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38documents trouvés

18179
23/05/2018

Simple Synthetic Molecular Hydrogels from Self- Assembling Alkylgalactonamides as Scaffold for 3D Neuronal Cell Growth

A.CHALARD, L.VAYSSE, P.JOSEPH, L.MALAQUIN, S.ASSIE-SOULEILLE, B.LONETTI, J.C.SOL, I.LOUBINOUX, J.FITREMANN

MILE, INSERM, ELIA, I2C, IMRCP

Revue Scientifique : ACS applied materials & interfaces, Vol.10, N°20, pp.17004-17017, Mai 2018 , N° 18179

Lien : https://hal.laas.fr/hal-01810656

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Abstract

In this work, we demonstrated that the hydrogel obtained from a very simple and single synthetic molecule, N-heptyl-galactonamide was a suitable scaffold for the growth of neuronal cells in 3D. We evidenced by confocal microscopy the presence of the cells into the gel up to a depth of around 200 µm, demonstrating that the latter was permissive to cell growth and enabled a true 3D colonization and organization. It also supported successfully the differentiation of adult human neuronal stem cells (hNSCs) into both glial and neuronal cells and the development of a really dense neurofilament network. So the gel appears to be a good candidate for neural tissue regeneration. In contrast with other molecular gels described for cell culture, the molecule can be obtained at the gram scale by a one-step reaction. The resulting gel is very soft, a quality in accordance with the aim of growing neuronal cells, that requires low modulus substrates similar to the brain. But because of its fragility, specific procedures had to be implemented for its preparation and for cell labeling and confocal microscopy observations. Notably, the implementation of a controlled slow cooling of the gel solution was needed to get a very soft but nevertheless cohesive gel. In these conditions, very wide straight and long micrometric fibers were formed, held together by a second network of flexible narrower nanometric fibers. The two kinds of fibers guided the neurite and glial cell growth in a different way. We also underlined the importance of a tiny difference in the molecular structure on the gel performances: parent molecules, differing by a one-carbon increment in the alkyl chain length, N-hexyl-galactonamide and N-octyl-galactonamide, were not as good as N-heptyl-galactonamide. Their differences were analysed in terms of gel fibers morphology, mechanical properties, solubility, chain parity and cell growth.

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18104
13/03/2018

Bone degradation machinery of osteoclasts: An HIV-1 target that contributes to bone loss

B.RAYNAUD-MESSINA, L.BRACQ, M.DUPONT, S.SOURIANT, S.M.USMANI, A.PROAG, K.PINGRIS, V.SOLDAN, C.THIBAULT, F.CAPILLA, T.AL SAATI, I.GENNERO, P.JURDIC, P.JOLICOEUR, J.L.DAVIGNON, T.R.MEMPEL, S.BENICHOU, I.MARIDONNEAU-PARINI, C.VEROLLET

IPBS, INSERM, Paris, Harvard Medical Sch, Multiscale Electron, ELIA, INSERM, Centre de Physiopathologie, ENS Lyon, IRCM, Montreal

Revue Scientifique : Proceedings of the National Academy of Sciences, Vol.115, N°11, pp.E2556-E2565, Mars 2018 , N° 18104

Lien : https://hal.laas.fr/hal-01764831

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Abstract

Bone deficits are frequent in HIV-1–infected patients. We report here that osteoclasts, the cells specialized in bone resorption, are infected by HIV-1 in vivo in humanized mice and ex vivo in human joint biopsies. In vitro, infection of human osteoclasts occurs at different stages of osteoclastogenesis via cell-free viruses and, more efficiently, by transfer from infected T cells. HIV-1 infection markedly enhances adhesion and osteolytic activity of human osteoclasts by modifying the structure and function of the sealing zone, the osteoclast-specific bone degradation machinery. Indeed, the sealing zone is broader due to F-actin enrichment of its basal units (i.e., the podosomes). The viral protein Nef is involved in all HIV-1–induced effects partly through the activation of Src, a regulator of podosomes and of their assembly as a sealing zone. Supporting these results, Nef-transgenic mice exhibit an increased osteoclast density and bone defects, and osteoclasts derived from these animals display high osteolytic activity. Altogether, our study evidences osteoclasts as host cells for HIV-1 and their pathological contribution to bone disorders induced by this virus, in part via Nef.

143360
18022
27/02/2018

Two-photon lithography and microscopy of 3D hydrogel scaffolds for neuronal cell growth

A.ACCARDO, MC.BLATCHE, R.COURSON, I.LOUBINOUX, C.VIEU, L.MALAQUIN

ELIA, I2C, TEAM, INSERM

Revue Scientifique : Biomedical Physics & Engineering Express, Vol.4, N°2, 027009p., Février 2018 , N° 18022

Lien : https://hal.laas.fr/hal-01698486

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3D fabrication techniques are rapidly expanding in the field of scaffold development for cell culture and tissue engineering. Herein we report the realization of free-standing PEGDA hydrogel architectures by using two-photon lithography. The morphological and immunofluorescence characterization of neuro2A cells revealed a tridimensional colonization featuring multiple neuritic extensions per cell as well as the expression of β-tubulin neuronal marker and actin microfilaments. The results open new perspectives in the continuous quest for structured biomaterials able to provide a favorable environment to cells and at the same time not interfering with imaging protocols necessary for a clear scenario of the cell seeding.

142468
18008
29/01/2018

On-chip conductometric detection of short DNA sequences via electro-hydrodynamic aggregation

B.VENZAC, M.DIAKITE, D.HERTHNEK, I.CISSE, U.BOCKELMANN, S.DESCROIX, L.MALAQUIN, J.L.VIOVY

UPMC, Univ of Stockholm, ESPCI, ELIA

Revue Scientifique : The Analyst, Vol.143, N°1, pp.190-199, Janvier 2018 , N° 18008

Lien : http://hal.upmc.fr/hal-01679664

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Abstract

Fluorescence measurement is the main technology for post-amplification DNA detection in automated systems. Direct electrical reading of DNA concentration in solution could be an interesting alternative to go toward more miniaturized or less expensive devices, in particular in the pathogen detection field. Here we present the detection of short bacterial biomarkers with a direct impedancemetric measurement, within solutions of amplified and elongated DNA sequences in a microchannel. This technology relies on the electrohydrodynamic instability occurring in solutions of long charged macromolecules in a strong electric field. This instability specifically induces the aggregation of long DNAs and triggers conductivity variations that can be monitored by on-contact conductometry. An innovative isothermal amplification and elongation strategy was developed, combining SDA and HRCA reactions, in order to yield long DNAs suitable to be detected by the above principle, from a dilute initial DNA target. In contrast with previous label-free detection methods, this new strategy is very robust to matrix effects, thanks to the unique molecular weight dependence of the instability, coupled with this specific DNA amplification strategy. We demonstrate the detection of a 1 pM gene sequence specific to Staphylococcus aureus, in a portable system.

142183
17397
01/01/2018

Fabrication of biomolecule microarrays for cell immobilization using automated microcontact printing

J.FONCY, A.ESTEVE, A.DEGACHE, C.COLIN, J.C.CAU, L.MALAQUIN, C.VIEU, E.TREVISIOL

ELIA, IMS Bordeaux, LPN, INNOPSYS

Ouvrage (contribution) : Cell-based microarrays: methods and protocols, Springer, Janvier 2018, pp.83-95 , N° 17397

Lien : https://hal.laas.fr/hal-01613897

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Abstract

Biomolecule microarrays are generally produced by conventional microarrayer i.e. by contact or inkjet printing. Microcontact printing represents an alternative way of deposition of biomolecules on solid supports but even if various biomolecules have been successfully microcontact printed, the production of biomolecule microarrays in routine by microcontact printing remains a challenging task and needs an effective, fast, robust and low-cost automation process. Here, we describe the production of biomolecule microarrays composed of extracellular matrix protein for the fabrication of cell microarrays by using an automated microcontact printing device. Large scale cell microarrays can be reproducibly obtained by this method

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17437
08/12/2017

Cell biology of microbes and pharmacology of antimicrobial drugs explored by Atomic Force Microscopy

C.FORMOSA, R.DUVAL, E.DAGUE

SRSMC, ELIA

Revue Scientifique : Seminars in Cell & Developmental Biology, 12p., Décembre 2017 , N° 17437

Lien : https://hal.univ-lorraine.fr/hal-01651186

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Abstract

Antimicrobial molecules have been used for more than 50 years now and are the basis of modern medicine. No surgery can nowdays be imagined to be performed without antibiotics; dreadful diseases like tuberculosis, leprosis, siphilys, and more broadly all microbial induced diseases, can be cured only through the use of antimicrobial treatments. However, the situation is becoming more and more complex because of the ability of microbes to adapt, develop, acquire, and share mechanisms of resistance to antimicrobial agents. We choose to introduce this review by briefly drawing the panorama of antimicrobial discovery and development, but also of the emergence of microbial resistance. Then we describe how Atomic Force Microscopy (AFM) can be used to provide a better understanding of the mechanisms of action of these drugs at the nanoscale level on microbial interfaces. In this section, we will address these questions: (1) how does drug treatment affect the morphology of single microbes?; (2) do antimicrobial molecules modify the nanomechanical properties of microbes, or do the nanomechanical properties of microbes play a role in antimicrobial activity and efficiency?; and (3) how are the adhesive abilitites of microbes affected by antimicrobial drugs treatment? Finally, in a second part of this review we focus on recent studies aimed at changing the paradigm of the single molecule/cell technology that AFM typically represents. Recent work dealing with the creation of a microbe array which can be explored by AFM will be presented, as these developments constitute the first steps toward transforming AFM into a higher throughput technology. We also discuss papers using AFM as NanoMechnanicalSensors (NEMS), and demonstrate the interest of such approaches in clinical microbiology to detect quickly and with high accuracy microbial resistance.

141700
17385
05/12/2017

Regenerative potential of primary adult human neural stem cells on micropatterned bioimplants boosts motor recovery

C.DAVOUST, B.PLAS, A.BEDUER, B.DEMAIN, ASALABERT, J.C.SOL, C.VIEU, L.VAYSSE, I.LOUBINOUX

INSERM, EXT, UPS, ELIA

Revue Scientifique : Stem Cell Research & Therapy, art ID 253, Vol.8, Décembre 2017 , N° 17385

Lien : https://hal.laas.fr/hal-01618948

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Abstract

Background: The adult brain is unable to regenerate itself sufficiently after large injuries . Therefore, hopes rely o n therapies using neural stem cell or biomaterial transplantation to sustain brain reconstruction. The aim of the present study was to evaluate the improvement in sensorimotor recovery brought about by human p rimary adult NSCs in combination with bio implant s . Methods: hNSCs were pre - seeded on implants micropatterned for neurite guidance and inserted intracerebrally 2 weeks after a primary motor cortex lesion in rats . Long - term behavio u r was significantly improved after hNSC - Implants versus cell engraf tment in the grip strength test. MRI and immunohistological studies were conducted to elucidate the underlying mechanisms of neuro implants integration . Results: hNSC - I mplants promoted tissue reconstruction and limited hemispheric atrophy and glial scar expansion. After 3 months , grafted hNSCs were detected on implants and expressed mature neuron al markers (NeuN, MAP2, and SMI 312). They also migrated over a short distance to the reconstructed and to the perilesional tissue s , where 26% integrated as mature neurons. Newly formed host neural progenitors ( n e stin, DCX) colonized the implants, notably in the presence of hNSCs, and participated to tissue reconstruction. The microstructured bioimplants sustain ed the guided maturation of both grafted hNSCs and endo genous progenitors. Conclusions: t hese immunohistological results are coherent with and could explain the late improvement observed in sensorimotor recovery. These findings provide novel insight s into the regenerative potential of primary adult hNSCs combined with microstructure d implants .

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17484
01/12/2017

Colon cancer cells adhesion on polymeric nanostructured surfaces

A.ACCARDO, V.SHALABAEVA, R.LA ROCCA

ELIA, LCC

Revue Scientifique : MRS Communications, Décembre 2017 , N° 17484

Lien : https://hal.laas.fr/hal-01675463

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Abstract

n this work, we report on the adhesion of HCT116 (human colon carcinoma cells) cultured on nanofibrillar polymethylmethacrylate (PMMA) and SU-8 micropillars substrates. Both surfaces enabled a good cell proliferation and promoted the formation of adherent interconnections with the fabricated nano- and microstructures. The three-dimensional immunofluorescence confocal characterization of the cells on nanotextured PMMA highlighted the expression of well-spread F-actin cytoskeletal networks as well as the presence of focal adhesions. This study provides thus interesting perspectives for further investigations on the force/adhesion mechanisms related to cancer cell growth and proliferation.

142075
17452
28/11/2017

Functionalized double-walled carbon nanotubes for integrated gas sensors

L.YANG

ELIA

Doctorat : Université de Toulouse III - Paul Sabatier, 28 Novembre 2017, 145p., Président: K.ARAUJO-GUERIN, Rapporteurs: C.DEJOUS, M.ARAB, Examinateurs: G.VIAU, Directeurs de thèse: V.VIEU, E.FLAHAUT , N° 17452

Lien : https://hal.laas.fr/tel-01675535

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Résumé

Nous proposons dans ce travail une méthode robuste et bas-coût afin de fabriquer des détecteurs de gaz à base de Nanotubes de Carbone bi-parois (DWCNTs) chimiquement fonctionnalisés. Ces nano-objets (DWCNTs) sont synthétisés par dépôt catalytique en phase vapeur (CCVD), puis purifiés avant d’être oxydés ou bien fonctionnalisés par des terminaisons fluorées ou aminées. Les dispositifs de détection électriques ont été fabriqués par lithographie douce en utilisant un pochoir de PDMS (Poly-DiMethyl Siloxane) et un dépôt en phase liquide à la pipette d’une suspension aqueuse contenant les nanotubes fonctionnalisés, rinçage puis séchage à l’azote sec. Chaque dispositif (1 cm X 2 cm) est équipé d’un jeu de 7 résistors à base de DWCNTs. Chaque résistor peut accueillir des nanotubes fonctionnalisés par une entité chimique différente afin de cibler un gaz spécifique, permettant ainsi une détection multiplexée. En raison de leur faible encombrement et la possibilité de les fabriquer sur tout type de substrat y compris des substrats souples, ces détecteurs pourraient être utilisés pour une large gamme d’applications et notamment les détecteurs de gaz portatifs et intégrés. La résistance électrique des résistors s’avère décroître avec la température suggérant une conduction électrique gouvernée par l’effet tunnel et les fluctuations au sein du tapis désordonné de nanotubes de carbone. Nous avons cependant montré dans ce travail que pour des applications réelles de détection de gaz, une régulation thermique des dispositifs n’est pas nécessaire car les variations de résistance engendrées par l’adsorption de molécules de gaz sont significativement plus grandes que les variations causées par de possibles fluctuations de température. Les dispositifs produits présentent un caractère métallique à température ambiante et pour des applications de détection de gaz nous avons sélectionné des dispositifs présentant des résistances inférieures à 100 kΩ. Le principe de base de la détection de gaz étant basé sur la mesure directe de la résistance électrique du dispositif, la consommation électrique de ces dispositifs reste faible (<1 μW). La réponse des dispositifs à base de nanotubes de carbone non fonctionnalisés aux analytes testés (éthanol, acétone, ammoniac et vapeur d’eau) est faible. Les nanotubes de carbone fonctionnalisés présentent quant à eux, une réponse modérée à la vapeur d’eau, à l’éthanol et à l’acétone mais montrent une sensibilité excellente à l’ammoniac. En particulier, les nanotubes de carbone oxydés se sont avérés capables de détecter des concentrations sub-ppm d’ammoniac en présence de vapeur d’eau en excès et à température ambiante et ont montré une grande stabilité dans le temps même pour des expositions de gaz répétées. Nous pensons que les groupes chimiques fonctionnels ancrés à la surface des nanotubes de carbone modifient les interactions entre les molécules de gaz et les nanotubes et que le transfert de charges induit provoque les modifications de la conductance électrique du système. Nous avons construit un modèle phénoménologique pour analyser les réponses électriques de nos dispositifs lors de l’exposition d’un gaz. Ce modèle prend en compte une variation exponentielle de la résistance au cours du temps puis un régime d’accroissement linéaire de cette résistance. Nous montrons en particulier que la constante de temps extraite du régime exponentiel est très informative sur la sensibilité et la sélectivité du détecteur de gaz. Nous avons finalement testé nos dispositifs pour des applications représentatives comme par exemple la détection de traces d’ammoniac qui ont pu être aisément réalisées à des concentrations bien inférieures au seuil de détection du nez humain (0.04ppm). En raison de leur grande stabilité, facilité de fabrication (design très simple, technologies de fabrication bas coût, intégration sur substrats souples), robustesse (détection possible en présence de vapeur d’eau et résiliente aux fluctuations thermiques) et en raison de la faible quantité de nanotubes de Carbone nécessaire, nous pensons que nos résultats sont intéressants pour des applications de masse concernant des détecteurs de gaz portables pour l’industrie des technologies de l’information et de la communication.

Abstract

We have successfully fabricated gas sensors based on chemically functionalized double-wall carbon nanotubes (DWCNTs) using a robust and low cost process. The DWCNTs were synthesized by catalytic chemical vapor deposition (CCVD) method. They were then purified before functionalization (oxidation, amination, and fluorination). The sensor devices were fabricated by soft lithography using PDMS (Poly-DiMethylSiloxane) stencils and liquid phase pipetting of a suspension of chemically functionalized DWCNTs in deionized water, rinsing and finally drying in a nitrogen flow. Each device (1 cm x 2 cm) is equipped with a set of 7 DWCNT based resistors. Each resistor can accommodate a precise chemical functionalization for targeting a specific gas species, allowing a multiplexed (up to 7) detection. Due to their small size and the possibility to fabricate them on soft substrates, they could be used for many kinds of applications including wearable devices. The electrical resistance of the produced resistors turned out to decrease with temperature, suggesting fluctuations induced tunneling conduction through the disordered network of metallic nanotubes. However, we have shown in our work that for realistic applications, gas sensing can be achieved without any temperature regulation of our devices, because the variations of electrical conductance caused by gas molecules adsorption are significantly larger than those caused by possible temperature fluctuations. The as fabricated devices exhibit at room temperature a metallic conducting behavior. Devices with a resistance less than 100 kΩ were selected for gas detection. Because the sensing principle is based on the direct measurement of the resistance, our scheme ensures low power consumption (<1 μW). Raw (not functionalized) DWCNTs-based gas sensors exhibited a low sensitivity to the tested analytes, including ethanol, acetone, ammonia and water vapor. Functionalized DWCNTs-based gas sensors exhibited a moderate sensitivity to ethanol, acetone and water vapor but the response to ammonia, even in the presence of additional water vapor, was excellent. In particular, oxidized DWCNTs based gas sensors exhibited a high stability in the case of prolonged and repeated gas exposures. The oxidized DWCNTs gas sensors were also able to detect ammonia vapor at sub-ppm concentration in the presence of water vapor at high concentration. We believe that the functional groups grafted to the DWCNTs modify the interaction between gas molecules and DWCNTs and that the induced charge transfer is responsible for the modification of the electrical conductivity. We have built a simple phenomenological model for the analysis of the sensing response curve. This model includes two components for the variations of electrical resistance during gas exposure, an exponential regime and a linear one. In particular, the time constant extracted from the exponential part of the response was found to be informative on devices' sensitivity and selectivity. Finally, we tested our sensors for realistic applications such as trace detection of ammonia, which could be easily detected while far below the detection threshold of human nose (0.04ppm) Due to the high stability, ease of fabrication (very simple design, use of low-cost technologies, integration on flexible substrates), robustness (detection in the presence of a large excess of water vapor and resilient to temperature fluctuations) and extremely low amounts of carbon nanotube required, we expect these results to have some potential for a wide range of mass applications in the field of wearable gas sensors for Information and Communication Technologies (ICT) industry.

141773
17594
22/11/2017

Fabrication of versatile calibration samples for 3D super-resolution optical imaging with <5nm Z resolution using greyscale e-beam lithography and Nano-Imprint replication

A.LAUVERGNE, F.CARCENAC, E.DARAN, J.B.DOUCET, C.VIEU

TEAM, ELIA

Manifestation avec acte : Journées Nationales sur les Technologies Emergentes en micronanofabrication ( JNTE ) 2017 du 20 novembre au 22 novembre 2017, Orléans (France), Novembre 2017, 2p. , N° 17594

Lien : https://hal.laas.fr/hal-01724242

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Abstract

We present a 3D mold fabrication process to produce high precision calibration samples with few nanometers steps in the vertical direction. After imprinting into a resist layer of adequate refractive index using nano-imprint lithography, these molds can produce calibration samples valuable for super-resolution Direct Optical Nanoscopy with Axially Localized Detection (DONALD). This novel method is dedicated to the 3D mapping of labeled biomolecules into biological samples by fluorescence and requires calibration samples with well-characterized steps ranging from 5 nm to 125 nm in height.

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