Géodynamique (UGD)" a été introduit. Un terrane est formé d'une ou plusieurs UGD et représente un fragment océanique ou continental défini pas sa propre cinétique et évolution géodynamique pour une période donnée. Parallèlement, la durée de vie et le type des océans disparus (c'est-à-dire principal ou secondaire) est déduite grâce à l'étude du matériel contenu dans les zones de sutures. L'interprétation des environnements tectoniques des UGD associés à la restauration des océans mène à l'élaboration de scénarios géodynamiques. Depuis que le Cycle de Wilson a été présenté en 1967, de nombreux travaux ont démontré que la croissance continentale peut résulter de divers scénarios géodynamiques. L'identification et l'ex-ploitation de ces scénarios permet finalement l'élaboration de modèles de tectonique des plaques. Les modèles sont auto-contraignants (c'est-à-dire contraintes spatiales et temporelles) et peuvent soit contester ou confirmer les scénarios géodynamiques initialement proposés. Les Altaïdes peuvent être divisées en trois domaines : (1) le Domaine Péri-Sibérien, (2) le Domaine Kazakh, et (3) le Domaine Tarim-Nord Chinois. Le Domaine Péri-Sibérien est composé de terranes déplacés (c'est-à-dire Terrane du Sayan, Tuva-Mongol et Lake-Khamsara) et exotiques (c'est-à-dire Terrane Altai-Mongol et Khangai-Argunsky) qui ont été accrétés au craton Sibérien durant la période du Vendien à l'Ordovicien. Suite à l'accrétion de ces terranes, la marge sud-est de la Sibérie nouvellement formée reste active jusqu'à sa collision partielle avec le Superterrane Ka-zakh au Carbonifère. La partie est de la marge active (c'est-à-dire Mongolie de l'est) continue son activité jusqu'au Permien lors de sa collision avec le Superterrane Tarim-Nord Chinois. L'évolu¬tion géodynamique de la partie est du Domaine Sibérien est compliquée par l'ouverture Silurienne de l'Océan Mongol-Okhotsk qui disparaîtra seulement au Jurassique. Le Domaine Kazakh est composé de plusieurs terranes d'origine est-Gondwanienne accrétés les uns avec les autres avant ou pendant le Silurien inférieur et leurs evolution successive sous la forme d'un seul superterrane. Le Superterrane Kazakh collisione avec un terrane Tarim-Nord Chinois (c'est-à-dire Terrane du Tianshan-Hanshan) durant le Dévonien et le continent Sibérien au Dévonien supérieur. Ce nouvel agencement des plaques induit une marge active continue le long des continents Sibérien, Kazakh et Nord Chinois et la fermeture de l'Océan Junggar-Balkash qui provoque le plissement oroclinal du Superterrane Kazakh durant le Carbonifère. L'histoire de la formation du Domaine Tarim-Nord Chinois est moins complexe. La marge passive nord Cambrienne devient active à l'Ordovicien et l'ouverture Silurienne du bassin d'arrière-arc du Tianshan sud mène à la formation du terrane du Tianshan-Hanshan. La collision Dévonienne entre ce dernier et le Superterrane Kazakh provoque la fermerture de l'Océan Tianshan sud. Finalement, la collision entre la Sibérie et la partie est du continent Tarim-Nord Chinois (c'est-à-dire Mongolie Intérieure) prend place durant le Permien suite à la fermeture de l'Océan Solonker. La majeure partie des Altaïdes est alors formée, seul l'Océan Mongol-Okhotsk est encore ouvert. Ce dernier se fermera seulement au Jurassique.

Cointegrase, a naturally occurring, truncated form of IS21 transposase, catalyzes replicon fusion rather than simple insertion of IS21.

The bacterial insertion sequence IS21 contains two genes, istA and istB, which are organized as an operon. IS21 spontaneously forms tandem repeats designated (IS21)2. Plasmids carrying (IS21)2 react efficiently with other replicons, producing cointegrates via a cut-and-paste mechanism. Here we show that transposition of a single IS21 element (simple insertion) and cointegrate formation involving (IS21)2 result from two distinct non-replicative pathways, which are essentially due to two differentiated IstA proteins, transposase and cointegrase. In Escherichia coli, transposase was characterized as the full-length, 46 kDa product of the istA gene, whereas the 45 kDa cointegrase was expressed, in-frame, from a natural internal translation start of istA. The istB gene, which could be experimentally disconnected from istA, provided a helper protein that strongly stimulated the transposase and cointegrase-driven reactions. Site-directed mutagenesis was used to express either cointegrase or transposase from the istA gene. Cointegrase promoted replicon fusion at high frequencies by acting on IS21 ends which were linked by 2, 3, or 4 bp junction sequences in (IS21)2. By contrast, cointegrase poorly catalyzed simple insertion of IS21 elements. Transposase had intermediate, uniform activity in both pathways. The ability of transposase to synapse two widely spaced IS21 ends may reside in the eight N-terminal amino acid residues which are absent from cointegrase. Given the 2 or 3 bp spacing in naturally occurring IS21 tandems and the specialization of cointegrase, the fulminant spread of IS21 via cointegration can now be understood.

Role of brain alpha 1B-adrenoceptors in modafinil-induced behavioral activity.

These studies show that either central pharmacological blockade or genetic ablation of alpha(1B)-adrenoceptors markedly attenuates the behavioral activation caused by modafinil, implicating these receptors in the drug's action.

The structure of nervous tissue and of Gram-positive bacteria studied by cryo-electron microscopy of vitreous sections

Résumé La structure, ou l'architecture, des êtres vivants définit le cadre dans lequel la physique de la vie s'accomplit. La connaissance de cette structure dans ses moindres détails est un but essentiel de la biologie. Son étude est toutefois entravée par des limitations techniques. Malgré son potentiel théorique, la microscopie électronique n'atteint pas une résolution atomique lorsqu'elle est appliquée ä la matièxe biologique. Cela est dû en grande partie au fait qu'elle contient beaucoup d'eau qui ne résiste pas au vide du microscope. Elle doit donc être déshydratée avant d'être introduite dans un microscope conventionnel. Des artéfacts d'agrégation en découlent inévitablement. La cryo-microscopie électronique des sections vitreuses (CEMOVIS) a ëté développée afin de résoudre cela. Les spécimens sont vitrifiés, c.-à-d. que leur eau est immobilisée sans cristalliser par le froid. Ils sont ensuite coupés en sections ultrafines et celles-ci sont observées à basse température. Les spécimens sont donc observés sous forme hydratée et non fixée; ils sont proches de leur état natif. Durant longtemps, CEMOVIS était très difficile à exécuter mais ce n'est plus le cas. Durant cette thèse, CEMOVIS a été appliqué à différents spécimens. La synapse du système nerveux central a été étudiée. La présence dans la fente synaptique d'une forte densité de molécules organisées de manière périodique a été démontrée. Des particules luminales ont été trouvées dans Ies microtubules cérébraux. Les microtubules ont servi d'objets-test et ont permis de démontrer que des détails moléculaires de l'ordre du nm sont préservés. La compréhension de la structure de l'enveloppe cellulaire des bactéries Grampositives aété améliorée. Nos observations ont abouti à l'élaboration d'un nouveau modèle hypothétique de la synthèse de la paroi. Nous avons aussi focalisé notre attention sur le nucléoïde bactérien et cela a suscité un modèle de la fonction des différents états structuraux du nucléoïde. En conclusion, cette thèse a démontré que CEMOVIS est une excellente méthode poux étudier la structure d'échantillons biologiques à haute résolution. L'étude de la structure de divers aspects des êtres vivants a évoqué des hypothèses quant à la compréhension de leur fonctionnement. Summary The structure, or the architecture, of living beings defines the framework in which the physics of life takes place. Understanding it in its finest details is an essential goal of biology. Its study is however hampered by technical limitations. Despite its theoretical potential, electron microscopy cannot resolve individual atoms in biological matter. This is in great part due to the fact. that it contains a lot of water that cannot stand the vacuum of the microscope. It must therefore be dehydrated before being introduced in a conventional mìcroscope. Aggregation artefacts unavoidably happen. Cryo-electron microscopy of vitreous sections (CEMOVIS) has been developed to solve this problem. Specimens are vitrified, i.e. they are rapidly cooled and their water is immobilised without crystallising by the cold. They are then. sectioned in ultrathin slices, which are observed at low temperatures. Specimens are therefore observed in hydrated and unfixed form; they are close to their native state. For a long time, CEMOVIS was extremely tedious but this is not the case anymore. During this thesis, CEMOVIS was applied to different specimens. Synapse of central nervous system was studied. A high density of periodically-organised molecules was shown in the synaptic cleft. Luminal particles were found in brain microtubules. Microtubules, used as test specimen, permitted to demonstrate that molecular details of the order of nm .are preserved. The understanding of the structure of cell envelope of Gram-positive bacteria was improved. Our observations led to the elaboration of a new hypothetic model of cell wall synthesis. We also focused our attention on bacterial nucleoids and this also gave rise to a functional model of nucleoid structural states. In conclusion, this thesis demonstrated that CEMOVIS is an excellent method for studying the structure of bìologìcal specimens at high resolution. The study of the structure of various aspects of living beings evoked hypothesis for their functioning.

Radiolarian biochronology and Pacific origin of Jurassic-Cretaceous oceanic terranes in Costa Rica and Puerto Rico

AbstractAs demonstrated during several recent geological conferences, there is still a large debate concerning the origins of the Mesozoic oceanic remnants on the Caribbean Plate. The geodynamic models describing the Mesozoic history of the Caribbean realm can be divided into two main categories based on the origin of the Caribbean Plate: 1) An in situ origin between the Americas; 2) A Pacific origin and an eastward transport relative to the Americas. The study of the ribbon-bedded radiolarite is a key in determining the origins of associated Mesozoic oceanic terranes and may help to achieve a general agreement regarding the basic principles on the evolution of the Caribbean Plate. The Early Jurassic to early Late Cretaceous Bermeja Complex of Puerto Rico, witch contains serpentinized peridotite, altered basalt, amphibolite, and chert (Mariquita Chert Formation), and the contemporaneous Santa Rosa Accretionary Complex, which crops out in several half-windows along the south shores of the Santa Elena Peninsula in northwestern Costa Rica, are two of these little-known and crucial ophiolitic mélanges. The Manzanillo and Matambú fore-arc Terranes of the Nicoya Peninsula in the northwestern Costa Rica, which contain Late Cretaceous to Early Paleogene radiolarian-bearing siliceous mudstones and cherts associated with arc-derived mafic to intermediate volcaniclastics, bring important information on the history of the western active margin of the Caribbean Plate. A systematic radiolarian study of these three regions is presented herein in three different articles.The radiolarian biochronology of the Mariquita Chert Formation of the Bermeja Complex presented in this work indicate an early Middle Jurassic to early Late Cretaceous (late Bajocian-early Callovian to middle Albian-middle Cenomanian) age for the Mariquita Chert Formation. The illustrated assemblages contain 150 species, of which 3 are new (Pantanellium karinae, Loopus bermejaense, and L. boricus), and belonging to 59 genera. A review of the previous radiolarian published works on this formation and the results of this study suggest that the Bermeja Complex ranges in age from Middle Jurassic to early Late Cretaceous (late Aalenian to middle Cenomanian) and also reveal a possible feature of the complex, which is the youngling of radiolarian cherts from north to south, evoking a polarity of accretion. On the basis of a currently exhaustive inventory of the ribbonbedded radiolaritic facies on the Caribbean Plate, a re-examination of the distribution of Middle Jurassic sediments associated with oceanic crust from the Caribbean realm, and a paleoceanographical argumentation on the water currents, we come to the conclusion that the radiolarite and associated Mesozoic oceanic terranes of the Caribbean Plate are of Pacific origin. The previous argument for a Pacific origin of the Bermeja Complex presented by Montgomery et al. (1994a), based on their radiolarian age and their estimation of the oldest Proto-Caribbean oceanic crust, is nowadays seriously questionable, owing to the recent progresses in radiolarian biostratigraphy and new discoveries on the age of the first oceanic crust spreading between the Americas. Furthermore, we interpret the radiolarian Parvicingulidae-rich assemblages in the low-latitude Caribbean context as potential indicators of upwelling or land nutrients inputs, instead of indicators of paleolatitudes,as firstly stated by Pessagno and Blome (1986). Eventually, a discussion on the origin of the cherts of the Mariquita Formation illustrated by Middle Jurassic to middle Cretaceous geodynamic models of the Pacific and Caribbean realms bring up the possibility that the rocks of the Bermeja Complex are remnants of two different oceans.The Santa Rosa Accretionary Complex contains various oceanic assemblages of alkaline basalt, radiolarite and polymictic breccias. The radiolarian biochronology (19 illustrated assemblages, 232 species belonging to 63 genera) presented in this work indicate an Early Jurassic to early Late Cretaceous (early Pliensbachian to earliest Turonian) age for the sediments associated with oceanic basalts or recovered from blocks in breccias or megabreccias from the Santa Rosa Accretionary Complex. This study brings to light the Early Jurassic age of a sequence of ribbon-bedded radiolarite, which was previously thought to be of Cretaceous age, intruded by alkaline basalts sills. The presence of Early Jurassic large reworked blocks of radiolarite in a polymictic megabreccia, firstly reported by De Wever et al. (1985) is confirmed. Therefore, the alkaline basalt associated with these radiolarites could be of Jurassic age. In the Carrizal tectonic window, Middle Jurassic radiolarian chert blocks and Early Cretaceous brick-red ribbon-bedded radiolarites overlying pillow basalts are interpreted as fragments of a Middle Jurassic oceanic basement accreted to an Early Cretaceous oceanic plate, in an intra-oceanic subduction context. Whereas, knobby radiolarites and black shale at Playa Carrizal are indicative of a shallower middle Cretaceous paleoenvironment. Other younger oceanic remnants documented the rapid approach of the site of sedimentation to a subduction trench during the late Early Cretaceous (AlbianCenomanian), maybe early Late Cretaceous (Turonian).In total, 60 species belonging to 34 genera were present in relatively well-preserved radiolarian faunas from volcaniclastics and associated pelagic and hemipelagic rocks of the Matambú and Manzanillo terranes, ranging in age from Late Cretaceous to Early Paleogene (middle Turonian-Santonian to late Thanetian-Ypresian). This study shows that radiolarians can provide significant biostratigraphic control in the Nicoya Peninsula where very similar lithologies of different ages are present. Two radiolarian samples directly date the Berrugate Formation for the first time (middle Turonian-Santonian and Coniacian-Santonian). These ages allow to determine a volcanic arc activity on the western edge of the future Caribbean Plate at least since the Santonian that could have lasted through the middle Turonian-early Campanian interval by stratigraphic superposition. Moreover on the basis of these radiolarian ages, the Loma Chumico Formation of Albian age, and the Berrugate Formation of middle Turonian-early Maastrichtian age, can now be clearly differentiated. Two samples from the Sabana Grande Formation give a Coniacian-Santonian age and a Coniacian-Campanian age and indicate that there is a stratigraphic gap of ~10 million years between this formation and the underlying Albian Loma Chumico Formation.RésuméComme cela a pu se vérifier à plusieurs reprises lors de conférences géologiques récentes, le débat sur l'origine des terrains océaniques mésozoïques de la Plaque Caraïbes est toujours d'actualité. Les modèles géodynamiques décrivant l'histoire de la région caraïbes peuvent être classés en deux catégories basées sur l'origine de la Plaque Caraïbes : 1) Une origine in situ entre les Amériques ; 2) Une origine Pacifique et un transport vers l'est, par rapport aux Amériques. L'étude des radiolarites rubanées est capitale pour la détermination de l'origine des terrains océaniques allochtones du Mésozoïque et peut être utile pour parvenir à un compromis général concernant les principes basiques de l'évolution de la Plaque Caraïbes. Le complexe de Bermeja à Porto Rico qui est constitué de péridotites serpentinisées, de basaltes altérés, d'amphibolites et de cherts (Formation des Cherts de Mariquita), et le Complexe d'Accrétion de Santa Rosa qui affleure dans plusieurs demi-fenêtres tectoniques au sud de la Péninsule de Santa Elena au nord-ouest du Costa Rica sont deux de ces mélanges ophiolitiques peu décrits et déterminants. Les terrains de fore-arc de Manzanillo et de Matambu dans la Péninsule de Nicoya au nord-ouest du Costa Rica qui sont composés de calcaires siliceux et de cherts riches en radiolaires associés à du matériel volcanique d'arc mafique à intermédiaire, apportent d'importantes informations sur l'histoire de la marge active occidentale de la Plaque Caraïbe. Une étude systématique des radiolaires de ces trois régions est présentée dans ce travail sous forme de trois articles.La biochronologie des radiolaires de la Formation des Cherts de Mariquita du Complexe d'Accrétion de Santa Rosa présentée dans ce travail indique un âge Jurassique Moyen inférieur à Crétacé Supérieur inférieur (Bajocien supérieur-Callovien inférieur à Albien moyen-Cénomanien moyen) pour la Formation des Cherts de Mariquita. Les assemblages illustrés contiennent 150 espèces, parmis lesquelles 3 sont nouvelles (Pantanellium karinae, Loopus bermejaense et L. boricus), et appartenant à 59 genres différents. Une révision des travaux publiés précédemment sur les radiolaires de cette formation, ainsi que les résultats de cette étude suggèrent que le Complexe de Bermeja a un âge allant du Jurassique moyen au Crétacé Supérieur inférieur (Aalénien supérieur à Cénomanien moyen) et révèle aussi une caractéristique éventuelle du complexe qui est le rajeunissement des radiolarites du nord au sud, évoquant une polarité d'accrétion. Sur la base d'un inventaire actuellement exhaustif du facies radiolaritique rubané sur la Plaque Caraïbes, d'un nouvel examen de la distribution globale des sédiments du Jurassique Moyen associés à de la croûte océanique et d'une argumentation paléocéanographique sur les courants, nous arrivons à la conclusion que les radiolarites et les unités tectoniques océaniques du Mésozoïque associées de la Plaque Caraïbes sont d'origine pacifique. L'argument antérieur pour une origine pacifique du Complexe de Bermeja présenté par Montgomery et al. (1994a), basé sur leur âge à radiolaire et leur estimation de l'âge de la plus vieille croûte océanique des Proto-Caraïbes, est sérieusement remis en question aujourd'hui, en raison des progrès récents de la biostratigraphie des radiolaires et des nouvelles découvertes concernant l'âge du début de l'océanisation entre les Amériques. En outre, dans le contexte de basses latitudes des Caraïbes, nous interprétons les assemblages à radiolaires riches en Parvicingulidae comme étant des indicateurs potentiels d'apports en nutriments des zones d'uppwelling ou des terres, plutôt que des indicateurs de paléolatitudes, comme exposer pour la première fois par Pessagno et Blome (1986). Finalement, une discussion sur l'origine des cherts de la Formation de Mariquita illustrée par des modèles géodynamiques du Jurassique Moyen au Crétacé moyen des régions pacifique et caraïbes, fait poindre la possibilité que les roches du Complexe de Bermeja proviennent de deux océans différents.Le Complexe d'Accrétion de Santa Rosa contient plusieurs assemblages océaniques différents de basaltes alcalins, radiolarites et brèches polymictes. La biochronologie des radiolaires (19 assemblages illustrés, 232 espèces appartenant à 63 genres) présentée dans ce second travail indique un âge Jurassique Inférieur à Crétacé Supérieur inférieur (Pliensbachien inférieur à Turonien initial) pour les sédiments associés aux basaltes océaniques ou provenant de blocs dans des brèches ou des mégabrèches du Complexe d'Accrétion de Santa Rosa. Cette étude met en évidence l'âge Jurassique Inférieur d'une séquence de radiolarites rubanées entrecoupée de sills de basaltes alcalins, dont l'âge estimé était précédemment le Crétacé.La présence de blocs plurimétriques de radiolarites d'âge Jurassique Inférieur remaniés dans une mégabrèche polymicte, dont la présence avait été signalée par De Wever et al. (1985), est confirmée. Par conséquent, les basaltes alcalins associés à ces radiolarites pourraient aussi être d'âge Jurassique. Dans la fenêtre tectonique de Carrizal, des blocs de radiolarites d'âge Jurassique Moyen et des radiolarites du Crétacé Inférieur recouvrant des basaltes en coussins sont interprétés comme des fragments d'une croûte océanique d'âge Jurassique Moyen accrétés à une plaque océanique d'âge Crétacé Inférieur, dans un contexte de subduction intra-océanique. Alors que dans la même zone, les radiolarites « noueuses » et les argiles noires associées sont interprétées comme des indicateurs d'un milieu peu profond au Crétacé. D'autres fragments océaniques plus jeunes documentent une approche rapide du lieu de sédimentation vers une fosse de subduction pendant le Crétacé Inférieur supérieur (Albien-Cénomanien), peut-être Crétacé Supérieur (Turonien).Au total, 60 espèces appartenant à 34 genres ont été déterminées à partir de faunes à radiolaires relativement bien préservées, extraites de roches volcanoclastiques et pélagiques à hémipélagiques associées, provenant des terrains de Matambu et Manzanillo et ayant des âges compris entre le Crétacé Supérieur et le Paléogène Inférieur (Turonien moyen-Santonien à Thanétien supérieur-Yprésien). Cette étude montre que les radiolaires peuvent fournir un contrôle stratigraphique significatif dans la Péninsule de Nicoya, où des lithologies similaires, mais d'âges différents sont présentes. Deux échantillons à radiolaires permettent de dater la Formation de Berrugate pour la première fois (Turonien moyen-Santonien et Coniacien-Santonien). Ces âges permettent d'établir une activité volcanique d'arc le long de la marge occidentale de la futur Plaque Caraïbes au moins depuis le Santonien et qui pourrait avoir durée jusqu'au Turonien moyen-Campanien inférieur. De plus, sur la base de ces âges à radiolaires, la Formation de Loma Chumico d'âge Albien, et la Formation de Berrugate d'âge Turonien moyen-Maastrichtien inférieur, peuvent maintenant être différenciées. Deux échantillons de la Formation de Sabana Grande donnent des âges Coniacien-Santonien et Coniacien-Campanien et indiquent qu'il existe une lacune stratigraphique d'environ 10 millions d'années entre cette formation et la Formation de Loma Chumico sous-jacente d'âge Albien.

WNT Signaling in Drosophila Neuromuscular Junctions

The Wnt -Wingless (Wg) in Drosophila- signaling is an evolutionary conserved, fundamental signal transduction pathway in animals, having a crucial role in early developmental processes. In the adult animal the Wnt cascade is mainly shut off; aberrant activation leads to cancer. One physiological exception in the adult animal is the activation of Wnt signaling in the nervous system. In the present work, we investigated Wg signaling in the Drosophila neuromuscular junctions (NMJs). The fly NMJs closely resemble the glutamatergic synapses in the mammalian central nervous system and serves as a model system to investigate the mechanism of synapse formation and stability. We demonstrate that the trimeric G-protein Go has a fundamental role in the presynaptic cell in the NMJ. It is implicated in the presynaptic Wg pathway, acting downstream of the ligand Wg and its receptor Frizzled2 (Fz2). Furthermore, we prove that the presynaptic Wg-Fz2-Gαo pathway is essential for correct NMJ formation. The neuronal protein Ankyrin2 (Ank2) localizes to the NMJ and has so far been considered to be a static player in NMJ formation, linking the plasma membrane to the cytoskeleton. We identify Ank2 as a direct target of Gαo. The physical and genetic interaction of Gαo with Ank2 represents a novel branch of the presynaptic Wg pathway, regulating the microtubule cytoskeleton in NMJ formation, jointly with the previously established Futsch-dependent branch, which controls microtubule stability downstream of the kinase Sgg (the homolog of GSK3ß). We moreover demonstrate that the Gαo-Ankyrin interaction to regulate the cytoskeleton is conserved in mammalian neuronal cells. Our findings therefore provide a novel, universally valid regulation of the cytoskeleton in the nervous system. Aberrant inactivation of the neuronal Wnt pathway is believed to be involved in the pathogenesis of the Aß peptide in Alzheimer's disease (AD). We modeled AD in Drosophila by expressing Aß42 in the nervous system and in the eye. Neuronal expression drastically shortens the life span of the flies. We prove that this effect depends on the expression specifically in glutamatergic neurons. However, Aß42 does not induce any morphological changes in the NMJ; therefore this synapse is not suitable to study the mechanism of Aß42 induced neurotoxicity. We furthermore demonstrate that genetic activation of the Wnt pathway does not rescue the Aß42 induced phenotypes - in opposition to the dominating view in the field. These results advice caution when interpreting data on the potential interaction of Wnt signaling and AD in other models. -- La voie de signalisation Wnt (Wingless (Wg) chez la drosophile) est conservée dans l'évolution et fondamentale pour le développement des animaux. Cette signalisation est normalement inactive chez l'animal adulte; une activation anormale peut provoquer le cancer. Or, ceci n'est pas le cas dans le système nerveux des adultes. La présente thèse avait pour but d'analyser le rôle de la voie de signalisation Wingless dans la plaque motrice de Drosophila melanogaster. En effet, cette plaque ressemble fortement aux synapses glutaminergiques du système nerveux central des mammifères et procure ainsi un bon modèle pour l'étude des mécanismes impliqués dans la formation et la stabilisation des synapses. Nos résultats montrent que la protéine trimérique Go joue un rôle fondamental dans la fonction de la cellule présynaptique de la plaque motrice. Go est en effet impliqué dans la voie de signalisation Wg, opérant en aval du ligand Wg et de son récepteur Frizzled2. Nous avons pu démontrer que cette voie de signalisation Wg-Fz2-Gαo est essentielle pour le bon développement et le fonctionnement de la plaque motrice. Fait intéressant, nous avons montré que la protéine neuronale Ankyrin2 (Ank2), qui est connue pour jouer un rôle statique en liant la membrane plasmique au cytosquelette dans la plaque motrice, est une cible directe de Gαo. L'interaction physique et génétique entre Gαo et Ank2 constitue ainsi une bifurcation de la voie de signalisation présynaptique Wg. Cette voie régule le cytosquelette des microtubules en coopération avec la branche liée à la protéine Futsch. Cette protéine est l'homologue de la protéine liant les microtubules MAP1B des mammifères et contrôle la stabilité des microtubules opérant en aval de la kinase Sgg (l'homologue de GSK3ß). De plus, la régulation du cytosquelette par l'interaction entre Gαo et Ankyrin est conservée chez les mammifères. Dans leur ensemble, nos résultats ont permis d'identifier un nouveau mode de régulation du cytosquelette dans le système nerveux, probablement valable de manière universelle. La voie de signalisation Wnt est soupçonnée d'être impliquée dans la toxicité provoquée par le peptide Aß dans le cadre de la maladie d'Alzheimer. Nous avons tenté de modéliser la maladie chez la drosophile en exprimant Aß42 spécifiquement dans le cerveau. Cette expérience a montré que l'expression neuronale d'Aß42 réduit la durée de vie des mouches de manière significative par un mécanisme impliquant les cellules glutamatergiques. Par contre, aucune modification morphologique n'est provoquée par Aß42 dans les plaques motrices glutamatergiques. Ces résultats montrent que ce modèle de Drosophile n'est pas adéquat pour l'étude de la maladie d'Alzheimer. De plus, l'activation génétique de la voie de signalisation Wg n'a pas réussi à restaurer les phénotypes de survie ou ceux des yeux causés par Aß42. Ces résultats indiquent que l'implication de la voie de signalisation Wg dans la maladie d'Alzheimer doit être considérée avec prudence.

Evidence for middle Cretaceous accretion at Santa Elena Peninsula (Santa Rosa Accretionary Complex), Costa Rica

An oceanic assemblage of alkaline basalts, radiolarites and polymictic breccias forms the tectonic substratum of the Santa Elena Nappe, which is constituted by extensive outcrops of ultramafic and mafic rocks of the Santa Elena Peninsula (NW Costa Rica). The undulating basal contact of this nappe defines several half-windows along the south shores of the Santa Elena Peninsula. Lithologically it is constituted by vesicular pillowed and massive alkaline basaltic flows, alkaline sills, ribbon-bedded and knobby radiolarites, muddy tuffaceous and detrital turbidites, debris flows and polymictic breccias and megabreccias. Sediments and basalt flows show predominant subvertical dips and occur in packages separated by roughly bed-parallel thrust planes. Individual packages reveal a coherent internal stratigraphy that records younging to the east in all packages and shows rapid coarsening upwards of the detrital facies. Alkaline basalt flows, pillow breccias and sills within radiolarite successions are genetically related to a mid-Cretaceous submarine seamount. Detrital sedimentary facies range form distal turbidites to proximal debris flows and culminate in megabreccias related to collapse and mass wasting in an accretionary prism. According to radiolarian dating, bedded radiolarites and soft-sediment- deformed clasts in the megabreccias formed in a short, late Aptian to Cenomanian time interval. Middle Jurassic to Lower Cretaceous radiolarian ages are found in clasts and blocks reworked from an older oceanic basement. We conclude that the oceanic assemblage beneath the Santa Elena Nappe does not represent a continuous stratigraphic succession. It is a pile of individual thrust sheets constituting an accretionary sequence, where intrusion and extrusion of alkaline basalts, sedimentation of radiolarites, turbidites and trench fill chaotic sediments occurred during the Aptian-Cenomanian. These thrust sheets formed shortly before the off-scraping and accretion of the complex. Here we define the Santa Rosa Accretionary Complex and propose a new hypothesis not considered in former interpretations. This hypothesis would be the basis for further research.

3

The hematopoietic stem cell (HSC) is probably the best characterized somatic stem cell and is still the only one regularly used in clinical practice. Nevertheless, expansion of HSCs in vitro has been surprisingly unsuccessful, limiting their full therapeutic potential. During homeostasis, the vast majority of HSCs are found in the bone marrow (BM) localized to specific microenvironments called stem cell "niches." Over the last few years our knowledge of cellular niche components and the signaling molecules that coordinate the crosstalk between HSCs and niche cells has dramatically increased. Here we review the two main niche types found in the BM: the endosteal and the vascular niches, and provide an overview of the different signaling and cell adhesion molecules that form the HSC-niche synapse. Signals from BM niches not only control HSC dormancy, but also regulate the balance between self-renewal and differentiation. In the future, successful expansion of HSCs for therapeutic use will require three-dimensional reconstruction of a stem cell-niche unit.