Directing Neural Stem Cell Differentiation Using Nanopatterned Substrates and Visualization of the Developing Nervous System

The development of neural stem cells is regulated by a variety of growth and cell adhesion factors. A promising approach to direct their differentiation in vitro is the generation of cell substrates that replicate the intricate physical and biochemical properties of their cellular environment. The primary aim of this work was to develop cell substrates that mimic these properties and explore their potential in directing the differentiation of embryonic neural stem cells and in supporting axonal outgrowth. For this, gold nanopatterned glass substrates were used for the controlled immobilization of proteins. The resulting substrates feature nanopatterned cell signaling proteins at variable surface density, as well as additional cell adhesive molecules. The role of both factors in regulating the differentiation of mouse embryonic neural stem cells was investigated independently. Differentiation of neural stem cells on substrates uniformly coated with the cell adhesion molecules laminin, fibronectin and N-cadherin showed no effect on the in vitro generation of newborn neurons in comparison to polyornithine controls. Moreover, a 2,5-fold increase in cell number was observed on all cell-adhesion molecules, indicating an increase in cell proliferation. Furhermore, the role of cell adhesion molecules in supporting axonal outgrowth of dorsal root ganglia explants was investigated. The longest axonal projections ranging up to 800µm could be observed on laminin-coated substrates. In contrast, outgrowth on fibronectin as well as fibronectin-derived peptide nanopatterned substrates resulted in 200-250µm long projections. The role of nanopatterned Notch cell receptor ligand Delta-like 1 (Dll-1) substrates in directing the differentiation of neural stem cell cultures was investigated using variable ligand densities. As a result, Notch activation resulted in increased neurite number, branching, and cell body size. The strongest response was observed using 340 ligands/µm2 (56nm interparticle spacing) in comparison to 735 ligand/µm2 (90nm) and uniformly coated Dll-1 substrates. Additionally, a small fraction of drastically enlarged neurons was observed on Dll-1 substrates, but no effect on the total number of newborn neurons. The next aim of this thesis was to develop a system for the non-invasive visualization of the embryonic nervous system. For this, a transgenic mouse line that expresses high levels of green fluorescent protein (GFP) specifically in newborn neurons was characterized. Imaging using light sheet fluorescence microscopy resulted in high- resolution visualization of the entire nervous system in whole embryos allowing for generation of three-dimensional models and virtual specimen sectioning. Additionally, this technique was successfully applied for the visualization of innervation defects caused by mutation of the axonal-guidance protein Semaphorin 3A. In summary, the nanopatterned protein substrates presented in this work constitute a powerful tool for influencing in vitro development of neural stem cells. Additionally, the introduced transgenic mouse featuring GFP-expressing neurons allows for highly detailed visualization of the developing nervous system in whole embryos

Primary cilia are critical for Sonic hedgehog-mediated dopaminergic neurogenesis in the embryonic midbrain

Gazea M, Tasouri E, Tolve M, et al. Primary cilia are critical for Sonic hedgehog-mediated dopaminergic neurogenesis in the embryonic midbrain. Developmental Biology. 2015;409(1):55-71

Antares Neutrino telescope and Deap Sea Observatory,

International audienceThe ANTARES detector consists of a multidisciplinary undersea observatory associated with a neutrino telescope. The neutrino telescope, with 12 mooring lines holding light detectors, was completed in May 2008 and is destined for research in the field of astroparticle physics, in particular in neutrino astronomy

Technical Design Report of the MEUST Infrastructure

MEUST (Mediterranean Eurocentre for Underwater Sciences and Technologies) is a second generation permanent submarine observatory to be deployed offshore of Toulon, France, as a follow up of the pioneering ANTARES neutrino telescope. The MEUST submarine network has a modular topology designed to connect up to 120 neutrino detection units,i.e. ten times more than ANTARES. This may allow to instrument one km3 of water for neutrino astronomy or, with a denser instrumentation, several Megatons for measurement of neutrino properties, and to deploy sensors for environmental sciences on an array of ten km. The topology and functionalities of the network comply with the specifications of the KM3NeT neutrino telescope, which plans to use MEUST as one of its 3 deployment sites, as well as with those of the environmental sensors developed for the Ligurian site of the EMSO submarine observatory network. The technical solutions developed for the MEUST infrastructure are adapted to any large deep sea detector array located within 50 km from the coast. After a brief reminder of the MEUST scientific motivation and submarine sensors, this document details the technical design of the infrastructure and summarizes the organization of the project

BathyBot - a Deep-sea Crawler to See the Unseen in the NW Mediterranean Sea

Ocean Sciences Meeting (OSM), 16-21 February 2020, San Diego, CA, USA.- 1 page, 3 figuresThe deep sea remains one of the less known environment on Earth and is characterized by high pressure, low availability of organic matter and absence of light. While there are still numerous discoveries concerning the diversity and adaptations of deep-sea organisms to their environement, this ecosystem is under an increasing anthropogenic pressure such as climate-related stressors (warming, acidification and deoxygenation), deep-sea fishing, human pollution (microplastics, POP), oil and gas extraction and could face new threats from emerging industries (e.g. mineral mining). How these changes will affect biodiversity and ecosystem functioning is one question of major importance for the future. In the darkness of the oceans, several organisms have the capability to emit light: called bioluminescence. Recent studies quantified that as much as 75% of pelagic and about 40% of benthic organisms are known to be bioluminescent. In this framework, we present a new deep-sea crawler, BathyBot, to be dedicated to the long-term exploration of deep-sea ecosystems allowing biological and-geochemical surveys. BathyBot will be deployed in 2020 in the Mediterranean Sea, at the MEUST-NUMerEnv/KM3NeT site, to strength the ecological-based monitoring capability of the European Multidisciplinary Seafloor and water column Observatory (EMSO ERIC) network. BathyBot will be able to explore an area of about 15 000 m2 at a depth of 2500m and will be devoted to 1) observe and monitor the dynamics of deep-sea pelagic and benthic organisms, 2) better define the occurrence and functions of bioluminescence in situ (increasing the dataset of bioluminescence records), 3) explore relationships between deep-sea organisms, biogeochemical (carbon content, oxygen concentrations) and environmental variables (temperature, salinity, current) in the context of global changes and their effects on the deep ocean, and 4) investigate benthic biogeochemical processes through the use of oxygen microprofiling in sediment porewatersThis work is funded by UE FEDER NUMerEnv project (number: 1166-39417, 2017), CNRS-INSU Moyens Mi-Lourd 2019 and VICAT-Tangram-MIO projectPeer reviewe