Damage Prediction Using Several Types of Macro-scale Damage Models in Different Cold Wire Production Lines

International audienceThe purpose of the present paper is to show how and to what extent the introduction of refined, shear sensitive models improves on previous ones, based on triaxiality only, for the phenomenological description of ductile damage in bulk cold metal forming processes. Wire-drawing and wire rolling are taken as examples. A set of mechanical tests has been conducted: round bar tension, notched bar tension, plane strain tension, and torsion for pure shear deformation. Both constitutive and damage model parameters have been carefully identified, with back-computation of the laboratory tests for validation. Application of the models to the cold forming processes, described here, shows the superiority of the shear-enhanced models for locating maximum damage in flat wire rolling, where a significant amount of shear is present (“blacksmith's cross” deformation pattern). On the contrary, it proves unnecessary for low-shear processes such as wire-drawing. The cavity-growth Gurson-Tvergaard-Needleman model seems to be the best basis for damage prediction in patented high carbon steel, a very ductile material

Scalable Production of AAV Vectors in Orbitally Shaken HEK293 Cells.

Adeno-associated virus (AAV) vectors are currently among the most commonly applied for in vivo gene therapy approaches. The evaluation of vectors during clinical development requires the production of considerable amounts of highly pure and potent vectors. Here, we set up a scalable process for AAV production, using orbitally shaken bioreactors and a fully characterized suspension-adapted cell line, HEKExpress. We conducted a proof-of-concept production of AAV2/8 and AAV2/9 vectors using HEKExpress cells. Furthermore, we compared the production of AAV2/9 vectors using this suspension cell line to classical protocols based on adherent HEK293 cells to demonstrate bioequivalence in vitro and in vivo. Following upstream processing, we purified vectors via gradient centrifugation and immunoaffinity chromatography. The in vitro characterization revealed differences due to the purification method, as well as the transfection protocol and the corresponding HEK293 cell line. The purification method and cell line used also affected in vivo transduction efficiency after bilateral injection of AAV2/9 vectors expressing a GFP reporter fused with a nuclear localization signal (AAV2/9-CBA-nlsGFP) into the striatum of adult mice. These results show that AAV vectors deriving from suspension HEKExpress cells are bioequivalent and may exhibit higher potency than vectors produced with adherent HEK293 cells

Revisiting the outcome of adult wild-type Htt inactivation in the context of HTT-lowering strategies for Huntington's disease.

Huntingtin-lowering strategies are central to therapeutic approaches for Huntington's disease. Recent studies reported the induction of age- and cell type-specific phenotypes by conditional huntingtin knockout, but these experimental conditions did not precisely mimic huntingtin-lowering or gene-editing conditions in terms of the cells targeted and brain distribution, and no transcriptional profiles were provided. Here, we used the adeno-associated delivery system commonly used in CNS gene therapy programmes and the self-inactivating KamiCas9 gene-editing system to investigate the long-term consequences of wild-type mouse huntingtin inactivation in adult neurons and, thus, the feasibility and safety of huntingtin inactivation in these cells. Behavioural and neuropathological analyses and single-nuclei RNA sequencing indicated that huntingtin editing in 77% of striatal neurons and 16% of cortical projecting neurons in adult mice induced no behavioural deficits or cellular toxicity. Single-nuclei RNA sequencing in 11.5-month-old animals showed that huntingtin inactivation did not alter striatal-cell profiles or proportions. Few differentially expressed genes were identified and Augur analysis confirmed an extremely limited response to huntingtin inactivation in all cell types. Our results therefore indicate that wild-type huntingtin inactivation in adult striatal and projection neurons is well tolerated in the long term

Limitations of dual-sgRNA CRISPR strategies for the treatment of CNS genetic disorders

International audienc

The Self-Inactivating KamiCas9 System for the Editing of CNS Disease Genes.

Neurodegenerative disorders are a major public health problem because of the high frequency of these diseases. Genome editing with the CRISPR/Cas9 system is making it possible to modify the sequence of genes linked to these disorders. We designed the KamiCas9 self-inactivating editing system to achieve transient expression of the Cas9 protein and high editing efficiency. In the first application, the gene responsible for Huntington's disease (HD) was targeted in adult mouse neuronal and glial cells. Mutant huntingtin (HTT) was efficiently inactivated in mouse models of HD, leading to an improvement in key markers of the disease. Sequencing of potential off-targets with the constitutive Cas9 system in differentiated human iPSC revealed a very low incidence with only one site above background level. This off-target frequency was significantly reduced with the KamiCas9 system. These results demonstrate the potential of the self-inactivating CRISPR/Cas9 editing for applications in the context of neurodegenerative diseases

Therapeutic efficacy of regulable GDNF expression for Huntington's and Parkinson's disease by a high-induction, background-free "GeneSwitch" vector.

Gene therapy is currently an irreversible approach, without possibilities to fine-tune or halt the expression of a therapeutic gene product. Especially when expressing neurotrophic factors to treat neurodegenerative disorders, options to regulate transgene expression levels might be beneficial. We thus developed an advanced single-genome inducible AAV vector for expression of GDNF, under control of the approved small molecule drug mifepristone. In the rat brain, GDNF expression can be induced over a wide range up to three hundred-fold over endogenous background, and completely returns to baseline within 3-4 weeks. When applied with appropriate serotype and titre, the vector is absolutely free of any non-induced background expression. In the BACHD model of Huntington's disease we demonstrate that the vector can be kept in a continuous ON-state for extended periods of time. In a model of Parkinson's disease we demonstrate that repeated short-term expression of GDNF restores motor capabilities in 6-OHDA-lesioned rats. We also report on sex-dependent pharmacodynamics of mifepristone in the rodent brain. Taken together, we show that wide-range and high-level induction, background-free, fully reversible and therapeutically active GDNF expression can be achieved under tight pharmacological control by this novel AAV - "Gene Switch" vector

Narration interactive ludique : les jeunes lecteurs se réapproprient la culture populaire sous forme de persona-fictions

En produisant une fiction de sa vie, l’homme évolue dans un entre-deux fantasmatique où se construit une identité narrative qui lui permet d’être présent à lui-même. Dans le cadre d’un jeu de rôle sur forum, écrire, jour après jour, la narration d’événements impliquant des personnages imaginés sur la base de récits littéraires populaires, et incarnés dans des récits de vie imaginée, sorte de persona-fiction, c’est, là aussi, se poser comme sujet en construction, vouloir donner à comprendre, à soi et aux autres, la psychologie de son personnage, avancer dans son histoire, intégrer les événements, et par là même, construire son identité

Les suffixes toponymiques français : atlas et base de données

Not availableL'étude des suffixes toponymiques occupe une place insignifiante dans les recherches toponymiques. Mais que recouvre donc la notion de "suffixe" ? En principe, les toponymes retenus sont constitués d'un segment de base suivi d'un autre élément qui se suffit à lui-même et ainsi appelé "suffixe", mais quelquefois la frontière est ténue car certains suffixes s'apparentent à des substantifs et ont une signification propre en dehors du terme auxquels ils se rattachent, c'est notamment le cas du suffixe déterminatif -olalos signifiant "clairière" en gaulois. Notre propos porte sur les communes de France ainsi que sur les hameaux et lieux-dits lorsqu'ils sont significatifs et apportent à notre recherche une précision supplémentaire. Cette thèse se matérialise sous forme de trois volumes : - une base de données - un atlas - un volume explicatif. Nous nous sommes attachés à inventorier dans une base de données les noms des lieux français suffixés en -(i)acus répondant à ces critères. Nous en avons dénombré 1545, cette base de données se veut avant tout un outil de travail pour les chercheurs, elle n'est en aucune mesure exhaustive, et peut, à tout moment être enrichie de nouveaux toponymes. La rédaction et la composition de l'atlas par suffixes obéissent à plusieurs motivations : créer un ouvrage permettant d'avoir des repères dans l'espace et une visualisation des noms de lieux suffixés. Tenter de synthétiser des connaissances éparses concernant les suffixes toponymiques et par la présentation cartographique, les rendre plus proches de notre vécu. Le volume explicatif, quant à lui, se propose de recenser les principaux suffixes toponymiques et d'expliquer leurs fonctionnements sous divers éclairages : étymologiques, sémantiques, syntagmatiques... L'onomastique n'est pas une science sclérosée, l'influence dialectale y est prépondérante dans l'évolution des formes, ainsi ce travail vivra par les remarques que ne manqueront pas de faire les onomasticiens et les dialectologues, observations qui nous permettront soit de compléter notre information, soit de redresser des erreurs d'interprétations